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  • A Halotolerant Endo-1,4-β-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-21
    Thiago Machado Pasin, José Carlos Santos Salgado, Ana Sílvia de Almeida Scarcella, Tássio Brito de Oliveira, Rosymar Coutinho de Lucas, Mariana Cereia, José César Rosa, Richard John Ward, Marcos Silveira Buckeridge, Maria de Lourdes Teixeira de Moraes Polizeli

    Abstract The use of non-potable water (such as seawater) is an attractive alternative for water intensive processes such as biomass pretreatment and saccharification steps in the production of biochemicals and biofuels. Identification and application of halotolerant enzymes compatible with high-salt conditions may reduce the energy needed for non-potable water treatment and decrease waste treatment costs. Here we present the biochemical properties of a halotolerant endo-1,4-β-xylanase produced by Aspergillus clavatus in submerged fermentation, using paper sludge (XPS) and sugarcane bagasse (XSCB), and its potential application in the hydrolysis of agroindustrial residues. The peptide mass fingerprint and amino acid sequencing of the XPS and XSCB enzymes showed primary structure similarities with an endo-1,4-β-xylanase from Aspergillus clavatus (XYNA_ASPCL). Both enzyme preparations presented good thermal stability at 50 °C and were stable over a wide range of pH and Vmax up to 2450 U/mg for XPS. XPS and XSCB were almost fully stable even after 24 h of incubation in the presence of up to 3 M NaCl, and their activity were not affected by 500 mM NaCl. Both enzyme preparations were capable of hydrolyzing paper sludge and sugarcane bagasse to release reducing sugars. These characteristics make this xylanase attractive to be used in the hydrolysis of biomass, particularly with brackish water or seawater.

    更新日期:2020-01-21
  • Glycerol Conversion to Lactic Acid with Unsupported Copper Salts and Bulk Cupric Oxide in Aqueous Alkali Media
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-21
    Kuo-Tseng Li, Hsin-Huey Li

    Abstract Glycerol conversion to lactic acid (LA) was investigated in aqueous alkali over eight unsupported copper compounds (CuBr2, CuBr, CuCl2, CuCl, CuF2, Cu(NO3)2,CuO, and Cu2O) for studying the effects of anion and valence. Powder X-ray diffraction and scanning electron microscopy measurements indicated that these copper compounds were reduced to metallic copper with different morphologies. Divalent copper compounds exhibited much better performances than the corresponding univalent species, ascribed to their greater reduction heat and higher local reaction temperature. Divalent copper species activity, ionic radius, and the reported reduction potential decreased in the same order: bromide > chloride > floride ≫ nitrate. With increasing reaction temperature, catalyst amount, NaOH concentration and reaction time, glycerol conversion, and LA selectivity increased (due to by-product conversions to LA). Kinetic studies indicated that glycerol disappearance rate was first-order with respect to its concentration. CuBr2 had greater activation energy and therefore exhibited better performance than CuO when reaction temperature was greater than 155 °C. At 185 °C, CuBr2 reached 95.7% lactic acid yield and 98.65% glycerol conversion.

    更新日期:2020-01-21
  • Homocysteine and Asymmetrical Dimethylarginine in Diabetic Rats Treated with Docosahexaenoic Acid–Loaded Zinc Oxide Nanoparticles
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-21
    Jihan Hussein, Mehrez El-Naggar, Ehsan Badawy, Nabila El-laithy, Maha El-Waseef, Hanan Hassan, Yasmin Abdel-Latif

    Abstract Hyperglycemia, the hallmark of diabetes mellitus, is considered one of the endothelial dysfunction risk factors, the main reason of vascular complication. In this study, we aimed to evaluate homocysteine (Hcy) and asymmetrical dimethylarginine (ADMA) levels in diabetic rats and the possibility to attenuate the elevation of these two parameters by supplementation of docosahexaenoic acid (DHA) alone or loaded zinc oxide nanoparticles (ZnONPs) to improve endothelial dysfunction in streptozotocin (STZ)-induced diabetic rats. Forty male albino rats weighing 180–200 g were classified as control, diabetic, diabetic treated with DHA, and diabetic treated with DHA-loaded zinc oxide nanoparticles (DHA/ZnONPs) groups. Fasting blood glucose, insulin, ADMA, Hcy, and nitric oxide (NO) were estimated. Fatty acids (linoleic acid (LA), arachidonic acid (AA), DHA, α-linolenic acid (ALA), and oleic acid (OA)) were also evaluated by reversed phase HPLC using a UV detector. The results showed that fasting blood sugar, insulin resistance, LA, AA, OA, ADMA, and Hcy increased significantly in diabetic rats compared with control while fasting insulin, DHA, ALA, and NO decreased significantly in diabetic rats. In both treated groups, fasting blood sugar, insulin resistance, LA, AA, OA, ADMA, and Hcy significantly decreased as compared with the diabetic group while fasting insulin, DHA, ALA, and NO were significantly increased. In conclusion, DHA and DHA/ZnONP supplementation protect against diabetic complications and improve endothelial dysfunction as well as hyperhomocysteinemia in diabetes. DHA/ZnONP-treated group appeared more efficient than DHA alone.

    更新日期:2020-01-21
  • Enhancement of Gluconobacter oxydans Resistance to Lignocellulosic-Derived Inhibitors in Xylonic Acid Production by Overexpressing Thioredoxin
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-20
    Yi Shen, Xin Zhou, Yong Xu

    Abstract Efficient utilization of lignocellulose is an economically relevant practice for improving the financial prospects of biorefineries. Lignocellulose contains significant levels of xylose that can be converted into valuable xylonic acid. However, some inhibitors of bioconversion processes are produced after pretreatment. Xylonic acid production in bacteria, such as Gluconobacter oxydans, is hindered by poor bacterial tolerance to contaminants. Therefore, in order to enhance bacterial resistance to inhibitors, a recombinant strain of G. oxydans was created by the introduction of the thioredoxin gene. Thioredoxin is a key protein responsible for maintaining cellular redox potential and is critical to the conversion of xylose to xylonate. Overexpression of thioredoxin was confirmed at the enzymatic level, while the recombinant strain showed increased catalytic activity when inhibitors, such as formic acid or p-hydroxybenzaldehyde (PHBA), were added to the synthetic xylose medium (17% and 7% improvement in xylonic acid yield, respectively). To probe the molecular mechanism behind the recombinant strain response to inhibitors, the expression levels of various genes were analyzed by qRT-PCR, which revealed five differentially expressed genes (DEGs) upon exposure to formic acid or PHBA.

    更新日期:2020-01-21
  • Isolation, Expression and Characterization of the Thermophilic Recombinant Esterase from Geobacillus thermodenitrificans PS01
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-20
    Po-Ting Chen, Cheng-Huan Liu, Yu-Ting Chen, Fang-Yu Hsu, Jei-Fu Shaw

    Abstract Esterases are widely used in the food industry. Here, a new thermophilic bacterium, Geobacillus thermodenitrificans PS01, was isolated and the esterase-encoding gene est1 was cloned, sequenced, and recombinant expressed in Escherichia coli Tuner (DE3). The highest activity of recombinant Est1 was detected at pH 8.0, and 40 °C and the extreme stability was observed at pH 6–9 over 30 days at 4 °C. In particular, Est1 can hydrolyze short- to medium-chain (C2–C10) triglycerides and p-nitrophenyl esters (C2–C12) and was not inhibited by most metal ions. Kinetic parameters of p-nitrophenyl butyrate hydrolysis under optimal conditions were determined: Km, 22.76 μM; kcat, 10,415 s−1; and kcat/Km, 457.53 μM−1 s−1. The outstanding specification of Est1 indicates its potential for use in industrial applications.

    更新日期:2020-01-21
  • SNHG3 Functions as miRNA Sponge to Promote Breast Cancer Cells Growth Through the Metabolic Reprogramming
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-20
    Yan Li, Zhenhui Zhao, Wei Liu, Xun Li

    Abstract Cancer-associated fibroblasts (CAFs) are important ingredient in tumor microenvironment. The dynamic interplay between CAFs and cancer cells plays essential roles during tumor development and progression. However, the mechanisms of intercellular communication between CAFs and cancer cells remain largely unknown. We characterized exosomes secreted from breast cancer patient-derived CAFs by transmission electron microscopy. The expression of SNHG3, miR-330-5p, and PKM (Pyruvate Kinase M1/M2) was examined by real-time QPCR and immunoblot. The function of SNHG3 on the growth and metabolism of tumor cells was used by CCK8 and mitochondrial oxygen consumption assays. The binding between SNHG3, miR-330-5p, and PKM was examined by dual luciferase reporter assays. Orthotopical xenograft of breast tumor experiments was performed to determine the function of SNHG3 in vivo. We demonstrated that exosomes secreted from CAFs reprogram the metabolic pathways after tumor cells uptake the exosomes. CAF-secreted exosomal lncRNA SNHG3 served as a molecular sponge for miR-330-5p in breast cancer cells. Moreover, PKM could be targeted by miR-330-5p and was controlled by SNHG3 in breast cancer cells. Mechanistically, SNHG3 knockdown in CAF-secreted exosomes suppressed glycolysis metabolism and cell proliferation by the increase of miR-330-5p and decrease of PKM expression in tumor cells. SNHG3 functions as a miR-330-5p sponge to positively regulate PKM expression, inhibit mitochondrial oxidative phosphorylation, increase glycolysis carboxylation, and enhance breast tumor cell proliferation. Overall, SNHG3 could play a major role in the development and progression of breast cancer and support the therapeutic potential of targeting communication between cancer cells and tumor microenvironment.

    更新日期:2020-01-21
  • Low-frequency Ultrasound with Short Application Time Improves Cellulase Activity and Reducing Sugars Release
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-20
    Maria Augusta de Carvalho Silvello, Julian Martínez, Rosana Goldbeck

    Abstract In this study, we investigated the effect of ultrasound (US) on the activity of commercial cellulase (Celluclast® 1.5 L) in the absence and in the presence of a cellulosic substrate (Avicel®, 2% w.v−1) using a central composite rotatable design. Sonication time (10 to 330 s), US intensity (120.6 to 263.7 W cm−2), and reaction temperature (25 to 50 °C) were varied using a horn-type ultrasound reactor, and endoglucanase (CMCase) and total cellulase (FPase) activities were determined. US intensity had a positive effect on enzyme activity. Under optimal conditions (170 s, 180.8 W cm−2, and 25 °C), CMCase activity was 13% higher than that of the control. In the presence of substrate, CMCase activity increased by 33.87% and KM reduced by 23% in relation to that of the control. The theoretical yield of cellulose was 42.08%. Cellulase activity can be improved by US treatment to maximize productivity gains and reduce costs in second-generation ethanol production, by the action of a low-frequency ultrasound with a short ultrasonication time of application.

    更新日期:2020-01-21
  • Removal of Bisphenol A and Its Potential Substitutes by Biodegradation
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-20
    Robert Frankowski, Agnieszka Zgoła-Grześkowiak, Wojciech Smułek, Tomasz Grześkowiak

    Abstract The possibility of removing bisphenol A and its five potential substitutes (bisphenols S, F, AF, E, and B) was tested using microorganism consortia from river water and activated sludge from municipal and rural wastewater treatment plants. For most bisphenols, biodegradation with activated sludge was faster than with river water and a greater extent of biodegradation was also achieved. However, only bisphenol A and bisphenol F underwent 100% primary biodegradation while other bisphenols degraded no more than about 50% which has some important implications in case of their increased usage. Metabolic activity in biodegradation liquors was also tested and it showed higher activity in the tests with activated sludge than with river water. However, there was no clear connection between the decline of metabolic activity and the extent of biodegradation as decreased activity was observed for two easily degrading bisphenols and two others with little biodegradability. It can be assumed that two different phenomena are involved in this process including depletion of nutrients for easily degradable bisphenol A and absence of nutrients for bacteria incapable of primary degradation of bisphenol AF and bisphenol S.

    更新日期:2020-01-21
  • MicroRNA Tough Decoy Knockdowns miR-195 and Represses Hypertrophy in Chondrocytes
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-20
    Pejman Abbasi Pashaki, Fakher Rahim, Mehryar Habibi Roudkenar, SMT Razavi-Toosi, Ammar Ebrahimi

    Abstract Cartilage hypertrophy is a condition in which the cells are completely differentiated, and new morphological changes and mineralization prevent proper cellular functions. The occurrence of hypertrophy during differentiation fails current regenerative strategies for treatment. Strategies to minimize hypertrophy in chondrocytes are categorized into two levels of protein and gene. Among these strategies, one way to affect multiple pathways involved in the development of hypertrophy is to manage microRNA activity in cells. Recent miRNA profiling studies have shown that miR-195-5p upregulates through the transition from chondrogenic to hypertrophic state. Bioinformatics assessment of microRNA targets also indicates that several genes repressed by miR-195-5p play important roles in processes related to hypertrophy. The aim of this study was to develop a microRNA Tough Decoy to suppress miR-195-5p and investigate whether it can prevent a hypertrophic state in chondrocytes. The Tough Decoy (TUD) was designed and evaluated bioinformatically and then cloned into the pLVX-Puro plasmid. The TUD function was validated by Dual-Luciferase assay and qRT-PCR. After delivering TUD to C28/I2 chondrocytes cultured in a hypertrophic medium, hypertrophic differentiation was assessed by histochemical staining, quantitative RT-PCR of hypertrophy marker genes, and alkaline phosphatase activity. Results showed that the TUD could inhibit miRNA efficiently and downregulate hypertrophic markers such as RUNX2, alkaline phosphatase, and collagen 10 significantly compared with the control group. Alcian blue and alizarin red staining also demonstrated the optimal effect of gene constructs on tissue properties and mineralization of the TUD group. Delivering the miR-195-5p Tough Decoy to the cartilage cells can prevent the occurrence of hypertrophy in chondrocytes and could be considered as a candidate for the treatment of other diseases such as osteoarthritis.

    更新日期:2020-01-21
  • Response of Isovalerate-Degrading Methanogenic Microbial Community to Inhibitors
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-17
    Jie Li, Hui-Zhong Wang, Yue Yi, Min Gou, Masaru Konishi Nobu, Ya-Ting Chen, Yue-Qin Tang

    Isovalerate is one of the key intermediates during anaerobic digestion treating protein-containing waste/wastewater. Investigating the effect of different kinds of inhibitors on isovalerate-degrading microbial community is necessary to develop measures for improving the effectiveness of the treatment plants. In the present study, dynamic changes in the isovalerate-degrading microbial community in presence of inhibitors (ammonium, sulfide, mixed ammonium and sulfide, and chlortetracycline (CTC)) were investigated using high-throughput sequencing of 16S rRNA gene. Our observations showed that the isovalerate-degrading microbial community responded differently to different inhibitors and that the isovalerate degradation and gas production were strongly repressed by each inhibitor. We found that sulfide inhibited both isovalerate oxidation followed by methanogenesis, while ammonium, mixed ammonium and sulfide, and CTC mainly inhibited isovalerate oxidation. Genera classified into Proteobacteria and Chloroflexi were less sensitive to inhibitors. The two dominant genera, which are potential syntrophic isovalerate oxidizers, exhibited different responses to inhibitors that the unclassified_Peptococcaceae_3 was more sensitive to inhibitors than the unclassified_Syntrophaceae. Upon comparison to acetoclastic methanogen Methanosaeta, hydrogenotrophic methanogens Methanoculleus and Methanobacterium were less sensitive to inhibitors.

    更新日期:2020-01-17
  • Kinetics Analysis of the Inhibitory Effects of Alpha-Glucosidase and Identification of Compounds from Ganoderma lipsiense Mycelium
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-17
    Tania Maria Costa, Diego Alex Mayer, Diogo Alexandre Siebert, Gustavo Amadeu Micke, Michele Debiasi Alberton, Lorena Benathar Ballod Tavares, Débora de Oliveira

    Abstract The studies on natural compounds to diabetes mellitus treatment have been increasing in recent years. Research suggests that natural components can inhibit alpha-glucosidase activities, an important strategy in the management of blood glucose levels. In this work, for the first time in the literature, the compounds produced by Ganoderma lipsiense extracts were identified and evaluated on the inhibitory effect of these on alpha-glucosidase activity. Four phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) to crude extract from G. lipsiense grown in red rice medium (RCE) and synthetic medium (SCE), being syringic acid identified in both extracts. Gas chromatography-mass spectrometry (GC-MS) analysis showed fatty acids and their derivatives, terpene, steroid, niacin, and nitrogen compounds to SCE, while RCE was rich in fatty acids and their derivatives. Both extracts demonstrated alpha-glucosidase inhibition (RCE IC50 = 0.269 ± 8.25 mg mL−1; SCE IC50 = 0.218 ± 9.67 mg mL−1), and the purified hexane fraction of RCE (RHEX) demonstrated the highest inhibition of enzyme (81.1%). Studies on kinetic inhibition showed competitive inhibition mode to RCE, while SCE showed uncompetitive inhibition mode. Although the inhibitory effects of RCE and SCE were satisfactory, the present findings identified some unpublished compounds to G. lipsiense in the literature with important therapeutic properties.

    更新日期:2020-01-17
  • Adipose Tissue–Derived Mesenchymal Stem Cells Protect Against Amiodarone-Induced Lung Injury in Rats
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-17
    Sara M. Radwan, Dalia Ghoneim, Manar Salem, Menna Saeed, Yara Saleh, Mohanad Elhamy, Kholoud Wael, Omnia Shokair, Sara A. Wahdan

    Pulmonary fibrosis (PF) is a progressive and irreversible lung disease, characterized by poor prognosis with limited treatment options. Mesenchymal stem cells (MSCs) are multi-potent cells having the ability to self-renew and differentiate into multiple tissues, thus considered a novel treatment option. The present study aimed to investigate the possible antifibrotic effect of undifferentiated adipose tissue–derived mesenchymal stem cell (AD-MSC) therapy on PF experimentally induced in rats using amiodarone (AMD). AMD (30 mg/kg) was given orally, once daily for 12 consecutive weeks to induce lung fibrosis. Following the confirmation of lung damage with histopathological examination, AD-MSCs (2 × 106 and 4 × 106 undifferentiated MSCs) were injected once intravenously, followed by 2 months for treatment. AMD induced focal fibroblastic cells proliferation in the peribronchiolar tissue, as well as in between the collapsed emphysematous alveoli. Also, AMD significantly increased serum and lung homogenate fibroblast growth factor-7 (FGF7), Clara cell protein-16 (CC16), and cytokeratin -19 (CK19) levels, as well as the expression of both iNOS and NFкB in the lung alveoli. Moreover, AMD caused excessive collagen deposition and increased alpha smooth muscle actin (α-SMA) expression. All findings significantly regressed on stem cell therapy in both doses, with superior effect of the high dose, providing evidence that adipose tissue–derived MSCs could be a promising approach for the treatment of PF.

    更新日期:2020-01-17
  • Effects of Different Energy Substrates and Nickel and Cadmium Ions on the Growth of Acidithiobacillus ferrooxidans and Its Application for Disposal of Ni-Cd Batteries
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-16
    Zi-Jing Yu, Huan Li, Jing-Hua Yao, Jian-Jun Wu, Yi-xin Zhang, Lei Xiao

    Abstract In the present work, the effects of different energy substrates and nickel ions (Ni2+) and cadmium ions (Cd2+) on the growth of Acidithiobacillus ferrooxidans (A. ferrooxidans) were investigated. Ferrous sulphate (FeSO4) was the optimum energy substrate for A. ferrooxidans growth, among the selected substrates. When cultured together with FeSO4 and sulphur (S), A. ferrooxidans first oxidised the ferrous ions (Fe2+), and the S was utilised as the concentration of Fe2+ decreased. After adapting to culture with Ni2+ and Cd2+, A. ferrooxidans presented good tolerance to both ions, with the maximum concentration reaching 4.11 g/L Ni2+ and 1.69 g/L Cd2+. A preliminary simulation of industrial application was also performed on used Ni-Cd batteries. With bioleaching, the highest concentrations of Cd2+ and Ni2+ were 3003 mg/L at day 8 and 1863 mg/L at day 14, respectively.

    更新日期:2020-01-16
  • Model Compounds Study for the Mechanism of Horseradish Peroxidase-Catalyzed Lignin Modification
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-16
    Dongjie Yang, Yalin Wang, Wenjing Huang, Zhixian Li, Xueqing Qiu

    Abstract Horseradish peroxidase (HRP) has demonstrated high activity for the modification of lignin. In this paper, several lignin model compounds with different functional groups and linkages are selected to investigate the reactivity of HRP-catalyzed lignin modification. The phenolic groups of lignin model compounds are indispensable for the HRP-catalyzed modification process. The introduction of the sulfomethylated methyl group or methoxyl group could facilitate or inhibit the modification, respectively. The oxidative coupling activity of α-O-4 lignin model compounds is higher than that of β-O-4 compounds. Meanwhile, the free energy obtained by density functional theory (DFT) is used to verify the results of the experimental study, and the order of preference for linkages is β-5 > β-β > β-O-4 in most cases. In addition, electron cloud density and steric hindrance of lignin model compounds have crucial effects on the oxidation and modification processes. Finally, the mechanism of HRP-catalyzed lignin modification is proposed.

    更新日期:2020-01-16
  • CRISPR-Cpf1-Assisted Engineering of Corynebacterium glutamicum SNK118 for Enhanced l -Ornithine Production by NADP-Dependent Glyceraldehyde-3-Phosphate Dehydrogenase and NADH-Dependent Glutamate Dehydrogenase
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-16
    Jinjun Dong, Baojun Kan, Hui Liu, Milin Zhan, Shuxian Wang, Guochao Xu, Ruizhi Han, Ye Ni

    Abstract Here, Corynebacterium glutamicum SNK118 was metabolically engineered for l-ornithine production through CRISPR-Cpf1-based genome manipulation and plasmid-based heterologous overexpression. Genes argF, argR, and ncgl2228 were deleted to block the degradation of l-ornithine, eliminate the global transcriptional repression, and alleviate the competitive branch pathway, respectively. Overexpression of CsgapC (NADP-dependent glyceraldehyde 3-phosphate dehydrogenases gene from Clostridium saccharobutylicum DSM 13864) and BsrocG (NADH-dependent glutamate dehydrogenase gene from Bacillus subtilis HB-1) resulted markedly increased ornithine biosynthesis. Eventually, the engineered strain KBJ11 (SNK118ΔargRΔargFΔncgl2228/pXMJ19-CsgapC-BsrocG) was constructed for l-ornithine overproduction. In fed-batch fermentation, l-ornithine of 88.26 g/L with productivity of 1.23 g/L/h (over 72 h) and yield of 0.414 g/g glucose was achieved by strain KBJ11 in a 10-L bioreactor. Our result represents the highest titer and yield of l-ornithine production by microbial fermentation. This study suggests that heterologous expression of CsgapC and BsrocG could promote l-ornithine production by C. glutamicum strains.

    更新日期:2020-01-16
  • Immobilization of Cholesterol Oxidase from Streptomyces Sp. on Magnetite Silicon Dioxide by Crosslinking Method for Cholesterol Oxidation
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-16
    Meka Saima Perdani, Muhamad Sahlan, Masafumi Yohda, Heri Hermansyah

    Abstract Enzymatic biosensor has been paid much attention to the research fields due to its advantage in medical application. As one of the application, we determined the optimum value of cholesterol oxidase against cholesterol. In this work, we studied the behavior of cholesterol oxidation by enzymatic reaction to get the optimum condition for cholesterol oxidation. The enzyme that used were in two form, free cholesterol oxidase, and immobilized cholesterol oxidase. Cholesterol oxidase was produced from Streptomyces sp. by using solid state fermentation method and identified had high enzyme activity to be 5.12 U/mL. Cholesterol oxidase was simultaneously crosslinked immobilized onto magnetite coated by silicon dioxide (M-SiO2). The support was characterized by Fourier transform infrared (FTIR) to determine the functional group of modified particle and scanning electron microscope (SEM) to observe the morphological or our prepared particle. Cholesterol oxidase sensitivity to substrate was analyzed by using HPLC with different interval time measurements. The oxidation of cholesterol by free enzyme and immobilized enzyme was also investigated. The best sensitivity of cholesterol oxidase was estimated to oxidize Cso (concentration of substrate) 1.46 mM of substrate with Ce (concentration of enzyme) 20 mg/mL for 180 min. Final oxidation value of cholesterol by immobilized enzyme was greater than 60%. The results of this study revealed that immobilized enzyme for cholesterol oxidation was stable, reproducible, and sensitive.

    更新日期:2020-01-16
  • Development of a Surfactant-Containing Process to Improve the Removal Efficiency of Phenol and Control the Molecular Weight of Synthetic Phenolic Polymers Using Horseradish Peroxidase in an Aqueous System
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-15
    Chao-Ling Yao, Che-Chi Lin, I-Ming Chu, Yi-Ting Lai

    To reduce phenolic pollutants in the environment, many countries have imposed firm restrictions on industrial wastewater discharge. In addition, the current industrial process of phenolic resin production uses phenol and formaldehyde as the reactants to perform a polycondensation reaction. Due to the toxicity of formaldehyde and phenolic pollutants, the main purpose of this research was to design a green process using horseradish peroxidase (HRP) enzymatic polymerization to remove phenols and to produce formaldehyde-free phenolic polymers. In this study, the optimal reaction conditions, such as reaction temperature, pH, initial phenol concentration and initial ratio of phenol, and H2O2, were examined. Then, the parameters of the enzyme kinetics were determined. To solve the restriction of enzyme inactivation, several nonionic surfactants were selected to improve the phenol removal efficiency, and the optimal operation conditions in a surfactant-containing system were also confirmed. Importantly, the molecular weight of the synthetic phenolic polymers could be controlled by adjusting the ratio of phenol and H2O2. The content of biphenols in the products was almost undetectable. Collectively, a green chemistry process was proposed in this study and would benefit the treatment of phenol-containing wastewater and the production of formaldehyde-free phenolic resin in the future.

    更新日期:2020-01-15
  • Improvement of the Thermostability and Activity of Pullulanase from Anoxybacillus sp. WB42
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-15
    Bo Pang, Li Zhou, Wenjing Cui, Zhongmei Liu, Zhemin Zhou

    Pullulanase is a commonly used starch-debranching enzyme with broad application in food, chemical and pharmaceutical industries. Since the starch-debranching process requires a high temperature, a thermostable pullulanase is desirable. In this study, based on the strategy of surficial residue replacement and disulfide bond introduction, a mutant pullulanase (PulAC) derived from the pullulanase (PulA) of Anoxybacillus sp. WB42 with higher thermostability and activity was isolated. The surficial residue Lys419 from the wild-type PulA was replaced by arginine, and two disulfide bonds were introduced between Thr245 and Ala326 and Trp651 and Val707. The specific activity and kcat/Km value of the PulAC reached 98.20 U/mg and 12.22 mL/mg/s respectively, 1.5 times greater than that of wild-type PulA. The optimum temperature of the mutant PulAC was 65 °C. The PulAC retained more than 85% activity after incubation at 65 °C for 30 min, which is much higher than the activity maintained by wild-type PulA. Due to its high optimum temperature, thermostability, and specific activity, the mutant PulAC reported here could play an important role in improving hydrolytic efficiency in the starch-debranching process.

    更新日期:2020-01-15
  • Enhanced Lipid Production in Yarrowia lipolytica Po1g by Over-expressing lro 1 Gene under Two Different Promoters
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-15
    Lita Amalia, Ya-Hui Zhang, Yi-Hsu Ju, Shen-Long Tsai

    Yarrowia lipolytica is a well-known oleaginous yeast that naturally accumulates lipids to more than 20% of their dry cell weight. Due to its brief doubling time and Generally Recognized as Safe (GRAS) properties, Y. lipolytica has been exploited for the production of commercially valuable lipids. Among the genes related to the lipid synthesis, the gene YALI0E16797g (LRO1) encoding a major triacylglycerol synthase of Y. lipolytica shows a significant impact during the acylation process. Thus, in the present work, we explore the contributions of hp4d or TEFintron promoters to the response of LRO1 expression on lipid accumulation by molecular cloning technology. Results showed that over-expression of LRO1 led to higher lipid content as well as lipid yield. The one with the hp4d promoter showed the highest lipid content of 12% wt. However, such an enhancement also caused a growth defect of cells. On the other hand, the lipid content of the cells over-expressing LRO1 with TEFintron promoter revealed only a modest increase in lipid content, but it promoted cell growth. Therefore, all things considered the one with the TEFintron promoter showed the highest lipid yield.

    更新日期:2020-01-15
  • Surface Functionalization of Poly( N -Vinylpyrrolidone) onto Poly(Dimethylsiloxane) for Anti-Biofilm Application
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-14
    Trong-Nghia Le, Cheng-Kang Lee

    Poly(dimethylsiloxane) (PDMS) has been widely used in the field of microfluidics, optical systems, and sensors. However, the hydrophobic nature of PDMS leads to low surface wettability and biofouling problems due to the nonspecific proteins–hydrophobic surface interactions and cell/bacterial adhesion. In this work, the PDMS surface was first introduced with amino groups (PDMS-NH2) via KOH-catalyzed reaction with 3-aminopropyltriethoxysilane (APTES). The PDMS-NH2 was then grafted with poly(N-vinylpyrrolidone) (PVP) based on the self-adhesion reaction between the amino surface and catechol-functionalized PVP (CA-PLL-PVP). CA-PLL-PVP as a comb-polymer was synthesized by conjugating PVP-COOH along with caffeic acid to the ε-polylysine backbone. A significantly enhanced water wettability was observed with contact angles dropped from 116° to 14° after coating with CA-PLL-PVP. The coated surface demonstrated excellent antifouling performance that no appreciable Staphylococcus epidermidis biofilm formation could be observed. This novel facile antifouling coating on PDMS surface may find greater biomedical applications to eliminate the potential adherence problems caused by natural biofouling.

    更新日期:2020-01-14
  • Biotechnological Applications of Paenibacillus sp. D9 Lipopeptide Biosurfactant Produced in Low-cost Substrates
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-14
    Abdullahi Adekilekun Jimoh, Johnson Lin

    The present study assesses the Paenibacillus sp. D9 lipopeptide biosurfactant synthesis in cheap substrates including functional properties and applicability for varying biotechnological processes. Different experimental setups were made for oil dispersion, heavy metals removals from contaminated environments, and washing performance. The study revealed surface tension activities of 31.7–32.7 mN/m, and maximum biosurfactant yield of more than 8 g/L. Removals of 85.90%, 98.68%, 99.97%, 63.28%, 99.93%, and 94.22% were obtained for Ca, Cu, Fe, Mg, Ni, and Zn, respectively from acid mine effluents. In comparison with chemical surfactants, there was pronounced removal of heavy metals from wastewater, contaminated sands, and vegetable matter, as well as improved oil dispersing activity. A comparative study revealed that biosurfactant was more efficient (> 60%) for removal of tomato sauce and coffee stains than chemical surfactants (< 50%). Thus, lipopeptide biosurfactants are green biomolecules reducing hazards and contaminations within the environment. The future use of this lipopeptide biosurfactant is greatly promising in biotechnology.

    更新日期:2020-01-14
  • Interesterification of Soybean Oil with Propylene Glycol in Supercritical Carbon Dioxide and Analysis by NMR Spectroscopy
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-09
    Nazanin Vafaei, Michael N. A. Eskin, Curtis B. Rempel, Peter J. H. Jones, Martin G. Scanlon

    The time course study of high monoester mixtures from soybean oil (HMMS) synthesis, as healthier alternatives to trans food products, in a supercritical CO2 (SCCO2) medium with and without enzyme, was investigated. Phosphorous nuclear magnetic resonance (31P-NMR) was used to quantify the absolute amount of partially esterified acylglycerols (PEGs). Carbon NMR was utilized to determine the type and position of the fatty acids (FAs) of HMMS. Enzyme and time significantly influenced the synthesis of 1-monoglycerides (1-MGs), 2-MGs, and 1,2-diglycerides (1,2-DGs) in this alcoholysis of soybean oil with 1,2-propanediol, based on high catalytic activity and operational stability of Novozym 435 in SCCO2 during short reaction time. Results suggest that 4 h is a suitable reaction time for this lipase-catalyzed interesterification (LIE) system for the synthesis of 2-MGs with a yield of 20%. The highest polyunsaturated fatty acid (PUFA) (65%) in the triglyceride (TG) of HMMS was produced after 4 h of reaction. After 6 h of reaction, a high level (20%) of saturated fatty acids (SFAs) was found in the TGs of HMMS, which were distributed between the sn-2 (5%) and sn-1, 3 (15%) positions.

    更新日期:2020-01-09
  • Optimization Strategies for Purification of Mycophenolic Acid Produced by Penicillium brevicompactum
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-06
    Shubhankar Anand, Pradeep Srivastava

    The microbial fermentation of Penicillium brevicompactum produces secondary metabolite mycophenolic acid (MPA), which exhibits antifungal, antiviral, antibacterial, and antitumor activity. It is also a potent, selective, non-competitive, and reversible inhibitor of the human inosine monophosphate dehydrogenase (IMPDH). This study is an attempt to optimize the MPA production through a fermentation process using Penicillium brevicompactum and its further purification process optimization. In the batch fermentation process, the maximum concentration of MPA (1.84 g/L) was attained in a 3.7 L stirred tank reactor. Response surface methodology (RSM) using central composite design (CCD) was employed as a statistical tool to investigate the effect of pH, the volume of eluent and flow rate of the mobile phase on MPA purification process. Under optimum conditions, the experimental yield was observed to be 84.12%, which matched well with the predictive yield of 84.42%. High-performance liquid chromatography (HPLC) and Fourier-transform infrared spectroscopy (FTIR) analysis of the fermented product was carried out to confirm the presence of mycophenolic acid. The MPA purification was done by using column chromatography technique. The purification of broth involved mycophenolic acid extraction by selecting different solvents on the basis of polarity and the extraction efficiency of solvent. Various solid support materials were used for MPA purification in column chromatography. The MPA recovery through alumina column was observed to be 84.12% under the optimum conditions, which was maximum elution as compared with other support materials. The optimized purification process yielded pure MPA crystals.

    更新日期:2020-01-06
  • Application of Invertase Immobilized on Chitosan Using Glutaraldehyde or Tris(Hydroxymethyl)Phosphine as Cross-Linking Agent to Produce Bioethanol
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-06
    Ishita Malhotra, Seemi Farhat Basir

    Abstract Invertase was immobilized on chitosan using glutaraldehyde or tris(hydroxymethyl)phosphine as cross-linker. The optimum pH for free and immobilized enzyme was found to be 4.5 and 5.5, respectively. The optimum hydrolysis temperature was 55 °C for both the free and immobilized forms. Km and Vmax values for free invertase, and invertase immobilized on glutaraldehyde- and THP-activated chitosan were 15, 19, and 20 mM, respectively, and 238, 204, and 212 mM min−1, respectively. The THP-immobilized enzyme had the highest pH and thermal stability, higher reusability with 70% retention in activity after 9 batches of reuse and higher storage stability with 90% retention in activity after 12 weeks at 4 °C, pH 4.5. Fermentation of cane molasses by yeast to form ethanol in the presence of free invertase at 30°C, pH 5.0 led to an increase in ethanol production by 3% and the production increased by 10.7% when immobilized invertase was used as catalyst. Graphical Abstract

    更新日期:2020-01-06
  • Efficiency of Ionic Liquids–Based Aqueous Two-phase Electrophoresis for Partition of Cytochrome c
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-06
    Phei Er Kee, John Chi-Wei Lan, Hip Seng Yim, Joo Shun Tan, Yin Hui Chow, Hui-Suan Ng

    Cytochrome c is a small water-soluble protein that is abundantly found in the mitochondrial intermembrane space of microorganism, plants and mammalians. Ionic liquids (ILs)–based aqueous two-phase electrophoresis system (ATPES) was introduced in this study to investigate the partition efficiency of cytochrome c to facilitate subsequent development of two-phase electrophoresis for the separation of cytochrome c from microbial fermentation. The 1-Hexyl-3-methylimidazolium bromide, (C6mim)Br and potassium citrate salt were selected as the phase-forming components. Effects of phase composition; position of electrodes; pH and addition of neutral salt on the partition efficiency of cytochrome c in the ATPES were evaluated. Highest partition coefficient (K = 179.12 ± 0.82) and yield of cytochrome c in top phase (YT = 99.63% ± 0.00) were recorded with IL/salt ATPES composed of 30% (w/w) (C6mim)Br and 20% (w/w) potassium citrate salt of pH 7 and 3.0% (w/w) NaCl addition with anode at the bottom phase and cathode at the top phase. The SDS-PAGE profile revealed that cytochrome c with a molecular weight of 12 kDa was preferably partitioned to the IL-rich top phase. Present findings suggested that the single-step ATPES is a potential separation approach for the recovery of cytochrome c from microbial fermentation.

    更新日期:2020-01-06
  • Carbon Neutral Electricity Production from Municipal Solid Waste Landfill Leachate Using Algal-Assisted Microbial Fuel Cell
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-06
    Rajendran Lakshmidevi, Nagarajan Nagendra Gandhi, Karuppan Muthukumar

    We demonstrate the feasibility of algal lipid production and bioelectricity generation with concurrent treatment of municipal solid waste (MSW) leachate in a double-chamber algal-assisted microbial fuel cell (AAMFC). The cathode chamber was loaded with Synechococcus sp. and MSW leachate whereas anode chamber was loaded with anaerobic microflora. While treating 50% MSW leachate, highest power density (95.63 mW/m2), current density (2.48 A/m2), and biomass concentration (2.54 g/l) were observed. The algal growth in the cathode chamber increased the oxygen concentration from 5.5 to 8.6 mg/l. Secondly, we studied the influence of salinity in AAMFC performance. The addition of 30 mM NaCl with 50% leachate increased the power and current density to 110.92 mW/m2 and 5.169 A/m2, respectively. It also increased the biomass concentration, protein, and lipid content. The analysis of fatty acid profile of algae confirmed the presence of palmitic acid, stearic acid, and linoleic acid. The proposed technique is effective for concurrent treatment of MSW leachate and power generation besides algal lipid production without external aeration.

    更新日期:2020-01-06
  • Characterization of Two Polyphosphate Kinase 2 Enzymes Used for ATP Synthesis
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2020-01-06
    Xing Zhang, Xiangwei Cui, Zhimin Li

    Abstract Enzymes used for adenosine triphosphate (ATP) synthesis play important roles in energy-dependent cascade reactions in vitro. In this study, two novel polyphosphate kinase 2 (PPK2) enzymes, HbPPK2 from Hydrogenophilaceae bacterium and NdPPK2 from Nocardioides dokdonensis, were characterized for ATP synthesis with the substrate polyphosphate (polyP). The optimum temperature and pH of both purified HbPPK2 and NdPPK2 were 30 °C and 6.5. HbPPK2 and NdPPK2 retained 30% and 14% of the initial activity at 30 °C for 12 h, respectively, whereas the presence of polyP significantly enhanced the stability of enzymes. The two PPK2s preferentially catalyzed the long-chain polyP hexametaphosphate as the phosphate donor. Adenosine monophosphate could not be used by HbPPK2 and NdPPK2 to synthesize ATP, indicating that they belonged to the class I subfamily of PPK2. HbPPK2 was used for ATP regeneration to produce glutathione by a two-enzyme cascade in vitro. 47.1 ± 0.4 mM glutathione was synthesized with a productivity of 13.5 ± 0.1 mM/h. ATP was regenerated approximately 471 times in the system within 3.5 h. HbPPK2 showed potential application for ATP regeneration in cascade reaction.

    更新日期:2020-01-06
  • Core-Shell Encapsulation of Lipophilic Substance in Jelly Fig ( Ficus awkeotsang Makino ) Polysaccharides Using an Inexpensive Acrylic-Based Millifluidic Device
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-26
    Thangavel Ponrasu, Ren-Fang Yang, Tzung-Han Chou, Jia-Jiuan Wu, Yu-Shen Cheng

    The polysaccharides extracted from the achenes of jelly fig, Ficus awkeotsang Makino, were mainly composed of low methyl pectin and used as a novel shell material for encapsulating lipophilic bioactives in the core of microcapsule. The polysaccharide microcapsules with oil core were prepared using a novel acrylic-based millifluidic device developed in this study. To investigate the physiochemical properties of and find the suitable formula of polysaccharide shells, the films casted with jelly fig polysaccharide were thoroughly characterized. For the preparation of microcapsules, the millifluidic device was optimized by controlling the flow rate to obtain uniform spherical shape with a core diameter of 1.4−1.9 mm and the outer diameter of 2.1–2.8 mm. The encapsulation efficiency was around 90%, and the microcapsules displayed a clear boundary between the polysaccharide shell and oil core. Encapsulation of curcumin in the microcapsules was prepared to test the applicability of the device and processes developed in this study, and the results showed that the microencapsulation could enhance the stability of curcumin against external environment. Overall, the results suggested that the jelly fig polysaccharides and the developed millifluidic device can be useful for the preparation of core-shell microcapsules for encapsulation of lipophilic bioactives.

    更新日期:2019-12-27
  • Therapeutic “Tool” in Reconstruction and Regeneration of Tissue Engineering for Osteochondral Repair
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-21
    Xueyan Hu, Jie Xu, Wenfang Li, Liying Li, Roxanne Parungao, Yiwei Wang, Shuangshuang Zheng, Yi Nie, Tianqing Liu, Kedong Song

    Abstract Repairing osteochondral defects to restore joint function is a major challenge in regenerative medicine. However, with recent advances in tissue engineering, the development of potential treatments is promising. In recent years, in addition to single-layer scaffolds, double-layer or multilayer scaffolds have been prepared to mimic the structure of articular cartilage and subchondral bone for osteochondral repair. Although there are a range of different cells such as umbilical cord stem cells, bone marrow mesenchyml stem cell, and others that can be used, the availability, ease of preparation, and the osteogenic and chondrogenic capacity of these cells are important factors that will influence its selection for tissue engineering. Furthermore, appropriate cell proliferation and differentiation of these cells is also key for the optimal repair of osteochondral defects. The development of bioreactors has enhanced methods to stimulate the proliferation and differentiation of cells. In this review, we summarize the recent advances in tissue engineering, including the development of layered scaffolds, cells, and bioreactors that have changed the approach towards the development of novel treatments for osteochondral repair.

    更新日期:2019-12-21
  • Evaluation of Iminodiacetic Acid (IDA) as an Ionogenic Group for Adsorption of IgG 1 Monoclonal Antibodies by Membrane Chromatography
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-21
    Igor Tadeu Lazzarotto Bresolin, Iara Rocha Antunes Pereira Bresolin, Sônia Maria Alves Bueno

    Iminodiacetic acid (IDA) is one of the chelating ligands most frequently employed in immobilized metal-ion affinity chromatography (IMAC) due to its ability to act as electron-pair donor, forming stable complexes with intermediate and borderline Lewis metal ions (electron acceptor). Thus, IDA can also be employed in ion exchange chromatography to purify positively charged proteins at neutral pH values. This study aimed to evaluate IDA as an ionogenic group (ion exchanger) immobilized on poly (ethylene vinyl alcohol) (PEVA) hollow fiber membranes for immunoglobulin G1 (IgG1) monoclonal antibody (MAb) purification. IDA-PEVA membranes showed considerable promise for MAb purification, since IgG1 was recovered in eluted fractions with traces of contaminants as confirmed by Western blotting and ELISA analysis. Quantification of IgG1 showed that a purity of 94.2% was reached in the elution step. Breakthrough curve and batch adsorption experiments showed that the MAb dynamic binding capacity (DBC) of 3.10 mg g−1 and the maximum adsorption capacity of 70 mg g−1 were of the same order of magnitude as those found in the literature. The results obtained showed that the IDA-PEVA hollow fiber membrane could be a powerful adsorbent for integrating large-scale processes for purification of MAb from cell culture supernatant.

    更新日期:2019-12-21
  • Recombinant Penicillium oxalicum 16 β-Glucosidase 1 Displays Comprehensive Inhibitory Resistance to Several Lignocellulose Pretreatment Products, Ethanol, and Salt
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-20
    Hanxin Li, Shi Yi, Eric W. Bell, Qiuxia Huang, Xihua Zhao

    β-Glucosidase (BGL) is a rate-limiting enzyme of lignocellulose hydrolysis for second-generation bioethanol production, but its inhibition by lignocellulose pretreatment products, ethanol, and salt is apparent. Here, the recombinant Penicillium oxalicum 16 BGL 1 (rPO16BGL1) from Pichia pastoris GS115 kept complete activity at 0.2–1.4 mg/mL furan derivatives and phenolic compounds, 50 mg/mL sodium chloride (potassium chloride), or 100 mg/mL ethanol at 40 °C. rPO16BGL1 retained above 50% residual activity at 30 mg/mL organic acid sodium, and 60% residual activity at 40 °C with 300 mg/mL ethanol. Sodium chloride and potassium chloride had a complicated effect on rPO16BGL1, which resulted in activation or inhibition. The inhibition kinetics of the enzyme reaction demonstrated that organic acids and organic acid sodium were non-competitive inhibitors and that ethanol was a competitive inhibitor at < 1.5 mg/mL salicin. Moreover, substrate inhibition of the enzyme was found at > 2 mg/mL salicin, and the Km/KI and Km/KSI average values revealed that the inhibitory strength was ranked as salicin-organic acids > organic acids > salicin-organic acid sodium salt > organic acid sodium salt > salicin > salicin-KCl > salicin-NaCl > salicin-ethanol > ethanol.

    更新日期:2019-12-20
  • Exploring Dual-Substrate Cultivation Strategy of 1,3-Propanediol Production Using Klebsiella pneumoniae
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-20
    Wei-Chuan Chen, Chi-Ju Chuang, Jo-Shu Chang, Li-Fen Wang, Po-Chi Soo, Ho-Shing Wu, Shen-Long Tsai, Yu-Hong Wei

    1,3-Propanediol (1,3-PDO) has numerous industrial applications in the synthesis of the monomer of the widely used fiber polytrimethylene terephthalate. In this work, the production of 1,3-PDO by Klebsiella pneumoniae is increased by dual-substrate cultivation and fed-batch fermentation. Experimental results indicate that the production of 1,3-PDO can be elevated to 16.09 g/L using a dual substrate ratio (of glucose to crude glycerol) of 1/30 and to 20.73 g/L using an optimized dual-substrate ratio of 1/20. Ultimately, the optimal dual-substrate feeding for a 5 L scale fed-batch fermenter that maximizes 1,3-PDO production (29.69 g/L) is determined. This production yield is better than that reported in most related studies. Eventually, the molecular weight and chemical structure of 1,3-PDO were obtained by FAB-MS, 1H-NMR, and 13C-NMR. Also, in demonstrating the effectiveness of the fermentation strategy in increasing the production and production yield of 1,3-PDO, experimental results indicate that the fermentation of 1,3-PDO is highly promising for commercialization.

    更新日期:2019-12-20
  • Biosynthesis of a Phycocyanin Beta Subunit with Two Noncognate Chromophores in Escherichia coli
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-19
    Huaxin Chen, Caiyun Zheng, Peng Jiang, Gengsheng Ji

    Abstract Recombinant phycobiliprotein can be used as fluorescent label in immunofluorescence assay. In this study, pathway for phycocyanin beta subunit (CpcB) carrying noncognate chromophore phycoerythrobilin (PEB) and phycourobilin (PUB) was constructed in Escherichia coli. Lyase CpcS and CpcT could catalyze attachment of PEB to Cys84-CpcB and Cys155-CpcB, respectively. However, PEB was attached only to Cys84-CpcB when both CpcS and CpcT were present in E. coli. A dual plasmid expression system was used to control the expression of lyases and the attachment order of PEB to CpcB. The production of PEB-Cys155-CpcB was achieved by L-arabinose-induced expression of CpcS, CpcB, Ho1, and PebS, and then the attachment of PEB to Cys84-CpcB was achieved by IPTG-induced expression of CpcS. The doubly chromophorylated CpcB absorbed light maximally at 497.5 nm and 557.0 nm and fluoresced maximally at 507.5 nm and 566.5 nm. An amount of light energy absorbed by PUB-Cys155-CpcB is transferred to PEB-Cys84-CpcB in doubly chromophorylated CpcB, conferring a large stokes shift of 69 nm for this fluorescent protein. There are interactions between chromophores of CpcB which possibly together with the help of lyases lead to isomerization of PEB-Cys155-CpcB to PUB-Cys155-CpcB.

    更新日期:2019-12-19
  • Increased Production of Dicinnamoylmethane Via Improving Cellular Malonyl-CoA Level by Using a CRISPRi in Escherichia coli
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-18
    Luan Luong Chu, Ramesh Prasad Pandey, Dipesh Dhakal, Jae Kyung Sohng

    Curcuminoids are natural phenylpropanoids that are biosynthesized via an L-phenylalanine metabolism pathway in turmeric (Curcuma longa L.). Curcuminoids have various chemopreventive activities and pharmaceutical applications in human life. In this study, we synthesized dicinnamoylmethane, one principal component of curcuminoids, from cinnamic acid by means of co-expression of Oryza sativa curcuminoid synthase and Petroselinum crispum 4-coumarate-CoA ligase in Escherichia coli BL21 (DE3). Moreover, we used CRISPRi systems to knock down the genes in a tricarboxylic acid cycle and fatty acid biosynthesis pathway. The repression of target genes led to an increase of up to 0.236 μmol g−1 DCW of malonyl-CoA in cytosol-engineered E. coli and subsequently increased the biosynthesis of dicinnamoylmethane. We found that the S10 strain containing a CRISPRi repression for three genes, fabF, fabD, and mdh, showed the highest amount of dicinnamoylmethane of 7.54 μM, which is 5.76-fold higher than that of the wild-type strain. Finally, 41.94 μM (~ 11.6 mg) of dicinnamoylmethane was obtained in a 3-L fermenter.

    更新日期:2019-12-18
  • Assessment of Post-thaw Quality of Dental Mesenchymal Stromal Cells After Long-Term Cryopreservation by Uncontrolled Freezing
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-18
    Shalini Raik, Ajay Kumar, Vidya Rattan, Saurabh Seth, Anupriya Kaur, Shalmoli Bhatta charyya

    Cryopreservation abilities of dental tissue-derived mesenchymal stromal cells (DMSCs) including dental pulp stem cells (DPSCs) and dental follicle stem cells (DFSC) play an important role in the applications of these cells in clinical settings. In this context, we checked whether storage at − 80 °C in 10% DMSO for a longer period has any adverse effect on the functionality and genetic stability. We carried our studies on DPSC and DFSC samples that were revived after a maximum of 5 years of cryopreservation. We observed that even after long-term uncontrolled freezing at − 80 °C, these cells survived and proliferated efficiently. The assessment was made based on their post-thaw morphology, immunophenotypes, differentiation potential, growth kinetics, and genetic features. These cells retained the expression of stemness markers, differentiation ability and maintained their normal karyotype. Our results indicated no significant morphological or immunophenotypic differences between the cryopreserved DMSCs and the fresh DMSCs. Our study implies that mesenchymal stromal cells derived from the dental tissue origin are very robust and do not require any sophisticated preservation protocols. Thus, these can be an ideal source for research, stem cell banking, as well as successful clinical applications in tissue engineering and cell-based therapeutics.

    更新日期:2019-12-18
  • Novel Copper Bearing Schiff Bases with Photodynamic Anti-Inflammatory and Anti-Microbial Activities
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-18
    Furkan Ayaz, Ilyas Gonul, Burcu Demirbag, Kasim Ocakoglu

    Schiff bases and their copper complexes have been previously studied for their anti-inflammatory, anti-tumor as well as anti-microbial activities. Schiff bases can be derivatized to gain photoluminiscence capacity. This property of the schiff bases enables the transfer of the electrons upon absorption of the light at a specific wavelength. In this study, we exploited this attribute of novel copper bearing schiff bases and tested their photodynamic biological activities. These compounds exerted photodynamic anti-inflammatory activities on the in vitro activated mammalian macrophages. Compared with salicylic acid control groups, these novel schiff bases had stronger activity which became more prominent with photo-induction. Moreover, they also had anti-microbial activity on gram negative bacteria E.coli and gram positive bacteria S.aureus.This anti-microbial activity was stronger than that of Neomycin on both bacterial strains. Our results suggest their potential use as anti-inflammatory and anti-microbial agents both in the dark as well as after photo-induction.

    更新日期:2019-12-18
  • Lipase Catalysis in Presence of Nonionic Surfactants
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-18
    Debajyoti Goswami

    Lipase can catalyze varieties of reactions at the interface of aqueous and organic phase. Among various alternatives to modify catalytic performance of lipase, the addition of surfactants, particularly nonionic surfactants, has been widely studied. Low concentrations of nonionic surfactants augment lipase catalysis; on increasing surfactant concentration, often the catalytic performance decreases. Mole ratio of water to (nonionic) surfactant also has a profound effect on lipase activity. Catalytic abilities of some lipases are either enhanced or reduced in the presence of all nonionic surfactants of the same type, whereas for some other lipases, nonionic surfactants of the same type have mixed effect. Nonionic surfactant even changes substrate specificity of lipase. Water-in-ionic liquid microemulsion involving nonionic surfactant often performs better than other systems in improving catalytic ability of lipase. Tween and Triton surfactants often enhance enantiomeric separation catalyzed by lipase. Nonionic surfactants significantly affect activities of immobilized lipase, being present either as a component during immobilization or as a component in reaction medium. Lipases coated with nonionic surfactants act better than reverse micelles and microemulsions containing lipase. Thus, nonionic surfactants help lipase catalyzed processes in various media to enhance production of useful compounds like flavor ester, structured lipids, optically pure compounds, and noncrystalline polymers.

    更新日期:2019-12-18
  • Enhanced Degradation of Diesel Oil by Using Biofilms Formed by Indigenous Purple Photosynthetic Bacteria from Oil-Contaminated Coasts of Vietnam on Different Carriers
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-18
    Le Thi Nhi-Cong, Do Thi Lien, Bhaskar Sen Gupta, Cung Thi Ngoc Mai, Hoang Phuong Ha, Nguyen Thi Minh Nguyet, Tran Hoa Duan, Dong Van Quyen, Hayyiratul Fatimah Mohd Zaid, Revathy Sankaran, Pau Loke Show

    Oil pollution in marine environment caused by oil spillage has been a main threat to the ecosystem including the ocean life and to the human being. In this research, three indigenous purple photosynthetic strains Rhodopseudomonas sp. DD4, DQ41, and FO2 were isolated from oil-contaminated coastal zones in Vietnam. The cells of these strains were immobilized on different carriers including cinder beads (CB), coconut fiber (CF), and polyurethane foam (PUF) for diesel oil removal from artificial seawater. The mixed biofilm formed by using CB, CF, and PUF as immobilization supports degraded 90, 91, and 95% of diesel oil (DO) with the initial concentration of 17.2 g/L, respectively, after 14 days of incubation. The adsorption of DO on different systems was accountable for the removal of 12–16% hydrocarbons for different carriers. To the best of our knowledge, this is the first report on diesel oil degradation by purple photosynthetic bacterial biofilms on different carriers. Moreover, using carriers attaching purple photosynthetic bacteria to remove diesel oil in large scale is considered as an essential method for the improvement of a cost-effective and efficient bioremediation manner. This study can be a promising approach to eliminate DO from oil-contaminated seawater.

    更新日期:2019-12-18
  • Development and Characterization of Nano-emulsions Based on Oil Extracted from Black Soldier Fly Larvae
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-18
    Tzung-Han Chou, Daniel Setiyo Nugroho, Yu-Shen Cheng, Jia-Yaw Chang

    Insect-based biorefinery is seen as a potential alternative approach to manufacturing foods, feeds, and fuel because of the increasing demand for renewable and sustainable products. Insect oil and protein are the two major components that can be quantitatively obtained from insect farming. However, very few attempts have been conducted to utilize insect oil for the production of value-added products. In this study, the oil extracted from the black soldier fly (Hermetia illucens) larvae (BSFL) was used as a novel feedstock for preparing nano-emulsions. The nano-emulsions were prepared with BSFL oil, hydrogenated lecithin (HL), and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) in water using pre-homogenization followed by ultrasonication. The morphology and the particle size of nano-emulsions were affected by ratios of HL to TPGS. Moreover, the nano-emulsions showed a nearly Newtonian liquid behavior and the presence of TPGS was able to improve the storage stability of HL nano-emulsions. The addition of TPGS could eliminate the phase transition region of HL nano-emulsions but did not provide a significant change for the molecular mobility in the HL nano-emulsions. In summary, the BSFL oil could be used as a renewable feedstock for formulating nano-emulsions from the aspect of high value-added applications and physicochemical characteristics of the nano-emulsions could be adjusted by the mixed surfactant ratio, surfactant to oil ratio, and oil content.

    更新日期:2019-12-18
  • Sustainable and Effective Chitosan Production by Dimorphic Fungus Mucor rouxii via Replacing Yeast Extract with Fungal Extract
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-17
    Leila Abasian, Razieh Shafiei Alavijeh, Behzad Satari, Keikhosro Karimi

    The effects of switching morphology and replacing supplementary nutrients with fungal extract (5 and 10 g/L) on the production of major metabolites and chitosan by Mucor rouxii were investigated. This approach was supposed to promote sustainability of the fermentation process and improve its economic feasibility. Different fungal morphologies, i.e., purely filamentous (PF), purely yeast-like (PY), mostly filamentous (MF), and mostly yeast-like (MY), were evaluated. The highest ethanol yields were obtained from the media supplemented with 10 g/L fungal extract for all morphologies, while adding nutrient salts did not make any improvements in these yields, except a slight decrease in the fermentation time. Except for PF morphology, the replacement of yeast extract favored the biomass production yields. Moreover, the alkali insoluble material (AIM) yields were higher as a result of the replacement for most cases. Furthermore, the replacement resulted in increased glucosamine and decreased N-acetyl-glucosamine content of AIM for almost all the morphologies. AIM yields of at least 0.25 g/g-glucose and maximum chitin/chitosan yield of 0.78 g/g-AIM were obtained from the solids remaining after autolysis process, which were higher than that obtained from the raw biomass. The maximum yield of 0.135 g/g-AIM purified chitosan with intact molecular weight was obtained from the biomass with PF morphology supplemented with 10 g/L fungal extract plus nutrients.

    更新日期:2019-12-18
  • Characteristics of an Acidic Phytase from Aspergillus aculeatus APF1 for Dephytinization of Biofortified Wheat Genotypes
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-17
    Abhishake Saxena, Meena Verma, Bijender Singh, Punesh Sangwan, Ajar Nath Yadav, Harcharan Singh Dhaliwal, Vinod Kumar

    Phytases are the special class of enzymes which have excellent application potential for enhancing the quality of food by decreasing its inherent anti-nutrient components. In current study, a protease-resistant, acidic phytase from Aspergillus aculeatus APF1 was partially purified by ammonium sulfate fractionation followed by chromatography techniques. The molecular weight of partially purified phytase was in range of 25–35 kDa. The purified APF1 phytase was biochemically characterized and found catalytically active at pH 3.0 and 50 °C. The Km and Vmax values of APF1 phytase for calcium phytate were 3.21 mM and 3.78 U/mg protein, respectively. Variable activity was observed with metal ions and among inhibitors, chaotropic agents and organic solvents; phenyl glyoxal, potassium iodide, and butanol inhibited enzyme activity, respectively, while the enzyme activity was not majorly influenced by EDTA, urea, ethanol, and hexane. APF1 phytase treatment was found effective in dephytinization of flour biofortified wheat genotypes. Maximum decrease in phytic acid content was noticed in genotype MB-16-1-4 (89.98%) followed by PRH3–30-3 (82.32%) and PRH3–43-1 (81.47%). Overall, the study revealed that phytase from Aspergillus aculeatus APF1 could be effectively used in food and feed processing industry for enhancing nutritional value of food.

    更新日期:2019-12-18
  • Production of Short Chain Fructo-oligosaccharides from Inulin of Chicory Root Using Fungal Endoinulinase
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-17
    Jayaram Chikkerur, Ashis Kumar Samanta, Atul P. Kolte, Arindam Dhali, Sohini Roy

    Short chain fructo-oligosaccharides (SC-FOS) are the potential prebiotics possessing diverse applications in both food and feed industries. The present study was aimed to extract inulin from chicory roots followed by its conversion into SC-FOS applying endoinulinase from Aspergillus fumigatus. The inulin was extracted from chicory roots through boiling in hot water, followed by precipitation with ethanol at room temperature or freezing condition. Maximum yield (42%) of inulin was obtained with three volumes of chilled absolute ethanol at room temperature. HPLC analysis of enzymatic hydrolysate detected kestose (GF2), nystose (GF3), and other FOS having higher degree of polymerization (DP). Maximum GF2 (5.79 mg/ml) was detected at temperature 50 °C, pH 5.5 with 2 U of enzyme dose after 6 h of hydrolysis; while maximum GF3 (4.33 mg/ml) was recorded at 60 °C, 5.5 pH with 0.5 U enzyme dose after 2 h of hydrolysis. Nevertheless, complete hydrolysis of inulin was noticed with 99% total oligosaccharide yield at 55 °C, 5.5 pH with 0.5 U enzyme dose after 4 h of hydrolysis with negligible amount of mono- and di-saccharides. The present finding demonstrated the process for higher yield of inulin from chicory roots followed by its conversion into SC-FOS applying fungal endoinulinase.

    更新日期:2019-12-18
  • A Modified, Efficient and Sensitive pH Indicator Dye Method for the Screening of Acid-Producing Acetobacter Strains Having Potential Application in Bio-Cellulose Production
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-16
    Manoj Kumar, Nipunta Tanoj, Saurabh Saran

    It is imperative that promising bacterial cellulose-producing bacteria mainly belongs to genera Acetobacter (acid-producing bacteria). In order to screen cellulose-producing Acetobacter, the isolated cultures from vinegar/rotten fruits were inoculated in Hestrin-Schramm (HS) medium containing ethanol and CaCO3. After the desired incubation, the positive cultures form a zone, which is observed around the bacterial growth, resulted from the solubilization of CaCO3 by acetic acid produced from the oxidation of ethanol during fermentation. However, in this method, the clarity of the solubilized zone is not very sharp and distinct. In the present, investigation, an improved method for screening, of the microorganisms producing acetic acid has been developed. In this method, methyl red (MR) is incorporated as a pH indicator in HS medium containing ethanol and CaCO3. Plates containing MR at alkaline pH are yellow and turn dark red at acidic pH. Thus, a distinctive, clear zone is formed around bacterial colonies producing acetic acid and is easy to differentiate between acid producers and non-producers. The present method is more rapid, accurate, and sensitive and can be successfully be used for the detection of acetic acid-producing bacteria particularly for the screening of potent cellulose producer Acetobacter sp.

    更新日期:2019-12-17
  • Prediction of Skin Disease with Three Different Feature Selection Techniques Using Stacking Ensemble Method
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-16
    Anurag Kumar Verma, Saurabh Pal

    Skin disease is the most common problem between people. Due to pollution and deployment of ozone layer, harmful UV rays of sun burn the skin and develop various types of skin diseases. Nowadays, machine learning and deep learning algorithms are generally used for diagnosis for various kinds of diseases. In this study, we have applied three feature extraction techniques univariate feature selection, feature importance, and correlation matrix with heat map to find the optimum data subset of erythemato-squamous disease. Four classification techniques Gaussian Naïve Bayesian (NB), decision tree (DT), support vector machine (SVM), and random forest are used for measuring the performance of model. Stacking ensemble technique is then applied to enhance the prediction performance of the model. The proposed method used for measuring the performance of the model. It is finding that the optimal subset of the erythemato-squamous disease is performed well in the case of correlation and heat map feature selection techniques. The mean value, slandered deviation, root mean square error, kappa statistical error, and area under receiver operating characteristics and accuracy are calculated for demonstrating the effectiveness of the proposed model. The feature selection techniques applied with staking ensemble technique gives the better result as compared to individual machine learning techniques. The obtained results show that the performance of proposed model is higher than previous results obtained by researchers.

    更新日期:2019-12-17
  • Enhanced 5-Aminolevulinic Acid Production by Co-expression of Codon-Optimized hemA Gene with Chaperone in Genetic Engineered Escherichia coli
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-16
    Tzu-Hsuan Yu, Ying-Chen Yi, I-Tai Shih, I-Son Ng

    5-Aminolevulinic acid (ALA) is an important metabolic intermediate compound with high value and has recently been used in agriculture and medicine. In this study, we have constructed six recombinant Escherichia coli (E. coli) strains that are involved in pET system under the regulation of the T7 promoter and LacI to express codon-optimized hemA gene from Rhodobacter capsulatus (RchemA) for ALA production via the C4 pathway. Due to codon optimization, hemA has a high transcriptional level; however, most RcHemA proteins were formed as inclusion body. To improve expression in soluble form, the vector with TrxA fusion tag was successfully used and co-expressed with partner GroELS as chaperone in another vector. As a result, ALA production increased significantly from 1.21 to 3.67 g/L. In addition, optimal ALA production was developed through adjustment of induction time and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, as well as substrate addition conditions. By adopting a two-stage induction strategy, the highest ALA reached 5.66 g/L when 0.1 mM of IPTG was added at early exponential phase (i.e., OD600 was equal to 0.7 to 0.8), while 6 g/L of glycine, 2 g/L of succinate, and 0.03 mM of pyridoxal 5′-phosphate (PLP) were provided in the mid-exponential phase in fermentation.

    更新日期:2019-12-17
  • A Kinetic Process to Determine the Interaction Type Between Two Compounds, One of Which Is a Reaction Product, Using Alkaline Phosphatase Inhibition as a Case Study
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-16
    Rui M. F. Bezerra, Paula A. Pinto, Albino A. Dias

    This study describes the development of a new methodology based on a new integrated equation which allows the determination of the kinetic parameters for two mutually non-exclusive inhibitors when one of which is produced during the time-course reaction. Alkaline phosphatase simultaneously inhibited by phosphate and urea is used to illustrate this methodology, including the evaluation of interaction effects between them. Data analyses were carried out using two integrated velocity equations: exclusive linear mixed inhibition (EMI) and non-exclusive linear mixed inhibition (NEMI). Kinetic parameters are estimated using non-linear regression and results show that (i) the interaction between enzyme and the inhibitors urea and phosphate exhibit a mutually non-exclusive behavior; (ii) more specifically, these inhibitors are non-exclusive only in free enzyme (E) species; (iii) the inhibitors also show an interaction with enzyme classified as facilitation; (iv) phosphate is a competitive inhibitor and urea a mixed inhibitor; (v) the inhibition constant for phosphate is much lower than that determined for urea. In addition, a functional Excel Spreadsheet which can be adapted to any kinetic study is also included as a supplement.

    更新日期:2019-12-17
  • Circulating miR-135 May Serve as a Novel Co-biomarker of HbA1c in Type 2 Diabetes
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-12
    Yousef Khazaei Monfared, Maryam Honardoost, Mohamad Reza Sarookhani, Seyed Amir Farzam

    Hemoglobin A1c (HbA1c) is a reliable marker of insulin resistance in normal glucose tolerance patients; however, several physiological, environmental, and genetic factors may affect HbA1c and cause false results. Therefore, it is essential to use new biomarkers due to increasing diabetes predictive value. Recently, it has been indicated that microRNAs (miRNAs) are involved in the pathophysiology of diabetes, particularly, in insulin resistance pathways. Therefore, miRNAs have the potential to be introduced as new glycemic control biomarkers. The aim of this study was to investigate the association between plasma level of miRNA-135a and HbA1c in patients with prediabetes and type 2 diabetes. In this case-control study, 120 samples were enrolled (healthy individuals, people with type 2 diabetes, and prediabetes) and HbA1c and miR-135a expression level in their plasma samples were evaluated. Multinomial logistic regression and ROC curve analysis were conducted to assess the diagnostic accuracy of plasma miR-135a in T2D , prediabetes, and healthy control groups. Data analysis indicated that miR-135a was significantly elevated in both diabetes/prediabetes samples. Then, subjects were reclassified based on the calculated cutoff value of miRNA. Logistic Regression analysis showed that an increased level of miRNA positively correlated with HbA1c level in prediabetes (Adjusted OR = 1.14, p value = 0.033) and diabetic status (Adjusted OR = 1.27, p value = 0.024 ). miR-135 may provide an assistant marker for HbA1c to detect type 2 diabetes.

    更新日期:2019-12-13
  • Laccase-catalyzed polymerization of hydroquinone incorporated with chitosan oligosaccharide for enzymatic coloration of cotton
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-11
    Rubing Bai, Yuanyuan Yu, Qiang Wang, Jinsong Shen, Jiugang Yuan, Xuerong Fan

    Chitosan oligosaccharide (COS), a water-soluble carbohydrate obtained from chemical or enzymatic hydrolysis of chitosan, has similar structure and properties to non-toxic, biocompatible, and biodegradable chitosan. However, COS has many advantages over chitosan due to its low molecular weight and high water solubility. In the current work, COS was incorporated in the laccase-catalyzed polymerization of hydroquinone. The laccase-catalyzed polymerization of hydroquinone with or without COS was investigated by using simple structure of glucosamine hydrochloride as an alternative to COS to understand the mechanism of COS-incorporated polymerization of hydroquinone. Although polyhydroquinone can be regarded as the polymeric colorant with dark brown color, there is no affinity or chemical bonding between polyhydroquinone and cotton fibers. Cotton fabrics were successfully in-situ dyed into brown color through the laccase-catalyzed polymerization of hydroquinone by incorporating with COS as a template. The presence of COS enhanced the dye uptake of polyhydroquinone on cotton fibers due to high affinity of COS to cotton and covalent bonding between COS and polyhydroquinone during laccase catalysis. This novel approach not only provides a simple route for the biological coloration of cotton fabrics but also presents a significant way to prepare functional textiles with antibacterial property.

    更新日期:2019-12-11
  • Immobilization of Thermoalkalophilic Lipase from Bacillus atrophaeus FSHM2 on Amine-Modified Graphene Oxide Nanostructures: Statistical Optimization and Its Application for Pentyl Valerate Synthesis
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-10
    Atefeh Ameri, Mojtaba Shakibaie, Mehdi Khoobi, Mohammad Ali Faramarzi, Alieh Ameri, Hamid Forootanfar

    Synthesis of (3-aminopropyl) triethoxysilane (APTES)-functionalized graphene oxide (GO) nanosheets, statistical optimization of conditions for immobilization of Bacillus atrophaeus lipase (BaL) on as-synthesized support, and application of the immobilized BaL for esterification of valeric acid were carried out in this investigation. The optimum specific activity of the immobilized BaL (81.60 ± 0.28 U mg−1) was achieved at 3 mg mL−1 of GO-NH2, 50 mM of phosphate buffer, pH 7.0, 60 min sonication time, 100 mM glutaraldehyde, 25 U mL−1 of enzyme, and 8 h immobilization time at 4 °C. The immobilized BaL retained about 90% of its initial activity after 10 days of storage. Moreover, about 70% of the initial activity of the immobilized BaL was retained after 10 cycles of application. The results of esterification studies exhibited that maximum pentyl valerate synthesis using the free BaL (34.5%) and the immobilized BaL (92.7%) occurred in the organic solvent medium (xylene) after 48 h of incubation at 60 °C.

    更新日期:2019-12-11
  • Electrospinning of Scaffolds from the Polycaprolactone/Polyurethane Composite with Graphene Oxide for Skin Tissue Engineering
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-10
    Ali Sadeghianmaryan, Yaghoub Karimi, Saman Naghieh, Hamed Alizadeh Sardroud, Mohsen Gorji, Xiongbiao Chen

    Creating scaffolds for skin tissue engineering remain challenging in terms of their mechanical and biological properties. In this paper, we present a study on the nanocomposite polyurethane (PU)/polycaprolactone (PCL) scaffolds with graphene oxide (GO), which were fabricated by using electrospinning method, for potential skin tissue engineering. For this, homogenous and soft PU nanofibers containing varying percent of polycaprolactone (12% and 15%) and nano GO (0.5–4%) were electrospun, respectively, and then characterized by different techniques/assays in vitro. For the scaffold characterization, scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) were used. The SEM results show the spun scaffolds have 3D porous structure (90%) with the fiber diameter increased with the GO concentration, while the FTIR results confirmed the presence of PU, PCL, and Go in the scaffolds. Also, the biocompatibility, via the cytotoxicity, of the scaffolds was examined by MTT assay with the human skin fibroblast cells, along with their wettability in terms of contact angle. Our results show that the scaffolds are biocompatible to the skin fibroblast cell, illustrating their potential use in skin tissue engineering. Also, our results illustrate that the addition of GO to the PU/PCL composite can increase the wettability (or hydrophilicity) and biocompatibility of scaffolds. Combined together, the nanocomposite PU/PCL scaffolds with GO are promising as biocompatible constructs for skin tissue engineering.

    更新日期:2019-12-11
  • Anthraquinone from Edible Fungi Pleurotus ostreatus Protects Human SH-SY5Y Neuroblastoma Cells Against 6-Hydroxydopamine-Induced Cell Death—Preclinical Validation of Gene Knockout Possibilities of PARK7, PINK1, and SNCA1 Using CRISPR SpCas9
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-09
    Bindhu J., Arunava Das, K. M. Sakthivel

    Parkinson’s disease (PD) results from the degeneration of the nervous tissue brought about by ecological and hereditary components which affects nerve cells in the brain. It is the world’s second most normal neurodegenerative issue, which can essentially weaken the personal satisfaction, make reliance, and trigger untimely mortality of affected people. The commonness pace of PD is 0.5–1% among individuals in the age group of 65–69 years and 1–3% among those 80 or more. Clinical appearances incorporate bradykinesia, tremors, unbending nature, and postural unsteadiness; spectrums of non-motor symptoms include psychological hindrance and passionate and behavioral brokenness. In this study, 6-OHDA-induced neurotoxicity was analyzed for various cytotoxicity analyses. The genes identified were PINK1 (PTEN-induced kinase 1), PARK7 (Parkinsonism-associated deglycase) and SNCA 1 (alpha synuclein1) validated using CRISPR spcas9 genome editing tool. In this study, Anthraquinone isolated from Pleurotus ostreatus was treated against a dopaminergic neurotoxin, 6-hydroxydopamine (6-OHDA), which induced neurotoxicity in SH-SY5Y cells. Experimental groups in SH-SY5Y neuroblastoma cells were treated with anthraquinone (50 nM) and 6-OHDA (100 nM). MTT and ROS assays were performed to assess the cell viability and oxidative stress within the cells, followed by mixed-member proportional (Mitochondrial membrane potential), dual staining, and immunoblotting. 6-OHDA-induced cell death in SH-SY5Y cells was dose-dependently attenuated by treatment with anthraquinone. The genes responsible for mutation were studied and the mutated RNAs knockout possibilities was studied using CRISPR spcas9 genome editing tool. Treatment with anthraquinone attenuated the level of oxidative stress and reduced the mitochondrial dysfunction associated with 6-OHDA treatment. Immunoblot analysis carried out with apoptotic markers showed that cytochrome C and caspase-3 expression increased significantly in anthraquinone-treated cells, whereas 6-OHDA-treated group showed a significant decrease when compared with an experimental control group. The mutated genes PARK7, PINK1, and SNCA1 were analyzed and found to exhibit four gene knock possibilities to treat PD. Reports demonstrate that other than following up on the biosynthesis of dopamine and its metabolites, these mixes counteract D2 receptors’ extreme touchiness. It is proposed that further examinations need be directed to better understand the activity of the bioactive mixes circulated in these edible fungi Pleurotus ostreatus. The gene knockout possibilities identified by CRISPR SpCas9 will pave a way for better research for PD treatment.

    更新日期:2019-12-11
  • Polydeoxyribonucleotide Activates Mitochondrial Biogenesis but Reduces MMP-1 Activity and Melanin Biosynthesis in Cultured Skin Cells
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-07
    Yeon-Ji Kim, Min-Jung Kim, Dong-Keon Kweon, Seung-Taik Lim, Sung-Joon Lee

    The regulation of mitochondrial biogenesis, melanogenesis, and connective tissue proteins is critical for homeostasis and aging skin cells. We examined the biological effects of polydeoxyribonucleotide (PDRN) on mitochondrial biogenesis, melanogenesis, and connective tissue proteins in vitro. In a radical scavenging assay, PDRN showed antioxidant activities in a dose-dependent manner, and those activities can suppress cellular oxidative stress in skin cells. PDRN directly inhibited mushroom tyrosinase activity and cellular tyrosinase activity, thus significantly reducing the cellular melanin content in B16-F10 melanocytes. The mRNA and protein expressions of the microphthalmia-associated transcription factor (MITF), which is a key melanogenic gene transcription factor, were significantly downregulated by PDRN. Accordingly, tyrosinase-related protein 1, dopachrome tautomerase, and tyrosinase, which gene expressions were regulated by MITF, were significantly downregulated by PDRN. Mitotracker-probed mitochondria image analysis suggested that PDRN enhanced mitochondrial density in both murine melanoma cells and in human skin fibroblast cells. In addition, PDRN strongly suppressed in vitro elastase enzyme activity in a dose-dependent manner and inhibited matrix metalloproteinase-1 gene expression in human skin fibroblast cells. Collectively, these findings indicate that PDRN has multiple beneficial biological activities in skin cells: hypopigmentation, induction of mitochondrial biogenesis, and the inhibition of collective tissue proteins.

    更新日期:2019-12-11
  • Astaxanthin Encapsulated in Biodegradable Calcium Alginate Microspheres for the Treatment of Hepatocellular Carcinoma In Vitro
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-07
    Xiujuan Zhang, Wenjuan Li, Xiang Dou, De Nan, Gaohong He

    Astaxanthin (AST) has attracted great interests in the scientific world because of its anti-oxidative, anti-inflammatory properties. And biodegradable materials, like chitosan, have been employed as the AST carrier to protect it from degradation and promote its bioavailability. However, the lack of pH responsiveness of these materials usually could not protect AST from the strong acidic gastric juices. In this study, calcium alginate (CA) microspheres, a pH responsive and biodegradable material, were prepared by a modified double emulsion technology and used as the AST encapsulation agent. Experimental results showed that the microparticles formed had a good degree of roundness, dispersity, encapsulation efficiency, and pH responsiveness. Cellular studies demonstrated that AST encapsulated in CA could inhibit hepatoma cells (HepG2 cell line) but it has relatively small or no impact on control hepatocytes (THLE-2 cell line). Furthermore, investigation of the underlying mechanism indicated that recovery of disorder of glucose metabolism by inhibiting aerobic glycolysis and promoting tricarboxylic acid cycle played an important part in the cell proliferation inhibition of hepatoma cells. As suggested above, AST could be a very promising therapeutic agent of liver cancer in clinical trials.

    更新日期:2019-12-11
  • Author Correction: Improving Substrate Consumption and Decrease of Growth Yield in Aerobic Cultures of Pseudomonas denitrificans By Applying Low Voltages in Bioelectric Systems
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-06
    Luis F. Cházaro-Ruiz, María Irene López-Cázares, Ignacio González, Yanet Toriz, Felipe Alatriste-Mondragon, Marcela Santana, Lourdes B. Celis

    The original version of this article unfortunately contained a mistake in Equation 1.

    更新日期:2019-12-11
  • Glycoprotein Prompted Plausible Bactericidal and Antibiofilm Outturn of Extracellular Polymers from Nostoc microscopicum
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-06
    Lavania Ramachandran, Narayanasamy Marappa, Kavitha Sethumadhavan, Thajuddin Nooruddin

    Nostoc microscopicum an effective extracellular polymers (EPs) synthesizer among cyanobacteria was isolated, and its elementary morphological features were defined with the aid of light microscope and CLSM. Bioseparation of EPs from 44 days-aged culture gave 0.90 g/L of the dry powdered extract. Chemical quantification of EPs showed the presence of 550 mg/g of carbohydrate and 395 mg/g of protein. HPLC results depicted the presence of mannose (monomer sugar), fucose (hexose deoxy sugar), mannitol (sugar alcohol) and N- acetylglucosamine (glycoprotein) in the EPs. The vibration-based spectrum produced by FT-IR proves the β-Sheet structure of EPs glycoprotein and the size as 45 kDa by performing SDS-PAGE. Bactericidal activity evaluation of EPs on Pseudomonas aeruginosa and Staphylococcus aureus co-expressed the MIC value as 125 μg/mL, while zone of inhibition was 12 mm for Gram-negative and 8 mm for Gram-positive bacteria. Biofilm inhibition assay was effective in 1.0 mg/mL concentration of EPs in both bacterial strains with a mean rate of 60 percentages which was further confirmed using confocal laser scanning microscopic imaging. This natural polymeric extract of Nostoc microscopicum indicates its possible applications in bactericidal and biofilm inhibition.

    更新日期:2019-12-11
  • The Protective Effects of Silymarin on Thioacetamide-Induced Liver Damage: Measurement of miR-122, miR-192, and miR-194 Levels
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-06
    Ozgun Teksoy, Varol Sahinturk, Mustafa Cengiz, Behcet İnal, Adnan Ayhancı

    This study aims to investigate the protective effects of silymarin (Sm) in thioacetamide (TAA)-related liver damage. What makes this study special is that it attempts to determine the expression of changes in the liver at the level of gene expression. Routine liver damage markers were compared with changes in the levels of microRNA (miRNA) known as new biomarkers. With this in mind, we divided the rats into four groups including control, TAA, Sm + TAA (50 + 50 mg/kg), and Sm + TAA (100 + 50 mg/kg). Blood and tissue samples belonging to the rats were collected in consideration of morphological, immunohistochemistry, miRNAs levels, and biochemical evaluations. Our study results showed that miR-122, miR-192, and miR-194 levels had decreased in the experimental groups given TAA, whereas miR-122, miR-192, and miR-194 levels had increased in the doses of Sm + TAA-given group. Therefore, Sm treatment undertaken before exposure to the toxin successfully altered its effects upon the study animals.

    更新日期:2019-12-11
  • Drug Delivery of Amphotericin B through Core-Shell Composite Based on PLGA/Ag/Fe 3 O 4 : In Vitro Test
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-04
    Shiva Sadat Akhavi, Shahram Moradi Dehaghi

    This research aimed at developing and designing a slow and targeted delivery of Amphotericin B (AmB) antibiotic by placing three types of shells containing different ratios of biodegradable and biocompatible polymers poly (D, L-lactide)-co-(glycolide) (PLGA), polyethylene glycol (PEG), and polyvinyl pyrrolidone (PVP) on core-shell structures including silver nanoparticles that were activated with magnetic nanoparticles (MNPs). Emulsion solvent evaporation technique was employed to synthesize three types of shells: (i) (PVP-PEG) (100:20, w/w), (ii) (PLGA-PEG) (100:20, w/w), and (iii) (PLGA-PEG) (50:10, w/w) introduced as D1, D2, and D3 respectively. The in vitro release of AmB was examined in aqueous medium phosphate buffer saline (PBS) in pH~ 7.2. Several spectroscopy methods characterized the structure and properties of the nanoparticles. In vitro antifungal activity of pure AmB and D1, D2, and D3 was studied against Candida albicans (C. albicans). The results explained that frequency of drug released from D2 at the first 10 h was (18%) that was compared with D1 (30%) and D3 (24%) at the same time. D2 had more efficient and longer targeted controlled release. The findings showed that D2 can be used as an effective carrier for in vitro targeted controlled release and D2 and D3 had powerful activity against C. albicans.

    更新日期:2019-12-11
  • Carnosic Acid Content Increased by Silver Nanoparticle Treatment in Rosemary ( Rosmarinus officinalis L.)
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-04
    Mojtaba Hadi Soltanabad, Mohammad B. Bagherieh-Najjar, Manijeh Mianabadi

    Biosynthesis of carnosic acid (CA), one of the most industrially valuable medicinal compounds present in rosemary (Rosmarinus officinalis L.) leaves, is affected by various plant stressors. In this study, effects of silver nanoparticle (AgNP) treatment on the secondary metabolism and CA production of rosemary plants were investigated. AgNP of 0, 25, 50, 100, and 200 ppm were utilized on hydroponically grown plants using foliar spray. Efficient absorbance and translocation of AgNPs to the plant roots were confirmed by XRF (X-ray fluorescence) analysis. The fluctuations of important antioxidant compounds such as CA content, phenolics, flavonoids, and acid ascorbic were analyzed and their correlations evaluated. Results revealed that application of 200 ppm AgNPs for 12 days increased CA level more than 11%, as compared to the control plants. Furthermore, significant positive correlations were observed between total flavonoids and CA content under AgNP treatment, suggesting that AgNP acted as an elicitor and triggered the enhancement of CA accumulation effectively. These data suggest that concentration-dependent AgNP may be used to boost antioxidant activity and phytochemical contents of other medicinal plants.

    更新日期:2019-12-11
  • Carbon-Phosphorus Lyase—the State of the Art
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-02
    Natalia Stosiek, Michał Talma, Magdalena Klimek-Ochab

    Organophosphonates are molecules that contain a very chemically stable carbon-phosphorus (C-P) bond. Microorganisms can utilize phosphonates as potential source of crucial elements for their growth, as developed several pathways to metabolize these compounds. One among these pathways is catalyzed by C-P lyase complex, which has a broad substrate specifity; therefore, it has a wide application in degradation of herbicides deposited in the environment, such as glyphosate. This multi-enzyme system accurately recognized in Escherichia coli and genetic studies have demonstrated that it is encoded by phn operon containing 14 genes (phnC-phnP). The phn operon is a member of the Pho regulon induced by phosphate starvation. Ability to degradation of phosphonates is also found in other microorganisms, especially soil and marine bacteria, that have homologous genes to those in E. coli. Despite the existence of differences in structure and composition of phn gene cluster, each of these strains contains phnGHIJKLM genes necessary in the C-P bond cleavage mechanism. The review provides a detailed description and summary of achievements on the C-P lyase enzymatic pathway over the last 50 years.

    更新日期:2019-12-11
  • Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails
    Appl. Biochem. Biotechnol. (IF 2.14) Pub Date : 2019-12-02
    Dhruv Agrawal, Neha Basotra, Venkatesh Balan, Adrian Tsang, Bhupinder Singh Chadha

    In this study, two novel thermostable lytic polysaccharide monooxygenases (LPMOs) were cloned from thermophilic fungus Scytalidium thermophilum (PMO9D_SCYTH) and Malbranchea cinnamomea (PMO9D_MALCI) and expressed in the methylotrophic yeast Pichia pastoris X33. The purified PMO9D_SCYTH was active at 60 °C (t1/2 = 60.58 h, pH 7.0), whereas, PMO9D_MALCI was optimally active at 50 °C (t1/2 = 144 h, pH 7.0). The respective catalytic efficiency (kcat/Km) of PMO9D_SCYTH and PMO9D_MALCI determined against avicel in presence of H2O2 was (6.58 × 10-3 and 1.79 × 10-3 mg-1 ml min-1) and carboxy-methylcellulose (CMC) (1.52 × 10-1 and 2.62 × 10-2 mg-1 ml min-1). The HRMS analysis of products obtained after hydrolysis of avicel and CMC showed the presence of both C1 and C4 oxidized oligosaccharides, in addition to phylogenetic tree constructed with other characterized type 1 and 3 LPMOs demonstrated that both LPMOs belongs to type-3 family of AA9s. The release of sugars during saccharification of acid/alkali pretreated sugarcane bagasse and rice straw was enhanced upon replacing one part of commercial enzyme Cellic CTec2 with these LPMOs.

    更新日期:2019-12-11
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中山大学化学工程与技术学院
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天合科研
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