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Production and partial purification by PEG/citrate ATPS of a β-galactosidase from the new promising isolate Cladosporium tenuissimum URM 7803
Preparative Biochemistry & Biotechnology ( IF 2.9 ) Pub Date : 2020-09-09 , DOI: 10.1080/10826068.2020.1815054
Anderson José Paulo 1 , Maria Carolina de Albuquerque Wanderley 2 , Rafael José Vilela de Oliveira 3 , Willie Anderson Dos Santos Vieira 4 , Luiz Carlos Alves 5 , Daniela de Araújo Viana Marques 6 , Attilio Converti 7 , Ana Lúcia Figueiredo Porto 8
Affiliation  

Abstract

β-Galactosidase production, partial purification and characterization by a new fungal were investigated. Partial purification was performed by aqueous two-phase system (ATPS) using polyethylene glycol (PEG) molar mass, PEG concentration, citrate concentration and pH as the independent variables. Purification factor (PF), partition coefficient (K) and yield (Y) were the responses. After identification by rDNA sequencing and classification as Cladosporium tenuissimum URM 7803, this isolate achieved a maximum cell concentration and β-galactosidase activity of 0.48 g/L and 462.1 U/mL, respectively. β-Galactosidase partitioned preferentially for bottom salt-rich phase likely due to hydrophobicity and volume exclusion effect caused in the top phase by the high PEG concentration and molar mass. The highest value of PF (12.94) was obtained using 24% (w/w) PEG 8000 g/mol and 15% (w/w) citrate, while that of Y (79.76%) using 20% (w/w) PEG 400 g/mol and 25% (w/w) citrate, both at pH 6. The enzyme exhibited optimum temperature in crude and ATPS extracts in the ranges 35–50 °C and 40–55 °C, respectively, and optimum pH in the range 3.0–4.5, with a fall of enzyme activity under alkaline conditions. Some metal ions and detergents inhibited, while others stimulated enzyme activity. Finally, C. tenuissimum URM 7803 β-galactosidase showed a profile suitable for prebiotics production.



中文翻译:

用PEG /柠檬酸ATPS从新的有希望的分离菌株克氏假单胞菌URM 7803生产和部分纯化β-半乳糖苷酶

抽象的

研究了新型真菌对β-半乳糖苷酶的生产,部分纯化和表征。使用聚乙二醇(PEG)摩尔质量,PEG浓度,柠檬酸盐浓度和pH作为自变量,通过水两相系统(ATPS)进行部分纯化。响应因子为纯化因子(PF),分配系数(K)和产率(Y)。通过rDNA测序鉴定后分类为小球藻(Cladosporium tenuissimum)URM 7803,该分离物的最大细胞浓度和β-半乳糖苷酶活性分别为0.48 g / L和462.1 U / mL。β-半乳糖苷酶可能优先分配给底部富盐相,这可能是由于较高的PEG浓度和摩尔质量在顶部相中引起的疏水性和体积排阻作用所致。使用24%(w / w)PEG 8000 g / mol和15%(w / w)柠檬酸盐可获得PF的最大值(12.94),而Y则为(79.76%)使用20%(w / w)PEG 400 g / mol和25%(w / w)柠檬酸盐,pH均为6。该酶在原油和ATPS提取物中的最佳温度为35–50°C分别在40-55°C和40-55°C的温度范围内,最佳pH在3.0-4.5范围内,碱性条件下酶的活性下降。一些金属离子和去污剂抑制,而另一些刺激酶活性。最后,腱糖衣原体URM 7803β-半乳糖苷酶显示出适合于益生元生产的概况。

更新日期:2020-09-09
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