Recombinant expression and characterization of a novel cold-adapted type I pullulanase for efficient amylopectin hydrolysis
Introduction
Due to increasing concerns about energy and the environmental crisis, it is generally accepted that the gradual replacement of oil with renewable biomass resources is an effective way to develop a sustainable society (Cinelli et al., 2015; Hii et al., 2012). Therefore, efficient biotransformation of inexpensive and environmentally friendly biomass resources into industrial raw materials or biofuels has attracted a great deal of attention. Starch, one of the most abundant biopolymers on Earth, has been used as a major raw material for foods, chemicals, and biofuels (van der Maarel et al., 2002; Lu et al., 2018; Wang et al., 2017). Most starch contains 70–95 % amylopectin, which is composed of α-1,4-linked glucose chains ramified with α-1,6-linked branch points. Since most amylases specifically attack the α-1,4-glycosidic linkage, enzymes that cleave the α-1,6-glycosidic linkages are indispensable for effective and complete decomposition of starch into small-molecule sugars (Chang et al., 2016; Duan et al., 2013; Sun et al., 2010).
Pullulanase (EC 3.2.1.41), a well-known starch-debranching enzyme, has been widely applied to catalyze the hydrolysis of α-1,6-glycosidic linkages at branching points in pullulan, amylopectin, and related saccharides (Wang et al., 2019b). Based on substrate preference and reaction products, pullulanases are classified into five groups: type I pullulanases, amylopullulanases, neopullulanases, isopululanases and pullulan hydrolases type III (Møller et al., 2016; Vincent Lombard et al., 2014). Type I pullulanases specifically attack the α-1,6-glycosidic linkages due to the consensus sequence YNWGYDP at the catalytic domain (Hatada et al., 1996; Yamashita et al., 1997). Pullulanases are members of the glycoside hydrolase family 13 (GH13) (CAZy; http://www.cazy.org) and consist of a highly conserved catalytic domain, C-terminal domain and additional carbohydrate-binding modules (CBMs) at the N-terminus (Xu et al., 2014; Zeng et al., 2019a).
In the saccharification process, the addition of pullulanase can lower the dosage of glucoamylase, reduce the reaction time, and effectively improve the conversion rate (Duan et al., 2013; Lu et al., 2018). In recent years, a great deal of research has been focused on isolating thermoactive pullulanases from various microbial sources, such as Staphylothermus marinus (Li et al., 2013), Geobacillus kaustophilus DSM7263 (Li et al., 2018a), Bacillus sp. CICIM 263 (Li et al., 2012), and Thermotoga neapolitana (Chang et al., 2016; Kang et al., 2011). Further research has been focused on improving thermostability through site-directed mutagenesis or domain modification based on structure or sequence information (Chang et al., 2016; Chen et al., 2015, 2019; Duan and Wu, 2015a; Li et al., 2015; Wang et al., 2018b). All of these studies have been aimed at making pullulanases suitable for the current starch saccharification process, which is performed at least at 60 °C (Hii et al., 2012).
However, it should be noted that starch hydrolysis at high temperature is expected to be replaced by new technologies functioning at relatively moderate temperatures owing to its high energy consumption and complex process. In the past few years, cold starch hydrolysis, also known as “granular starch hydrolysis”, which is enabled by enzymes that synergistically hydrolyze starch at 35 °C and pH 5–6, has been an emerging technology (Lu et al., 2018). Since the granular starch hydrolysis process entails direct hydrolysis of the starch-based substrate, without high-temperature incubation, gelatinization, and subsequent cooling, the operational costs of industrial plants have been estimated to be 51 % lower (Cinelli et al., 2015; Lu et al., 2018). The best conversion rate for starch into glucose hitherto achieved is 98.6 % (Cinelli et al., 2015). In recent years, some cold-adapted α-amylases (Yang et al., 2017), that randomly attack the α-1,4-glycosidic linkages in a linear amylose chain, and raw-starch-digesting glucoamylases (Sun et al., 2007), capable of releasing glucose from the non-reducing ends of starch and related substrates, have been studied. Pullulanase plays a fundamental role as a debranching enzyme in the hydrolysis of starch. In the research of using corn starch as substrate to produce ethanol through deep enzymolysis and fermentation, the addition of pullulanase accelerates the corn starch hydrolysis at 55 °C and pH 5.5, leading to lower amounts of dextrins and concomitantly higher ethanol yield (Klosowski et al., 2010; Lu et al., 2018). Therefore, cold-adapted pullulanases showing high activity and stability at around 40 °C have huge potential for application in granular starch hydrolysis.
In the last few years, only a few cold-adapted pullulanases exhibiting maximum activity at around 40 °C have been reported (Elleuche et al., 2015; Lu et al., 2018; Rajaei et al., 2015; Wei et al., 2015). Pul703 isolated from Bacillus pseudofirmus 703 and Pul-SH3 from Exiguobacterium sp. SH3, with maximum activity at 45 °C and pH 7.0–8.0, are current candidates for cold starch hydrolysis (Lu et al., 2018; Rajaei et al., 2014). However, the exhibition of optimal activity in alkaline environments and low catalytic efficiency greatly restrict their application. Herein, we present an excellent type I pullulanase (PulPB1) isolated from Bacillus methanolicus PB1 with great activity and stability over a wide temperature range 30–50 °C at pH 5.5. PulPB1 can be used for efficient cold starch hydrolysis without adjusting the temperature or pH, indicating its great potential in the granular starch hydrolysis industry.
Section snippets
Bacterial strains, plasmids, and media
Escherichia coli DH5α and BL21 (DE3) hosts were used for gene cloning and expression of pullulanase PulPB1, respectively. The strain of Bacillus methanolicus PB1 was preserved in our laboratory. The plasmid pET-28a was used as an expression vector. Luria–Bertani (LB) medium containing 1 % peptone, 0.5 % yeast extract, and 1% NaCl was used for the cultivation of E. coli DH5α and BL21 (DE3). Bacillus methanolicus PB1 was grown in SOBsuc medium, which was composed of SOB medium (2 % peptone, 0.5 %
Cloning and sequence analysis of pulPB1
Through PCR amplification, a 2148 base-pair pullulanase gene was successfully cloned from the genome of Bacillus methanolicus PB1 (GenBank accession number: NZ_AFEU01000003). The GenBank accession number of PulPB1 is WP_004439017.1.
Homology analysis revealed that PulPB1 showed low identity with reported cold-adapted pullulanases (Table 1). In particular, only 30.6 % homology was identified with pullulanase Pul703, which has been characterized as a good candidate for granular starch hydrolysis (
Conclusions
A novel cold-adapted pullulanase PulPB1 from Bacillus methanolicus PB1 strain was successfully expressed, purified, and characterized. The PulPB1 was identified as a type I pullulanase that can specifically hydrolyze α-1,6-glycosidic bonds efficiently at relatively low temperature range of 30−50 °C. PulPB1 was highly active and stable at ambient temperatures and relatively acidic surroundings. Its half-life at 50 °C and pH 5.5 was as long as about 137 h. The N-terminal domain of PulPB1 plays a
CRediT authorship contribution statement
Shi-Yu Zhang: Investigation, Data curation, Writing - original draft, Writing - review & editing. Ze-Wang Guo: Conceptualization, Investigation. Xiao-Ling Wu: Software, Validation. Xiao-Yang Ou: Software, Writing - review & editing. Min-Hua Zong: Methodology, Writing - review & editing. Wen-Yong Lou: Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
The authors gratefully acknowledge the National Natural Science Foundation of China (21878105, 21908070), the National Key Research and Development Program of China (2018YFC1603400, 2018YFC1602100), the Science and Technology Program of Guangzhou (201904010360), the Fundamental Research Funds for the Central Universities (2019PY15, 2019MS100), and the China Postdoctoral Science Foundation (BX20180102, 2019M652902) for partially funding this work.
References (42)
- et al.
Protein engineering of Bacillus acidopullulyticus pullulanase for enhanced thermostability using in silico data driven rational design methods
Enzyme Microb. Technol.
(2015) - et al.
Hydrogen-bond-based protein engineering for the acidic adaptation of Bacillus acidopullulyticus pullulanase
Enzyme Microb. Technol.
(2019) - et al.
A brief review on the emerging technology of ethanol production by cold hydrolysis of raw starch
Fuel
(2015) - et al.
Triton X-100 enhances the solubility and secretion ratio of aggregation-prone pullulanase produced in Escherichia coli
Bioresour. Technol.
(2015) - et al.
Cloning, expression and characterization of the recombinant cold-active type-I pullulanase from Shewanella arctica
Journal Mol Catal B-Enzym.
(2015) - et al.
Amino acid sequence and molecular structure of an alkaline amylopullulanase from Bacillus that hydrolyzes alpha-1,4 and alpha-1,6 linkages in polysaccharides at different active sites
J. Biol. Chem.
(1996) - et al.
Molecular cloning and biochemical characterization of a heat-stable type I pullulanase from Thermotoga neapolitana
Enzyme Microb. Technol.
(2011) - et al.
Characterisation of fermentation of high-gravity maize mashes with the application of pullulanase, proteolytic enzymes and enzymes degrading non-starch polysaccharides
Biosci. Bioeng.
(2010) - et al.
Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp. LM18-11 for improved thermostability
J. Biotechnol.
(2015) - et al.
A pH-stable, detergent and chelator resistant type I pullulanase from Bacillus pseudofirmus 703 with high catalytic efficiency
Int J Biol Macromol.
(2018)
Properties and applications of starch-converting enzymes of the α-amylase family
J. Biotechnol.
Improvement of extracellular secretion efficiency of Bacillus naganoensis pullulanase from recombinant Escherichia coli: peptide fusion and cell wall modification
Protein Epression Purif.
Industrially produced pullulanases with thermostability: discovery, engineering, and heterologous expression
Bioresour. Technol.
Amino acid residues specific for the catalytic action towards α-1,6-glucosidic linkages in Klebsiella pullulanase
J. Ferment. Bioeng.
Effects of different carbohydrate-binding modules on the enzymatic properties of pullulanase
Int. J. Biol. Macromol.
Identification and analysis of binding residues in the CBM68 of pullulanase PulA from Anoxybacillus sp
LM18-11. J Biosci Bioeng.
Advances in molecular engineering of carbohydrate-binding modules
Proteins
Improving the thermostability of acidic pullulanase from Bacillus naganoensis by rational design
PLoS One
Downsizing a pullulanase to a small molecule with improved soluble expression and secretion efficiency in Escherichia coli
Microb. Cell Fact.
Pullulan degrading enzymes of bacterial origin
CRC Crit. Rev. Microbiol.
Enhancing the secretion efficiency and thermostability of Bacillus deramificans pullulanase mutant D437H/D503Y by N-terminal domain truncation
Appl. Environ. Microbiol.
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