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Improvement of the thermostability and catalytic efficiency of a highly active ?-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge-charge interactions.

ABSTRACT: BACKGROUND:?-Glucanase is one of the most extensively used biocatalysts in biofuel, food and animal feed industries. However, the poor thermostability and low catalytic efficiency of most reported ?-glucanases limit their applications. Currently, two strategies are used to overcome these bottlenecks, i.e., mining for novel enzymes from extremophiles and engineering existing enzymes. RESULTS:A novel endo-?-1,3-1,4-glucanase of GH16 (Tlglu16A) from the thermophilic fungus Talaromyces leycettanus JCM12802 was produced in Pichia pastoris and characterized. For potential industrial applications, recombinant TlGlu16A exhibits favorable enzymatic properties over most reported glucanases, i.e., remarkable stability over a wide pH range from 1.0 to 10.0 and superior activity on glucan substrates (up to 15,197 U/mg). The only weakness of TlGlu16A is the thermolability at 65 °C and higher. To improve the thermostability, the enzyme thermal stability system was then used to engineer TlGlu16A through optimization of residual charge-charge interactions. Eleven mutants were constructed and compared to the wild-type TlGlu16A. Four mutants, H58D, E134R, D235G and D296K, showed longer half-life time at 80 °C (31, 7, 25, 22 vs. 0.5 min), and two mutants, D235G and D296K, had greater specific activities (158.2 and 122.2 %, respectively) and catalytic efficiencies (k cat/K m, 170 and 114 %, respectively). CONCLUSIONS:The engineered TlGlu16A has great application potentials from the perspectives of enzyme yield and properties. Its thermostability and activity were apparently improved in the engineered enzymes through charge optimization. This study spans the genetic, functional and structural fields, and provides a combination of gene mining and protein engineering approaches for the systematic improvement of enzyme performance.

SUBMITTER: You S

PROVIDER: S-EPMC4906821 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge-charge interactions.

You Shuai S Tu Tao T Zhang Lujia L Wang Yuan Y Huang Huoqing H Ma Rui R Shi Pengjun P Bai Yingguo Y Su Xiaoyun X Lin Zhemin Z Luo Huiying H Yao Bin B

Biotechnology for biofuels 20160613

<h4>Background</h4>β-Glucanase is one of the most extensively used biocatalysts in biofuel, food and animal feed industries. However, the poor thermostability and low catalytic efficiency of most reported β-glucanases limit their applications. Currently, two strategies are used to overcome these bottlenecks, i.e., mining for novel enzymes from extremophiles and engineering existing enzymes.<h4>Results</h4>A novel endo-β-1,3-1,4-glucanase of GH16 (Tlglu16A) from the thermophilic fungus Talaromyce ...[more]

PMID: 27303445

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Project description:BACKGROUND:The development of sustainable technologies for plant cell wall degradation greatly depends on enzymes with hydrolytic activities against carbohydrates. The waste by-products of agricultural cereals are important biomass sources because they contain large amounts of saccharides. Achieving efficient debranching and depolymerization are two important objectives for increasing the utilization of such renewable bioresources. GH51 ?-L-arabinofuranosidases are important in biomass pretreatment because they act synergistically with other enzymes during hemicellulose hydrolysis. RESULTS:A GH51 ?-L-arabinofuranosidase from Talaromyces leycettanus JCM12802 was heterologously expressed in Pichia pastoris GS115 and characterized. The recombinant ?-L-arabinofuranosidase, TlAbf51, showed an optimum temperature and pH of 55-60 °C and 3.5-4.0, respectively, and remained stable at 50 °C and pH 3.0-9.0. TlAbf51 showed a higher catalytic efficiency (5712 mM-1 s-1) than most fungal ?-L-arabinofuranosidases towards the substrate 4-nitrophenyl-?-L-arabinofuranoside. Moreover, TlAbf51 preferentially removed 1,2- or 1,3-linked arabinofuranose residues from arabinoxylan and acted synergistically with the bifunctional xylanase/cellulase TcXyn10A at an activity ratio of 5:1. The highest yields of arabinose and xylooligosaccharides were obtained when TlAbf51 was added after TcXyn10A or when both enzymes were added simultaneously. High-performance anion-exchange chromatography analyses showed that (i) arabinose and xylooligosaccharides with low degrees of polymerization (DP1-DP5) and (ii) arabinose and xylooligosaccharides (DP1-DP3) were the major hydrolysates obtained during the hydrolysis of sodium hydroxide-pretreated cornstalk and corn bran, respectively. CONCLUSIONS:In contrast to other fungal GH51 ?-L-arabinofuranosidases, recombinant TlAbf51 showed excellent stability over a broad pH range and high catalytic efficiency. Moreover, TlAbf51 acted synergistically with another hemicellulase to digest arabino-polysaccharides. These favorable enzymatic properties make TlAbf51 attractive for biomass pretreatment and biofuel production.

Dataset Information

Improvement of the thermostability and catalytic efficiency of a highly active ?-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge-charge interactions.

Publications

Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge-charge interactions.

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