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

Abstract

Backgroundβ-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.ResultsA novel endo-β-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 (kcat/Km, 170 and 114 %, respectively).ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0544-8) contains supplementary material, which is available to authorized users.

A Novel Thermostable GH3β-Glucosidase fromTalaromyce leycettanuswith Broad Substrate Specificity and Significant Soybean Isoflavone Glycosides-Hydrolyzing Capability

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Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency

“…6C , middle panel), and decreasing the negative surface charge of E230G ( Fig. 6C , right panel) may improve the stability of the enzyme to high temperature and low pH 49 50 51 . Metal ions can bind to proteins at some atoms, i.e., C and H, inhibiting enzymatic activity 52 53 54 .…”

A novel thermostable aspartic protease fromTalaromyces leycettanusand its specific autocatalytic activation through an intermediate transition state

“…Extremophiles are excellent sources of novel enzymes that can retain their integrity and function under extreme reaction conditions (32). Talaromyces leycettanus JCM12802, a thermotolerant fungus, that has an optimal growth temperature of 42 °C, is the source of various thermostable hydrolases including β -mannanase (33), xylanase (34), β -glucanase (35), and α -Amylase (36). In this study, a novel aspartic protease precursor comprising of 424 amino acid residues was identified in Talaromyces leycettanus and was determined to be a member of the A1 family of aspartic proteases.…”