Directed Evolution of a β-mannanase from to Improve Catalytic Activity in Acidic and Thermophilic Conditions

Overview

Journal Biotechnol Biofuels

Publisher Biomed Central

Specialty Biotechnology

Date 2017 Jun 8

PMID 28588644

Citations 6

Authors

Yan-Xiao Li

Ping Yi

Qiao-Juan Yan

Zhen Qin

Xue-Qiang Liu

Zheng-Qiang Jiang

Affiliations

Soon will be listed here.

Abstract

Background: β-Mannanase randomly cleaves the β-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of β-mannanase have tremendously expanded in recent years, the wild-type β-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 β-mannanase (Man5A) from shows many outstanding properties, such as high specific activity and hydrolysis property. However, owing to the low catalytic activity in acidic and thermophilic conditions, the application of Man5A to the biorefinery of mannan biomasses is severely limited.

Results: To overcome the limitation, Man5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated β-mannanase (mMan5A) with high catalytic activity in acidic and thermophilic conditions was obtained, and then characterized. The mutant displayed maximal activity at pH 4.5 and 65 °C, corresponding to acidic shift of 2.5 units in optimal pH and increase by 10 °C in optimal temperature. The catalytic efficiencies (/) of mMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of Man5A were inherited by the mutant mMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mMan5A, namely Tyr233His, Lys264Met, and Asn343Ser. To identify the function of each substitution, four site-directed mutations (Tyr233His, Lys264Met, Asn343Ser, and Tyr233His/Lys264Met) were subsequently generated, and the substitutions at Tyr233 and Lys264 were found to be the main reason for the changes of mMan5A.

Conclusions: Through directed evolution of Man5A, two key amino acid residues that controlled its catalytic efficiency under acidic and thermophilic conditions were identified. Information about the structure-function relationship of GH family 5 β-mannanase was acquired, which could be used for modifying β-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a β-mannanase from zygomycete engineered by directed evolution.

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