Dataset Information

Directed evolution of a ?-mannanase from Rhizomucor miehei to improve catalytic activity in acidic and thermophilic conditions.

ABSTRACT: BACKGROUND:?-Mannanase randomly cleaves the ?-1,4-linked mannan backbone of hemicellulose, which plays the most important role in the enzymatic degradation of mannan. Although the industrial applications of ?-mannanase have tremendously expanded in recent years, the wild-type ?-mannanases are still defective for some industries. The glycoside hydrolase (GH) family 5 ?-mannanase (RmMan5A) from Rhizomucor miehei 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 RmMan5A to the biorefinery of mannan biomasses is severely limited. RESULTS:To overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated ?-mannanase (mRmMan5A) 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 (kcat/Km) of mRmMan5A towards many mannan substrates were enhanced more than threefold in acidic and thermophilic conditions. Meanwhile, the high specific activity and excellent hydrolysis property of RmMan5A were inherited by the mutant mRmMan5A after directed evolution. According to the result of sequence analysis, three amino acid residues were substituted in mRmMan5A, 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 mRmMan5A. CONCLUSIONS:Through directed evolution of RmMan5A, 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 ?-mannanase was acquired, which could be used for modifying ?-mannanases to enhance the feasibility in industrial application, especially in biorefinery process. This is the first report on a ?-mannanase from zygomycete engineered by directed evolution.

SUBMITTER: Li YX

PROVIDER: S-EPMC5457547 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Directed evolution of a β-mannanase from <i>Rhizomucor miehei</i> to improve catalytic activity in acidic and thermophilic conditions.

Li Yan-Xiao YX Yi Ping P Yan Qiao-Juan QJ Qin Zhen Z Liu Xue-Qiang XQ Jiang Zheng-Qiang ZQ

Biotechnology for biofuels 20170602

<h4>Background</h4>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 (<i>Rm</i>Man5A) from <i>Rhizomucor miehei</i> shows many outstanding properties, such as high specific activit ...[more]

PMID: 28588644

Similar Datasets

Project description:Cavities are created by hydrophobic interactions between residue side chain atoms during the folding of enzymes. Redesigning cavities can improve the thermostability and catalytic activity of the enzyme; however, the synergistic effect of cavities remains unclear. In this study, Rhizomucor miehei lipase (RML) was used as a model to explore volume fluctuation and spatial distribution changes of the internal cavities, which could reveal the roles of internal cavities in the thermostability and catalytic activity. We present an inside out cavity engineering (CE) strategy based on computational techniques to explore how changes in the volumes and spatial distribution of cavities affect the thermostability and catalytic activity of the enzyme. We obtained 12 single-point mutants, among which the melting temperatures (Tm) of 8 mutants showed an increase of more than 2°C. Sixteen multipoint mutations were further designed by spatial distribution rearrangement of internal cavities. The Tm of the most stable triple variant, with mutations including T21V (a change of T to V at position 21), S27A, and T198L (T21V/S27A/T198L), was elevated by 11.0°C, together with a 28.7-fold increase in the half-life at 65°C and a specific activity increase of 9.9-fold (up to 5,828 U mg-1), one of the highest lipase activities reported. The possible mechanism of decreased volumes and spatial rearrangement of the internal cavities improved the stability of the enzyme, optimizing the outer substrate tunnel to improve the catalytic efficiency. Overall, the inside out computational redesign of cavities method could help to deeply understand the effect of cavities on enzymatic stability and activity, which would be beneficial for protein engineering efforts to optimize natural enzymes. IMPORTANCE In the present study, R. miehei lipase, which is widely used in various industries, provides an opportunity to explore the effects of internal cavities on the thermostability and catalytic activity of enzymes. Here, we execute high hydrostatic pressure molecular dynamics (HP-MD) simulations to screen the critical internal cavity and reshape the internal cavities through site-directed mutation. We show that as the global internal cavity volume decreases, cavity rearrangement can improve the stability of the protein while optimizing the substrate channel to improve the catalytic efficiency. Our results provide significant insights into understanding the mechanism of action of the internal cavity. Our strategy is expected to be applied to other enzymes to promote increases in thermostability and catalytic activity.

Dataset Information

Directed evolution of a ?-mannanase from Rhizomucor miehei to improve catalytic activity in acidic and thermophilic conditions.

Publications

Directed evolution of a β-mannanase from <i>Rhizomucor miehei</i> to improve catalytic activity in acidic and thermophilic conditions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets