Abstract
Backgroundβ-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 (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.ResultsTo overcome the limitation, RmMan5A was successfully engineered by directed evolution. Through two rounds of screening, a mutated β-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.ConclusionsThrough 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 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.
Highlights
As the huge consumption of fossil fuels results in enormous emission of greenhouse gases, more and moreLi et al Biotechnol Biofuels (2017) 10:143 into mono- and oligosaccharides through chemical, physical, and enzymatic methods [1, 4, 5]
According to the results of DNA sequencing, mRmMan5A contained 5 base substitutions, two of which led to silent mutations (Ala261: gct-gcc, Leu318: tta-ttg), while the others led to three non-silent mutations, namely Tyr233His, Lys264Met, and Asn343Ser
Amino acid sequence alignment of RmMan5A with the homologous β-mannanases was performed and revealed that residue Try233 was entirely conserved, Lys264 was partly conserved, and Asn343 was non-conserved in glycoside hydrolase (GH) family 5 β-mannanases (Fig. 1)
Summary
As the huge consumption of fossil fuels results in enormous emission of greenhouse gases, more and moreLi et al Biotechnol Biofuels (2017) 10:143 into mono- and oligosaccharides through chemical, physical, and enzymatic methods [1, 4, 5]. The industrial applications of β-mannanases have developed rapidly in food, feed, and biorefinery industry [3, 7]. Β-mannanases from subfamily 8 display higher activity than subfamilies 7 and 10, which usually act at acidic and neutral pH [16]. Even though wild-type enzymes have various desirable properties, their biochemical properties display many limitations, which make them not suitable for industrial applications in some cases [17]. Many wild-type β-mannanases exhibit the maximal activity under acidic and/or thermophilic conditions, their specific activities maintain at low levels in their optimal pH and temperatures [20, 21], causing the high cost in production and the limitation in biorefinery industry
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