Cloning and expression of a β-mannanase gene from Bacillus sp. MK-2 and its directed evolution by random mutagenesis.
A β-mannanase gene was cloned from Bacillus sp. MK-2 and expressed in Bacillus subtilis WB800. The ORF of the β-mannanase gene was 1104 bp in length, encoding 367 aa. The deduced amino acid sequence shared high sequence identity with the β-mannanase from Bacillus subtilis, and belongs to glycosyl hydrolase family 26. The purified recombinant enzyme had a specific activity of 2802 U/mg and displayed optimum activity at pH 6.0 and 55 °C. To obtain an enzyme with high specific activity and facilitate its industrial applications, molecular engineering of Bman26 was undertaken using random mutagenesis in Bacillus subtilis WB800. Three positive mutants with substantially improved specific activities were selected and studied. The best performing mutant was K291E, for which the single amino acid substitution led to a 3.5-fold increase in kcat/Km. Mutants Q112R and L211I also exhibited an apparently increased kcat/Km towards konjac glucomannan, approximately 200% and 80% improvement, respectively. Structural-functional analysis indicated that a slight conformational change could dramatically affect certain enzyme characteristics. In addition, three amino acid sites (Gly88-Leu212-Lys288) in Bman26 were found to have close relationships with the enzyme's thermal stability. These new findings will help promote the development of industrially useful β-mannanase, with both good thermal stability and high specific activity.
['Zhang|Wen|W|', 'Liu|Zhemin|Z|', 'Zhou|Sijia|S|', 'Mou|Haijin|H|', 'Zhang|Ruifu|R|']
D000595:Amino Acid Sequence D001407:Bacillus D001412:Bacillus subtilis D019020:Directed Molecular Evolution D004795:Enzyme Stability D015870:Gene Expression D006863:Hydrogen-Ion Concentration D007477:Ions D007700:Kinetics D008351:Mannans D009154:Mutation D016366:Open Reading Frames D013329:Structure-Activity Relationship D013696:Temperature D044902:beta-Mannosidase
β-mannanase Directed evolution Bacillus subtilis Structural-functional analysis Catalytic efficiency