Title:Production and Functional Characterization of a Novel Mannanase from
Alteromonadaceae Bacterium Bs31
Volume: 29
Issue: 8
Author(s): Rui Ding, Huifang Xie, Zhenggang Han*Jiangke Yang
Affiliation:
- College of Biology and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan 430023, China
Keywords:
Auto-induction, Escherichia coli expression, glycoside hydrolase family 5, mannanase, mannooligosaccharides, Pichia pastoris expression.
Abstract:
Background: Mannans are the main components of hemicellulose in nature and serve as
the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their
functional characteristics are an important basis for mannan biotechnological applications.
Objective: In this study, a putative mannanase gene (ManBs31) from the genome of the marine
bacterium Alteromonadaceae Bs31 was characterized.
Methods: Amino acid sequence analysis and protein structural modeling were used to reveal the
molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced
using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the
enzymes were determined by reducing sugar assay and thin-layer chromatography.
Results: Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a
catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding
domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and
pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity
up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain
modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and
ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides,
and no monosaccharide was generated. Structural analysis suggested that
ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases.
Conclusion: ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative
for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.
