Expression of a β-mannosidase from Paenibacillus polymyxa A-8 in Escherichia coli and characterization of the recombinant enzyme.

Xi Bai,Zeshen Yang,Qianming Huang,Quanbin Wei,Hong Hu,Huaping Chen

doi:10.1371/journal.pone.0111622

Abstract

Paenibacillus polymyxa A-8, which secretes β-mannosidase, was isolated from the soil sample under a pine tree located in the “Laoban” mountain region of Sichuan, China. The β-mannosidase gene (MANB) was isolated from P. polymyxa A-8, using primers according to the complete genome. The MANB (2,550 bp) encoding 849 amino acid residues was expressed in Escherichia coli. The specific activities of β-mannosidase produced by P. polymyxa A-8 and E. coli pET30a-MANB were 12 nkat/mg and 635 nkat/mg respectively. SDS-PAGE analysis indicated that the molecular mass of the recombinant MANB was approximately 96 kDa. The recombinant MANB was active between pH 7.0–8.5 with the maximum activity at pH 7.0. It had good pH stability and adaptability. The MANB had the optimal temperature of 35°C and was relatively stable at 35–40°C. In addition, the MANB activity was enhanced by K+, Ca2+, Mn2+, and Mg2+ and inhibited by Zn2+, Cu2+, and Hg2+.

Highlights

Mannan is the second largest component of the hemicellulose and is distributed in plant cell walls [1,2]
The results indicated that the specific MANB activity of strain A-8 was 12 nkat/mg, which was higher than the b-mannosidase activity in Thermotoga neapolitana 5068 reported by Duffaud et al (1997) [25]
The GeneBank database and Mega 5.1 software were used to build a phylogenetic tree according to the alignment of the 16S rRNA sequence, and the results indicated that the A-8 strain was genetically more similar to P. polymyxa L1-9 than to the other microorganisms (Figure 2)

Summary

Introduction

Mannan is the second largest component of the hemicellulose and is distributed in plant cell walls [1,2] It is classified into four subfamilies: galactoglucomannan, galactomannan, glucomannan and linear mannan. Expression and Characterization of b-Mannosidase in Escherichia coli synergistic action of mannanases, galactosidases and mannosidases One of these important mannan-degrading enzymes is b-mannosidase [4,5,6,7]. The pET vectors are the most effective plasmids for the expression of proteases in Escherichia coli under the control of the bacteriophage T7 promoter, which can greatly increase their production level [20,21]. The b-mannosidase gene (MANB) from P. polymyxa A-8 was cloned and expressed in E. coli with the vector pET30a. The biochemical properties of the recombinant MANB were fully determined

Materials and Methods

Results and Discussion

Conclusions