Cloning, Expression, and Characterization of Xylanase G2 from Aspergillus oryzae VTCC-F187 in Aspergillus niger VTCC-F017

Do Thi Tuyen,Nguyen Sy Le Thanh,Nguyen Thi Trung,Nguyen Tien Cuong,Nguyen Thi Thao,Nguyen Thi Hien Trang,Le Thanh Hoang,Dao Thi Mai Anh,Gustavo Graciano Fonseca

doi:10.1155/2021/8840038

Abstract

The study focuses on engineering of recombinant Aspergillus niger to produce highly active xylanase. The xylanase G2 encoding gene originating from Aspergillus oryzae VTCC-F187 was cloned, amplified, and inserted into the pAN7.1GluA vector with specific primers possessing BamHI. The recombinant plasmid was introduced into Aspergillus niger VTCC-F017 by chemical methods. The recombinant strain was checked by polymerase chain reaction method and Southern blot. Next, the recombinant protein was expressed and purified by His-tag column. The molecular mass of the purified xylanase G2, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), was 21 kDa with a specific activity of 1025 IU/mg towards 0.5% (w/v) of birchwood xylan. The optimal temperature and pH were 55°C and pH 6.5, respectively. The enzyme was stable in a temperature ranges 25–40°C and a pH ranges 5–7. The presence of Tween 80 enhanced xylanase activity. Triton X-100, however, had no impact on the function of the enzyme. The xylanase activity was reduced by Tween 20, SDS, and organic solvents. The enzyme was completely inhibited by Hg2+ and partially by Zn2+, Fe2+, and Ag+, while it was slightly stimulated by K+ and EDTA.

Highlights

Xylanase is a class of enzymes produced by microorganisms to breakdown xylans, the most abundant hemicellulosepolysaccharides found in plant cell walls
Kit ProBondTM Nickel-chelating resin was from Invitrogen Corp. (Carlsbad, CA, USA). 3,5,5-Dinitrosalisilic acid (DNS), birchwood xylan, and SDS were from Sigma-Aldrich Co
The PCR product had a size of 699 bp, which corresponded to the size of the xlnG2 (Supplement 1C). These results suggested that the xlnG2 was successfully cloned in to pJET1.2 and transformed into E. coli DH5α

Summary

Introduction

Xylanase is a class of enzymes produced by microorganisms to breakdown xylans, the most abundant hemicellulosepolysaccharides found in plant cell walls. They are used as a pre-bleaching agent which facilitates subsequent chemical bleaching, reduces the toxic chemical demand (especially organochlorine compounds), and improves the brightness of the pulp [1]. Xylanases are used in the clarification of fruit juice, wine, beer, and forming xylitol in the confectionery industry [2]. The enzymes hydrolyze food containing xylan and, at the same time, help to reduce the viscosity in the digestive system followed by many positive effects such as improved food absorption, improved microorganism populations of the intestine in the advantageous direction, and reduced digestion disorder [3, 4]

Methods

Results

Conclusion