Characterization of xylanase produced by genetically engineered bacillus subtilis

Abstract

A hydrolytic enzyme xylanase was produced using a genetically modified Bacillus subtilis strain, and its properties were characterized. The strain Bacillus subtilis(pJX18) used was previously constructed in our laboratory. This strain contains a xylanase gene from Clostridium thermocellum artificially connected to a strong native Bacillus promoter. The xylanase hydrolyzes xylan, which is the second abundant organic compound next to cellulose in nature, into xylooligosaccharides. The xylooligosaccharides, especially xylobiose and xylotriose, are industrially attractive, because they are known to stimulate the growth of Bifidobacterium selectively in human and animal intestines and, in turn, suppress the growth of undesirable intestinal flora. The extracellular xylanase was produced by growing B. subtilis(pJX18) in a 7-liter fermentor at $37^\circ C$ and pH 8. The culture medium was composed of maltose(40 g/l), yeast extract(1.5 g/l), $K_2HPO_4$(3.5 g/l), $KH_2PO_4$(1.5 g/l), and trace metal solutions. When the growth approached the stationary phase, the xylanase production started and reached a maximum level of 74.3 U/ml, after 12 hours of cultivation. Under the culture temperature of $37^\circ C$ and the pH range of 6 to 9, the plasmid showed a 100\% stability. The xylanase produced was purified by acetone precipitation and column chromatographies on Sepharose 6B and DEAE-Sepharose. SDS-PAGE and isoelectric focusing of purified xylanase were carried out. The molecular weight and pI value of enzyme were determined to be 59 kilodalton and 4.7, respectively. The xylanase showed a sharp temperature preference at $60^\circ C$ for its activity. However, at temperatures above $60^\circ C$, this enzyme lost its stability rapidly, while the enzyme was quite stable as far as it was stored at temperatures below $55^\circ C$. Under the optimal reaction temperature of $60^\circ C$, the enzyme preferred pH 5.4 sharply for its activity. The enzyme was stable in a broad range ...