Cloning and DNA shuffling of xylanase genes from Bacillus subtilis

• Main Authors: Chia-Liang, Tsai, 蔡佳良
• Other Authors: Min-Jen Tseng
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/34332609663711163392

碩士 === 台北醫學院 === 細胞及分子生物研究所 === 89 === Xylan is a major component of hemi-cellulose and is characterized by a ß-1,4-linked D-xylopyranosyl main chain that carries a variable number of neutral or uronic monosaccharide substituents. The hydrolysis of its characteristic backbone involves ß-1,4-xylanases and ß-xylosidases. Xylanases attack internal xylosidic linkages on the backbone to produce several xylooligomers. Microorganisms including fungi and bacteria often produce more than one type of xylanase. Xylanases are used as bleach boosters in the pulp industry. In paper production, the pulp must be treated to remove the lignin component which gives the unwanted colour. The standard method of paper whitening uses environmentally unfriendly chlorine bleaches. Xylanases pretreatment reduces the quantity of chloride needed. As industrial pulping is conducted under conditions of high temperatures and high pH, the ideal xylanase for pulp bleaching would be thermostable and active at alkaline pH. So in this experiment, the specific aim is to obtain a thermostable xylanase by manipulating its gene with DNA shuffling. From the genome sequence, a Bacillus subtilis might encode 5 different xylanases. Firstly, we used PCR to clone two xylanase genes, xynA and ynfF; which are 0.68 kb and 1.28 kb, respectively. Over-express these two genes in pKK223-3 vector and showed the molecular weight of xynA protein is 22 kDa and that of ynfF protein is 42 kDa in SDS-PAGE. Also these two proteins showed xylanase activity in zymogram gel. On the zymogram gel the ynfF protein showed discrete protease processed bands. Truncated ynfF protein with deletion of 127 amino acid at C- terminus from the HindIII recognition site did not showed protein band in SDS-PAGE. The deletion might cause the truncated protein folded wrongly and was degraded by protease immediately. The xynA DNA was fragmented by DNaseI to produce DNA pieceses smaller than 300 bp and reassembled by PCR. In xylan-plate and RBB-xylan plate showed only 30 % of these mutated clones had xylanase activity. By measuring the size of clear zones in xylan or RBB-xylan plate, one clone, named xynA-1, had the largest clear zone. Comparing the DNA and amino acid sequence between xynA and xynA-1 showed there are three nucleotides changes in xynA-1 DNA sequence and only one amino acid change in xynA-1 protein. The changed amino acid is the 41th amino acid from glycine to serine. In temperature effect experiment xynA-1 protein exhibited broadened optimal temperature ranges than xynA protein. Then in the thermostability experiment, the Tm value shifted from 45oC of xynA protein to 49oC of xynA-1 protein (Tm is the temperature at which 50 % inactivation occurs after heat treatment for 30 min). These two proteins have the same pH optimum at 7. Bacillus circulans xylanase (BCX) was used as the structure model for xynA-1 protein because it has only one amino acid difference from xynA protein. The BCX structure resembles a partially closed right-hand. The increased thermostability of xynA-1 protein might be due to the newly formed hydrogen bond between the side chains of the 41th Ser and the 39th Asp residues in the first b-turn. This bonding might stabilize this b-turn which in turn can strengthen the structure of b-sheets 1 and 2 that formed the thumb. The Km, initial rate and Vmax of xynA-1 protein are similar with the wild-type protein so it shows this mutant did not affect the catalytic site. This could be predicted because the changed amino acid in xynA-1 protein is not close to the active site.