The cloning and biochemical characterization of β-1,3-1,4-glucanase genes from Neocallimastix frontalis J11

• Main Authors: Yu-Lung Hung, 洪玉龍
• Other Authors: Yo-Chia Chen
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/81447005465634943536

碩士 === 國立屏東科技大學 === 生物科技研究所 === 95 === Abstract Student ID：M9318015 Title of Thesis:：The cloning and biochemical characterization of β-1,3-1,4-glucanase genes from Neocallimastix frontalis J11 Total Page：66 Name of Institute:National Pingtung University of Science Technoogy Name of Department:Graduate Institute of Biotechnology Graduate Date：July, 2007 Degree Conferred：Master Name of Student：Yu-Lung Hung Adviser：Yo-Chia Chen The Contents of Abstract in This Thesis： β-1,3-1,4-glucans constitute a non-substituted group of hemicellulosess. Structurally, they are linear glucans of up to about 1200 β-D-glucosyl residues linked through β-1,3 and β-1,4 glycosidic bonds, with variations in the proportion of both types of linkages. β-1,3-1,4-glucanase exhibits a strict substrate specificity for cleavage of β-1,4 glycosidic bonds in 3-O-substituted glucopyranose units, and the main final hydrolysis products of barley β-glucans are the trisaccharide (3-O-β-cellobiosyl-D-glucopyranose) and the tetrasaccharide (3-O-β-cellotriosyl-D-glucopyranose), and are widely applied in the beer brewing and animal feedstuff industries. In this study, four β-1,3-1,4-glucanase genes, m2, a13, a16 and a51 , were isolated from cDNA libraries of an anaerobic rumen fungus Neocallimastix frontalis J11 and sorted by three- dimensional protein structure from seven β-1,3-1,4-glucanase genes . Based upon the results of the DNA sequence alignment, the most similar sequence with the four recombinant enzymes was Orpinomyces sp. PC-2 licA gene and it showed 81.8 to 82.3 percent sequence similarity to the known licA gene. The four β-1,3-1,4-glucanase genes, m2, a13 a16 and a51, expressed in Escherichia coli BL21(DE3) had the same molecular weight about 27 kDa, and the specific activities which using barley β-glucans as substrate were 41,209, 32,095, 39,524 and 26,529 U/mg, respectively. Optimal temperature for enzyme activity was 45℃ except for M2 which was 50℃, and optimal pH was 6 besides A51 which was 6.5. A13 had better thermal stability than others, and M2 was the best pH stability enzyme. The effect of various metal ions on the activity indicated that the activities of all of the recombinant enzymes were significant increased by Co2+. The results of substrate specificity analysis showed that the four recombinant enzymes were only able to hydrolyse barley β-glucans and lichenans. Comparing with the diversities of protein structures and biochemical properties within the four recombinant enzymes, there was probably no direct relationship between them. Key words: β-1,3-1,4-glucan, β-1,3-1,4-glucanase , Neocallimastix frontalis