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Cloing and Sequence Analysis of Key Genes in Metabolism from L.bulgaricus

Author: TanWenJun
Tutor: WenJianPing
School: Tianjin University
Course: Biochemical Engineering
Keywords: L.bulgaricus PCR acetic acid kinase gene ack L-lactate dehydrogenation enzyme gene ldhL D-lactate dehydrogenase gene ldhD
CLC: Q78
Type: Master's thesis
Year: 2009
Downloads: 87
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Abstract


D-lactic acid is precursor of many chiral substances, widely used in many fields. Production of high pure D-lactic acid is always a problem need to be resolved. We studied the key enzymes in lactic acid metabolic pathway from L.bulgaricus DBLB28 and found valuable functional genes and regulatory sites.We studied the glucose metabolism of L.bulgaricus and found acetic acid is main by-product in fermentation broth. Based on the above study, we built major metabolic pathway of L.bulgaricus under anaerobic conditions, and found the key functional genes: L-lactate dehydrogenation enzyme gene, D-lactate dehydrogenase gene and acetic acid kinase gene which could control the production of acetic acid.Based on the related genes in Genbank, we cloned three key genes of glucose metabolism from L.bulgaricus, and analysed function of these genes.We cloned, sequenced and analysis part of ack gene, and found four highly conservative regions. The region CHLGSGASITAVDGKSFDSMGFS full of glycine belongs to the acetate kinase, butyrate kinase family. Under anaerobic condition, acetate kinase catalyzes reaction between acetyl phosphate and acetate acid with production of ATP. The conservative G-rich region may be the substrate or ATP binding site. In addition, the conservative sequences YAPHNPAE and YGAHGTSVR Y contain H play the role of proton donors.The length of ldhD is 1002 bp; it encodes a dehydrogenation enzyme of 333 amino acids. The active site of LdhD may be PLVDTDA in 247 points; the coenzyme NAD+ binding site may be VGVIGTGHIGQVFMQIMEGFGAKVIAYDI (from 162 points to 190 points). Highly conserved His existes in the 219 points and 310 points; we speculated that the two sites are proton donors of D-lactate dehydrogenase.L-lactate dehydrogenase gene ldhL encodes 307 amino acids. The protein molecular weights 32631.38 Dalton and there are a large number ofβ-turns in its secondary structure. The number of conservative regions in amino acid sequence is 11, and the active domains may be NPVDILT (NAD+ binding site) and LGEHGD that contains highly conserved region of H.

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