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High-effective Catalytic Characteristics of Glycerol Dehydratase from K.pneumoniae XJPD-Li

Author: LvBo
Tutor: LiChun
School: Shihezi University
Course: Agricultural Products Processing and Storage
Keywords: Glycerol dehydratase Site-directed mutagenesis 1,3 - propanediol yqhD The K. pneumoniae XJPD - Li
CLC: Q814
Type: Master's thesis
Year: 2009
Downloads: 9
Quote: 1
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Abstract


Glycerol or glucose from renewable resources such as raw materials in the biological synthesis of 1,3 - propanediol (1,3-PD) with production of clean, environmental pollution \Task Force preliminary screening strain Klebsiella the pneumoniae XJPD-Li having a higher temperature (40 ° C) and high pH (8.0), efficient and rapid synthesis of 1,3 - propanediol characteristics. The paper by Klebsiella pneumoniae XJPD-Li 1,3 - propylene glycol key enzyme gene cloning, expression and site-directed mutagenesis, the the glycerol dehydratase efficient catalytic characteristics with the following results: 1. By PCR from K. pneumoniae XJPD The-Li total DNA was successfully cloned glycerol dehydratase gene fragments and construct the expression vector pET28a-dhaBCE. In 1.0mmIPTG, 30 ° C conditions induced by 5 hours, successful expression of recombinant glycerol dehydratase. Of recombinant glycerol dehydratase catalytic reaction optimum reaction temperature to 45 ℃, the optimum pH of 8.0, the specific activity of 40.2U mg -1 , approximately 80 times of the original strain. Sequence alignment K. pneumoniae glycerol dehydratase reported in GenBank (U60992) gene similarity was 99.48% and 99.55% amino acid similarity. 2 large primer PCR and plasmid fast mutation K. pneumoniae XJPD-Li glycerol dehydratase alpha subunit amino acid mutation was successfully constructed the mutant M193α (S193C) M407α to (E40A), in 1.0mmIPTG, 30 ℃ for 5 hours induced conditions, M193α glycerol dehydratase enzyme-catalyzed reaction optimum temperature of 45 ℃, the optimum pH was 8.0, the specific activity of 37.8 U · mg -1 ; M407α optimum reaction temperature to 40 ° C, compared with the native enzyme decreased by 5 ° C, the optimum pH of 8.0, the specific activity of 14.7 U · mg -1 , is 36.6% of the original enzyme. (3) the use of plasmid fast mutagenesis, K. the pneumoniae XJPD-Li recombinant glycerol dehydratase beta subunit differences in amino acid mutations. The successfully constructed the mutant M47β (N47I), M189β, (V189I), in 1.0mmIPTG 30 ° C conditions induced by 5 hours under the conditions the the glycerol dehydratase for M47β and M189β specific activity were the 38.7U mg -1 and 39.2 U · mg -1 , and mutant recombinant glycerol dehydratase close to the specific activity. M47β and M189β glycerol dehydratase optimum temperature is 40 ° C and 45 ° C, the optimum pH were 8.0, optimum temperature for recombinant bacteria compared with the unmutated, M47β catalytic reaction decreased by about 5 degrees. 4 by PCR from E. coli K -1 2 in successfully cloned the yqhD genes with expression vectors the pET28a-dhaBCE series expression, protein electrophoresis analysis showed that the 61 kD, 43kD, 24KD and 16KD at specific bands. By glycerol dehydratase gene of Klebsiella pneumoniae XJPD-Li Cloning and expression of fixed-point mutation research provide the basis for the study of the glycerol dehydratase efficient catalytic mechanism, and also provide theoretical guidance for the directed evolution of glycerol dehydratase.

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