Dissertation > Industrial Technology > Chemical Industry > Other chemical industries > Fermentation industry > Enzyme preparation ( enzyme )

Isolation and Characterization of Transglutaminase Producing Strain Mepe0134 and Cloning of TGase Gene

Author YuXiaoNa
Tutor CuiZhongLi
School Nanjing Agricultural College
Course Microbiology
Keywords Bacillus sp. transglutaminase protein polymerization purifieation
Type Master's thesis
Year 2011
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Transglutaminase(TGase; protein-glutamineγ-glutamyl transferase, EC2.3.2.13) is a recently developed enzyme and is capable of catalyzing acyl transfer reactions, which introduces covalent cross-links between proteins, peptides and various primary amines. So it has potential application in food processing, cosmetic, pharmacy and other industries. In order to obtain higher transglutaminase producing strain, transglutaminase produced strains were screened from soil in this research, and its encoding gene cloned by molecular biology methods expressed in Escherichia coli massively.Strain MEPE0134, which could produce transglutamiase, was selected from the soil. In addition, we also tried to construct high transglutaminase-producing E.coli by genetic engineering. In order to identify this strain, the strain patterns, culture characteristics, Physiological and biochemical characteristics were studied, as well as 16S rDNA sequences. The strain MEPE0134 had typical morphology of Bacillus species. The strain MEPE0134 has a similar characteristic to Bacillus amyloliquefaciens in strain patterns, culture characteristics, physiological and biochemical characteristics and 16S rDNA sequences.First of all, studies the fermentation condition ventilation, temperature, inoculation quantity, cultivate nutrient-containing medium of initial pH value of MEPE0134 enzyme production factors, especially ventilation, cultivate temperature, broth of MEPE0134 initial pH enzyme production has certain influence. Therefore, through optimizing fermentation conditions can improve enzyme production quantity.Second, The genome DNA was extracted from the strain MEPE0134. The full-length of the transglutaminase gene was amplified. According to compare the tgl sequence of MEPE0134 with other Bacillus strains. sequence analysis showed that tgl of MEPE0134 exhibited a higher similarity to that of than to other Bacillus amyloliquefaciens sequenced by PCR amplifieation and cloned into pET-22b(+) to constrant a expression plasmid pET22-tgl, then the plasmid pET22-tgl was transformed into E.coli BL21 and expressed TGase successfully induced by IPTG. The bacteria with IPTG induetion were collected and lysed by ultrasonic. TGase fusion protein was isolated and purified by metale helate affinity chromatography from the Supernatant of the baeterial lysate.The reaetion conditions of TGase were studied in this paper by polymerizing BSA. Almost all BSA could be cross-linked into polymers after 12 hours. On the temperature stability, TGase could be stored in 4℃with no activity loss.In conelusion, This TGase producted strains MEPE0134 was screened from soil, and the gene of TGase from Bacillus sp. MEPE0134 was cloned and expressed in Escherichia coli successfully, and then TGase had been purified by metal-chelating chromatography. The characteristics of TGase were studied with the substrate of BSA. All the results were very valuable for further studies on the TGase characteristics and application.

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