【作者】 余小娜；

【导师】 崔中利；

【作者基本信息】 南京农业大学 ， 微生物学， 2011， 硕士

【摘要】 谷氨酰胺转胺酶(Microbial Transglutaminase,简称TGase,EC 2.3.2.13)(?)色催化多种蛋白质分子间和分子内的酰基转移反应,改善蛋白质的功能特性,在食品、化妆品、制药等工业领域有着广阔的应用前景。本研究从土壤中筛选出产谷氨酰胺转胺酶TGase菌株,以期获得有较高产酶能力的菌株,并克隆其编码基因,通过分子生物学手段在大肠杆菌中大量表达谷氨酰胺转胺酶。从土壤中筛选得到一株产酶的菌株MEPE0134,并根据菌株形态、培养特征、生理生化特性以及16S rDNA序列等进行多项分类研究。发现该菌株具有典型的芽孢杆菌属的形态学特征,在16S rDNA序列比较分析的基础上,并综合形态特征、培养特征及生理生化特性,初步判定该菌株与芽孢杆菌属中的解淀粉芽孢杆菌菌种相近。首先,研究了发酵条件通气量,接种量,培养温度,培养液初始pH值对MEPE0134产酶的影响,其中通气量,培养温度,培养液初始pH对MEPE0134产酶都有一定影响,因此,通过优化发酵条件可以提高产酶量。其次,从产酶菌株MEPE0134中获得其基因组DNA,利用PCR方法扩增出TGase全长基因(tgl)并进行测序分析,通过比较分析MEPE0134菌株的tgl序列与芽孢杆菌属其他tgl的序列,结果表明MEPE0134的tgl与解淀粉芽孢杆菌的TGase相似性最高。将tgl克隆到表达载体pET-22b(+)中,构建重组质粒pET22-tgl,将重组质粒pET22-tgl成功转化E.coli BL21,在IPTG的诱导下成功表达重组蛋白。取诱导表达的菌经超声波破菌后离心,上清液通过金属鳌合层析进行分离纯化,获得的样品在电泳中显示为单一条带,证明成功地获得TGase蛋白纯品。接着对TGase的部分性质进行了研究,通过对牛血清白蛋白(BSA)的交联,发现随着蛋白浓度的增加,被交联的蛋白量逐渐增多,而且约12小时后绝大部分的蛋白都被交联掉了。在温度稳定性方面,TGase蛋白于4℃保存一月相当稳定,活性损失很少。综上所述,本文从土壤中筛选得到一株产酶的菌株MEPE0134,利用分子手段从MEPE0134染色体中克隆了tgl序列。并在大肠杆菌系统中获得了可溶性表达,进一步通过亲和层析得到蛋白纯品后,以BSA为底物对TGase的性质进行了研究,取得较理想的结果,为深入研究产TGase的芽孢杆菌菌株的性质及应用奠定了良好的基础。更多还原

【Abstract】 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.更多还原