產(chǎn)谷氨酰胺轉(zhuǎn)胺酶菌株的鑒定及其誘變選育研究.pdf

1、中南民族大學(xué)碩士學(xué)位論文產(chǎn)谷氨酰胺轉(zhuǎn)胺酶菌株的鑒定及其誘變選育研究姓名：陳潔申請(qǐng)學(xué)位級(jí)別：碩士專(zhuān)業(yè)：生化與分子生物學(xué)指導(dǎo)教師：何冬蘭20080522產(chǎn)谷氨酰胺轉(zhuǎn)胺酶菌株的鑒定及其誘變選育研究 IIABSTRACT Transglutaminase could improve the characteristics of protein foods by catalyzing protein cross-linking within or

2、 between protein molecules, and is very valuable for the food industry. To obtain higher transglutaminase producing strain, the transglutaminase -producing strain H197 from soil by the laboratory was treated by UV and ge

3、ne cloning of transglutaminase from the strain H197 was tried. In addition, we also tried to conduct high transglutaminase-producing E. coli by genetic engineering. Strain H197, which could produce transglutamiase, was s

4、elected from the soil. In order to identify this strain, were studied the strain patterns, culture characteristics, physiological and biochemical characteristics, as well as 16SrDNA sequences. The results showed as follo

5、wing. First, the strain H197 had typical morphology of Streptomyces species. Second, the strain H197 has a similar characteristic to Streptomyces hygroscopicus in strain patterns, culture characteristics, physiological a

6、nd biochemical characteristics and 16SrDNA sequences. According to the multi- classification principle and the phylogenetic analysis, the strain H197 was identified as Streptomyces hygroscopicus. In addition, sequence an

7、alysis showed that TG gene of H197 exhibited a higher similarity to that of Streptomyces hygroscopicus than to other Streptomyces strains. H197 (transglutaminase activity for 0.34 u/mL) was used as the original strain, a

8、fter two ultraviolet repeat mutation, high TGase producting strains was selected by gel-producing and measuring the enzyme activity. Finally, a high TGase producting strain (transglutaminase activity for 1.25 u / mL) was

9、 selected, with which the activity ratio of MTG got 3.68 to fold that of H197. The genome DNA was extracted from the strain H197. The full-length of the transglutaminase gene was amplified and sequenced. The analysis of

10、gene sequence revealed that the full-length sequence of transglutaminase gene was 1257 bp in length with 61.4% GC content, and encoded 418 amino acids with was molecular mass of 47 kD and a pI value of 7.08. BamH I and H

11、ind III was selected for the restriction enzyme to produce the cohesive end. The full length of the transglutaminase gene with an additional restriction site was conducted by ligating the enzyme gene into the vector pET-