Screening Of TGase Producing Strains,Construction Of A Secretory Shuttle Expression Vector, Cloning And Expression Of Tgl

Microbial transglutaminase(transglutaminase, TGase) can catalyze crosslinking reaction of protein to improve the structure of protein, gel formation and protein stability,so it has wide application prospect in food industry as well as others. This study carried out the following experiments: screening of TGase producing strains from the soil samples,cloning and characterization of the transglutaminase gene(tgl), construction of a new secretory shuttle expression vector, expression of the tgl gene in Escherichia coli and Bacillus subtilis. This research will provide evidence for characterization of TGase and its application in food industry.1. Three TGase producing strains were isolated from the soil samples, including two actinomycetes strains that its activity were similar and one bacteria strain. These strains were given the number respectively A109.5, A110.4 and TGase1318. The strain Bacillus subtilis subsp. natto TGase1319 that stored in this laboratory was also identified as a TGase producing strain. The strain TGase1318 was identified as B. subtilis according to its morphological features, physiological and biochemical characteristics, as well as 16 S r DNA sequences anaylsis that demonstrated it shared 99% homology with that of other B.subtilis strains.2. Using primers designed based on the conserved sequence of the published tgl gene of B. subtilis, two tgl genes, which were 738 bp long nucleotides, were cloned from the B.subtilis strains TGase1318 and TGase1319 by PCR. These two tgl genes encoded two proteins which each had 245 amino acids and there were only two different amino acids between the proteins. The isoelectric point, secondary structure, tertiary structure and other characteristics were analyzed respectively by molecular biology software. The results indicated that these TGases had similar structure characteristics, the same molecular weight(28.29 k Da) and the same p I(p I 6.71). Homological analysis of the two TGase peptide sequences revealed that it shared 94~100% conservation with TGases of other B.subtilis strains released by NCBI under BLAST alignments.3. A DNA fragment containing the sequence of signal peptide(Apre) from B. subtilis natto alkaline protease gene E and the sequence of xylose promoter(Pxylr) that derived from the E. coli-B. subtilis shuttle expression vector p TL were amplified by overlap PCR.Then the fusion DNA fragment replaced the sequence of T7 peptide of the shuttle vector p TL to construct an E. coli-B. subtilis secretory shuttle expression vector p TZ which could be induced the exogenous protein expression by D-xylose. The genes gus and lip A encoding β-glucuronidase(GUS), lipase(from Serratia marcescens) separately, were amplified by PCR, then were inserted into the multiple cloning site in the vector p TZ generating two recombinant plasmids. The engineered strains W8-gus, W8-lipa were obtained after these two recombinant plasmids were transformed into the strain B. subtilis WB800. Using gus as a reporter gene, the preliminary optimization for GUS expression was done by observation of fluorescence intensity under a microscope after W8-gus was induced by D-xylose and the optimal condition was as follows: the culture was adding 2%D-xylose as a final concentration and incubated at 37 oC, 200 r/min for 24 h, the supernatant of W8-gus strain turned blue. Uner this condition, the expression of Lip A was induced and the lipase activity of supernatant from the strain W8-lipa was 195.2±0.05U/mg, which was increased nearly 300 times compared to the sample of non-induced strain W8-lipa, assayed with the p-NPP colorimetry. The results revealed that the two genes were highly expressed and the facts demonstrated the vector p TZ was an E. coli-B. subtilis secretory shuttle expression vector with the characters of easy to handle and highly efficient expression.4. The two tgl genes from the strains TGase1318, TGase1319 were cloned into the secretory expression vector p TZ and the E. coli expression vector p ET-21 b. After that, the four recombinant plasmids(two p TZ-tgl and two p ET-21b-tgl) were constructed and the TGases were induced by D-xylose after they were transformed into the corresponding strains B. subtilis WB800 and E. coli BL21. The BSA could be cross-linked under the supernatant of the strains WB800-p TZ-t8, WB800-p TZ-t9 and the cultures of the strains BL21-p ET21b-t8, BL21-p ET21b-t9 catalyzed at 70°C and the TGase activity of BL21-p ET21b-t8 and BL21-p ET21b-t9 cell lysate was 0.45±0.06 U/m L. The results showed that the tgl genes from B. subtilis were successfully expressed.The TGase activity is higher than those have been reported and even show significant activity of BSA cross-linking at 70 °C, which the characteristic may have potential value in food processing under high temperature.