Screening Of Microbial Transglutaminase And Cloning And Expression Of The Objective Gene

The transglutaminase was usually called "super binding agent"that improved the functional properties of protein as a kind of new food additive.It play a positive role in developing innovative food and enhancing the product quality in many fileds.however, transglutaminase have not used widly in Chinese food industry yet, the main reasons were that:firstly, imported-transglutaminase was very expensive, the production-cost was greatly increased by using this kind of transglutaminase; secondly, the domestic strain of producing-transglutaminase was in low production as well as the low enzyme activity, that made a higher production cost. In order to acquire the genetic engineering strain of high production, the wild strain of producing-transglutaminase should be screened firstly. In this work, we designed a protein cross-linked-flocculation precipitate method as the primary screening. A wild strain was finally selected in which the transglutaminase activity reached0.47U/ml. After the identification of physiology-biochemistry and16SrDNA, the strain was taxonomically identified as to be Streptomyces. In this study, the transglutaminase gene was cloned from Streptomyces St4, and then expressed in Escherichia coli, in order to get a high-producing transglutaminase. Main study results are as following:1. Screening transglutaminase-producing strain. We gathered several soil samples and designed a protein cross-linked-flocculation precipitate method as the primary screening, avoiding to directly assay the enzyme activity using a lot of the very expensive substrate CBZ-Gln-Gly, then assayed the enzyme activity at the second screening phase, and finally got a relatively higher strain Streptomyces St4. The transglutaminase activity reached0.47U/ml.2. The identification of the strain. According to several research of categorization identification on the strain appearance, cultural characteristic, physiology-biochemistry character, the16S rDNA sequence analysis, we initially get the conclusion that St4belong to Hygroscopicus of Streptomyces of Actinomycetes.3. The cloning of MTG gene. We initially clone the centre sequence including the acticity centre using degenerate primer, then clone the upper and downstream sequence respectively using sitefinding-PCR technology, and finally get the whole sequence. The coding region of MTG gene of Hygroscopicus St4is1251bp. Comparing with the transglutaminase gene sequence EU477523that was publicated the origin of Hygroscopicus, there are214bp disparity, and the homology of nucleotide sequence is83%. The active region site is the149th Cys, the catalytic triple copula linguae of mature enzyme is Cysl49-His359-Asp346that coincidence with homologous enzyme from other Streptomyce.4. The optimum of fermentation conditions. MTG gene was fused with the expression vector pET-23(a) and pET-32(a), and constucted the recombinant pET-23(a)-MTG/pET-32(a)-MTG, then transformed into Escherichia coli BL21(DE3) and expressed in Escherichia coli. It was certified that mtg was expressed correctly through SDS-PAGE electrophoresis, then optimized the fermention conditions. The optimum surveys indicated that initial pH at6.5, cultivated for2.5h, then added the induction IPTG by75μg/mL,30℃for4h, the recombinase activity reached up to10.23U/ml. After the optimization, the enzyme activity improved1.85times and22times compared with original strain and wild strain respectively.