| Cheese is a fermented dairy food derived from milk that is produced by adding milk-clotting enzyme(MCE) and some microorganism to coagulate and obtain the unique flavor. Besides high content of protein, fat, calcium, phosphorus and vitamins, cheese is also rich in bioactive peptides, such as casomorphins, phosphopeptides and immunopeptides.Traditionally, milk-clotting enzymes were extracted from the fourth stomach of young ruminants. In past few decades, engineered bacteria are increasingly applied to commercial rennet production and only 20-30% of milk-clotting enzyme obtains from calf rennet nowadays. Currently, microbial MCE especially bacterial source still have some questions, including strict culture conditions, complicated separation technology and poor enzymatic specificity. Therefore expression of MCE gene in hosts which were suitable for process condition by the recombinant technology may be an effective method to solve those questions. In this study, MCE encoding gene from a high MCE producing strain, Bacillus amyloliquefaciens JNU002, was cloned and identified by expressing in E. coli and B. subtilis. MCE production in E. coli DE3 was optimized and the enzymatic properties of the purified enzyme were studied.The amino sequence of purified MCE were analyzed by MALDI-TOF-MS/MS and identified in NCBI data base. Two gene of MCEamy and pro MCE were amplified by PCR and then inserted into p ET28a(+) vector. After transforming to E.coli DE3, the MCE activity(MCA) was detected in recombinant E. coli BL21(p ET-28a-pro MCE) inducing by IPTG. However, MCE without propeptide was an inactive protein in E. coli BL21(p ET-28a-MCEamy). Identification of recombinant MCE expression and gene sequences show that pro MCE contained encoding gene of propeptide and mature peptide. Moreover, the propeptide played an essential role similar to “chaperone” in expression of MCE.The inducing condition for the recombinant E. coli BL21(p ET-28a-pro MCE) was optimized to enhance the MCE expression. The highest production was obtained under the followed condition: 0.2 mmol·L-1 IPTG was adding to recombinant E. coli BL21(p ET-28a-pro MCE) cultured for 5 h at 37 °C, 200 r·min-1 and then the strain was cultured under 22°C and 150 r·min-1 for 18 h. Under this condition, a maximum MCA was observed for 117.24±2.28 SU·m L-1.The primers were designed according to the homologous sequence alignment and the gene of c MCE containing signal peptides was amplified from B. amyloliquefaciens JNU002. The obtained gene was inserted into the plasmid p MA5 and the constructed plasmid was transformed into B. subtilis WB600. The recombinant protein was expressed by detecting hydrolysis circle on milk plate. The MCE activity of the recombinant enzyme was 4.67 SU·m L-1. Results validated the MCE encoding gene c MCE which contains the signal peptide, propeptide and mature MCE sequence.The gene c MCE encoding the signal peptide, propeptide and mature MCE protein was amplified from p MD-19T-c MCE and then was inserted to the plasmid p MA5. The constructed p MA5-c MCE was transformed to Bacillus subtilis WB600. The recombinant protein was expressed by detecting hydrolysis circle on milk plate. The MCE activity of the recombinant enzyme was 4.67 SU·m L-1.The recombinant MCE was purified by His-trap colume and a molecular weight of 28.5 k Da protein with the yield of 66.51 % was detected by SDS-PAGE. The results of enzymatic properties show that the optimum p H for MCA was 5.0. Meanwhile, the optimum for MCA and proteolytic activity(PA) was 70 and 65 °C. The purified enzyme was thermolabile with 20% activity remaining after incubating at 60 °C for 25 min. The recombinant enzyme was stable at p H 5-7 which remained 70% MCE activity. The coagulating rate reached a maximum when the substrate contained 0.04 mol·L-1 Ca Cl2 and common metal ions had little effect on both MCA and PA. |
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