| Chymosin with the unique milk-clotting characterization has a wide application value in the food and pharmaceutical industries. In recent years, the supply of rennin is increasingly inadequate, so chymosin alternatives have been vigorously developed through genetic engineering technology to obtain recombinant rennet as a new research direction of. Although recombinant chymosin has important applications in the fields of medicine, cheese industry, some problems limit large-scale industrial production of recombinant chymosin. The main problems are as follows:(?) low production and enzymatic activity of recombinant chymosin;(?) the high cost due to the extraction and purification processes. In this study, the recombinant bovine rennin was selected as the research object because of its good enzymatic properties, extracellular secretion, commercial application and high expression level.To improve expression and secretion efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene with full length of 1095 bp by using optimized codons. The synthesized prochymosin gene was then cloned into the expression vector pKLAC1, resulting in Bchymo290-1. The Bchymo290-1 was linearized by Sac? and transformed into K. lactis GG799 by electro-transformation. The four excellent transformants were obtained and named as bPC-1?bPC-2?bPC-3 and bPC-4 through screening from the nearly 5000 recombinant strains.Response Surface Methodology (RSM) was employed to optimize fermentation medium for enhancing the ability of the recombinant strain bPC-1 to produce recombinant chymosin. Based on the Plackett-Burman design, the concentration of glucose, corn syrup and yeast extract were selected as significant factors affecting the activity of recombinant chymosin. The center points of these three factors were determined by using the Steepest Ascent, and these three points were optimized by the Box-Behnken design. A maximum of chymosin activity value 452.62 SU/mL was predicted by original glucose concentration of 46.8 g/L, corn syrup of 28.7 g/L and yeast extract of 7.5 g/L. Using this optimized fermentation medium,448.62 SU/mL chymosin activity value was obtained from fermentation, Which was 22.65% higher than that before optimization.The pH of the fermentation process of bPC-1 was optimized by setting pH 4.0, pH 5.0, pH 6.0 and natural pH in 1 L quadruple fermenter (glass tank). Recombinant chymosin activity was achieved to be 571.43 SU/mL, and therefore the pH of the fermentation process can be maintained under natural fermentation pH. After 60 hours of fermentation, the maximum activity of chymosin was 648.65 SU/mL, which was higher than that of other experimental groups, by setting four dissolved oxygen (DO) parameter gradients DO 20%, DO 30%, DO 40%, DO 50%, and therefore the DO parameters in the fermentation process can be controlled at 40%. The recombinant recombinant strain bPC-1 was expanded in 5L fermentor, and the maximum recombinant rennet yield was 700 SU/mL at 66 hours, which was 56% higher than that of shaking flask fermentation.The supernatant obtained from the 5 L fermentor was subjected to acidification, which was concentrated and purified by using a 10 kDa,50 kDa ultrafiltration membranes. The total activity value of the 100 mL crude enzyme solution was 70000 SU before ultrafiltration. By recycling and concentrating, the total activity value reached 54857 SU, recombinant chymosin recovery rate was 78.36%.In conclusion, we exploited a new chymosin with high enzymatic activity, easy purification, good enzymatic characteristics and safe production process. The results provided a new method for the development of bovine chymosin alternatives and laid the foundation for the large-scale application of chymosin in food and other industries. |
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