Studies On Factors Affecting Of The Production Of A Milk-Clotting Enzyme From Mucor Pusillus And Its Recombinant Enzyme, And Their Enzymatic Properties

Cheese is a kind of nutritional, convenient dairy product with unique flavor. With the improvement of people's living standard, the production of cheese in China will increase a new development of dairy products in the coming years. The milk-clotting enzyme (MCE) is critical in the process of cheese making for milk clotting, and subsequently for excluding the whey. The source of MCE is wide, including animals, plants and microbes. The MCE from animals is the primary source for cheese making, since the ratio between activity and proteolytic activity of the enzyme is high. With increasing of cheese production, but short supply calves in the world, people are seeking for the substitute to calf chymosin which is widespread and economical. The MCE from plants is widespread, but the proteolytic activity of the enzyme is high, and it's hard to develop for the limitation of growth time, growth area and growth cycle; The MCE from microbes is potential, because of the short growth cycle; high production; low limitation of climate, area and growth time; lower production costs; easily enzyme extracted; and high economic returns. Although the proteolytic activity of the enzyme of Mucor pusillus MCE is higher than the calves', but it's lower than others and powerful to milk-clotting, so the Mucor pusillus MCE is widely used.But the proteolytic activity of the enzyme of Mucor pusillus MCE is high in the cheese yield, which results in the lower production and bitter taste when it's ripe; meanwhile the high thermostability makes it inconvenient to use the whey. And genetic engineering technique has the ideal way to solve these problems. Currently chymosin, which is needed for cheese production, still depends on import, resulting in the higher price of enzyme (approximately 800 yuan/Kg). Moreover, the cost of chymosin production is high when it is extracted from the abomasum of calf. Recent years, with the development of dairy industry and the increase of consumer's demand for cheese in China, it is significant to research and produce the chymosin by our own technology.In the present study, the MCE produced by Mucor pusillus which has been applied in the production of cheese was studied. The conditions for production of MCE by Mucor pusillus and the recombinant Pichia pastoris transformed with the MCE gene from Mucor pusillus were studied. Some enzymatic properties between the two were compared. Conclusions can be made as follows:(1).The fermentation media of Mucor pusillus were optimized. The effects of factors (carbon, whey power, nitrogen, phosphate, CaCl2, yeast powder, MgSO4 etc.) on the milk-clotting enzyme production were investigated based on the single factor test. It was found that the enzyme activity was increase when added to the bran based medium with 1% glucose, 0.8% whey power, 0.6% NaH2PO4·2H2O, 0.6%CaCl2, 0.2% yeast power, 0.5% MgSO4·7H2O. The cultivation conditions of Mucor pusillus were optimized as follows: studied and the results followed: the solid-liquid ratio 1:0.7, the inoculation percentage 2ml/250ml, and the medium quantity in the flask 15g/250ml.(2).Based on the single factor tests, three ingredients'(CaCl2 X1, whey power X2, glucose X3) of the medium were optimized with Response Surface Analysis. The functional relationship between the main factors and response value (milk clotting enzyme activity, Y) was established, and the regression equation was obtained. The regression equation was: Y=1118.53+136.98X1+14.39X2+33.84X3+25.85X1X2+61.25X1X3+41.13X2X3-205.67X12-78.94X22-130.14X32The model was used to evaluate the key factors related to milk-clotting activity of the key factors and their interactions, and the optimal values of the factors were obtained: CaCl20.58%, whey power0.64%,glucose 1.13%. Under the optimized conditions, the theory value of Mucor pusillus MCE activity is 1150.81 SU/ml, and the average value of tested is 1109.7 SU/ml near the theory value. Therefore, the model is reliable and can be used obtained from the fermentation expenriments of this study for practical prediction. (3).Factors affecting the production of recombinant chymosin in Pichia pastoris (GS115/pPIC9K) constructed in our laboratory. The fermentation conditions for the recombinant strain engineering microbe were optimized, including the induction time of methanol, amount of methanol added, change of media and inducing period choice when BMGY is not centrifugal and so on. By single factor test, the results showed: the inducing time with methanol 96h, the methanol addition value 1.3%/12h, there was no significant effect on the MCE activity by change of media during fermentation, the yeast original concentration 2.0 OD600/ml.(4).The properties of milk-clotting enzyme from Mucor pusillus and the recombinant MCE were studied. The optimal temperature of the two enzymes was 55℃; The Mucor pusillus MCE was inactivated when treated at 50℃for 30min or 55℃for 10min. The recombinant MCE was inactivated at 55℃for 20min,thermal instability of recombinant MCE meet the request of whey processing whivh happens in the process of cheese production; The milk-clotting activity of Mucor pusillus MCE was stable at pH 6.2 and the recombinant MCE was stable at pH 6.07; When the substrate pH was 5.43, the two enzymes showed best clotting activity; Metal ions such as Ca2+and Na+ could increase the milk-clotting activity, but K+,Mg2+ and Cu+ inhibited the milk-clotting activity.