The Screening Of Bacillus Subtilis Produing Chymosin And Studies On Enzymic Properties

Cheese is fresh or ripe dairy products by milk-clotting and separating whey,and milk, butter,skim milk,butter milk or mixture of the milk are raw materials.Cheese has plenty of nutrient substance.For the past few years,it's prodution and consumption has constantly maintained adscendent tendency.Chymosin is an important enzyme which has milk-clotting reaction in production of cheese.There are many sources to get chymosin,such as from animal,plants and microbe.Because of the merits,such as wide source,simple culture method,extracting the chymosin conveniently and low cost,microbe has become an important source of chymosin.This paper is a reseach about the microbe,and mainly deals with four aspects:1.The screening of the bacteria which could produce chymosinQL-2 was obtained by screening on plate,which could produce chymosin among 34 microbes isolated from ten samples.QL-2 was isolated from "ten years cheese".The fermentation broth of QL-2 has milk-clotting activity which was 62.83SU.2.The phylogenetic analysis of QL-2Through a series studies of their Characteristics,including the appearance observation,enviroment factor influence,growing characteristic,physiological and biochemical determination,sequence analysis of 16S rRNA,the determination show that QL-2 is a new strain of Bacillus subtilis,which is already conserved in CCTCC with number M208023,named Bacillus subtilis QL-2.Now we have applied the patent on the bacteria in SIPO of the PRC,and the patent number is 2008103011093.3.Optimization of culture condition and study of enzymic propertiesSome researches were studied about the influence of temperature,pH,volume of culture media on milk-clotting activity by orthogonal experiment.The optimum culture condition test showed:culture temperature 35℃,the initial pH 7.0,volume of culture media 40ml.As for the enzymic properties:Bacillus subtilis QL-2 could produce chymosin and the fermentation broth had milk-clotting activity.Ca²⁺could significantly increase the milk-clotting activity;when the milk's pH was 5.8,the fermentation broth had the fastest reaction velocity;the optimal reaction temperature was 60℃.