Screening Of Yeast High-yielding Lactase, Medium Optimization And Separation And Purification Of Lactase

14strains of yeast separated from koumiss in XiMeng region of Inner Mongolia was selected to study their ability of producing lactase through qualitative reaction and quantitative measurement in this paper. As a result, the yeast J2was screened as the strain of high yield enzyme lactase. Then the culture medium was optimized by single factor and orthogonal test. Based on the optimized medium, the enzyme production and growth rule of J2was studied. Another, the method separating and purificating of the enzyme from crude enzyme liquid also be studied.In initial screening the strains of high-yield lactase. X-gal and ONPG fermentation tube qualitative test was tried. The results showed that two methods of qualitative results are consistent, eight strains of yeast which can yield lactase was acquired. They are the yeast of J2, J8, J13, J14, J16, J20, J21andJ24. Moreover, the tuth two methods are consistent proved that ONPG fermentation tube qualitative method could be used for identificating the ability to producting lactase of fungus. After re-screening measuring8strains of positive yeast enzyme activity of intracellular and extracellular, the results showed that enzyme activity reached140.9U/ml,55.24U/ml in fermentation liquid of J2and J21. On the contrary, the enzyme activity of other strains were lower. At the same time, three ways to break the cell wall of J2and J21that the ice bath ultrasonic, freeze-thaw at-80℃for three times and keeping at-20℃all the night of-serial open-style way of producing enzyme was used to, to eliminate the influence of the wall-breaken methods. Results show that the enzyme from J2, J21was out of the cell.Based on the results of MI culture medium carbon source, nitrogen source and metal ion species and adding level after the single factor experiment, the orthogonal experiment was tried to get the optimum culture medium. The results as showed:lactose and sucrose to1:1to join, content is1%, urea and peptone3:7, content is0.5%, yeast powder1%, with0.05%MgSO4. Based on medium optimization, enzyme production capacity of J2from140U/mL increased to more than400U/mL, increased nearly three times. After the optimization of the medium of J2enzyme production and made a research on the growth rule. Results showed that the J2began to secrete lactase after24h, reach the highest at36h. Enzyme activity in fermented liquid dropped to zero at60h. Through the determination of the effect of different degree of saturation of ammonium sulfate, determine55%saturated ammonium sulfate as the best saturation. Crude enzyme liquid after55%ammonium sulfate, SephadexG-100chromatographic column. After the two step purification crude enzyme liquid was purified by1.28times and1.28times respectively.