Isolation And Purification Of A Ketone Reductase From Candida Parapsilosis ATCC 7330 And Its Enzymatic Properties

Candida Parapsilosis ATCC 7330 was used as the experimental strain,after homogenous crushing,centrifugation,and two aqueous phase treatments,the crude keto reductase contained in Candida parapsilosis ATCC 7330 was finally obtained,and a simple The high-efficiency enzyme activity detection method has created a convenient and quick experimental scheme for obtaining ketoreductase,and also laid a solid experimental foundation for the further related research of ketoreductase.The results of this experiment are as follows:1.Researched the method for measuring the enzyme activity of Candida parapsilosis ATCC 7330,and through continuous improvement and optimization,the final determination of the use of homogeneous crushing,the detailed method of enzyme activity detection is:the total reaction volume is 1000?L,and 100?L are added respectively 0.2m M NADH,100?L 4 m M substrate acetophenone,700?L 100 m M phosphate buffer(p H=7.5),30°C water bath for 2 min,add appropriate amount of enzyme solution and start recording at 340 nm with a spectrophotometer Changes in absorbance.Enzyme activity is defined as the amount of enzyme that consumes 1?mol of NADH per minute under the above conditions is 1 unit of enzyme activity.2.The high-pressure homogenization crushing process of Candida parapsilosis ATCC 7330 was studied.The orthogonal temperature was used to examine the homogenization temperature,yeast mass fraction,homogenization pressure and homogenization frequency.Enzyme activity.The results show that the influence of the four factors is in order of homogenization pressure>yeast mass fraction>homogenization temperature>homogenization times.Among them,homogenization pressure is a significant factor affecting the crushing effect of near-smooth Candida parapsilosis,and the best crushing process is The homogenization temperature is 6?,the yeast mass fraction is20%,the homogenization pressure is 180 MPa,and the number of homogenization times is3.After the verification test,the method of homogenizing the yeast cells by high-pressure homogenization can obtain 1.572 U/m L of crude enzyme liquid,which is more than 150%higher than before optimization.3.The process of extracting ketoreductase from crude enzyme of Candida parapsilosis ATCC 7330 by polyethylene glycol(PEG)/ammonium sulfate[(NH₄)₂SO₄]two-phase system was studied,and the molecular weight of PEG and its molecular weight were systematically investigated.The influence of concentration,(NH₄)₂SO₄concentration,temperature,extraction time and other factors on the purification factor of ketoreductase.The results show that the optimal extraction conditions of the two aqueous phases are(NH₄)₂SO₄concentration of 15%,PEG concentration of 19%,temperature of16?,extraction time of 15min,and the purification factor of ketoreductase can reach 6times the above.This study laid the foundation for the application of the enzyme in Candida parapsilosis ATCC 7330 in the green synthesis of chiral compounds.