Studies On The Inhibitors Of Yeast Proteinase A

The inhibitors of proteinase A(PrA) have great prospect in food, medicine andagriculture field. In this paper, PrA was purified from yeast. An inhibitor of PrA wasextracted from broth of Ganoderma Lucidum and its basal character was studied. Themethod of separating and purifying PrA was improved. An effective method screening theinhibitor of PrA by immobilized enzyme affinity chromatography was set up.The mainresults were given as follows:1. The method of purifying PrA was improved that the step of ion-exchangechromatography was substituted for hydrophobic chromatography. The specific activityof the product PrA was 12.6μ/mg and the purified multiple was 25.2. After improvingpurification method, the period of purification was kept down.2. After alcohol fractional precipitation, molecular sieving chromatography by UltrogelAcA44 and strong anion-exchange chromatography by Source Q, an inhibitor of PrAnamed GLPAI , which showed 75 percentage inhibition to PrA, was obtained from thebroth of Ganoderma Lucidum.3. The molecular weight of GLPAI was about 37000.In GLPAI, the content of protein andglucose was relative high. GLPAI had great temperature stability;the optimal pH andreaction time of GLPAI to PrA were pH 3.5～5.5 and one hour. GLPAI showed someinhibition to pepsin, trypsin, papaya enzyme and neutral protease.4. The kinetics studies of GLPAI showed that :the Ki of GLPAI to pepsin was 4.64μmol/Land the inhibition type was the complex of non-competitive and anti-competitiveinhibition;the Ki of GLPAI to trypsin was 33.5μmol/L and the inhibition type was thecomplex of non-competitive and competitive inhibition;the Ki of GLPAI to PrA was2.7μmol/L and the inhibition type was the complex of non-competitive andanti-competitive inhibition.5. The optimal conditions of immobilizing reaction were as follows: pH of reaction was4.0;the concerntration of the glutaraldehyde was 0.6% and the addition of PrA was40mg/0.2g chitin. The yield of immobilized PrA was 45.9% in the optimal conditions.6. The inhibitor of PrA was obtained by single affinity chromatography step when theimmobilized PrA was as fixed phase and the broth of Ganoderma Lucidum as fluid phase.We can make a conclusion that the inhibitor purified by affinity chromatography andGLPAI were the same matter because they showed little difference in the molecularweight and the proportion of protein to glucose.