Study On Screening Of Heat-tolerant Acidic-pectinase Producing Strain,fermenta-Tion Optimizing And The Enzymatic Properties

Pectinase is a collective call for any enzyme,which can be divided into acidic pectinase and alkaline pectinase.The acidic pectinase has been widely used in the aspects of clarifying fruit and vegetable juice,fruit wine brewing.With the development of fruit wine juice industry in China,its production has been far can not meet the need of food industry.Current domestic,enzymatic activity of the acidic pectinase-producing by the bacterial fermentation,is generally low.Current studies have been focused on the screening of acidic pectinase-producing strains,especially bacterium,optimizing the fermentation conditions and so on.In this paper,we used the iodine solution to produce transparent circle,which could filter out some of strains that producing pectinase in selective medium.Pectin from citrus peel was used as a substrate for screening of the strain producing pectinase fi-om soil samples under different fruit tree.After preliminary screening,22 strains which could produce pectinase were obtained.Then with DNS detection,the highest enzyme activity strain was selected to conduct the following experiment.By morphological observative,physiological and biochemical character-istics,and 16SrDNA analysis,the strain named ZJ1407 was identified as Bacillus methylotrophicus,which produced the highest pectinase activity of 338.95U/mL.To improve the production of-pectinase from Bacillus sp.ZJ1407,the best fermentation conditions was optimized by single-fractor test.Then,the response surface methodology(RSM)was explored to optimize the medium compositions.A 2-level Factorial design was reseai-ched to estimate the main ef-fects and interactions,and screen the main factors.And a central composite design(CCD)was used for analysis of the results to obtain the optimal medium compositions.Three significant factors such as:lactose,tryptone and(NH4)2SO4.The final optimized medium compositions were as follows:lactose 44.8g/L,tryptone 30.9g/L,(NH4)2S04 1.35μg/L,MnSO4 H2 0.2g/L,MgSO4 0.4g/L,NaCl 3.5g/L.The optimum enzyme producing conditions were:temperature 34℃,pH4.5,inoculum concentration 8%,liquid volume in 250mL flask of 50mL.At optimum conditions,the pectinase activity was obviously improved to 737.61 U/mL after fermented 48 h.The ZJ1407 was induced under the optimal culture conditions.We used ammonium sulfate precipitation,Cellulose DE-52 ion-exchange column chromatography and Sephadex G-100 column chromatography to purify enzymatic proteins.Then,we got in 39.49-fold pectinase purificat-ion with a recovery rate of 10.68%,a specific activity of 6942 9.51U/mg.The purified pectinase had a molecular mass of 23kDa.The optimal pH and temperature of the purified pectinase were pH5.0 and 37℃.The pec-tinase was stable within a pH range of 3.0-6.0,and showed high thermo-stability at 80℃,retaining 13.3%of its maximum activity.Ba2+could slightly promote the pectinase activity.And Mn2+seriously inhibits this enzyme activity.