Screening, Fermentation And Enzymatic Characteristics Of Producing Polygalacturonase Strain

The research on production and application on Polygalacturonase is later in china. Nowadays, most of the polygalacturonase are producted by Aspergillus niger fermentatio-n, the enzyme production and quality can't be satisfyed with the needs of food industry, and the price is expensive.One hundred and twenty strains which isolated from rotten fruits, soil, rice straw compost and biologic fertilizer are as the original strains. Finally, we got one Aspergillus terreus mutant strain for the polygalacturonase high production after a series of screening, identification and mutation.Then we researched the fermentati-on conditions and enzyme characteristic. The experimental results are summarized as follows.All the strains were positive for pectinase activity in a translucent circle assay, as evid-enced by clear translucent halos. Twenty strains presented considerable polygalacturonas-e activity.The cultivation of the best five strains (C1,C2,C3,G1,H4) by liquid-state fermentation, resulted in high activities of polygalacturonase enzyme.The enzyme activity were 17.89U/mL,17.68U/mL,18.37U/mL,14.04U/mL,13.56U/mL respectively.Then they were classified respectively as Aspergillus terreus (C1,C2,C3), Alternaria alternate (G1), Aspergillus niger (H4) by the analysis of their colonymorphology, micrology morph-ology, and physiology characteristic.UV and microwave mutagenesis were applied to the strain of Aspergillus terreus C3,we got the best result in360s and 50s,the lethality rate were 86.89% and 83.05%, the obverse variability were 33.33% and 20% respectively.We got the mutant strain C3-A8 and C3-B5, the polygalacturonase activity of them increased 12.79% and 11.27% respectively. The genetic characteristic of C3-A8 was stable,but C3-B5 was not.Then strain C3-A8 and C3-B5 conidia were treated by different concentration and time of LiCl,we found we could get the best result in 5% LiCl to deal with 5 minutes,the lethality rates were 33.90% and 32.14%, the obverse variability were 8.33% and 9.09% respectively.This process resulted in the isolation of two new mutants which produced higher activity of polygalacturonase.The polygalacturonase activity of C3-A8-A6 and C3-B5-D5 reached 21U/mL and 20.88U/mL, and they increased 14.32% and 13.66% respectively. The genetic characteristics of strain C3-A8-B6 was stable, but the result of strain C3-B5-D5 was not satisfactory.The fermentation medium and conditions of enzyme production were investigated by single factor and orthogonal experiment.The optimal medium was determined as follows: 5% bean cake powder as carbon source,5% orange peel powder as inducing substance, 0.6% NaNO2 as nitrogen source,MgSO47H2O 0.5mmol/L,MnSO4·H2O 0.75mmol/L, K2HPO4 0.1%, CaCl2 0.75mmol/L, BaCl2 lmmol/L,FeSO4·7H2O 0.5mmol/L.The optimal culture temperature was 30℃, the optimal initial pH was 5.0,the volume of medium of 45mL in measuring flask of 250mL and the inoculation amount was 7%(V/V) spore suspension whose concentration was 106cfu/mL.Under the above optimum conditions, the strain reached the highest polygalacturonase activity of 44.642U/mL in shaking flaskafter 4d cultivation, and the results showed significant increasing.Impure polygalacturonase from Aspergillus terreus C3-A8-B6 was purified by ultraf-iltration and SephadexG-100 gel column chromatography exchange techniques.The enzy-me had a relative molecular mass of 39.87KDa by SDS polyacrylamide gel electrophores-is. The optimal temperature in enzymolysis of polygalacturonase was 38℃, the polygala-cturonase activity was stable in the range of temperature from 38 to 53℃and kept above 90% enzyme activity when treated for 1h. The optimal pH in enzymolysis of polygalactu-ronase was from 5.0 to 6.0,and the polygalacturonase activity was remained above 90% in the range of pH from 4.5-6.0. Metal ions such as Fe2+,Mg2+,Ca2+,Ba2+ could activate the polygalacturonase activity significantly. Cu2+,Al3+ were special inhibitors. Na+,K+ stimulated the polygalacturonase activity feebly, and Li+,Zn2+,Si2+inhibited the polygala-cturonase activity feebly.The Michaelis-Mentenkinetic parameters of the polygalacurona-se (Km and Vmax) were 9.24×10-3g/L and1.027×l03μg/min respectively.