Screening And Identification Of Pectinase-producing Microorganism And The Enzymatic Properties Of Its Thermophilic Pectinase

Pectinases are the groups of related enzymes which cancleave the pectin substances. Due to its versitale function, pectinases has become one of the most important industrial microbial enzymes, and shown more and more applications in many fields, such as biofuel, food industries, textile, environmental protection, etc. In the few past years, most of researches on pectinases have been centred on mesophilic microorganisms. Little information on pectinase from thermophilic microbials has been available, especially its enzymatic characteristics are scare so far. Therefore, it is necessary to isolate and identify the thermophilic pectinase-producing microorganisms as well as investigate itss enzymatic properties. The findings in this work are very helpful to enlarge thermophilic pectinase origins and its applications. Herein, we reported the isolation and identification of thermophilic pectinase-producing microorganism based on its morphology, physico-chemical characters and molecular biology. Also we optimize the parameters affecting thermophilic pectinase production through submerge culture via response surface methology. Futhermore, we addressed the enzymology properties of pectinase and its applications in apple and orange juice processings. The main results obtained in this work are listed as follows:1. Isolation and identification of thermophilic pectinase-producing strain. Combined with tranditional fermentation, DGGE was employed to analyze the microbial communities in the piles during pu’er tea fermentation in Yunnan province, and eight thermophilic bacteria and eight thermophilic fungi were isolated and identified. Seven pectinase-producing thermophilic strains were screened by congo red medium for the first step, and then through the second screening, one thermophilic pectinase-producing strain was obtained and identified as Aspergillus fumigates according to its morphology, physico-chemical characters and18S rDNA molecular biology analysis.2. Optimization of fermentation parameters affecting pectinase production from Aspergillus fumigates. According to the single factor experiments, main fermentation factors influencing pectinase production from Aspergillus fumigates were obtained in this work. And then, three factors and three levels of Box-Behnken Design were employed to optimize the main factors through response surface mothodology (RSM), and the optimal parameters wer achieved:temperature45℃, pH5.0, inocubated volume0.07%. Under this optimal condition, the highest pectinase activity of360.0U/mL was obtained after36h fermentation.3. Elucidation of enzymatic properties of the thermophilic pectinase. Enzymatic properties of thermophilic pectinase, such as temperature, pH, stability, reaction kinetics were investigated. Results showed that the pectinase presented high stability and activity in the ranges of temperature30-70℃, and the optimum temperature was50℃with the highest enzyme activity of462.0U/mL the optimal pH of the thermophilic pectinase was5.0, and it showed satisfactory activity in the ranges of pH4.0-5.0and high stability in the ranges of pH2.0-7.0. Interestingly, thermophilic pectinase represented storing stability, and over80%residue activity was obtained after storage24h. In additon, the reaction kinetic parameters of the thermophilic pectinase was studied, and Km and Vmax value were found to be35mg/mL and7.69μmol·mL-1·min-1, respectively.4. Evaluation of the thermophilic pectinase applications in apple and orange juices processing. Juice clarification experiments were carried out to evaluate the application of the thermophilic pectinase in apple and orange juice processings. For apple juice processing, the optimal conditons were:pectinase dosage300U, temperature ranges20℃to0℃, and pH5.0. Under this optimal condition, the juice clarification could be enhanced by19.4%after20min treatment. While in view of orange juice processing, the optimal conditions were:pectinase dosage300U, temperature range20℃to70℃, and pH5.0. Under this optimal condition, the juice clarification could be increased by18.3%after40min treatment.