Optimization Of Fermentation Conditions And Immobilization Of Pectinase Pel-4A

Pectin substances is an important component of plant’s cell wall in biosphere. Pectinase refers to those kinds of enzymes which can cleave the pectin substances. Bacillus subtilis is one of the most commonly used host bacteria for high level expression of heterologous proteins. Comparing with Escherichia coli expressed proteins, Bacillus subtilis expresses proteins had an outstanding advantage which can secret heterologous proteins out of cells in media. Moreover, Bacillus subtilis with characteristic of high expression, strong resistance, and nonpathogenic has drawn more and more attention. so, Bacillus subtilis has been one of the most promising host bacterias for expressing heterologous proteins.This article by construct reconbinant Bacillus subtilis WB700/pAUB110-Pel-4A, is mainly aimed to optimize the fermentation conditions of recombination Bacillus subtilis WB700producing alkaline pectinase Pel-4A. Yeast extract, tryptone, and phosphate were screened as three significant factors by operating single factor test and Plackett-Burman test, and their concentration were determined by steepest ascent test, then we optimized these three significant factors through Box-Behnken test. Finally, the reconbinant strain were cultured for expressing in bottles, and the optimized production of alkaline pectinase Pel-4A reached up to1500U/mL. After that, we did fermentation experiment, and the highest pectinase activity went to3100U/mL from357U/mL, and the maximum fermentation production went to2.9g/L from0.22g/L, which was a big improvement. The optimum temperature/pH of alkaline pectinase Pel-4A is60℃/pH10.0. This pectinase also exhibited great stability at pH10.0. To strengthen repeating usage, the recombinant alkaline pectinase Pel-4A was immobilized onto amino support(ZH-HA) using Glutaraldehyde. The immobilized pectinase activity reached to200U/g, and retained more than80%of its initial activity after reusing30times, which showed its great operational stability comparing that reusing10times retained76%activity. These results made a big contribution to alkaline pectinase’s application.