Optimization On The Production Of Alkaline Protease By Exiguobacterium Aurantiacum And Its Enzymatic Properties

One alkaline protease-produced bacterial strain named EX0was isolated fromsample of polluted alkaline silica sol. The isolate was characterized and identified asspecies of Exiguobacterium aurantiacum. This research focused on the ability ofalkaline protease production, conditions and its enzymatic properties, and provided thebasis to explore new sources of alkaline protease as well as its industrialization.This bacterial strain was proved to have the ability of hydrolyzing casein by usingthe casein flat medium. The protease activity could reach13.361U/mL.The culturemedium composition and fermentation conditions for protease producing fromExiguobacterium aurantiacum had been studied with univariate tests and orthogonalexperiment in this paper. The optimal medium compositions was as follows10g/Lyeast extract,10g/L corn starch and5g/L NaCl, and the best fermentation conditionswas as C/N for1:1, time for21h, temperature for25℃, inoculum8%and initial pH8.8.The protease activity could reach37.793U/mL after optimization.This experiment precipitated protease by different saturation of ammoniumsulfate.The result showed that the best ammonium sulfate saturation is50%. Afterisolation and purification, strain EX0could secrete alkaline protease which molecularweight was42kDa.Differernt concentration of EDTAand β-mercaptoethanol showedvarious degrees of inhibition. The enzyme may contain certain metal ion could bededuced from the inhibition of EDTA.The optimum condition for the protease activitywas50℃and pH was8.0. Ions such as Cu²⁺、Zn²⁺、Mn²⁺could greatly inhibit proteaseactivity. Mg²⁺promoted the activity of protease.In recent years,the research on alkaline protease mainly focus on Bacilluslicheniformis,Bacillus subtilis etc, and whether other species of microbes couldproduce alkaline protease which has potential industrial value remain to be furtherexplored. Alkaline silica sol has low nutrition and the high pH condition and E.aurantiacum could grow in low nutrition environment, thus this bacteria has thecharacteristics of easy to cultivate. The result showed that E. aurantiacum couldsecrete alkaline protease and reached the highest production at24h. This reasearch laidthe foundation for getting efficient expression of alkaline protease gene by means of genetic engineering and enzyme engineering,and also provided theoretical basis tomodify gene by using protein engineering techniques.