Production And Partial Of Esterase By Lichtheimia HSM Strain

As an important industrial enzyme, with catalytic capacity of hydrolysis andsynthesis, esterase has a broad prospect market in the food, pharmaceutical, chemicaland military industry. This test of screening esterase-producing strain HSM,identification and optimization of the fermentation conditions of the strain, the effectsofinducers on enzyme-producing and isolation and purification of esterase, andenzymatic properties has done a more comprehensive research.First use of glycerol ester of n-plate method of transparent circle, as hydrolysis speed and the diameter of the circle appears on the transparent plate for the basis preliminary screening, and esterase activity in fermentation broth for screening basis, has screened a high yielding esterase producing strain HSM fromLuzhou Laojiao and Zhijiang Daqu include high-temperature, medium-temperature and medium-high-temperature Daqu, enzyme activities can reache5.38U/mLon average. Through the strain morphologic identification and sequence analysisof18S rDNA-ITS combined with phylogenetic analysis, the HSM has been identified as Lichtheimia.Optimized for HSM esterase produced by liquid fermentation, first bysingle-factor test, identification of soluble starch as best carbon sources, concentrationsand ratio of yeast extract powder with KNO3at3:1for the best nitrogen source. By PBexperimental design, have effects on enzyme production results of the most significantare the three factors MgSO4, KNO3and yeast extract powder. By Box-Behnkenexperimental design to further optimize the three significant factors are the optimalculture medium formula for: soluble starch1.8%, yeast extract powder1.8%, KNO30.5%, K2HPO40.04%, MgSO40.04%, KCl0.04%, FeSO40.001%and MnSO40.04%.Got the HSM optimal esterase-producing fermentation conditions by univariateoptimization method: fermentation temperature of34℃, inoculation volume10%,outfit fluid volume45mL/200mL, agitation speed180rpm, initial pH value of6.5.Within these simple combination optimal parameters of the fermentation, the esteraseactivities can reach8.90U/mL, which was65.43%higher than the original conditions.The research on different effects of different type of inducers on esterase-producing strain HSM, draw a conclusion that the esterase is inducible enzyme, induction of acetic ester, olive oil and Tween-80on esterase-producing is comparison strongly and effect of acetic ester is best, the results showed that the addition concentration of0.2%can increase the productivity of esterase by65.43%,while the esterase activities of fermentation liquid can reach15.58U/mL.The crude chondroitinase was initially isolated with40%and80%ammoniumsulfate two step precipitation, then the crude chondroitinase was dissolved and dialyzed, and purified by PEG20000, then again to purify through DEAE SepharoseFast Flow ion-exchange chromatography, found that the HSM esterase was preliminarypurified, the specific activity is up to6209.06U/mg, concentrated about26.79-foldwith a yield of2.17%.The esterase has an optimum reaction temperature of50℃,and more sensitive tohigh temperatures, and poor thermal stability; the optimum pH value is7.4and the pHreaction range(7.2～7.8) is quite narrow, but it has good alkali-resistant. Esterase hasthe highest relative enzyme activities when NaCl concentration is8%(W/V), and havea high salt tolerance. Mg2+, Ca2+has no significant effect on esterase activities,whereas Mn2+, Zn2+and Cu2+presents a different degree of inhibition, and inhibitoryeffects of Cu2+is the most evident.