Research On Screening And Characterization Of Esterase Producing Strain

Esterase?Esterase,EC3.1.1.1?which is a collection for enzymes hydrlzing carboxylic acid esters is a kind of important biological catalyst.Plasticizer is a kind of polymer additives that is added in plastics processing.Citric acid ester plasticizer is worldwide recognized as environmentally friendly plasticizer.Currently,the production of citric acid ester plasticizer is produced by chemical synthesis.It is one of the green biotechnology research directions synthezing of citric acid ester plasticizer by esterase for its dural function of hydrolysis in water phase and synthesis in organic phase.This study,citric acid ester plasticizer was served as sole carbon source adding bromocresol purple indicator in the flask to enrich soil sample.The bacteria with higher esterase activity will have shorter time for discoloration.A bacterium strain was screened out,which was rod-shaped,gram-negative and non-spore.The strain 16S rDNA had 99%identity with Stenotrophomonas maltophilia 16S rDNA.Thherfore,the strain was named Stenotrophomonas maltophilia SF-H?abbreviated as S.mal SF-H?.A mutant strain S.mal SF-H1 was obtained which esterase activity was 20%higher than the control strain by plasma mutagenesis,taking S.mal SF-H as the starting strain.The fermentation condition of strain S.mal SF-H1 was optimized and the composition of fermentation medium as following?L?:maltose 30.0 g,peptone 5.0 g,yeast extract 2.5 g,?NH4?₂SO₄ 4.0 g,KH₂PO₄ 1.0 g,MgSO₄·7H₂O 1.0 g,NaCl 0.5 g,Fe²⁺1.0 mg,Zn²⁺1.0 mg,Mn²⁺0.5 mg,Cu²⁺0.5 mg,sterile GF?1 mL/400 mL?,inducer agent citric acid tri butyl acrylate?LM30?5.0g,initial pH 7.0;at 30°C and 180 r/min,shake flask culture 30 h,strain S.mal SF-H1 reached the maximum esterase activity.Esterase properties of strain S.mal SF-H1 were studied in detail.The results showed that the esterase hydrolytic activity of short chain ester was significantly higher than the long ones.The esterase had obvious substrate specificity.With p-nitrophenyl acetate as a substrate,the enzyme showed a good resistance to high temperature.The optimum temperature was 80°C and enzyme activity was still had 95%when it was kept at 70°C for2 h.The optimum pH value was 9.0 and the best stability in pH 8.0.When esterase in volume fraction in 66%of butanol and 66%ethanol,more enzyme activity was lost,and the residual enzyme activity was about 60%.The fermentation broth was condensed 5 times by vacuum rotary at 60°C because S.mal SF-H1 esterase had the good thermal stability.The concentrated fermentation broth still have 97%more esterase activity comparing with untreated sample.The results showed that the esterase stability was good.To increase the enzyme efficiency and catalytic synthesis in organic phase the esterase was immobilized by precipitation of the adsorbent for the first time.The results showed that 55.4%of the esterase in the solution can be fully immobilized.The immobilized enzyme still had 85%hydrolysis activity after 30 days usage.Our research laid theoretical foundation for the practical application.