Study On Catalytic Properties Of Esterase From Bacillus.sp And Its Application In Cellarless Esterification Process

In order to establish a foundation for entire mechanization of brewing Luzhou flavor liquor,the strains producing esterase were screened and applied in brewing Luzhou flavor liquor without cellar mud.A bacteria producing esterase was screened from sediments and identified as Bacillus subtilis.The optimum conditions of the strain producing esterase in solid state:wheat bran is the optimal matrix,the initial water content of 40%,32?,initial pH 6.5,inoculum 10%,culture time 5 d,enzyme activity was 100.42 U.whereas the optimum conditions in liquid state:25 g wheat bran,10 g tryptone,1 g K2HPO4,0.5 g MgSO4 and distilled water 1 L,initial pH 6.5,25?,culture time 3 d,enzyme activity reached 3.32U.The optimum esterified conditions of ethyl caproate hexanoate:1.5%caproic acid8%ethanol,pH 7.5,35?,8%koji-bran,esterified time 5 d,the esterifying capacity is1.61 mg/g.The content of ethyl ester was synthesized by esterfying acetic acid,lactic acid,butyric acid or hexanic acid is 170.14 mg/100mL,18.95 mg/100mL,25.52mg/100mL and 256.68 mg/100mL,respectively.The optimum temperature of esterase hydrolase was 40?,and the optimum pH value of enzyme activity was 6.5.When the metal ions concentration was 5mmol/L,the enzyme activity of Fe²⁺was 0%,and Cu²⁺,Ca²⁺,Mg²⁺,Zn²⁺and Mn²⁺was 5%,27%,83%,16%and 55%,respectively.The hydrolytic enyme activity of C₂?p-nitrophenol acetate?was 3.78 U,C4?p-nitrophenol butyrate?1.89 U,C8?p-nitrophenol octanoic acid ester?0.67 U,while C₁₂?p-nitrophenol laurate?was only 0.014 U.When glucose concentration is 2%,enzyme activity is 60%,and ethanol concentration is between2%⁴%,enzyme activity is still maintained at 95%.When the concentration of acetic acid,lactic acid,butyric acid and caproic acid is 0.2%,the enzyme activity is 40%,60%,50%and 60%,respectively.When the concentration of acetic acid,lactic acid,butyric acid and hexanic acid is more than 0.5%,the relative enzyme activity is 0%.This showed that glucose,ethanol and organic acids can inhibit the enyme activity of esterase hydrolase.The esterification power of bran-koji that was preparated by ester producing yeast and bacillus was 1.81 mg/g.The esterification power of bran-koji that was preparated by saccharomyces cerevisiae and bacillus was 1.98 mg/g.The bran-koji was preprated from mixture of Saccharomyces cerevisiae,ester producing yeast and Bacillus,its esterification capacity is 1.21 mg/g.The esterification power of bran-koji that was preparated by single bacillus was 2.95 mg/g.The test,the crude enzyme preparation of bacillus producing esterase were applied in brewing of Luzhou flavor liquor without cellar mud,showed:15%for caproic acid bacteria inoculum size,30 d for fermentation time.the content of ethyl caproate in blank group was only 97.08 mg/L,whrease the content of ethyl caproate was 2236.13 mg/L in experimental group,which was added 15%for crude enzyme preparation.This experiment have screened bacillus that is efficiently able to produce esterase.Esterase with stable enzymatic properties had higher substate selectivity for caproic acid.The stain was applied in brewing Luzhou liquor without cellar mud,which improved the content of ethyl caproate in a large part.The job have achieved some success in application of brewing Luzhou flavor liquor without cellar mud.