Study On Biocatalytic Synthesis Of Short-chain Flavor Esters By A Whole-cell Lipase From Aspergillus Oryzae

Flavor and fragrance compounds,which are the products supporting industries of food,tobacco,pharmaceutical and feed,etc,have been extensively used in daily chemicals,cosmetics and pharmaceuticals.Nowadays,some short-chain flavor esters have been successfully synthesized with lipases from Bacillus,Burkholderia,Candida,Pseudomonas,Rhizopus,Mucor,Fusarium,Saccharomyces,Thermomyces,etc.However,no study have been conducted focusing on the lipase from Aspergillus oryzae?AOL?.So far,AOL is one of the most important lipases for it retains high enzymatic activity in organic solvents media,in particular,it has been recognized as a GRAS?generally regarded as safe?enzyme by the FDA.We aim to synthesize different short-chain flavor esters under high substrate concentration with Aspergillus oryzae whole cells as the biocatalyst.The optimization of Aspergillus oryzae lipase fermentation conditions was studied as well.1.AOL catalytic efficiency remains very high when the substrate concentration evolves to 2.0 M high.In terms of the synthesis of ethyl heptanoate,the conversion rate of acid could reach 88%by 48 h.While the substrate loading was increased to 3.0 M high,the conversion rate of acid could also reach 80%by 144 h.AOL exhibited high catalytic activity toward a series of short-chain acids and alcohols.The conversion rates of acids were all higher than 80%in the synthesis of a majority of heptanoic acid esters,octanoic acid esters and n-propyl hexanoate at 30? in cyclohexane by 48 h with molar ratio of alcohol to acid of 1:1,enzyme loading of 20 g·L^-1 and substrate concentration of 2.0 M.2.The kinetics of esterification of heptanoic acid and ethanol catalyzed by AOL was found to follow the Ping-Pang Bi-Bi mechanism with an alcohol inhibition.The enzymatic kinetics equation was:The initial rate value calculated by the equation is highly similar to the experimental value.The relative error is within 10%.For the esterification,the excess of alcohol-acyl receptor could reduce the reaction rate,but the acid-acyl donor wouldn't.3.For the esterification catalyzed by AOL,excessive alcohol-acyl receptor could inhibit the reaction rate but not deactivate it,while excessive product water could decrease the final conversion rate.By increasing the adding times and reducing the adding quantity to a small amount could alleviate the inhibition of excessive alcohol on AOL.The reaction time then shortened to a third of the original value while the equilibrium endpoint remained the same.The final conversion rate of acid will increase from 88%to 96%by a second-set reaction?by removing the water and adding the biocatalyst?.4.For agro-industrial residues medium,olive oil cannot promote the enzyme production,but there is an enhancement for the production of lipase by adding olive oil when using Czapek s medium and PDA medium.The production of lipase depends on the composition and content of nitrogen source in the medium.The less the amount of organic nitrogen source,the less the production of intracellular and extracellular hydrolytic enzyme.However,the production of intracellular esterifying enzyme will not change when the content of organic nitrogen source reduced to half of the original amount.Of course,continuing to reduce the content of organic nitrogen source will influence the production of intracellular esterifying enzyme.Aspergillus oryzae will not grow in the medium with no organic nitrogen source.5.The conditions of enzyme production?esterification activity?by Aspergillus oryzae in the medium of 1%organic nitrogen source?peptone?in 5 L fermentation process was studied.The results showed that Aspergillus oryzae grew very fast,and reached the stable growth phase by 24 h,the pH variation range of the fermentation broth is within about 0.5 during the first 48 h.The optimal fermentation time for extracellular hydrolytic enzyme,intracellular esterifying enzyme and intracellular hydrolytic enzyme were different.The optimal fermentation process was obtained:the age of cell,agitation rate and airflow rate were 24 h,400 rpm and 1.0 vvm respectively.Under such condition,the maximum enzyme production of extracellular hydrolytic enzyme,intracellular esterifying enzyme and intracellular hydrolytic enzyme were 1.51 U·mL^-1 by 18 h,3200 U·g^-1 by 36 h and 145 U·g^-1 by 48 h,respectively.They were improved respectively by 216.7%,64%and 21.7%compared with that obtained under the shake flask condition.