| Cutinase(EC 3.1.1.74), a kind of serine esterase, can catalyze the esterification of a variety of fatty acids and alcohols, and interesterification of different types of esters and alcohols, the products obtained including short chain fatty acid ester, linear alkyl esters and multipolyesters, have been widely used in food, cosmetics, beverages and other industries. In this research, an acidic cutinase(ScCutA) from Sirococcus conigenus VTT D-04989 was cloned and expressed in P. pastoris KM71, and enzyme properties were studied; this study also optimized the conditions for the production of ScCutA by recombinant strain in the 3 L fermentor and coexpressed N-acetyltransferase(MprI) to improve the expression level of ScCutA; the application of recombinant ScCutA in synthesis of short chain ester was researched. The main results are as follows:(1) The recombinant plasmid pPIC9K-ScCutA carrying the S. conigenus cutinase gene was transferred into P. pastoris KM71 after linearized by Sac I, and successfully obtained the recombinant P. pastoris KM71/pPIC9K-ScCutA. After 120 h of induced by methanol, the ScCutA activity was 28.6 U·mL-1. After various purification steps to obtain the purified ScCutA, and enzymological qualitative research showed that the purified enzyme exhibited a specific activity from 90.6 units·mg-1 to 149.1 units·mg-1 for pNPB; the purification fold is 1.6 and recovery yield is 31.1% the temperature for optimal activity of the ScCutA was determined to be 65℃ and the half-life in 50℃ and 45℃were 3 h and 12 h, relatively; the optimum pH was 4.5, stable in the range of pH 3.5-6.5. ScCutA demonstrated great stability in many organic solvents such as ethanol and butanol. The optimization of ScCutA production from engineered P. pastoris KM71/pPIC9K-ScCutA in the 3 L fermentor was investigated. Investigation of different induction strategies revealed that induction was optimal when the temperature was maintained at 28℃, the inducer(methanol) was fed at 1%(v·v-1), and protein expression was induced when the cell density reached 38.4 g·L-1(OD600 was 100). Under these conditions, the ScCutA activity of 419.2 U·m L-1 was achieved after 138 h of induce.(2) N-acetyltransferase(MprI) has the physiological function of reducing the intracellular reactive oxygen species(ROS) levels. In order to optimize the growth environment of recombinant P. pastoris and the expression of ScCutA was further improved, the recombinant plasmid pPICZA-MprI carrying the P. pastoris N-acetyltransferase gene was linearized and transformed into P. pastoris KM71/pPIC9K-ScCutA to achieve recombinant P. pastoris KM71/pPIC9K-ScCutA/pPICZA-MprI. Shake flask cultures results showed that ScCutA activity reached 39.4 U·mL-1, 1.4-fold of the control group strain. The optimization of ScCutA production from engineered P. pastoris KM71/pPIC9K-ScCut A/pPICZA-Mpr I in the 3 L fermentor was investigated. Investigation of different induction strategies revealed that when the cell density reached 38.4 g·L-1( OD600 was 100),the inducer(methanol) was fed at 1.5%(v·v-1), and protein expression was induced when the temperature was maintained at 28℃, and then 138 h later the ScCutA activity was up to 483.3 U·mL-1, was 1.2 times higher than that of control group.(3) The effects of short chain ester synthesis by recombinant ScCutA in organic phase, water phase, wine and solid-state fermentation of Fen flavor liquor were studied. The optimization of ester production in the organic phase was as follows: the initial water content, caproic acid concentration, molar ratio of caproic acid/alcohol, temperature, optimum enzyme dosage was 0.04%, 0.1 mol·L-1, 1:1.5, 50℃, 40 U·m L-1,conversion of ethyl caproate reached 98.3% after 16 h reaction. The highest synthesis rates in aqueous phase was 5.7%-15.9%. When ScCutA was used for wine aging, aromatic ester content in liquor was decreased, the content of ethyl caproate decreased most significantly lower than the control group of 34.3%, aromatic ester content in Yellow Wine markedly increased in different degree, which changes the content of ethyl lactate was higher than the control group of 152.3%, therefore, adding ScCutA accelerated the changes of aromatic ester content in the aging process of wine. In addition, the effects of ScCutA on the changes of main aromatic esters in the short term fermentation process of Fen flavor liquor were studied. The results showed that 2%(kg·kg-1) of ScCutA was supplement to fermented grains, total ester, ethyl acetate and ethyl lactate content increased significantly, increased by 29.6%, 25.3% and 17.1% than the control group. |
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