| Cinnamon Cassia is an important and special oil tree in Guangxi China, which was mainly planted in the south foots of Shiwan Mountains and Xijiang River Valley. Cinnamon Cassia in Guangxi yielded60%cinnamon and90%cinnamon oil domestic products. Cinnamon was applied widely in the field of food and medicine, and its essential oils (cinnamon oil) used to be an important natural spices. In this project, cinnamon slag (the residue of cinnamon bark after extracting its volatile oil), cinnamaldehyde (the main component of cinnamon oil), cinnamyl alcohol (a derivative of cinnamaldehyde) and some kind of prochiral aromatic ketones were used as the reaction substrate catalyzed by microbe and a series of biocatalyzed reactions were researched. The main research contents and results are shown as follows:1. Preparation of cinnamide from cinnamon slag by biodegradation and optimization of the process conditions.The residue of cinnamon bark after extracting volatile oil was usually used as fertilizer or just discharged as waste, which has not been effectively utilized. Based on the present situation, a study on microbial biotransformation of cinnamon slag was carried out to provided experimental basis for the comprehensive utilization of cinnamon bark resources.The strain CG19which could convert some components of cinnamon slag to cinnamide was screened from soil samples, thus it could prepare cinnamide from cinnamon slag. CG19was identified as Streptomyces sp. according to the16S rDNA gene sequences and designated as Streptomyces sp. CG19. The optimization of growth conditions showed that the heaviest wet mycelia was obtained when Streptomyces sp. CG19was cultured at pH5.0,30℃for72h using30g·L-1maltose as carbon source and10g·L-1ammonium sulfate as nitrogen source. The optimal reaction conditions for the biodegradation of cinnamon slag with Streptomyces sp. CG19were as follows:8g·L-1cinnamon slag,40g·L-1wet mycelia,30mL·L-1cosubstrate ethanol,100mL of phosphate buffer solution (pH5.0),30℃, reacted for3d. Under these conditions, the maximum yield was0.32%.2. The screen of strains which could biodegradated cinnamyl alcohol (CMO) to3-phenylpropanol (HCMO) and the optimization of the process conditions.The strain CG10which could biodegradated CMO to HCMO was screened from special soil samples and was identified as Mucor sp. according to the16S rDNA gene sequences and named as Mucor sp. CG10.The optimal reaction conditions for the bioreduction of CMO to HCMO with Mucor sp. CG10were as follows:After cultured at pH5.0,30℃for2d using30g·L-1dextrin as carbon source and10g-L"1ammonium sulfate as nitrogen source, CMO was added to the broth as substrate in three steps. The initial addition amount was2mL·L-1and reacted for24h; the secondary addition amount was0.5mL·L-1and reacted for8h of reaction, and the third addition amount was also0.5mL·L-1, but for16h of reaction. Under the optimized conditions, the conversion of CMO and the yield of HCMO were99%and92%, respectively. The maximum HCMO content was2.9g·L-1, which was123%higher than the former one.3. The bioreduction of cinnamaldehyde (CMA) by microbe and the optimization of the process conditions. It was proved that CMA was prior reduced to CMO, rather than3-phenylpropyl aldehyde (HCMA) and then further reduced to HCMO when it was bioreduction with microbe as catalyst.(1)7strains which could selectively hydrogenate CMA to CMO was screened from soil samples by enriching and purifying method using enriched medium added with CMA (Conv.CMA>20%). The strain CG08could catalyzed hydrogenation selectively of CMA to CMO with high reaction rate and selectivity. According to the16S rDNA gene sequences, CG08was identified as Citrobacter freundii and named as Streptomyces sp. CG08.The optimal reaction conditions for the selectively bioreduction of CMA to CMO with Streptomyces sp. CG08were as follows:After cultured at pH6.0,30℃for72h using30g·L-1glucose as carbon source and16g-L"1peptone as nitrogen source, CMA was added to the broth as substrate in two steps. The initial addition amount was2.0mL·L-1and reacted for40h; the secondary addition amount was1.0mL·L-1for32h of reaction. Under the optimized conditions, the conversion of CMA and the yield of CMO were100%and90%, respectively. The maximum CMO content was2.9g·L-1. Streptomyces sp. CG08could selectively reduced CMA to CMO with high efficiency and good conversion, but could not further reduced CMO to HCMO.(2) The bioreduction of CMA catalyzed by Mucor sp. CG10was studied. The optimal reaction conditions were as follows:Mucor sp. CG10was cultured in100mL culture medium composed of20g dextrin and20g peptone which pH was adjusted to5.0with hydrochloric acid, at pH5.0,30℃for72h. Then,2.0mL·L-1CMA was added to the broth as substrate, the reaction was reduced for27h and the best results were obtained with CMA conversion of92%, CMO selectivity of88%. The result showed that Mucor sp. CG10could reduced CMA to CMO with high efficiency and good conversion by two-step reaction.If24g-L"1more wet mycelia of Mucor sp. CG10were added to above transformation, the product CMO was further reduced to HCMO. At last, the concentration of HCMO in the later transformation came to1.9g·L-1. In the whole reaction, the conversion of CMA was100%and the yield of HCMO was86%, remaining few amount of CMO not been reduced.4. An efficient method for the asymmetric reduction of prochiral aromatic ketones to the corresponding (S)-configuration chiral aromatic alcohols with whole cells of Mucor sp. CG10was developed. Mucor sp.CG10from slant was incubated into a500ml conical flask containing100mL culture medium composed of3g dextrin and2.4g peptone which pH was adjusted to5.0with hydrochloric acid and allowed to grow at30℃under shaking condition (180r·min-1) for72h. After that period, mycelia were separated from the culture broth by filtration and wet mycelia were then obtained.2.4g wet mycelia were put into a500ml conical flask containing100mL of phosphate buffer solution (pH4.0), then20mM acetophenone and its nine kinds of aromatic ring substituents (la-10a) were added to the medium as substrate. Bioreductions were performed at30℃,180r·min-1for3d and the asymmetric reaction mixtures were then obtained.Mucor sp. CG10used glucose for cofactor regeneration and proved to be an excellent biocatalyst in the asymmetric reduction of prochiral aromatic ketones to their corresponding (S)-configuration chiral aromatic alcohols. The conversion rate of substrate (Abbreviated as:Conv.) in the range of52%to99%, and the enantiom ericexcess (Abbreviated as:e.e) of product were in the range of38%to99%. The results showed that the electronic effects of substituting group on the benzene ring of prochiral aromatic ketones could affected the conversion of the substrates and the enantioselectivity of the reaction, as well as the microbial tolerance of the substrate. The trend observed was as followed:As the halogen substituting group at the p-position became more electron donating from F, to Cl, Br and I, the conversion of the substrates6a,7a,8a,9a decreased in proper order, while the enantioselectivity of the product6b,7b,8b,9b increased successively. |
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