Zhong-Hua Yang

Asymmetric reduction of prochiral ketones to chiral alcohols catalyzed by plants tissue

As an important organic compound, chiral alcohols are the key chiral building blocks to many single enantiomer pharmaceuticals. Asymmetric reduction of the corresponding prochiral ketones to produce the chiral alcohols by biocatalysis is one of the most promising routes. Asymmetric reduction of different kinds of non-natural prochiral ketones catalyzed by various plants tissue was studied in this work. Acetophenone, 4′-chloroacetophenone and ethyl 4-chloroacetoacetate were chosen as the model substrates for simple ketone, halogen-containing aromatic ketone and β-ketoesters, respectively. Apple (Malus pumila), carrot (Daucus carota), cucumber (Cucumis sativus), onion (Allium cepa), potato (Soanum tuberosum), radish (Raphanus sativus) and sweet potato (Ipomoea batatas) were chosen as the biocatalysts. It was found that these kinds of prochiral ketoness could be reduced by these plants tissue with high enantioselectivity. Both R- and S-form configuration chiral alcohols could be obtained. The e.e. and chemical yield could reach about 98 and 80% respectively for acetophenone and 4′-chloroacetophenone reduction reaction with favorable plant tissue. And the e.e. and yield for ethyl 4-chloroacetoacetate reduction reaction was about 91 and 45% respectively.

Production of chiral alcohols from prochiral ketones by microalgal photo-biocatalytic asymmetric reduction reaction

Microalgal photo-biocatalysis is a green technique for asymmetric synthesis. Asymmetric reduction of nonnatural prochiral ketones to produce chiral alcohols by microalgal photo-biocatalysis was studied in this work. Acetophenone (ACP) and ethyl acetoacetate (EAA) were chosen as model substrates for aromatic ketones and β-ketoesters, respectively. Two prokaryotic cyanophyta and two eukaryotic chlorophyta were selected as photo-biocatalysts. The results proved that nonnatural prochiral ketones can be reduced by microalgal photo-biocatalysis with high enantioselectivity. Illumination is indispensable to the photo-biocatalysis. For aromatic ketone, cyanophyta are eligible biocatalysts. For ACP asymmetric reduction reaction, about 45% yield and 97% e.e. can be achieved by the photo-biocatalysis reaction with Spirulina platensis as biocatalyst. On the contrary, chlorophyta are efficient biocatalysts for β-ketoester asymmetric reduction reaction among the four tested algae. For EAA asymmetric reduction reaction, about 70% yield and 90% e.e. can be achieved with Scenedesmus obliquus as biocatalyst. The microalgae used in this study outperformed other characterized biocatalysts such as microbial and plant cells.

Production of Chiral Aromatic Alcohol by Asymmetric Reduction with Vegetable Catalyst

Abstract Chiral aromatic alcohols are the key chiral building block for many important enantiopure pharmaceuticals. In this work, we studied asymmetric reduction of prochiral aromatic ketone to produce the corresponding chiral alcohol using vegetables as the biocatalyst. Acetophenone was chosen as the model substrate. The results indicate that acetophenone can be reduced to the corresponding chiral alcohols with high enantioselectivity by the chosen vegetables, i.e. apple (Malus pumila), carrot (Daucus carota), cucumber (Cucumis sativus), onion (Allium cepa), potato (Soanum tuberosum), radish (Raphanus sativus), and sweet potato (Ipomoea batatas). In the reaction, R-1-phenylethanol is produced with apple, sweet potato and potato as the catalyst, while S-1-phenylethanol is the product with the other vegetables as the catalyst. In term of the enantioselectivity and reaction yield, carrot (D. carota) is the best catalyst for this reaction. Furthermore, the reaction characteristics were studied in detail using carrot (D. carota) as the biocatalyst. The effects of various factors on the reaction were investigated and the optimal reaction conditions were determined. Under the optimal reaction conditions (reaction time 50 h, substrate concentration 20 mmol·L−1, reaction temperature 35 °C and pH 7), 95% of e.e. (to S-1-phenylethanol) and 85% chemical yield can be obtained. This work extends the biocatalyst for the asymmetric reduction reaction of prochiral aromatic ketones, and provides a novel potential route to produce enantiopure aromatic alcohols.

A Novel Cellulase Produced by a Newly Isolated Trichoderma virens

Screening and obtaining a novel high activity cellulase and its producing microbe strain is the most important and essential way to improve the utilization of crop straw. In this paper, we devoted our efforts to isolating a novel microbe strain which could produce high activity cellulase. A novel strain Trichoderma virens ZY-01 was isolated from a cropland where straw is rich and decomposed, by using the soil dilution plate method with cellulose and Congo red. The strain has been licensed with a patent numbered ZL 201210295819.6. The cellulase activity in the cultivation broth could reach up to 7.4 IU/mL at a non-optimized fermentation condition with the newly isolated T. virens ZY-01. The cellulase was separated and purified from the T. virens culture broth through (NH4)2SO4 fractional precipitation, anion-exchange chromatography and gel filtration chromatography. With the separation process, the CMC specific activity increased from 0.88 IU/mg to 31.5 IU/mg with 35.8 purification fold and 47.04% yield. Furthermore, the enzymatic properties of the cellulase were investigated. The optimum temperature and pH is 50 °C and pH 5.0 and it has good thermal stability. Zn2+, Ca2+ and Mn2+ could remarkably promote the enzyme activity. Conversely, Cu2+ and Co2+ could inhibit the enzymatic activity. This work provides a new highly efficient T. virens strain for cellulase production and shows good prospects in practical application.

Strengthening NADPH Regeneration for Improving Photo-biocatalytic Ketones Asymmetric Reduction Reaction by Synechocystis Through Overexpression of FNR

AbstractMicroalgae are excellent biocatalyst candidates for photo-biocatalytic-asymmetric reduction of prochiral ketones to produce enantiomer alcohols. In the biocatalysis asymmetric reduction of carbonyl group process, the cofactor, NADPH, plays a key role. So the current study focused on the effect of NADPH availability on the reduction. Photosynthesis is a main pathway for NADPH regeneration in microalgae, and the ferredoxin-NADP+ oxidoreductase (FNR) is the key enzyme in this process. In this work, we constructed an engineered cyanobacterium Synechocystis sp. PCC6803::Ω-PpetE-petH to overexpress FNR gene petH to strengthen NADPH regeneration. The results show that the intracellular NADPH content was increased 80.8% compared to the wild type. Applying the Synechocystis sp. PCC6803::Ω-PpetE-petH to catalyze the model asymmetric reduction reaction (reduction of acetophenone to S-phenylethanol), the yield was improved 61.2% compared to the wild-type. This work makes the microalgal biocatalysis asymmetric reduction process become more preponderant via providing a new route to enhance the regeneration of cofactor NADPH.Graphical AbstractPutative metabolic process of nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and the photo-biocatalytic-asymmetric reduction reaction of prochiral ketones in microalgal cells.