Meiling Chen

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

Surface display is effectively utilized to construct a whole-cell biocatalyst. Codon optimization has been proven to be effective in maximizing production of heterologous proteins in yeast. Here, the cDNA sequence of Rhizopus oryzae lipase (ROL) was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, and based on the Saccharomyces cerevisiae cell surface display system with α-agglutinin as an anchor, recombinant yeast displaying fully codon-optimized ROL with high activity was successfully constructed. Compared with the wild-type ROL-displaying yeast, the activity of the codon-optimized ROL yeast whole-cell biocatalyst (25 U/g dried cells) was 12.8-fold higher in a hydrolysis reaction using p-nitrophenyl palmitate (pNPP) as the substrate. To our knowledge, this was the first attempt to combine the techniques of yeast surface display and codon optimization for whole-cell biocatalyst construction. Consequently, the yeast whole-cell ROL biocatalyst was constructed with high activity. The optimum pH and temperature for the yeast whole-cell ROL biocatalyst were pH 7.0 and 40 °C. Furthermore, this whole-cell biocatalyst was applied to the hydrolysis of tributyrin and the resulted conversion of butyric acid reached 96.91% after 144 h.

Application of a Novel Freeze-Dried Aerotolerant Bifidobacterium animailis Subsp. Lactis Qq08 (Bl Qq08) in Probiotic Yoghurt Production

Probiotics are of great beneficial functions, especially on gastrointestinal health. Probiotic yoghurt is gaining popularity for it is the main way to supplement probiotics. In this study, a novel probiotic yoghurt was made by adding a freeze-dried aerotolerant Bifidobacterium animailis subsp. lactis Qq08 (Bl Qq08) to traditional yoghurt. Microbial, physical, chemical and flavor changes of the yoghurt during cold storage were analyzed weekly to study the effects of Bifidobacterium addition on the yoghurt quality. Results showed that the addition of Bl Qq08 didn’t significantly affect the cell viabilities in yoghurt which kept above the recommended level. Chemical and flavor characteristics of the probiotic yoghurt kept similar or turned better, indicating that the lyophlized Bl Qq08 was an excellent probiotic bacteria to product yoghurt.

The Preparation of a Hyper-Thermostable Whole-Cell Biocatalyst and its Application for Biosynthesis of Biodiesel

In this study, a hyper-thermostable lipase whole-cell biocatalyst has been developed. The hydrolytic activity of the codon-optimized ROL displayed on the yeast cell surface was 19.5 U /g dried cells cultured in SD medium, while the biomass was 13 g/L. Moreover, the whole-cell biocatalyst presented great thermostability. After 10 min incubation at 90 °C, 95 °C and 100 °C, the residual activity of lipase still remained 89%, 86% and 68%, respectively. Therefore, the whole-cell biocatalyst was applied in bioconversion of FAME and biodiesel. In our study, 50 mM oleic acid was most reasonable for bioconversion of FAME, and 50 mM soy oil was most reasonable for bioconversion of biodiesel, when the reaction were carried out in hexane as the solvent, and the molar ratio of oleic acid (or soy oil) and methanol was 1:4. Reactions were catalyzed by 0.5 g lyophilized bioimprinted whole-cell biocatalyst and incubated at 45 °C for 12 h with shake.

Optimization of the Freeze-Drying Media and Survival throughout Storage of Freeze-Dried Aerotolerant Bifidobacterium animailis Subsp. Lactis Qq08

The aim of the present work was to optimize the formula of cryoprotectants for freeze drying process of the previous selected Bifidobacteriumanimailis subsp.lactis Qq08 (Bl Qq08). Single factor and orthogonal experiments were conducted to optimize the cryoprotectants formula. The formula of cryoprotectants was optimized as follows: skim milk 15%, lactose 8%, Vc 1.5% and glycerol 3%. In the triplicate lyophilization tests, the average survival rate of Bl Qq08 reached 91.23%.The optimized formula of cryoprotectants was suitable for freeze drying of the Bl Qq08.