Constitutive display of Candida antarctica lipase B on the cell surface of Aspergillus niger and regulation of its fermentation / 生物工程学报

Displaying Candida antarctica lipase B (CALB) on the cell surface of Aspergillus niger is effectively applied for the industries of food, cosmetics, pharmaceutical and so on. Displaying CALB using induced promoter of glucoamylase on the cell surface of A. niger SH-1 has some problems such as inhibiting its expression under high concentration of glucose, mycelium cleavage and decreasing enzyme activity in the later period of fermentation process. Displaying CALB manipulated by constitutive promoter from glyceraldehyde-3-phosphate dehydrogenase instead of glucoamylase on the cell surface of A. niger SH-1, called AN-GpdA, could solve the above problems effectively. Furthermore, it can not only use glucose, but also xylose as a sole carbon source. Enzyme activity of AN-GpdA using xylose for fermentation reached 1 100.28 U/g of dry cell. We also used lignocellulose such as the hydrolysate of bagasse for fermentation with good performance. The result would provide a novel strategy for the utilization of bagasse.

Synthesis of diisooctyl adipate catalyzed by lipase-displaying Pichia pastoris whole-cell biocatalysts / 生物工程学报

An enzyme-displaying yeast as a whole-cell biocatalyst is an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, lipase-displaying Pichia pastoris whole-cell was used as a biocatalyst to synthesize diisooctyl adipate in the non-aqueous system. The maximum productivity of diisooctyl adipate was obtained as 85.0% in a 10 mL reaction system. The yield could be reached as high as 97.8% when the reaction system was scaled up to 200 mL. The purity obtained is 98.2% after vacuum distillation. Thus, the lipase-displaying P. pastoris whole-cell biocatalyst was promising in commercial application for diisooctyl adipate synthesis in non-aqueous phase.

Synthesis of glucose laurate monoester catalyzed by Candida antarctica lipase B-displaying Pichia pastoris whole-cells / 生物工程学报

We developed a new enzymatic-catalyzing producing process of glucose laurate monoester. In the process we used Candida antarctica lipase B-displaying Pichia pastoris whole-cells as biocatalyst, glucose as the acyl acceptor and lauric acid as the acyl donor. The product glucose laurate monoester was purified by silica gel column chromatography and preparative liquid chromatography, and identified by liquid chromatography-mass spectrometry. Then we optimized the process from various aspects, such as solvent composition, ratio of dmethyl sulfoxide to 2-Methyl-2-butanol (V/V), catalyst dosage, substrate concentration, water activity and temperature. The optimal reaction conditions were: glucose 0.5 mmol/L, lauric acid 1.0 mmol/L, ratio of 2-Methyl-2-butanol to Dmethyl sulfoxide is 7:3 in 5 mL volume, temperature 60 degrees C, the best initial water activity of whole-cells biocatalyst is 0.11. The maximum glucose conversion could be 48.7% after 72 h.

Expression of Candida antarctica lipase B on yeast surface and synthesis of ethyl hexanoate catalyzed by CALB / 生物工程学报

Short-chain esters play a significant role in the food industry as flavor and aroma constituents. Candida antarctica lipase B (CALB) is one of the most effective catalysts for organic synthesis. We constructed a CALB-displaying yeast whole-cell biocatalyst and applied it to esterification from caproic acid and ethanol. CALB was fused with the alpha-agglutinin C-terminal and the signal peptide of Glucoamylase in pICAS, a yeast surface display vector, to construct plasmid pICAS-CALB. An extremely Asn-rich linker, named celAL was inserted in the Xho I of pICAS-CALB to construct plasmid pICAS-celAL-CALB. The fused gene was under the control of GAPDH promoter. After incubated at 30 degrees C for 96 h the lipase hydrolytic activity of the yeast whole cells reached a plateau, 26.26 u/(g x dry cell). In nonaqeous media, the yield of 98.0% ethyl hexanoate was obtained after 24 h esterification from caproic acid and ethanol (the molar ratio of caproic acid : ethanol = 1 : 1.25) using lyophilized CALB displaying yeast whole cells.

Global expression profiling of Saccharomyces cerevisiae: metabolic remodeling in post-log phase / 生物工程学报

For the purpose of revealing the mechanism of the reduction of yeasts ethanol production rate after entrance of post-log phase, we used microarray to study expression profiles of the yeast Saccharomyces cerevisiae during the transition from mid-log growth phase to post-log growth. The results demonstrate that the global pattern of gene expression is very stable during the mid-log phase. However, a dramatic metabolic remodeling was found when the yeast entries post-log phase, during which many of amino acid synthesis and metabolism related genes are up-regulated, moreover, ion transport, energy generation and storage related genes are also up regulated during this phase, while a large number of genes involved in transposition and DNA recombination are repressed. Central metabolic pathways also engage in metabolic remodeling, within which the genes involved in succinate and a-ketoglutarate synthesis pathways are up regulated, accordance with those of amino acid synthesis and metabolism. These results demonstrate that the increasing demand for amino acids in post-log phase lead to a metabolic transition into TCA cycle and glyoxylate cycle, which subsequently reduce the ethanol production rate. This suggests a global insight into the process of yeast ethanol fermentation.

Selection of Ginkgo biloba cell line for suspension culture and studies on stability of ginkgolide B in subculture / 中草药

Object To select the cell line of Ginkgo biloba L that may produce high yield of ginkgolide B (GB) in suspension culture and to study the stability of GB in subculture Methods Calculus was induced with stem, root and leaf of selected elite species and high yield suspension cell lines chosen by hypoxia stress Results Seven suspension cell lines with improved yield of GB were obtained Among which cell line MH 3 gave a 3 71 fold increase of cellular bioproduct with an increased content up to 302 ?g/g DW after culturing for 18 d, a leading record country wide In shaking bottle culture, it showed a consistent yield of GB with a content of 291 ?g/g DW in 6 successive subcultures, coefficient of variation=0 131 Conclusion The suspension cell line selected by hypoxia stress gave a higher yield and stability in successive transfer culture