L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.
Aminobutyrates ; chemical synthesis ; Bacillus thuringiensis ; enzymology ; Candida ; enzymology ; Escherichia coli ; enzymology ; Formate Dehydrogenases ; metabolism ; Leucine Dehydrogenase ; metabolism ; Threonine ; metabolism ; Threonine Dehydratase ; metabolism

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.
Candida ; genetics ; isolation & purification ; metabolism ; Genetic Vectors ; genetics ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; biosynthesis ; Metabolic Engineering ; Recombination, Genetic ; Rhizopus ; enzymology ; genetics ; Transformation, Bacterial

This paper reports the progress of biodiesel production with enzymatic catalysis in Beijing University of Chemical Technology, one of the leaders in biodiesel R & D in China, which includes screening of high-yield lipase production strains, optimization and scale-up of the lipase fermentation process, lipase immobilization, bioreactor development and scale-up, biodiesel separation and purification and the by-product glycerol utilization. Firstly, lipase fermentation was carried out at industrial scale with the 5 m3 stirred tank bioreactor, and the enzyme activity as high as 8 000 IU/mL was achieved by the species Candida sp. 99-125. Then, the lipase was purified and immobilized on textile membranes. Furthermore, biodiesel production was performed in the 5 m3 stirred tank bioreactor with an enzyme dosage as low as 0.42%, and biodiesel that met the German biodiesel standard was produced. And in the meantime, the byproduct glycerol was used for the production of 1,3-propanediol to partly offset the production cost of biodiesel, and 76.1 g/L 1,3-propanediol was obtained in 30 L fermentor with the species Klebsiella pneumoniae.
Biofuels ; Bioreactors ; Biotechnology ; economics ; methods ; Candida ; enzymology ; Catalysis ; China ; Enzymes, Immobilized ; metabolism ; Esterification ; Fermentation ; Lipase ; metabolism ; Plant Oils ; chemistry

We immobilized Candida sp. lipase onto seven kinds of industrial adsorption and ion exchange resins. By determining the activity of each immobilized enzyme, the weakly basic anionic exchange resin of D301 showed the best results for the immobilization of Candida sp. lipase. Comparing the scanning electron micrographs of D301 with Novozym 435 (immobilized Candida antarctica lipase B from Novo Nordisk Corp.), we selected D301 as a carrier for the immobilization of Candida sp. lipase. And we pretreated the resin D301 with the bifunctional agent glutaraldehyde and crosslinked it with Candida sp. lipase. The optimal conditions for the immobilization of Candida sp. lipase were as follows: 8 mL of the amount of 5% glutaraldehyde solution, five hours of the time pretreated D301 with glutaraldehyde, 1.0 g/L the concentration of Candida sp. lipase used, pH of the phosphate buffered, 6.0 and 10 hours of time for immobilization, respectively. The activity of immobilized enzyme was over 35 U/mg and the efficiency of immobilization was around 3.5 Ul(mg x h).
Candida ; enzymology ; Enzyme Stability ; Enzymes, Immobilized ; chemistry ; drug effects ; metabolism ; Ion Exchange Resins ; pharmacology ; Lipase ; chemistry ; metabolism

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.
Biocatalysis ; Candida ; enzymology ; Esters ; chemistry ; metabolism ; Fungal Proteins ; Genetic Engineering ; Glucose ; chemistry ; metabolism ; Laurates ; chemistry ; metabolism ; Lipase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

An enzyme-displaying yeast as a whole-cell biocatalyst seemed an alternative to immobilized enzyme, due to its low-cost preparation and simple recycle course. Here, we tried to use a recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) to catalyze the synthesis of short chain flavor esters in n-heptane. We studied some major influential factors of esterification reactions, such as carbon chain length of the substrates, alcohol structure, enzyme concentration, substrates concentration, molar ratio of the substrates. The acid conversions were determined by titration and gas chromatography analysis. About ten kinds of esters were synthesized successfully, and the acid conversions of eight esters reached as high as 90% after reaction for 6 h. The result also indicated that ethanol and hexanoic acid were the most suitable substrates for this whole-cell catalyst. Under the optimal reaction conditions (the amount of lipase 20 g/L (306.0 U/g-dry cell), hexanoic acid concentration 0.8 mol/L, the molar ratio of hexanoic acid to ethanol 1:1.1), hexanoic acid conversion reached 97.3% after reaction for 1.5 h. To our knowledge, the CALB-displaying P. pastoris whole-cell biocatalyst showed good tolerance for high substrates concentration and exhibited high reaction rate on esterification of short chain flavor esters among the present enzyme/cell reported. Thus, CALB-displaying P pastoris whole-cell biocatalyst was promising in commercial application for flavor esters synthesis in non-aqueous phase.
Biocatalysis ; Candida ; enzymology ; Enzymes, Immobilized ; Esters ; metabolism ; Fungal Proteins ; Lipase ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

To improve the expression level and catalytic efficiency of (R)-carbonyl reductase from Candida parapsilosis in Escherichia coli, we optimized the mRNA secondary structure of (R)-carbonyl reductase gene in translation initiation region (from +1 to +78), and constructed the corresponding variant. The formation of hairpin structure was significantly reduced and the Gibbs free energy was dramatically decreased from -9.5 kcal/mol to -5.0 kcal/mol after optimization. As a result, the expression level of (R)-carbonyl reductase in the variant was increased by 4-5 times and its specific activity in cell-free extract was enhanced by 61.9% compared to the wild-type strain. When using the whole cells as catalyst and 2-hydroxyacetophenone as substrate with a high concentration of 5.0 g/L, the variant showed excellent performance to give (R)-1-phenyl-1, 2-ethanediol with optical purity of 93.1% enantiomeric excess and a yield of 81.8%, which were increased by 27.5% and 40.5% respectively than those of the wild-type. In conclusion, the optimization of mRNA secondary structure in translation initiation region can overcome the steric hindrance of translation startup, promote translation smoothly to acquire high expression of target protein, and favor protein folding correctly to efficiently improve the enzyme specific activity and biotransformation function.
Alcohol Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Base Sequence ; Biocatalysis ; Candida ; enzymology ; Catalysis ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Messenger ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; Stereoisomerism

Yeast surface display involves that the exogenous protein, which was fused with the yeast outer shell cell wall protein, was genetically anchored on the yeast cell surface. It has been widely used in expression and screening of functional protein. Here, we focused on the construction of lipase-displaying systems and its application in enzymatic biosynthesis, such as fatty acid methyl esters, short-chain flavour esters and sugar esters applications, and so on.
Candida ; enzymology ; genetics ; Lipase ; biosynthesis ; genetics ; Pichia ; enzymology ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Solvents ; Yeasts ; enzymology ; genetics

Candida glycerinogenes WL2002-5 (C.g) is an important industrial strain for glycerol production. To further improve glycerol production, we reconstructed a binary vector pCAM3300-zeocin-CgGPD1, introduced it to Agrobacterium tumefaciens LBA4404 by electroporation, and then transformed the T-DNA harboring the CgGPD1 to Candida glycerinogenes by Agrobacterium tumefaciens-mediated transformation (ATMT). After 96 h fermentation with glucose as the substrate, we screened a transformant named C.g-G8 with high glycerol production. Compared with the wild strain, the glucose consumption rate of C.g-G8 and the glycerol production were 12.97% and 18.06% higher, respectively. During the fermentation, the activity of glycerol-3-phosphate dehydrogenase of C.g-G8 was 27.55% higher than that of the wild strain. The recombinant Candida glycerinogenes with high glycerol production was successful constructed by ATMT method.
Agrobacterium tumefaciens ; enzymology ; genetics ; Candida ; genetics ; metabolism ; Electroporation ; Fermentation ; Glycerol ; analysis ; metabolism ; Glycerolphosphate Dehydrogenase ; genetics ; Recombination, Genetic ; Transformation, Genetic

We used immobilized lipase from Candida sp. 99-125 to produce fatty acid methyl esters (FAMEs) from crude oil and methanol. We studied the effects of phospholipids on activity of immobilized lipase, reaction velocity, stability of immobilized lipase and the stability of immobilized lipase in crude and refined oil. Results showed that the activity of the lipase immersed in petroleum ether with 1% phospholipids dropped more quickly than the lipase in petroleum ether without phospholipids. When soybean oil was used without phospholipids as material, the FAMEs yield of 15 min was 26.2%, whereas the yield decreased to 12.4% when there were 5% phospholipids in the soybean oil. However when the phospholipids content was below 1%, the stability of the lipase did not change obviously. The lipase was stable when used to catalyze crude soybean oil and crude jatropha oil, after 10 cycles the FAMEs yield was still above 70%. This lipase showed great potential for industrial production of biodiesel from crude oil.
Biofuels ; analysis ; Candida ; enzymology ; metabolism ; Enzymes, Immobilized ; Fatty Acids ; metabolism ; Lipase ; metabolism ; Methanol ; metabolism ; Methyl Ethers ; metabolism ; Petroleum ; metabolism ; Phospholipids ; metabolism