Upscale production of ethyl (S)-4-chloro-3-hydroxybutanoate by using carbonyl reductase coupled with glucose dehydrogenase in aqueous-organic solvent system.

(S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate to synthesize the side chain of cholesterol-lowering drug atorvastatin. To biosynthesize the (S)-CHBE, a recombinant Escherichia coli harboring the carbonyl reductase and glucose dehydrogenase was successfully constructed. The recombinant E. coli was cultured in a 500-L fermentor; after induction and expression, the enzyme activity and cell biomass were increased to 23,661.65 U/L and 13.90 g DCW/L which was 3.24 and 2.60-folds compared with those in the 50 L fermentor. The biocatalytic process for the synthesis of (S)-CHBE in an aqueous-organic solvent system was constructed and optimized with a substrate fed-batch strategy. The ethyl 4-chloro-3-oxobutanoate concentration reached to 1.7 M, and the (S)-CHBE with yield of 97.2 % and enantiomeric excess (e.e.) of 99 % was obtained after 4-h reaction in a 50-L reactor. In this study, the space-time yield and space-time yield per gram of biomass (dry cell weight, DCW) were 413.17 mM/h and 27.55 mM/h/g DCW for (S)-CHBE production, respectively, which were the highest values as compared to previous reports. Finally, (S)-CHBE was extracted from the reaction mixture with 82 % of yield and 95 % of purity. This study paved the foundation for the upscale production of (S)-CHBE by biocatalysis method.

Mutagenesis breeding of high echinocandin B producing strain and further titer improvement with culture medium optimization.

A combination of microbial strain improvement and statistical optimization is investigated to maximize echinocandin B (ECB) production from Aspergillus nidulans ZJB-0817. A classical sequential mutagenesis was studied first by using physical (ultraviolet irradiation at 254 nm) and chemical mutagens (lithium chloride and sodium nitrite). Mutant strain ULN-59 exhibited 2.1-fold increase in ECB production to 1583.1 ± 40.9 mg/L when compared with the parent strain (750.8 ± 32.0 mg/L). This is the first report where mutagenesis is applied in Aspergillus to improve ECB production. Further, fractional factorial design and central composite design were adopted to optimize the culture medium for increasing ECB production by the mutant ULN-59. Results indicated that four culture media including peptone, K2HPO4, mannitol and L-ornithine had significant effects on ECB production. The optimized medium provided another 1.4-fold increase in final ECB concentration to 2285.6 ± 35.6 mg/L compared to the original medium. The results of this study indicated the combined application of a classical mutation and medium optimization can improve effectively ECB production from A. nidulans and could be a promising tool to improve other secondary metabolites production by fungal strains.

Expression, characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin.

A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was closer in mutant which improved the catalytic activity. The expressed Wt-HHDH and Mut-HHDH were purified and characterized using 1,3-dichloro-2-propanol (1,3-DCP) as substrates. The specific activity of the mutant was enhanced 26-fold and the value of k cat was 18.4-fold as compared to the Wt-HHDH, respectively. The Mut-HHDH showed threefold extension of half-life at 45 °C than that of Wt-HHDH. Therefore it is possible to add 1,3-DCP concentration up to 100 mM and epichlorohydrin (ECH) was produced at a relatively high conversion and yield (59.6 %) using Mut-HHDH as catalyst. This Mut-HHDH could be a potential candidate for the upscale production of ECH.

Efficient production of methionine from 2-amino-4-methylthiobutanenitrile by recombinant Escherichia coli harboring nitrilase.

Methionine as an essential amino acid has been attracting more attention for its important applications in food and feed additives. In this study, for efficient production of methionine from 2-amino-4-methylthiobutanenitrile, a codon-optimized nitrilase gene was newly synthesized and expressed, and the catalytic conditions for methionine production were studied. The optimal temperature and pH for methionine synthesis were 40 °C and 7.5, respectively. The recombinant nitrilase was thermo-stable with half-life of 5.52 h at 40 °C. The substrate loading was optimized in given amount of catalyst and fixed substrate/catalyst ratio mode to achieve higher productivity. Methionine was produced in 100 % conversion within 120 min with a substrate loading of 300 mM. The production of methionine with the immobilized resting cells in packed-bed reactor was investigated. The immobilized nitrilase exhibited good operation stability and retained over 80 % of the initial activity after operating for 100 h. After separation, the purity and the total yield of methionine reached 99.1 and 97 %, respectively. This recombinant nitrilase could be a potential candidate for application in production of methionine.

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase.

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0-7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni(2+), Ba(2+) and Fe(2+). The apparent K (m) and V (max) values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 µmol min(-1) mg(-1), for 2-deoxyribose were 0.033 mM and 2.59 µmol min(-1) mg(-1), respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.

Enzymatic production of 2-amino-2,3-dimethylbutyramide by cyanide-resistant nitrile hydratase.

A novel enzymatic route for the synthesis of 2-amino-2,3-dimethylbutyramide (ADBA), important intermediate of highly potent and broad-spectrum imidazolinone herbicides, from 2-amino-2,3-dimethylbutyronitrile (ADBN) was developed. Strain Rhodococcus boritolerans CCTCC M 208108 harboring nitrile hydratase (NHase) towards ADBN was screened through a sophisticated colorimetric screening method and was found to be resistant to cyanide (5 mM). Resting cells of R. boritolerans CCTCC M 208108 also proved to be tolerant against high product concentration (40 g l(-1)) and alkaline pH (pH 9.3). A preparative scale process for continuous production of ADBA in both aqueous and biphasic systems was developed and some key parameters of the biocatalytic process were optimized. Inhibition of NHase by cyanide dissociated from ADBN was successfully overcome by temperature control (at 10°C). The product concentration, yield and catalyst productivity were further improved to 50 g l(-1), 91% and 6.3 g product/g catalyst using a 30/70 (v/v) n-hexane/water biphasic system. Furthermore, cells of R. boritolerans CCTCC M 208108 could be reused for at lease twice by stopping the continuous reaction before cyanide concentration rose to 2 mM, with the catalyst productivity increasing to 12.3 g product/g catalyst. These results demonstrated that enzymatic synthesis of ADBA using whole cells of R. boritolerans CCTCC M 208108 showed potential for industrial application.

Isolation of enantioselective α-hydroxyacid dehydrogenases based on a high-throughput screening method.

To isolate enantioselective α-hydroxyacid dehydrogenases (α-HADHs), a high-throughput screening method was established. 2,4-Dinitrophenylhydrazine solution forms a red-brown complex with ketoacid produced during the α-HADH-mediated oxidation of α-hydroxyacid. The complex can be easily quantified by spectrophotometric measurement at 458 nm. The enantioselectivity of α-HADH in each strain can be measured with this colorimetric method using (R)- and (S)-α-hydroxyacid concurrently as substrates to evaluate the apparent enantioselectivity (E (app)). The E (app) closely matches the value of true enantioselectivity (E (true)) determined by HPLC analysis. With this method, a total of 34 stains harboring enantioselective α-HADHs were selected from 526 potential α-HADH-producing microorganisms. Pseudomonas aeruginosa displayed the highest (S)-enantioselective α-HADH activity. This strain appears promising for potential application in industry to produce (R)-α-hydroxyacids. The method described herein represents a useful tool for the high-throughput isolation of enantioselective α-HADHs.

Improvement of 1,3-dihydroxyacetone production from Gluconobacter oxydans by ion beam implantation.

Improvement of dihydroxyacetone (DHA) production by mutagenesis of ion beam implantation and medium optimization using response-surface methodology (RSM) were investigated in this work. More than 1000 mutant strains were selected through a mutagenesis method using N(+) ions implantation with a dose of 60 × (2.6 × 10(13)) ions/cm(2) and energy of 10 keV. Several high-yield mutant strains were showed the potent application for DHA production and the genetically stable mutant strain G. oxydans ZJB09113 was selected for optimization of cultivation condition by RSM. The optimal medium for DHA fermentation is composed (in g/L) of yeast extract 4.88, CaCO(3) 2.00, and glycerol 52.86 mL/L (initial pH 4.89). The maximal DHA concentration of 40.0 g/L was achieved after 24 hr of shaken flask fermentation at 30°C with 150 rpm, and 196.3% increase in DHA production in comparison with unoptimized conditions.

Biosynthesis of 2-amino-2,3-dimethylbutyramide by nitrile hydratase from a newly isolated cyanide-resistant strain of Rhodococcus qingshengii.

Cells of a new isolate of Rhodococcus qingshengii harboring nitrile hydratase converted 2-amino-2,3-dimethylbutyronitrile into 2-amino-2,3-dimethylbutyramide (ADBA). Cells also hydrated a broad range of substrates including saturated, unsaturated and cyclic aliphatic nitriles. The microorganism tolerated KCN and ADBA up to 5 mM and 40 g/l, respectively. At 10 °C, ADBA reached 33.8 g/l (yield 84.5%) which was 2.5 times that at 30 °C.

Enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile for production of (R)-(-)-mandelic acid by a newly isolated mutant strain.

(R)-(-)-Mandelic acid (R-MA) is an important intermediate with broad uses. Recently, R-MA production using nitrilase has been gaining more and more attention due to its higher productivity and enantioselectivity. In this work, a new bacterium WT10, which exhibited favorable nitrilase activity and excellent enantioselectivity for production of R-MA by enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile, was isolated and identified as a strain of Alcaligenes faecalis. In order to improve its nitrilase activity for industrial application, the wild-type strain WT10 was further subjected to mutagenesis using a combined LiCl-ultraviolet irradiation and low energy N(+) ion beams implantation technique. A valuable mutant strain A. faecalis ZJUTB10 was obtained. The nitrilase specific activity of the mutant strain was greatly improved up to 350.8 U g(-1), in comparison with wild-type strain WT10 of 53.09 U g(-1). The reaction conditions for R-MA production by mutant strain A. faecalis ZJUTB10 were also optimized. Nitrilase activity in mutant strain showed a broad pH optimum at pH 7.7-8.5. The optimal temperature was 35°C. The highest production rate reached 9.3 mmol h(-1) g(-1). The results showed that mutant strain A. faecalis ZJUTB10 was a new candidate for efficient R-MA production from (R,S)-mandelonitrile and could potentially be used in industrial production.

Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis.

(R)-ethyl-3-hydroxyglutarate with highly optical purity (> or = 99%) can be used as a novel precursor for synthesis of chiral side chain of rosuvastatin. In this study, a novel synthesis route of (R)-ethyl-3-hydroxyglutarate by whole microorganism cells from racemic ethyl 4-cyano-3-hydroxybutyate was created. A strain ZJB-0910 capable of transforming racemic beta-hydroxy aliphatic nitrile was isolated by employing a screening method based on a colorimetric reaction of Co(2+) ion with ammonia, and identified as Rhodococcus erythropolis based on its morphology, physiological tests, Biolog, and the 16S rDNA sequence. After cultivation in a sterilized medium with composition of 20 g glucose, 5 g yeast extract, 0.5 g KH(2)PO(4), 0.5 g K(2)HPO(4), 0.2 g MgSO(4).7H(2)O per liter at 30 degrees C and 150 rpm for 48 h, the whole cells of R. erythropolis ZJB-0910 were prepared as a catalyst in (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate for synthesis of (R)-ethyl-3-hydroxyglutarate, without bearing hydrolase activity for the ester bond of ethyl 4-cyano-3-hydroxybutyate. Under the optimized biotransformation conditions of pH 7.5, 30 degrees C,and 20 mM substrate concentration, (R)-ethyl-3-hydroxyglutarate with 46.2% yield (ee>99%) was afforded, and its chemical structure was determined by ESI-MS, NMR, and IR. The apparent Michaelis constant K(m) and maximum rate Vmax for this biocatalytic reaction were 0.01 M and 85.6 micromol min(-1)g(-1), respectively.

Enantioselective hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide by immobilized cells of an R-amidase-producing bacterium, Delftia tsuruhatensis CCTCC M 205114, on an alginate capsule carrier.

Immobilized cells of Delftia tsuruhatensis CCTCC M 205114 harboring R-amidase were applied in asymmetric hydrolysis of (R)-2, 2-dimethylcyclopropane carboxamide (R - 1) from racemic (R, S)-2, 2-dimethylcyclopropane carboxamide to accumulate (S)-2, 2-dimethylcyclopropane carboxamide (S - 1). Maximum R-amidase activity of 13.1 U/g wet cells (0.982 U/g beads) was obtained under conditions of 3% sodium alginate, 2.5% CaCl(2), 15 h crosslinking and 2 mm bead size, which was 53.9% of that of free cells (24.3 U/g wet cells). In addition, characterization of the immobilized cells was examined. The optimum R - 1 hydrolysis conditions were identified as follows: substrate concentration 10 mM, pH 8.5, temperature 35 degrees C and time course 40 min. Under optimum conditions, the maximum yield and enantiomeric excess of (R)-2, 2-dimethylcyclopropanecarboxylic acid were 49.5% and >99%, respectively. This afforded S - 1 with a yield >49% and an e.e. of 97.7%. With good operational stability and excellent enanotioselectivity, the immobilized cells could be potentially utilized in industrial production of S - 1.

Production of 1,3-dihydroxyacetone from glycerol by Gluconobacter oxydans ZJB09112.

The culture variables were optimized to increase 1,3-dihydroxyacetone (DHA) production by Gluconobacter oxydans ZJB09112 in shake flask and bubble column bioreactor. After fermentation in the optimized medium (g/L: yeast extract 5, glycerol 2.5, mannitol 22.5, K(2)HPO(4) 0.5, KH(2)PO(4) 0.5, MgSO(4) x 7H(2)O 0.1, CaCO(3) 2.0, pH 5.0), when five times of glycerol feeding were made, 161.9 + or - 5.9 g/l of DHA was attained at 88.7 + or - 3.2% conversion rate of glycerol to DHA.

Production of acrylic acid from Acrylonitrile by immobilization of Arthrobacter nitroguajacolicus ZJUTB06-99.

Immobilized cells of Arthrobacter nitroguajacolicus ZJUTB06-99 capable of producing nitrilase was used for biotransformation of acrylonitrile to acrylic acid. Six different entrapment matrixes were chosen to search for a suitable support in terms of nitrilase activity. Ca-alginate proved to be more advantageous over other counterparts in improvement of the biocatalyst activity and bead mechanical strength. The effects of sodium alginate concentration, CaCl2 concentration, bead diameter, and ratio by weight of cells to alginate, on biosynthesis of acrylic acid by immobilized cells were investigated. Maximum activity was obtained under the conditions of 1.5% sodium alginate concentration, 3.0% CaCl2 concentration, and 2-mm bead size. The beads coated with 0.10% polyethylenimine (PEI) and 0.75% glutaraldehyde (GA) could tolerate more phosphate and decrease leakage amounts of cells from the gel. The beads treated with PEI/ GA could be reused up to 20 batches without obvious decrease in activities, which increased about 100% compared with the untreated beads with a longevity of 11 batches.

Biotransformation of p-methoxyphenylacetonitrile into p-methoxyphenylacetic acid by resting cells of Bacillus subtilis.

Resting cells of Bacillus subtilis ZJB-063 were used for the direct transformation of MOPAN (p-methoxyphenylacetonitrile) to MOPAA (p-methoxyphenylacetic acid), which is an important pharmaceutical intermediate. The B. subtilis ZJB-063 culture conditions for the production of nitrilase and the reaction conditions for this nitrilase-mediated conversion were optimized. The maximum production of nitrilase was achieved when glucose and a combination of ammonium sulfate and yeast powder were added as carbon and nitrogen sources respectively. Previously reported inducers were found to be unnecessary for the production of nitrilase from B. subtilis ZJB-063, which indicated that this nitrilase appeared to be constitutive. However, when epsilon-caprolactam (6-hexanolactam) was added as the inducer, B. subtilis ZJB-063 exhibited nitrile hydratase and amidase activity. The maximum conversion of MOPAN into MOPAA (specific activity 17.03 units.g(-1)(DCW); DCW is dry cell weight) was observed in a solution containing 50 mM phosphate buffer (pH 7.0), 10 mM MOPAN, 2.7 mg DCW.ml(-1) wet resting cells and 5% (v/v) DMSO for 4 h at 32 degrees C. MOPAN (10 mM) was completely converted into MOPAA (9.65 mM) in 5 h in shake flasks without the formation of p-methoxyphenylacetamide. The small deviation of MOPAA (9.65 mM) from the theoretical amount (10 mM) may be due to partial consumption of the products by B. subtilis ZJB-063. Both MOPAN and MOPAA inhibited the hydrolysis at concentrations above 15 mM. Scale up of the reaction to 200 ml in a bubble bioreactor shortened the reaction time compared with the reactions performed in shake flasks.

Improvement of amidase production by a newly isolated Delftia tsuruhatensis ZJB-05174 through optimization of culture medium.

The R-amidase production by a newly isolated strain of Delftia tsuruhatensis ZJB-05174 was optimized in this paper. Effects of factors such as carbon sources, nitrogen sources, and inducers on amidase production were investigated. The medium composition was optimized using central composite designs and response surface analysis. The optimal medium components for enhanced amidase production were found to be as follows: glucose, 8.23 g/l; yeast extract, 11.59 g/l; 2,2-(R,S)-dimethylcyclopropane carboxamide, 1.76 g/l; NaCl, 1 g/l; KH2PO4, 1 g/l; and K2HPO4, 1 g/l. A maximum enzyme production of 528.21 U/l was obtained under the optimized conditions, which was 4.7 times higher than that obtained under initial conditions.

Isolation and characterization of Delftia tsuruhatensis ZJB-05174, capable of R-enantioselective degradation of 2,2-dimethylcyclopropanecarboxamide.

R-Enantioselective amidases are of considerable industrial interest due to potential applications in the production of optically active compounds. Strain ZJB-05174, capable of R-enantioselective degradation of 2,2-dimethylcyclopropanecarboxamide, was isolated employing a newly established colorimetric screening method. Based on morphology, physiological tests, ATB system (ID 32 GN) and the 16S rRNA sequence, this strain was identified as Delftia tsuruhatensis. The intracellular amidase exhibited excellent thermostability and was not inhibited by urea, a known inhibitor of many amidases. The enantiomeric ratio of intracellular amidase decreased from 27 to 5 when the reaction temperature was increased from 30 to 46 degrees C. A novel temperature-dependent reversal of stereospecificity was observed when the temperature was increased to 56 degrees C. Interestingly, when the reaction was performed at 46 degrees C, cell suspensions pre-incubated at 56 degrees C exhibited the same inversion of stereospecificity.

Quantitative determination of valienamine and validamine by thin-layer chromatography.

A simple and valid thin-layer chromatographic method for the separation and quantitative determination of valienamine and validamine is described. The two compounds are separated using a Silica gel G plate as the stationary phase and a mixture of 1-PrOH-AcOH-H2O (4:1:1, v/v/v) as the mobile phase. The plate is developed for 1 h at 25 degrees C and dried by a hairdrier, then immersed in 0.1% ninhydrin aqueous solution and heated for 5 min at 121 degrees C. The reacted spots are scanned with a single wavelength at 420 nm in the measurement mode of absorption. The limits of detection of the two compounds are both 0.4 microg. The responses of the densitometry are highly correlated with the amounts of valienamine and validamine in the range of 0.4-2.8 pg. Moreover, the method shows good accuracy and high precision.

Purification and characterization of R-stereospecific amidase from Brevibacterium epidermidis ZJB-07021.

A R-stereospecific amidase was purified from Brevibacterium epidermidis ZJB-07021 and characterized in detail. The amidase was purified to homogeneity by three chromatographic steps for up to 328.9-fold with specific activity of 31.9 U mg(-1). The enzyme was a homodimer with a molecular mass of 94 kDa. It exhibited maximum activity at 40 °C and pH 7.5. The enzyme was strongly inactivated by serine protease inhibitor PMSF. The values of Km and Vmax for racemic 2,2-dimethylcyclopropane carboxamide (DMCPCA) were 4.58 mM and 35.03 µmol min(-1) mg(-1) protein, respectively. The amidase showed a broad substrate spectrum toward aliphatic, aromatic and heterocyclic amides, but could hardly hydrolyze the bulky side-chain-containing amides. Furthermore, kinetic resolution of racemic DMCPCA by the amidase afforded S-DMCPCA in 46.3% yield and 99% ee with an average E-value of 67. These unique properties of the amidase imply that it is a promising biocatalyst for the production of chiral amides and carboxylic acids.

Preparation of trehalase inhibitor validoxylamine A by biocatalyzed hydrolysis of validamycin A with honeybee (Apis cerana Fabr.) beta-glucosidase.

Validoxylamine A is structurally similar to trehalose and acts a potent competitive inhibitor of trehalase. It has recently been receiving increased attention as a potential material for the development of new insecticides or drugs. In this study, beta-glucosidase extracted from honeybees (Apis cerana Fabr.) was used as a catalyst to produce validoxylamine A through enzymatic hydrolysis of validamycin A. Beta-glucosidase was separated and purified from honeybees, and its characteristics were examined. The results showed that beta-glucosidase was stable across a range of temperatures from 30 to 40 degrees C and across a relatively wide range of pH values from 5.0 to 7.5. Investigation of the biocatalyzed hydrolysis process from validamycin A to validoxylamine A with beta-glucosidase revealed that both the substrate (validamycin A) and the product (validoxylamine A) inhibited beta-glucosidase activity. The inhibition constant of the substrate Kis value was 5.01 mM, and that of the product Kip value was 1.32 mM. This product inhibition was competitive.