Characterization and constitutive expression of an acidic mesophilic endo-1,4-β-d-xylanohydrolase with high thermotolerance and catalytic efficiency in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl11 from Aspergillus niger, encoding a 188-residue xylanase of glycosyl hydrolase family 11, was constitutively expressed in Pichia pastoris. The recombinant Xyl11 exhibited optimal activity at pH 5.0 and 50 °C, and displayed more than 68 % of the maximum activity over the temperature range 35–65 °C and 33 % over the pH range 2.2–7.0. It maintained more than 40 % of the original activity after incubation at 90 °C (pH 5.0) for 10 min and more than 75 % of the original activity after incubation at pH 2.2–11.0 (room temperature) for 2 h. The specific activity, K _m and V _max of purified Xyl11 were 22,253 U mg^?1, 6.57 mg ml^?1 and 51,546.4 μmol min^?1 mg^?1. It could degrade xylan to a series of xylooligosaccharides and no xylose was detected. The recombinant enzyme with high stability and catalytic efficiency could work over wide ranges of pH and temperature and thus has the potential for various industrial applications.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Xylan-hydrolyzing thermotolerant <Emphasis Type="Italic">Candida tropicalis</Emphasis> HNMA-1 for bioethanol production from sugarcane bagasse hydrolysate

Sugarcane bagasse is one of the low-cost substrates used for bioethanol production. In order to solubilize sugars in hemicelluloses like xylan, a new thermotolerant isolate of Candida tropicalis HNMA-1 with xylan-hydrolyzing ability was identified and characterized. The strain showed relative tolerance to high temperature. Our results demonstrated 0.211 IU ml^?1 xylanase activity at 40 °C compared to 0.236 IU ml^?1 at 30 °C. The effect of high temperature on the growth and fermentation of xylose and sugarcane bagasse hydrolysate were also investigated. In both xylose or hydrolysate medium, increased growth was recorded at 40 °C. Meanwhile, the efficiency of ethanol fermentation was adversely affected by temperature since yields of 0.088 g g^?1 and 0.076 g g^?1 in the xylose medium, in addition to 0.090 g g^?1 and 0.078 g g^?1 in the hydrolysate medium were noticed at 30 °C and 40 °C, respectively. Inhibitory compounds in the hydrolysate medium demonstrated negative effects on fermentation and productivity, with maximum ethanol concentration attained after 48 h in the hydrolysate, as opposed to 24 h in the xylose medium. Our data show that the newly thermotolerant isolate, C. tropicalis HNMA-1, is able to efficiently ferment xylose and hydrolysate, and also has the capacity for application in ethanol production from hemicellulosic sources.     

Characteristics of thermostable endoxylanase and β-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans TSAA1 and its applicability in generating xylooligosaccharides and xylose from agro-residues

An extremely thermophilic bacterial isolate that produces a high titer of thermostable endoxylanase and β-xylosidase extracellularly in an inducible manner was identified as Geobacillus thermodenitrificans TSAA1. The distinctive features of this strain are alkalitolerance and halotolerance. The endoxylanase is active over a broad range of pH (5.0–10.0) and temperatures (30–100 °C) with optima at pH 7.5 and 70 °C, while β-xylosidase is optimally active at pH 7.0 and 60 °C. The T _1/2 values of the endoxylanase and β-xylosidase are 30 min at 80 °C, and 180 min at 70 °C, respectively. The endoxylanase activity is stimulated by dithiothreitol, but inhibited strongly by EDAC and Woodward’s reagent K. N-BS and DEPC strongly inhibited β-xylosidase. MALDI-ToF (MS/MS) analysis of tryptic digest of β-xylosidase revealed similarity with that of G. thermodenitrificans NG 80-2, and suggested that this belongs to the GH 52 glycosyl hydrolase super family. The action of endoxylanase on birch wood xylan and agro-residues such as wheat bran and wheat straw liberated xylooligosaccharides similar to endoxylanases of the family 10 glycoside hydrolases, while the enzyme preparation having both endoxylanase and β-xylosidase liberated xylose as main hydrolysis product.     

Themoanaerobacterium calidifontis sp. nov., a novel anaerobic, thermophilic, ethanol-producing bacterium from hot springs in China

A novel thermophilic Gram staining positive strain Rx1 was isolated from hot springs in Baoshan of Yunnan Province, China. The strain was characterized as a hemicellulose-decomposing obligate anaerobe bacterium that is rod-shaped (diameter: 0.5–0.7 μm; length: 2.0–6.7 μm), spore-forming, and motile. Its growth temperature range is 38–68 °C (optimum 50–55 °C) and pH range is 4.5–8.0 (optimum 7.0). The maximum tolerance concentration of NaCl was 3 %. Rx1 converted thiosulfate to elemental sulfur and reduced sulfite to hydrogen sulfide. The bacterium grew by utilizing xylan and starch, as well as a wide range of monosaccharide and polysaccharides, including glucose and xylose. The main products of fermentation were ethanol, lactate, acetate, CO₂, and H₂. The maximum xylanase activity in the culture supernatant after 30 h of incubation at 55 °C was 16.2 U/ml. Rx1 DNA G + C content was 36 mol %. 16S rRNA gene sequence analysis indicated that strain Rx1 belonged to the genus Thermoanaerobacterium of the family ‘Thermoanaerobacteriaceae’ (Firmicutes), with Thermoanaerobacterium aciditolerans 761–119 (99.2 % 16S rRNA gene sequence similarity) being its closest relative. DNA–DNA hybridization between Rx1 and T. aciditolerans 761–119 showed 36 % relatedness. Based on its physiological and biochemical tests and DNA–DNA hybridization analyses, the isolate is considered to represent a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium calidifontis sp. nov. is proposed, with the type strain is Rx1 (=JCM 18270 = CCTCC M 2011109).     

Purification and characterization of a novel thermo-active amidase from Geobacillus subterraneus RL-2a

A thermostable amidase produced by Geobacillus subterraneus RL-2a was purified to homogeneity, with a yield of 9.54 % and a specific activity of 48.66 U mg^?1. The molecular weight of the native enzyme was estimated to be 111 kDa. The amidase of G. subterraneus RL-2a is constitutive in nature, active at a broad range of pH (4.5–11.5) and temperature (40–90 °C) and has a half-life of 5 h and 54 min at 70 °C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co²⁺, Hg²⁺, Cu²⁺, Ni²⁺, and thiol reagents. The presence of mid-chain aliphatic and amino acid amides enhances the enzymatic activity. The acyl transferase activity was detected with propionamide, butyramide and nicotinamide. The enzyme showed moderate stability toward toluene, carbon tetrachloride, benzene, ethylene glycol except acetone, ethanol, butanol, propanol and dimethyl sulfoxide. The K _m and V _max of the purified amidase with nicotinamide were 6.02 ± 0.56 mM and 132.6 ± 4.4 μmol min^?1 mg^?1 protein by analyzing Michaelis–Menten kinetics. The results of MALDI-TOF analysis indicated that this amidase has homology with the amidase of Geobacillus sp. C56-T3 (gi|297530427). It is the first reported wide-spectrum thermostable amidase from a thermophilic G. subterraneus.     

A novel isolate of <Emphasis Type="Italic">Clostridium butyricum</Emphasis> for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ _m ) and the specific butyric acid yield were 0.1466 h^?1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.     

Description of a new anaerobic thermophilic bacterium,Thermoanaerobacterium butyriciformans sp. nov.

Strain USBA-019^T, an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019^T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4 × 3.0–7.0 μm. Optimal growth occurs at 50–55 °C (35–65 °C). Optimum pH is 5.0–5.5 (4.0–8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019^T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019^T, Th. thermostercoris DSM22141^T and Th. aotearoense DMS10170^T found DNA–DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019^T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019^T (=CMPUJ U-019^T = DSM 101588^T).     

Characterization and constitutive expression of a novel endo-1,4-β-d-xylanohydrolase from Aspergillus niger in Pichia pastoris

A putative endo-1,4-β-d-xylanohydrolase gene xyl10 from Aspergillus niger, encoding a 308-residue mature xylanase belonging to glycosyl hydrolase family 10, was constitutively expressed in Pichia pastoris. The recombinant Xyl10 exhibited optimal activity at pH 5.0 and 60 °C with more than 50 % of the maximum activity from 40 to 70 °C. It retained more than 90 % of the original activity after incubation at 60 °C (pH 5.0) for 30 min and more than 74 % after incubation at pH 3.0–13.0 for 2 h (25 °C). The specific activity, K _m and V _max values for purified Xyl10 were, respectively, 3.2 × 10³ U mg^?1, 3.6 mg ml^?1 and 5.4 × 10³ μmol min^?1 mg^?1 towards beechwood xylan. The enzyme degraded xylan to a series of xylooligosaccharides and xylose. The recombinant enzyme with these properties has the potential for various industrial applications.     

Paenibacillus thermophilus sp. nov., a novel bacterium isolated from a sediment of hot spring in Fujian province, China

A Gram-positive, thermophilic, strictly aerobic bacterium, designated WP-1^T, was isolated from a sediment sample from a hot spring in Fujian province of China and subjected to a polyphasic taxonomic study. Cells of strain WP-1^T were rods (~0.6–0.8 × 2.5–3.5 μm) and motile by means of peritrichous flagella. Endospores were ellipsoidal in terminal or subterminal positions. Strain WP-1^T grew at 37–60 °C (optimum 42–45 °C), 0–3 % NaCl (optimum 1 %, w/v) and pH 3.0–9.0 (optimum pH 6.5–7.0). The predominant menaquinone was MK-7. The major fatty acids were anteiso-C_15:0, iso-C_16:0, C_16:0 and anteiso-C_17:0. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, two glycolipids, two unidentified phospholipids and two unknown polar lipids. The cell-wall peptidoglycan contained meso-diaminopimelic acid (meso-DAP). The G + C content of the genomic DNA was 52.5 %. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain WP-1^T is a member of the genus Paenibacillus and exhibited sequence similarity of 99.3 % to Paenibacillus macerans DSM 24^T and both strains represented a separate lineage from all other Paenibacillus species. However, the level of DNA–DNA relatedness between strain WP-1^T and P. macerans DSM 24^T was 34.0 ± 4.7 %. On the basis of phylogenetic, physiological and chemotaxonomic analysis data, strain WP-1^T is considered to represent as a novel species of the genus Paenibacillus, for which the name Paenibacillus thermophilus sp. nov., is proposed, with the type strain WP-1^T (=DSM 24746^T = JCM 17693^T = CCTCC AB 2011115^T).     

Paludibacter jiangxiensis sp. nov., a strictly anaerobic,propionate-producing bacterium isolated from rice paddy field

A mesophilic, obligately anaerobic, propionate-producing fermentative bacterium, designated strain NM7^T, was isolated from rural rice paddy field. Cells of strain NM7^T are Gram-negative, non-motile, non-spore-forming, short rods, and negative for catalase. The strain grew optimally at 37 °C (the range for growth 15–40 °C) and pH 7.0 (pH 5.0–7.5). The strain could grow fermentatively on various sugars, including arabinose, xylose, fructose, galactose, glucose, mannose, cellobiose, lactose, maltose, sucrose, pectin and starch. The main end products of glucose fermentation were acetate and propionate. Yeast extract was not required but stimulated the growth. Nitrate, sulfate, thiosulfate, elemental sulfur, sulfite, and Fe(III) nitrilotriacetate were not used as terminal electron acceptors. The G+C content of genomic DNA was 42.8 mol%. The major cellular fatty acids were C_15:0, anteiso-C_15:0, C_16:0, and C_17:0. The most abundant polar lipid of strain NM7^T was phosphatidylethanolamine. 16S rRNA gene sequence analysis revealed that it belongs to the family Porphyromonadaceae of the phylum Bacteroidetes. The closest recognized species was Paludibacter propionicigenes (91.4 % similarity in 16S rRNA gene sequence). A novel species, Paludibacter jiangxiensis sp. nov., is proposed to accommodate strain NM7^T (=JCM 17480^T = CGMCC 1.5150^T = KCTC 5844^T).     

Desulfotomaculum tongense sp. nov., a moderately thermophilic sulfate-reducing bacterium isolated from a hydrothermal vent sediment collected from the Tofua Arc in the Tonga Trench

A novel, strictly anaerobic, moderately thermophilic, endospore-forming, sulfate-reducing bacterium, designated TGB60-1^T, was isolated from a hydrothermal sediment vent collected from the Tofua Arc in the Tonga Trench. The strain was characterized phenotypically and phylogenetically. The isolated strain was observed to be Gram-positive, with slightly curved rod-shaped cells and a polar flagellum. Strain TGB60-1^T was found to grow anaerobically at 37–60 °C (optimum, 50 °C), at pH 6.0–8.5 (optimum, pH 7.0) and with 1.0–4.0 % (w/v) NaCl (optimum, 3.0 %). The electron acceptors utilised were determined to be sulfate, sulfite, and thiosulfate. Strain TGB60-1^T was found to utilise pyruvate and H₂ as electron donors. Strain TGB60-1^T was determined to be related to representatives of the genus Desulfotomaculum and the closest relatives within this genus were identified as Desulfotomaculum halophilum SEBR 3139^T, Desulfotomaculum alkaliphilum S1^T and Desulfotomaculum peckii LINDBHT1^T (92.7, 92.1, and 91.8 % 16S rRNA gene sequence similarity, respectively). The major fatty acids (>20 %) were identified as C_16:0 and C_18:1 ω7c. The G+C content of the genomic DNA of this novel bacterium was determined to be 53.9 mol%. Based on this polyphasic taxonomic study, strain TGB60-1^T is considered to represent a novel species in the genus Desulfotomaculum, for which the name Desulfotomaculum tongense sp. nov. is proposed. The type strain of D. tongense is strain TGB60-1^T (= KTCT 4534^T = JCM 18733^T).     

Cloning,over-expression and characterization of a thermo-tolerant xylanase from Thermotoga thermarum

The xyn10B gene, encoding the endo-1,4-β-xylanase Xyn10B from Thermotoga thermarum, was cloned and expressed in Escherichia coli. The ORF of the xyn10B was 1,095 bp and encoded to mature peptide of 344 amino acids with a calculated MW of 40,531 Da. The recombinant xylanase was optimally active at 80 °C, pH 6.0 and retained approx. 60 % of its activity after 2 h at 75 °C. Apparent K _m , k _cat and k _cat /K _m values of the xylanase for beechwood xylan were 1.8 mg ml^?1, 520 s^?1 and 289 ml mg^?1 s^?1, respectively. The end products of the hydrolysis of beechwood xylan were mainly oligosaccharides but without xylose after 2 h hydrolysis.     

Xylose Transport by the Anaerobic Thermophile Thermoanaerobacter ethanolicus and the Characterization of a D-Xylose-Binding Protein

Thermoanaerobacter ethanolicus is a xylose-utilizing thermophilic anaerobe that produces considerable amounts of ethanol. A protein in xylose-growing cells was solubilized from cell membranes by extraction with octyl-β-glucoside. Internal peptide sequencing revealed that the protein was the product of a gene, xylF, encoding a putative D-xylose-binding protein. Metabolic labeling with ¹⁴C palmitic acid suggested that this is a lipoprotein that is anchored to the cell membrane via a cysteine residue. Binding was highly specific for xylose as evident by the lack of competition by sugars with structures similar to xylose. The apparent K _d of the protein for xylose was approximately 1.5 μM, and this value was very similar to the affinity constant determined for xylose transport by whole cells at low substrate concentrations. Uptake experiments with cells also suggested the presence of a separate low-affinity system. Binding activity varied less than 20% over a pH range of 4–8, and the level of activity was virtually unaffected when temperature was varied between 40°C and 80°C. This is the first biochemical characterization of a D-xylose-binding protein from a thermophilic organism. Received: 22 April 1998 / Accepted: 21 May 1998     

Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1

The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0–12.0 with T _1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol^?1. The K _m, V _max and k _cat (birchwood xylan) are 2.05 mg ml^?1, 333.33 μmol mg^?1 min^?1 and 3.33 × 10⁴ min^?1, respectively. The pKa₁ and pKa₂ of ionizable groups of the active site that influence V _max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.     

Geodermatophilus saharensis sp. nov., isolated from sand of the Saharan desert in Chad

A novel Gram-positive, aerobic, actinobacterial strain, CF5/5, was isolated from soil in the Sahara desert, Chad. It grew best at 20–35 °C and at pH 6.0–8.0 and with 0–4 % (w/v) NaCl, forming black-colored colonies. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G + C content was 75.9 mol%. The peptidoglycan contained meso-diaminopimelic acid; galactose and xylose were detected as diagnostic sugars. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, and phosphatidylinositol; MK-9(H₄) was the dominant menaquinone. The major cellular fatty acids were: iso-C_16:0 and iso-C_15:0. The 16S rRNA gene showed 95.6–98.3 % sequence similarity with the other named members of the genus Geodermatophilus. Based on the polyphasic taxonomy data, the isolate is proposed to represent a novel species, Geodermatophilus saharensis with the type strain CF5/5^T = DSM 45423 = CCUG 62813 = MTCC 11416.     

GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509

A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K _m and k _cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s^?1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K _m and k _cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s^?1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K _m and k _cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s^?1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.     

Characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with high space-time yield

A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg^?1. The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K _m and V _max values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min^?1 mg^?1, respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous–organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3 % and e.e. of 99 % was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP⁺ to (S)-CHBE were 26.5 mmol L^?1 h^?1 g^?1 DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.