A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression

A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol?min^?1?mg^?1, and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.     

Purification and characterization of acetophenone reductase with excellent enantioselectivity from Geotrichum candidum NBRC 4597

NADH-dependent enzyme reducing acetophenone derivatives with high stereoselectivities and wide substrate specificities from Geotrichum candidum NBRC 4597 was isolated, purified, characterized, and used for asymmetric synthesis. Through five-step purification including ammonium sulfate fractionation and a series of chromatographies, the enzyme was purified about 150-fold with a yield of 5.6%. The active enzyme has a molecular mass of 73 kDa determined by gel filtration chromatography, and the SDS-PAGE result reveals that the molecular size of the subunit is 36 kDa. These results indicate that the enzyme consists of a homodimer of a 36 kDa subunit. The acetophenone reductase exhibited the highest activity at 50°C and optimal pH at 5.5. The enzyme was the most stable at 40°C. No metal ions considerably activated the enzyme, and such metal ions as Cu²⁺, Cd²⁺, and Zn²⁺ strongly inhibited the activity of the enzyme. The V _max and the apparent K _m value of the reductase were 77.0 μmol/min per milligram of protein and 0.296 mM for acetophenone, respectively. The N-terminal and internal amino acid sequences were determined by peptide sequencer. Furthermore, the purified enzyme was used for asymmetric reduction of acetophenone, resulting in the formation of corresponding (S)-alcohol with 99% ee.     

Efficient biocatalytic synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol by a newly isolated Trichoderma asperellum ZJPH0810 using dual cosubstrate: ethanol and glycerol

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol is a crucial intermediate for the synthesis of Aprepitant. An efficient biocatalytic process for (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol was developed via the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone, catalyzed by whole cells of newly isolated Trichoderma asperellum ZJPH0810 using ethanol and glycerol as dual cosubstrate for cofactor recycling. A fungal strain ZJPH0810, showing asymmetric biocatalytic activity of 3,5-bis(trifluoromethyl) acetophenone to its corresponding (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, was isolated from a soil sample. Based on its morphological and physiological characteristics and internal transcribed spacer sequence, this isolate was identified as T. asperellum ZJPH0810, which afforded an NADH-dependent (R)-stereospecific carbonyl reductase and was a promising biocatalyst for the synthesis of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol. Some key reaction parameters involved in the bioreduction catalyzed by T. asperellum ZJPH0810 were subsequently optimized. The effectiveness of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol production was significantly enhanced by employing a novel dual cosubstrate-coupled system for cofactor recycling. The established efficient bioreduction system contained 50 mM of 3,5-bis(trifluoromethyl) acetophenone and 60 g l^?1 of resting cells, employing ethanol (6.0 %, v/v) and glycerol (0.5 %, v/v) as dual cosubstrate. The bioreduction was performed in distilled water medium, at 30 °C and 200 rpm. Under the above conditions, a best yield of 93.4 % was obtained, which is nearly a 3.5-fold increase in contrast to no addition of cosubstrate. The ee value of the product reached above 98 %. This biocatalytic process shows great potential in the production of (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol, a valuable chiral building block in the pharmaceutical industry.     

Cloning,expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) is a valuable chiral intermediate for the synthesis of antiemetic drug Aprepitant and Fosaprepitant. A Leifsonia xyli HS0904-derived carbonyl reductase (LXCAR), an effective biocatalyst for the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to (R)-BTPE, was overexpressed in Escherichia coli BL21 (DE3). Bioinformatics analysis indicated that the amino acid sequence of recombinant LXCAR showed 89 % similarity to short-chain dehydrogenase/reductase. E. coli recombinant carbonyl reductase crude extract showed a specific activity of 1.54 U/mg, which was 62 times higher than that of L. xyli HS0904 crude extract. By using error-prone polymerase chain reaction and site-directed mutagenesis, the engineered LXCAR demonstrated superior catalytic activity toward BTAP, and the obtained mutant LXCAR-S154Y exhibited nearly 13-fold, 5.4-fold, and 2.3-fold increase in k _cat/K _m value, k _cat value, and specific activity toward BTAP, respectively, compared to the recombinant LXCAR. Additionally, the reduction of BTAP by whole cells of mutant LXCAR-S154Y afforded a best yield of 99.6 % for (R)-BTPE within 2 h at 200 mM BTAP, which was shortened by 28 and 2 h compared to those catalyzed by L. xyli HS0904 cells and recombinant E. coli cells expressing LXCAR, respectively. Moreover, a yield of 82.5 % for (R)-BTPE was achieved within 12 h at an increased BTAP concentration of up to 1,000 mM (256 g/l), representing a 1.9-fold increase over the recombinant LXCAR. Homology modeling and docking analysis revealed the molecular basis for the high catalytic activity of mutant LXCAR-S154Y toward BTAP. The results present here provide a promising alternative for economical and efficient production of chiral alcohols by engineered LXCAR.     

Molecular cloning, characterization, and heterologous expression of a new κ-carrageenase gene from marine bacterium Zobellia sp. ZM-2

κ-Carrageenases exhibit apparent distinctions in gene sequence, molecular weight, enzyme properties, and posttranslational processes. In this study, a new κ-carrageenase gene named cgkZ was cloned from the marine bacterium Zobellia sp. ZM-2. The gene comprised an open reading frame of 1,638 bp and encoded 545 amino acids. The natural signal peptide of κ-carrageenase was used successfully for the secretory production of the recombinant enzyme in Escherichia coli. A posttranslational process that removes an amino acid sequence of about 20 kDa from the C-terminal end of κ-carrageenase was first discovered in E. coli. An increase in enzyme activity by 167.3 % in the presence of 5 mM DTT was discovered, and Na⁺ at a certain concentration range was positively correlated with enzyme activity. The κ-carrageenase production of E. coli was 9.0 times higher than that of ZM-2. These results indicate the potential use of the enzyme in the biotechnological industry.     

Cloning,purification and characterization of novel Cu-containing nitrite reductase from the <Emphasis Type="Italic">Bacillus firmus</Emphasis> GY-49

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K _m and K _cat of NirK on sodium nitrite were 0.27 mM and 0.36?×?10³ s^?1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.     

Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol

Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K _m values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.     

Differential sensitivities of the growth of Escherichia coli to acrylate under aerobic and anaerobic conditions and its effect on product formation

The effect of acrylate on the growth of Escherichia coli was determined under aerobic and anaerobic conditions in glucose-defined medium. Growth occurred with up to 35 mM acrylate under aerobic conditions but ceased at 5 mM acrylate under anaerobic conditions. This differential sensitivity can be attributed to inhibition of pyruvate formate lyase and/or pflB gene repression, as this enzyme is necessary for anaerobic growth of E. coli. The effect of acrylate on end-product distribution was also determined by growing E. coli first aerobically, then switching to anaerobic conditions. In the absence of acrylate, E. coli generated the typical distribution of mixed-acid products, with about 12 % of pyruvate being metabolically converted to lactate. In contrast, in the presence of 5 mM acrylate, E. coli converted 83 % of pyruvate to lactate, consistent with a reduction in pyruvate formate lyase activity.     

Expression, purification and characterization of flavin reductase from Citrobacter freundii A1

Flavin reductase plays an important biological role in catalyzing the reduction of flavin by NAD(P)H oxidation. The gene that codes for flavin reductase from Citrobacter freundii A1 was cloned and expressed in Escherichia coli BL21(DE3)pLysS. In this study, we aimed to characterize the purified recombinant flavin reductase of C. freundii A1. The recombinant enzyme was purified to homogeneity and the biochemical profiles, including the effect of pH, temperature, metal ions and anions on flavin reductase activity and stability, were determined. This enzyme exhibited optimum activity at 45 °C in a 10-min reaction at pH 7.5 and was stable at temperatures up to 30 °C. At 0.1 mM concentration of metal ions, flavin reductase activity was stimulated by divalent cations including Mn²⁺, Sr²⁺, Ni²⁺, Sn²⁺, Ba²⁺, Co²⁺, Mg²⁺, Ca²⁺ and Pb²⁺. Ag⁺ was noticeably the strongest inhibitor of recombinant flavin reductase of C. freundii A1. This enzyme should not be defined as a standard flavoprotein. This is the first attempt to characterize flavin reductase of C. freundii origin.     

A novel surfactant-, NaCl-, and protease-tolerant β-mannanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp. HJ14

A glycoside hydrolase family 5 β-mannanase-encoding gene was cloned from Bacillus sp. HJ14 isolated from saline soil in Heijing town. Coding sequence of mature protein (without the predicted signal peptide from M1 to A30) was successfully expressed in Escherichia coli BL21 (DE3). Purified recombinant mannanase (rMan5HJ14) exhibited optimal activity at pH 6.5 and 65 °C. The enzyme showed good salt tolerance, retaining more than 56 % β-mannanase activity at 3.0–30.0 % (w/v) NaCl and more than 94 % of the initial activity after incubation with 3.0–30.0 % (w/v) NaCl at 37 °C for 60 min. Almost no mannanase activity was lost after incubation of rMan5HJ14 with trypsin, proteinase K, and Alcalase at 37 °C for 60 min. Surfactants and chelating agents, namely SDS, CTAB, Tween 80, Triton X-100, EDTA, and sodium tripolyphosphate, showed little or no effect (retaining >82.4 % activity) on enzymatic activity. Liquid detergents, namely Tupperware, Walch, Bluemoon, Tide, and OMO, also showed little or no effect (retaining >72.4 % activity) on enzymatic activity at 0.5–2.0 % (v/v). The enzyme further presents a high proportion (11.97 %) of acidic amino acid residues (D and E), which may affect the SDS and NaCl tolerance of the enzyme. Together, the mannanase may be an alternative for potential use in liquid detergent industry.     

Simple enzymatic procedure for preparation of 15N-labeled l-glutamic acid

The application of a number of immobilized Procion dyes to the purification of inosine 5′-monophosphate dehydrogenase (EC 1.2.1.14) from Escherichia coli has been investigated. The enzyme is adsorbed to a number of these immobilized dyes and can be eluted by salt gradients with very substantial increases in specific activity. For example, adsorption of the enzyme from a crude cell-free extract of E. coli to immobilized Procion yellow MX-8G in the presence of 15% ($\text{v}\text{v}$) ethylene glycol and subsequent elution with a salt gradient yields an enzyme preparation approximately 90% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is quantitatively recovered with an overall increase in specific activity of 14-fold compared to the enzyme in the cell-free extract.     

Efficient synthesis of (R)-3-hydroxypentanenitrile in high enantiomeric excess by enzymatic reduction of 3-oxopentanenitrile

(R)-3-Hydroxypentanenitrile (HPN) is an important intermediate in the synthesis of an immunosuppressive inosine 5′-monophosphate dehydrogenase inhibitor. An efficient enzymatic procedure for the synthesis of (R)-HPN with over 99 % enantiomeric excess using a novel acetoacetyl-CoA reductase (AdKR) from Achromobacter denitrificans was successfully established. Many microorganisms are known to reduce 3-oxopentannitrile (KPN) to (R)-HPN. An enzyme from A. denitrificans partially purified using ion exchange chromatography reduced KPN to (R)-HPN with high enantioselectivity. The AdKR gene was cloned and sequenced and found to comprise 738 bp and encode a polypeptide of 26,399 Da. The deduced amino acid sequence showed a high degree of similarity to those of other putative acetoacetyl-CoA reductases and putative 3-ketoacyl-ACP reductases. The AdKR gene was singly expressed and coexpressed together with a glucose dehydrogenase (GDH) as a coenzyme regenerator in Escherichia coli under the control of the lac promoter. (R)-HPN was synthesized with over 99 % e.e. using a cell-free extract of recombinant E. coli cells coexpressing AdKR and GDH.     

Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization

A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLac? cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn²⁺ and Mg²⁺. The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k _m?=0.227 mmol/L, v _max= 29.9 U/mg, and k _cat/k _m=5.44?×?10⁴ per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.     

Structural Characterization and Functional Validation of Aldose Reductase from the Resurrection Plant Xerophyta viscosa

Aldose reductases are key enzymes in the detoxification of reactive aldehyde compounds like methylglyoxal (MG) and malondialdehyde. The present study describes for first time the preliminary biochemical and structural characterization of the aldose reductase (ALDRXV4) enzyme from the resurrection plant Xerophyta viscosa. The ALDRXV4 cDNA was expressed in E. coli using pET28a expression vector, and the protein was purified using affinity chromatography. The recombinant protein showed a molecular mass of ~36 kDa. The K _M (1.2 mM) and k _cat (14.5 s^?1) of the protein determined using MG as substrate was found to be comparable with other reported homologs. Three-dimensional structure prediction based on homology modeling suggested several similarities with the other aldose reductases reported from plants. Circular dichroism spectroscopy results supported the bioinformatic prediction of alpha–beta helix nature of aldose reductase proteins. Subcellular localization studies revealed that the ALDRXV4-GFP fusion protein was localized both in the nucleus and the cytoplasm. The E. coli cells overexpressing ALDRXV4 exhibited improved growth and showed tolerance against diverse abiotic stresses induced by high salt (500 mM NaCl), osmoticum (10 % PEG 6000), heavy metal (20 mM CdCl₂), and MG (5 mM). Based on these results, we propose that ALDRXV4 gene from X. viscosa could be a potential candidate for developing stress-tolerant crop plants.     

The attractive recombinant phytase from Bacillus licheniformis: biochemical and molecular characterization

The phyL gene encoding phytase from the industrial strain Bacillus licheniformis ATCC 14580 (PhyL) was cloned, sequenced, and overexpressed in Escherichia coli. Biochemical characterization demonstrated that the recombinant enzyme has an apparent molecular weight of nearly 42 kDa. Interestingly, this enzyme was optimally active at 70–75 °C and pH 6.5–7.0. This enzyme is distinguishable by the fact that it preserved more than 40 % of its activity at wide range of temperatures from 4 to 85 °C. This new phytase displayed also a high specific activity of 316 U/mg. For its maximal activity and thermostability, this biocatalyst required only 0.6 mM of Ca²⁺ ion and exhibited high catalytic efficiency of 8.3 s^?1 μM^?1 towards phytic acid.     

Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.     

Expression,purification, and characterization of recombinant mangrove glutamine synthetase

To expand our knowledge about the relationship of nitrogen use efficiency and glutamine synthetase (GS) activity in the mangrove plant, a cytosolic GS gene from Avicennia marina has been heterologously expressed in and purified from Escherichia coli. Synthesis of the mangrove GS enzyme in E. coli was demonstrated by functional genetic complementation of a GS deficient mutant. The subunit molecular mass of GSI was ~40 kDa. Optimal conditions for biosynthetic activity were found to be 35 °C at pH 7.5. The Mg²⁺-dependent biosynthetic activity was strongly inhibited by Ni²⁺, Zn²⁺, and Al³⁺, whereas was enhanced by Co²⁺. The apparent K _m values of AmGLN1 for the substrates in the biosynthetic assay were 3.15 mM for glutamate, and 2.54 mM for ATP, 2.80 mM for NH₄ ⁺ respectively. The low affinity kinetics of AmGLN1 apparently participates in glutamine synthesis under the ammonium excess conditions.     

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 Ni²⁺, Ba²⁺ and Fe²⁺. 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.     

Characterization of a serine hydroxymethyltransferase for l-serine enzymatic production from Pseudomonas plecoglossicida

Pseudomonas plecoglossicida, a bacterium strain that exhibits high Serine hydroxymethyltransferase (SHMT) activity, was isolated from the seawater. A full-length glyA encoding SHMT was obtained by a modified thermal asymmetric interlaced-PCR (TRIL-PCR), which consisted of 1,254 bp, encoded a 417 amino acid polypeptide, and shared the highest identity (75 %) with a glyA gene from Acinetobacter radioresistens CMC-1. Recombinant glyA gene was expressed in Escherichia coli BL21 (DE3) and purified by electrophoretic homogeneity. The enzyme showed the optimal activity at pH 8.0 and 40 °C, and remained stable in high alkali conditions. Using SHMT to produce l-serine by catalyzing the reaction of glycine and tetrahydrofolate is one of the most promising routes to synthesize l-serine, achieving 33.4 mM l-serine at the 12th h of the enzymatic reaction with the substrates of glycine (133 mM) and formaldehyde (13.3 mM). The properties make the SHMT a candidate for further enzymatic studies and industrial applications.     

Production of chiral compound using recombinant Escherichia coli cells co-expressing reductase and glucose dehydrogenase in an ionic liquid/water two phase system

(S)-3-Chloro-1-phenyl-1-propanol ((S)-CPPO) is a useful chiral building block for the synthesis of anti-depressant drugs. The yeast reductase, YOL151W, evidences enantioselective reduction activity, converting 3-chloro-1-phenyl-1-propanone (3-CPP) into (S)-CPPO. Escherichia coli whole cells co-expressing YOL151W and Bacillus subtilis glucose dehydrogenase were employed for the synthesis of CPPO following permeabilization treatment. A reaction system employing these recombinant E. coli whole cells could convert 30 mM 3-CPP enantioselectively into (S)-CPPO. In an effort to enhance substrate solubility and to prevent substrate/product inhibition during the enzyme reaction process, a variety of ionic liquids were tested and [Bmim][NTf₂] was ultimately selected for the ionic liquid/water two phase system. Tween 40 was added to accomplish the efficient mixing of the two phases. Using these recombinant E. coli whole cells and the [Bmim][NTf₂]/water two phase system, 100 mM (S)-CPPO was generated with an enantiomeric excess of >99%.