Single-Step Purification and Characterization of Recombinant Aspartase of Aeromonas media NFB-5

Aspartase (L-aspartate ammonia-lyase; EC 4.3.1.1) catalyzes the reversible amination of fumaric acid to produce L-aspartic acid. Aspartase coding gene (aspA) of Aeromonas media NFB-5 was cloned, sequenced, and expressed with His tag using pET-21b(+) expression vector in Escherichia coli BL21. Higher expression was obtained with IPTG (1.5?mM) induction for 5?h at 37?°C in LB medium supplemented with 0.3% K₂HPO₄ and 0.3% KH₂PO₄. Recombinant His tagged aspartase was purified using Ni?CNTA affinity chromatography and characterized for various biochemical and kinetic parameters. The purified aspartase showed optimal activity at pH?8.5 and 8.0 in the presence and absence of magnesium ions, respectively. The optimum temperature was determined to be 35?°C. The enzyme showed apparent K _m and V _max values for L-aspartate as 2.01?mM and 114?U/mg, respectively. The enzyme was stable in pH range of 6.5?C9.5 and temperature up to 45?°C. Divalent metal ion requirement of enzyme was efficiently fulfilled by Mg²⁺, Mn²⁺, and Ca²⁺ ions. The cloned gene (aspA) product showed molecular weight of approximately 51?kDa by SDS-PAGE, which is in agreement with the molecular weight calculated from putative amino acid sequence. This is the first report on expression and characterization of recombinant aspartase from A. media.     

l-myo-Inositol-1-Phosphate Synthase Expressed in Developing Organ: Isolation and Characterisation of the Enzyme from Human Fetal Liver

l-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) activity has been detected and partially purified for the first time from human fetal liver. Crude homogenate from the fetal liver was subjected to streptomycin sulphate precipitation and 0?C60?% ammonium sulphate fractionation followed by successive chromatography through DEAE cellulose and BioGel A 0.5-m columns. After the final chromatography, the enzyme was purified 51-fold and 3.46?% of MIPS could be recovered. The human fetal liver MIPS specifically utilised d-glucose-6-phosphte and NAD⁺ as its substrate and coenzyme, respectively. It shows pH optima between 7.0 and 7.5 while the temperature maximum was at 40?°C. The enzyme activity was remarkably stimulated by NH ₄ ⁺ , slightly stimulated by K⁺ and Ca²⁺ and highly inhibited by Zn²⁺, Cu²⁺ and Hg²⁺. The K _m values of MIPS for d-glucose-6-phosphate and NAD⁺ were found to be as 1.15 and 0.12?mM respectively while the V _max values were 280?nM and 252?nM for d-glucose-6-phosphate and NAD⁺ correspondingly. The apparent molecular weight of the native enzyme was determined to be 170?kDa.     

Biochemical Characterization and Antitumor Study of l-Glutaminase from Bacillus cereus MTCC 1305

l-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for l-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na⁺, K⁺) and phosphate ion activated the enzyme activity, while divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, Pb²⁺, Ca²⁺, Co²⁺, Hg²⁺, Cd²⁺, Cu²⁺) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, l-ascorbic acid, and β-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K _m, V _max, K _cat, of purified enzyme were found to be 6.25 mM, 100 μmol/min/mg protein and 2.22?×?10² M^?1s^?1, respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC₅₀ value of 82.27 μg/ml in the presence of different doses of l-glutaminase (10–100 μg/ml).     

Purification and Characterization of 3-Ketovalidoxylamine A C-N Lyase Produced by Stenotrophomonas maltrophilia

A soluble 3-ketovalidoxylamine A C-N lyase from Stenotrophomonas maltrophilia was purified to 367.5-fold from the crude enzyme, with a yield of 16.4% by column chromatography on High S IEX, Methyl HIC, High Q IEX, and Sephadex G 100. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the enzyme was a neutral protein having an isoelectric point value at pH?7.0. The optimal pH of 3-ketovalidoxylamine A C-N lyase was around 7.0. The enzyme was stable within a pH range of 7.0–10.5. The optimal temperature was found to be near 40?°C, and the enzyme was sensitive to heat. The enzyme was completely inhibited by ethylenediaminetetraacetic acid, and it was reversed by Ca²⁺. The product, p-nitroaniline, inhibited the enzyme activity significantly at low concentration. The enzyme has C-N lyase activity and C-O lyase activity, and need 3-keto groups. The apparent K _m value for p-nitrophenyl-3-ketovalidamine was 0.14 mM.     

Purification and Partial Characterization of Lignin Peroxidase from <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890 and Its Application in Decolorization of Textile Dyes

Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at pH 1.0, between a broad temperature range of 50 and 70°C, at H₂O₂ concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn²⁺, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes.     

Expression,Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina

Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)⁺ to NAD(P)H. The NADP⁺-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP⁺ as the cofactor. The K _m values for l-malate and NADP⁺ were 2.19?±?0.01 and 0.38?±?0.02 mM, respectively, while the V _max values were 147?±?2 and 302?±?14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn²⁺, Mg²⁺, Co²⁺, Ni²⁺, and low concentrations of Zn²⁺ rather than Ca²⁺, Cu²⁺, or high concentrations of Zn²⁺. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K _i values were 0.08 and 0.6 mM, respectively.     

Purification and Characterization of Agarase from Marine Bacteria <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>

Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52 U increased to 45.76 U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24 kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40 °C, respectively. The K_m and V_max values for agarase were 4.69 mg/ml and 0.5 μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5 mM concentration. The enzyme activity was stimulated by the presence of Fe²⁺, Mn²⁺, and Ca2⁺ ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30–40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.     

Purification and Characterization of the Glucoside 3-Dehydrogenase Produced by a Newly Isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251

A soluble glucoside 3-dehydrogenase (G3DH) was purified from a newly isolated Sphingobacterium faecium ZJF-D6 CCTCC M 2013251. The enzyme was purified to 35.71-fold with a yield of 41.91 % and was estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis with a molecular mass of 62 kDa. The sequences of two peptides of the enzyme were all contained in a GMC family oxidoreductase (EFK55866) by mass spectrometry analysis. The optimal pH of the enzyme was around 6.2. The enzyme was stable within a pH range of 5.0–6.6 and was sensitive to heat. G3DH from S. faecium exhibited extremely broad substrate specificity and well regioselectivity to validoxylamine A. The enzyme was completely inhibited by Hg₂Cl₂ and partly inhibited by Cu²⁺, Fe²⁺, Ca²⁺, and Cd²⁺. The apparent K _m values for D-glucose, sucrose, and validoxylamine were calculated to be 1.1, 1.7, and 2.1 mM, respectively. With this purified enzyme, 3-keto sucrose was prepared at pH 5.0, 30 °C for 10 h with a yield of 28.7 %.     

Purification and Characterization of a Zinc-Dependent Cinnamyl Alcohol Dehydrogenase from Leucaena leucocephala,a Tree Legume

A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13 % and specific activity of 812. 5 nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ～76 and ～38 kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75 μM^?1 s^?1) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH 8.8 and 40 °C, respectively. The enzyme activity was enhanced in the presence of 2.0 mM Mg²⁺, while Zn²⁺ at the same concentration exerted an inhibitory effect. The inclusion of 2.0 mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.     

A Feruloyl Esterase (FAE) Characterized by Relatively High Thermostability from the Edible Mushroom Russula virescens

A monomeric feruloyl esterase (FAE) with a molecular mass of 62 kDa was acquired from fresh fruiting bodies of the edible mushroom Russula virescens. The isolation procedure involved ion exchange chromatography on CM-cellulose, Q-Sepharose, and SP-Sepharose and finally fast protein liquid chromatography–gel filtration on Superdex 75. Two amino acid sequences were obtained after tryptic digestion, and they both showed some homology with the esterase of some fungi. Maximal activity was observed at pH 5.0 and at 50 °C. The enzyme displayed relatively high thermostability as evidenced by over 70 % residual activity at 70 °C and about 34 % residual activity at 80 °C. The K _m and V _max for this enzyme on methyl ferulate were 0.19 mM and 1.65 U/mg proteins, respectively. The purified FAE prefers methyl ferulate over methyl caffeate and is least active on methyl p-coumarate. The FAE activity was not significantly affected by the presence of cations such as Mn²⁺, Ca²⁺, Cd²⁺, Zn²⁺, Mg²⁺, Cu²⁺, and K⁺ ions but inhibited by Al³⁺, Hg²⁺, Fe²⁺, and Pb²⁺ ions at a tested concentration of 2. 5 mM.     

Production, Purification, and Characterization of a β-Glucosidase of Penicillium funiculosum NCL1

Penicillium funiculosum NCL1, a filamentous fungus, produced significantly higher levels of ??-glucosidase. The effect of initial pH, incubation temperature, and different carbon sources on extracellular ??-glucosidase production was studied in submerged fermentation. At 30?°C with initial pH 5.0, enzyme production was increased by 48-fold upon induction with paper mill waste, as compared to commercial cellulose powder. In zymogram analysis, four isoforms of ??-glucosidases were observed with wheat bran whereas a minimum of one isoform was observed with other carbon sources. A major ??-glucosidase (Bgl3A) with the apparent molecular weight of ~120?kDa, induced by paper mill waste, was purified 19-fold to homogeneity, with a specific activity of 1,796 U/mg. Bgl3A was a monomeric glycoprotein with 29% of neutral carbohydrate content. It showed optimum activity at pH 4.0 and 5.0, optimum temperature at 60?°C, and exhibited a half-life of 1?h at 60?°C. K _m of Bgl3A was found to be 0.057?mM with p-nitrophenyl ??-d-glucoside and V _max was 1,920 U/mg. The purified enzyme exhibited glucose tolerance with a K _i of 1.5?mM. Bgl3A readily hydrolyzed glucosides with ??-linkage. Bgl3A activity was enhanced (156%) by Zn²⁺ and was not affected by other metal cations and reagents. The supplementation of Bgl3A (5 U/mg) with Trichoderma reesei cellulase complex (5 FPU/mg) resulted in about 70% of enhanced glucose production, which emphasizes the industrial importance of Bgl3A.     

NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

NADP⁺-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg²⁺ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP⁺ and no NAD-dependent activity could be detected. The K _m values displayed for NADP⁺ and isocitrate were 59 and 31 μM (Mg²⁺), 120 μM and 58 μM (Mn²⁺), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K _m values for NADP⁺ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD⁺-dependent activity was detected for R322D mutant and the K _m and k _cat values for NAD⁺ were 47,000 μM and 0.38 s^?1, respectively. Although the R322D mutant showed low activity with NAD⁺, it revealed the feasibility of engineering an eukaryotic IDP to a NAD⁺-dependent one.     

Purification of Peroxidase from Red Cabbage (Brassica oleracea var. capitata f. rubra) by Affinity Chromatography

Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9 % from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K _m and V _max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC₅₀ and K _i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702?±?0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.     

Stability and Detergent Compatibility of a Predominantly β-Sheet Serine Protease from Halotolerant B. aquimaris VITP4 Strain

The present study deals with the characterization of halotolerant protease produced by Bacillus aquimaris VITP4 strain isolated from Kumta coast, Karnataka, India. The studies were performed at 40 °C and pH 8 in Tris buffer. Metal ions such as Mn²⁺ and Ca²⁺ increased the proteolytic activity of the enzyme by 34 and 30 %, respectively, at 10 mM concentration. Cu²⁺ at 1 mM concentration was found to enhance the enzyme activity by 16 %, whereas inhibition was observed at higher concentration (>5 mM). Slight inhibition was observed even with lower (>1 mM) concentrations of Zn²⁺, Hg²⁺, Fe³⁺, Ni²⁺, and Co²⁺.The activity of protease was completely inhibited by phenylmethylsulfonyl fluoride, indicating that the VITP4 protease is a serine protease. The presence of ethylenediaminetetraacetic acid and 1,10-phenanthroline (>5 mM) moderately inhibited the activity, suggesting that the enzyme is activated by metal ions. The protease was purified to homogeneity with a purification fold of 15.7 with ammonium sulfate precipitation and 46.65 with gel filtration chromatography using Sephadex G-100, resulting in a specific activity of 424?±?2.6 U mg^?1. The VITP4 protease consists of a single polypeptide chain with a molecular mass of 34.7 kDa as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization–time of flight. Among the different substrates used (casein, egg albumin, gelatin, and bovine serum albumin), the activity was higher with casein with V _max, K _m, and k _cat values of 0.817 mg ml min^?1, 0.472 mg ml^?1, and 2.31 s^?1, respectively. Circular dichroism studies revealed that the VITP4 protease has a predominantly β-sheet structure (51.6 %) with a temperature for half denaturation of 85.8 °C in the presence of 1 mM CaCl₂. Additionally, the VITP4 protease was found to retain more than 70 % activity in the presence of 10 mM concentration of different detergents (CTAB, urea, and sodium dodecyl sulfate) and surfactants (Triton X-100, Tween-20, and Tween-80), and the results of wash performance test with various commercial detergents confirmed that it can be used in detergent formulations.     

Purification and Properties of a Thermostable Xylanase GH 11 from Penicillium occitanis Pol6

An extracellular, endo-??-1,4-xylanase was purified to homogeneity from the culture filtrate of the filamentous fungus Penicillium occitanis Pol6, grown on oat spelt xylan. The purified enzyme (PoXyn2) showed a single band on SDS?CPAGE with an apparent molecular weight of 30?kDa. The xylanase activity was optimal at pH?3.0 and 65?°C. The specific activity measured for oat spelt xylan was 2,368?U?mg^?1. The apparent K _m and V _max values were 8.33?mg?ml^?1 and 58.82???mol?min^?1?ml^?1, respectively, as measured on oat spelt xylan. Thin-layer chromatography experiments revealed that purified PoXyn2 degrades xylan in an endo-fashion releasing xylobiose as main end product. The genomic DNA and cDNA encoding this protein were cloned and sequenced. This PoXyn2 presents an open reading frame of 962?bp, not interrupted by any introns and encoding for a mature protein of 320 amino acids and 29.88?kDa.     

Purification and Characterization of Alkaline Pectin Lyase from a Newly Isolated Bacillus clausii and Its Application in Elicitation of Plant Disease Resistance

Alkaline pectin lyase (PNL) shows potential as a biological control agent against several plant diseases. We isolated and characterized a new Bacillus clausii strain that can produce 4,180?U/g of PNL using sugar beet pulp as a carbon source and inducer. The PNL was purified to apparent homogeneity using ultrafiltration, ammonium sulfate fractionation, DEAE Sepharose Fast Flow, and Sephadex G-75 gel filtration. The purified PNL was found to be a monomeric protein with a molecular weight of 35?kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It demonstrated optimal activity with K _m of 0.87?mg/ml at pH?10.0 and 60?°C. The enzyme is stable in the pH range of 8.0?C10.0 and temperature ??40?°C. Ca²⁺ was found to stimulate the enzymatic activity of the PNL by up to 410?%. Mass spectrometric results gave 38?% match coverage with pectate lyase from B. clausii KSM-K16 (gi|56961845). The PNL was found to elicit disease resistance in cucumber seedlings, suggesting that it may have applications in biocontrol and sustainable agriculture.     

Gene Cloning,Heterologous Expression,and Characterization of a High Maltose-Producing α-Amylase of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4–6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5–6.5 and temperatures below 50 °C. Purified RoAmy had a K _m and V _max of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu²⁺ and 5 mM Fe²⁺, whereas 5 mM Ca²⁺ showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K _m = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.     

Purification,Gene Cloning,and Characterization of a Novel Halohydrin Dehalogenase from Agromyces mediolanus ZJB120203

A novel halohydrin dehalogenase (HHDH), catalyzing the transformation of 1,3-dichloro-2-propanol (1,3-DCP) to epichlorohydrin (ECH), was purified from Agromyces mediolanus ZJB120203. The molecular mass of the enzyme was estimated to be 28 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A 735-bp nucleotide fragment was obtained based on the N-terminal and internal amino acid sequences of the purified HHDH. The gene codes a protein sequence with 244 amino acid residues, and the protein sequence shows high similarity to Hhe A_AD2 (HHDH from Arthrobacter sp. AD2), defined as Hhe A_Am, which is the seventh reported HHDH. Expression of Hhe A_Am was carried out in Escherichia coli and purification was performed by nickel-affinity chromatography. The recombinant HheA_Am possessed an optimal pH of 8.5 and an optimal temperature of 50 °C and manifested a K _m of 4.58 mM and a V _max of 3.84 μmol/min^/mg. The activity of Hhe A_Am was not significantly affected by metal ions such as Zn²⁺, Ca²⁺, Cu²⁺, and EDTA, but was strongly inhibited by Hg²⁺ and Ag⁺. In particular, the Hhe A_Am exhibits an enantioselectivity for the conversion of prochiral 1,3-DCP to (S)-ECH. The applications of the Hhe A_Am as a catalyst for asymmetric synthesis are promising.     

Cloning, Expression, and Characterization of a Novel Methylglyoxal Synthase from Thermus sp. Strain GH5

A gene encoding methylglyoxal synthase from Thermus sp. GH5 (TMGS) was cloned, sequenced, overexpressed, and purified by Q-Sepharose. The TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3 kDa. The K _m and k _cat values of TMGS were 0.56 mM and 325 (s^?1), respectively. The enzyme exhibited its optimum activity at pH?6 and 75?°C. Comparing the amino acid sequences and Hill coefficients of Escherichia coli MGS and TMGS revealed that the loss of Arg 150 in TMGS has caused a decrease in the cooperativity between the enzyme subunits in the presence of phosphate as an allosteric inhibitor. Gel filtration experiments showed that TMGS is a hexameric enzyme, and its quaternary structure did not change in the presence of phosphate.     

Expression and Characterization of a Novel Propionyl-CoA Dehydrogenase Gene from Candida rugosa in Pichia pastoris

The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from Candida rugosa by the cDNA RACE technique. The 6× His-tagged recombinant PACD gene was expressed in Pichia pastoris GS115 and purified with Ni-NTA affinity chromatography. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified PACD was 49 kDa. The results showed that the recombinant protein had the activity of catalyzing propionyl-CoA to acrylyl-CoA. The K _m, k _cat, and V _max values of the purified PACD were calculated to be 40.86 μM, 0.566 s^?1 and 0.693 U?mg^?1 min^?1. The optimal temperature and pH of the purified PACD were 30 °C and 7.0, respectively. The recombinant PACD maintained 76.3%, 30.1%, and 4.3% of its original activity after 2 h incubation in standard buffer at 30, 40, and 50 °C, respectively. Mg²⁺ had an activating effect on the enzyme, while Mn²⁺, Ca²⁺, Zn²⁺, and Cu²⁺ had weak inhibition. Since PACD catalyzed the key step (from propionyl-CoA to acrylyl-CoA) in the modified β-oxidation pathway from glucose to 3-hydroxypropionic acid (3-HP), the integration of recombinant PACD could benefit the engineered strains for effective production of 3-HP from the most abundant biomass–sugars.