Cloning, Sequencing, and Characterization of a Heat- and Alkali-Stable Type I Pullulanase from Anaerobranca gottschalkii

The gene encoding a type I pullulanase was identified from the genome sequence of the anaerobic thermoalkaliphilic bacterium Anaerobranca gottschalkii. In addition, the homologous gene was isolated from a gene library of Anaerobranca horikoshii and sequenced. The proteins encoded by these two genes showed 39% amino acid sequence identity to the pullulanases from the thermophilic anaerobic bacteria Fervidobacterium pennivorans and Thermotoga maritima. The pullulanase gene from A. gottschalkii (encoding 865 amino acids with a predicted molecular mass of 98 kDa) was cloned and expressed in Escherichia coli strain BL21(DE3) so that the protein did not have the signal peptide. Accordingly, the molecular mass of the purified recombinant pullulanase (rPulAg) was 96 kDa. Pullulan hydrolysis activity was optimal at pH 8.0 and 70°C, and under these physicochemical conditions the half-life of rPulAg was 22 h. By using an alternative expression strategy in E. coli Tuner(DE3)(pLysS), the pullulanase gene from A. gottschalkii, including its signal peptide-encoding sequence, was cloned. In this case, the purified recombinant enzyme was a truncated 70-kDa form (rPulAg′). The N-terminal sequence of purified rPulAg′ was found 252 amino acids downstream from the start site, presumably indicating that there was alternative translation initiation or N-terminal protease cleavage by E. coli. Interestingly, most of the physicochemical properties of rPulAg′ were identical to those of rPulAg. Both enzymes degraded pullulan via an endo-type mechanism, yielding maltotriose as the final product, and hydrolytic activity was also detected with amylopectin, starch, β-limited dextrins, and glycogen but not with amylose. This substrate specificity is typical of type I pullulanases. rPulAg was inhibited by cyclodextrins, whereas addition of mono- or bivalent cations did not have a stimulating effect. In addition, rPulAg′ was stable in the presence of 0.5% sodium dodecyl sulfate, 20% Tween, and 50% Triton X-100. The pullulanase from A. gottschalkii is the first thermoalkalistable type I pullulanase that has been described.     

Characterization of Methylglyoxal Synthase from Clostridium acetobutylicum ATCC 824 and Its Use in the Formation of 1,2-Propanediol

A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824. The enzyme was overexpressed in Escherichia coli and purified. Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5. The K_m and V_max values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min⁻¹ μg⁻¹, respectively. When E. coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E. coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.     

Enhanced Heterologous Expression of Two Streptomyces griseolus Cytochrome P450s and Streptomyces coelicolor Ferredoxin Reductase as Potentially Efficient Hydroxylation Catalysts

The herbicide-inducible, soluble cytochrome P450s CYP105A1 and CYP105B1 and their adjacent ferredoxins, Fd1 and Fd2, of Streptomyces griseolus were expressed in Escherichia coli to high levels. Conditions for high-level expression of active enzyme able to catalyze hydroxylation have been developed. Analysis of the expression levels of the P450 proteins in several different E. coli expression hosts identified E. coli BL21 Star(DE3)pLysS as the optimal host cell to express CYP105B1 as judged by CO difference spectra. Examination of the codons used in the CYP1051A1 sequence indicated that it contains a number of codons corresponding to rare E. coli tRNA species. The level of its expression was improved in the modified forms of E. coli BL21(DE3), which contain extra copies of rare codon E. coli tRNA genes. The activity of correctly folded cytochrome P450s was further enhanced by cloning a ferredoxin reductase from Streptomyces coelicolor downstream of CYP105A1 and CYP105B1 and their adjacent ferredoxins. Expression of CYP105A1 and CYP105B1 was also achieved in Streptomyces lividans 1326 by cloning the P450 genes and their ferredoxins into the expression vector pBW160. S. lividans 1326 cells containing CYP105A1 or CYP105B1 were able efficiently to dealkylate 7-ethoxycoumarin.     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Substrate Specificity of Purified Recombinant Human β-Carotene 15,15′-Oxygenase (BCO1)

Humans cannot synthesize vitamin A and thus must obtain it from their diet. β-Carotene 15,15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15–15′ double bond to yield retinal (vitamin A). In this work, we quantitatively describe the substrate specificity of purified recombinant human BCO1 in terms of catalytic efficiency values (k_cat/K_m). The full-length open reading frame of human BCO1 was cloned into the pET-28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a V_max = 197.2 nmol retinal/mg BCO1 × h, K_m = 17.2 μm and catalytic efficiency k_cat/K_m = 6098 m⁻¹ min⁻¹. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin, and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene. The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that BCO1 favors full-length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.     

Biochemical and Genetic Characterization of trans-2′-Carboxybenzalpyruvate Hydratase-Aldolase from a Phenanthrene-Degrading Nocardioides Strain

trans-2′-Carboxybenzalpyruvate hydratase-aldolase was purified from a phenanthrene-degrading bacterium, Nocardioides sp. strain KP7, and characterized. The purified enzyme was found to have molecular masses of 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography. Thus, the homotrimer of the 38-kDa subunit constituted an active enzyme. The K_m and kcat values of this enzyme for trans-2′-carboxybenzalpyruvate were 50 μM and 13 s⁻¹, respectively. trans-2′-Carboxybenzalpyruvate was transformed to 2-carboxybenzaldehyde and pyruvate by the action of this enzyme. The structural gene for this enzyme was cloned and sequenced; the length of this gene was 996 bp. The deduced amino acid sequence of this enzyme exhibited homology to those of trans-2′-hydroxybenzalpyruvate hydratase-aldolases from Pseudomonas putida PpG7 and Pseudomonas sp. strain C18.     

Improving the Expression of Recombinant Proteins in E. coli BL21 (DE3) under Acetate Stress: An Alkaline pH Shift Approach

Excess acetate has long been an issue for the production of recombinant proteins in E. coli cells. Recently, improvements in acetate tolerance have been achieved through the use of genetic strategies and medium supplementation with certain amino acids and pyrimidines. The aim of our study was to evaluate an alternative to improve the acetate tolerance of E. coli BL21 (DE3), a popular strain used to express recombinant proteins. In this work we reported the cultivation of BL21 (DE3) in complex media containing acetate at high concentrations. In the presence of 300 mM acetate, compared with pH 6.5, pH 7.5 improved cell growth by approximately 71%, reduced intracellular acetate by approximately 50%, and restored the expression of glutathione S-transferase (GST), green fluorescent protein (GFP) and cytochrome P450 monooxygenase (CYP). Further experiments showed that alkaline pHs up to 8.5 had little inhibition in the expression of GST, GFP and CYP. In addition, the detrimental effect of acetate on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) by the cell membrane, an index of cellular metabolic capacity, was substantially alleviated by a shift to alkaline pH values of 7.5–8.0. Thus, we suggest an approach of cultivating E. coli BL21 (DE3) at pH 8.0±0.5 to minimize the effects caused by acetate stress. The proposed strategy of an alkaline pH shift is a simple approach to solving similar bioprocessing problems in the production of biofuels and biochemicals from sugars.     

Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

Endonuclease IV encoded by denB of bacteriophage T4 is implicated in restriction of deoxycytidine (dC)-containing DNA in the host Escherichia coli. The enzyme was synthesized with the use of a wheat germ cell-free protein synthesis system, given a lethal effect of its expression in E.coli cells, and was purified to homogeneity. The purified enzyme showed high activity with single-stranded (ss) DNA and denatured dC-substituted T4 genomic double-stranded (ds) DNA but exhibited no activity with dsDNA, ssRNA or denatured T4 genomic dsDNA containing glucosylated deoxyhydroxymethylcytidine. Characterization of Endo IV activity revealed that the enzyme catalyzed specific endonucleolytic cleavage of the 5′ phosphodiester bond of dC in ssDNA with an efficiency markedly dependent on the surrounding nucleotide sequence. The enzyme preferentially targeted 5′-dTdCdA-3′ but tolerated various combinations of individual nucleotides flanking this trinucleotide sequence. These results suggest that Endo IV preferentially recognizes short nucleotide sequences containing 5′-dTdCdA-3′, which likely accounts for the limited digestion of ssDNA by the enzyme and may be responsible in part for the indispensability of a deficiency in denB for stable synthesis of dC-substituted T4 genomic DNA.     

Enhancement of camptothecin-induced topoisomerase I cleavage complexes by the acetaldehyde adduct N2-ethyl-2'-deoxyguanosine

The activity of DNA topoisomerase I (Top1), an enzyme that regulates DNA topology, is impacted by DNA structure alterations and by the anticancer alkaloid camptothecin (CPT). Here, we evaluated the effect of the acetaldehyde-derived DNA adduct, N²-ethyl-2′-deoxyguanosine (N²-ethyl-dG), on human Top1 nicking and closing activities. Using purified recombinant Top1, we show that Top1 nicking-closing activity remains unaffected in N²-ethyl-dG adducted oligonucleotides. However, the N²-ethyl-dG adduct enhanced CPT-induced Top1–DNA cleavage complexes depending on the relative position of the N²-ethyl-dG adduct with respect to the Top1 cleavage site. The Top1-mediated DNA religation (closing) was selectively inhibited when the N²-ethyl-dG adduct was present immediately 3′ from the Top1 site (position +1). In addition, when the N²-ethyl-dG adduct was located at the −5 position, CPT enhanced cleavage at an alternate Top1 cleavage site immediately adjacent to the adduct, which was then at position +1 relative to this new alternate Top1 site. Modeling studies suggest that the ethyl group on the N²-ethyl-dG adduct located at the 5′ end of a Top1 site (position +1) sterically blocks the dissociation of CPT from the Top1–DNA complex, thereby inhibiting further the religation (closing) reaction.     

Expression and single-step purification of mercury transporter (merT) from <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l⁻¹.     

A candidate prostate cancer susceptibility gene encodes tRNA 3' processing endoribonuclease

tRNA 3′ processing endoribonuclease (3′ tRNase) is an enzyme responsible for the removal of a 3′ trailer from precursor tRNA (pre-tRNA). We purified ~85 kDa 3′ tRNase from pig liver and determined its partial sequences. BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown. We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli. The recombinant ELAC2 was able to cleave human pre-tRNA^Arg efficiently. The 3′ tRNase activity of the yeast ortholog YKR079C was also observed. The C-terminal half of human ELAC2 was able to remove a 3′ trailer from pre-tRNA^Arg, while the N‐terminal half failed to do so. In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3′ tRNase from ELAC2. We showed that human ELAC1 also has 3′-tRNase activity. Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3′-tRNase activity. Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3′-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.     

Production and purification of a novel antibiotic peptide,adenoregulin, from a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Adenoregulin is a member of dermaseptin family which are vertebrate antibiotic peptides having lethal effects against a broad spectrum of bacteria, fungi and protozoa. The 99 bp adenoregulin gene was cloned in the expression vector pET32a and transformed into Escherichia coli BL21(DE3). In fed-batch cultivation of BL21(DE3)/pET32a-adr, an exponential feeding strategy was applied to gain 60 g dry cells l^–1. The recombinant fusion protein Trx-ADR was expressed in a soluble form. The fusion protein was isolated by Ni²⁺-chelating chromatography, cleaved with CNBr and purified to homogeneity through reverse phase-HPLC and size exclusion-HPLC. The purified recombinant adenoregulin had antibacterial activity against Escherichia coli K12D31 with apparent Mr of 3.4 kDa, identical to the anticipated value.     

Cloning,expression of b-1,3-1,4 glucanase from Bacillus subtilis SU40 and the effect of calcium ion on the stability of recombinant enzyme: in vitro and in silico analysis

A new glucanolytic bacterial strain, SU40 was isolated, and identified as Bacillus subtilis on the basis of 16S rRNA sequence homology and phylogenetic tree analysis. The gene encoding β-1,3-1,4-glucanase was delineated, cloned into pET 28a+ vector and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme was about 24 kDa. The enzyme exhibited maximum activity (36.84 U/ml) at 60°C, pH 8.0 and maintained 54% activity at 80°C after incubation for 60 min. The enzyme showed activity against β-glucan, lichenan, and xylan. Amino acid sequence shared a conserved motif EIDIEF. The predicted three-dimensional homology model of the enzyme showed the presence of catalytic residues Glu105, Glu109 and Asp107, single disulphide bridge between Cys32 and Cys61 and three calcium binding site residues Pro9, Gly45 and Asp207. Presence of calcium ion improves the thermal stability of SU40 β-1,3-1,4-glucanase. Molecular dynamics simulation studies revealed that the absence of calcium ion fluctuate the active site residues which are responsible for thermostability. The high catalytic activity and its stability to temperature, pH and metal ions indicated that the enzyme β-1,3-1,4-glucanase by B. subtilis SU40 is a good candidate for biotechnological applications.     

Cloning and Expression of a Phloretin Hydrolase Gene from Eubacterium ramulus and Characterization of the Recombinant Enzyme

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37°C and 7.0, respectively. The K_m for phloretin was 13 ± 3 μM and the k_cat was 10 ± 2 s⁻¹. The enzyme did not transform phloretin-2′-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl₂ to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.     

Candida albicans NADPH-P450 reductase: Expression, purification, and characterization of recombinant protein

Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. In this study, we report the characterization of the functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein harboring a 6×(His)-tag was expressed heterologously in Escherichia coli, and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm, indicating the feature of an oxidized flavin cofactor, which was decreased by the addition of NADPH. It also evidenced NADPH-dependent cytochrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results indicate that purified C. albicans NPR is an orthologous reductase protein that supports cytochrome P450 or heme oxygenase enzymes in C. albicans.     

Gene cloning and biochemical characterization of 4-N-trimethylaminobutyraldehyde dehydrogenase II from Pseudomonas sp. 13CM

The gene encoding 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABaldehyde-DH) from Pseudomonas sp. 13CM, responsible for the conversion of 4-N-trimethylaminobutyraldehyde (TMABaldehyde) to γ-butyrobetaine in the carnitine biosynthesis pathway, isolated by shotgun cloning and expressed in Escherichia coli DH5α. The recombinant TMABaldehyde-DH was purified 19.5 fold to apparent homogeneity by hydrophobic and affinity chromatography and biochemically characterized. The enzyme was found to be a trimer with identical 52 kDa subunits. The isoelectric point was found to be 4.5. Optimum pH and temperature were found respectively as pH 9.5 and 40 °C. The K_m values for TMABaldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were respectively, 0.31, 0.62, and 1.16 mM. The molecular and catalytic properties differed from those of TMABaldehyde-DH I, which was discovered initially in Pseudomonas sp. 13CM. The new enzyme, designated TMABaldehyde-DH II, structural gene was inserted into an expression vector pET24b (+) and over-expressed in E. coli BL21 (DE3) under the control of a T7 promoter. The recombinant TMABaldehyde-DH from Pseudomonas sp. 13CM can now be obtained in large quantity necessary for further biochemical characterization and applications.     

High-level expression of a soluble snake venom enzyme,gloshedobin, in E. coli in the presence of metal ions

The mature gene of gloshedobin, a snake venom thrombin-like enzyme from the snake, Gloydius shedaoensis, was cloned and expressed in strain E. coli BL21(DE3). Having been induced by IPTG, the recombinant gloshedobin was in both soluble and insoluble forms. To avoid inclusion body formation, expression was optimized at 25 °C. Furthermore, a 50% increase in solubilization of the target protein was obtained by adding 0.1 mM Mg²⁺ to the medium. The purified recombinant gloshedobin gave a 44 kDa band on SDS-PAGE gel.     

Cloning, Expression, and Characterization of an GHF 11 Xylanase from Aspergillus niger XZ-3S

A xylanase gene (xynZF-2) from the Aspergillus niger XZ-3S was cloned and expressed in Escherichia coli. The coding region of the gene was separated by only one intron with the 68 bp in length. It encoded 225 amino acid residues of a protein with a calculated molecular weight of 24.04 kDa plus a signal peptide of 18 amino acids. The amino acid sequence of the xynZF-2 gene had a high similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The mature peptide encoding cDNA was subcloned into pET-28a(+) expression vector. The resultant recombinant plasmid pET-28a-xynZF-2 was transformed into E. coli BL21(DE3), and finally the recombinant strain BL21/xynZF-2 was obtained. A maximum activity of 42.33 U/mg was gained from cellular of E. coli BL21/xynZF-2 induced by IPTG. The optimum temperature and pH for recombinant enzyme which has a good stability in alkaline conditions were 40 °C and 5.0, respectively. Fe³⁺ had an active effect on the enzyme obviously.     

Purification and characterization of a cytosolic cytochrome P450 from the yeast Trichosporon cutaneum

A soluble cytochrome P450 from the yeast Trichosporon cutaneum was purified to homogeneity, using ammonium sulfate fractionation followed by fast protein liquid chromatography (FPLC) with DEAE-cellulose and phenyl-Sepharose columns. This procedure resulted in a 45-fold increase in specific activity with an activity yield of 6.8%. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified enzyme was homogeneous and had a molecular mass of 45 kDa. The purified enzyme contained a heme group and had a characteristic absorption peak at 448 nm in the reduced carbon monoxide difference spectrum. This enzyme was a monomeric protein and catalyzed the conversion of salicylic acid to catechol in the presence of NADH or NADPH. The N-terminal amino acid sequence indicated that the Trichosporon cutaneum cytochrome P450 did not show homology to most eukaryotic cytochromes P450, but had a high degree of homology to one cytochrome P450, the nitric oxide reductase, of Fusarium oxysporum.     

Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942

A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44°C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 ± 2.15 U mg⁻¹) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 ± 0.49 U mg⁻¹) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).