Modified ferric hydroxamate spectrophotometry for assaying glycolic acid from the hydrolysis of glycolonitrile by Rhodococcus sp. CCZU10-1

We successfully modified a ferric hydroxamate spectrophotometry method for assaying glycolic acid. Comparable to the high-performance liquid chromatography (HPLC)-based method, ferric hydroxamate spectrophotometry can be used to accurately monitor the time course of glycolonitrile bioconversion. Glycolic acid was assayed simply and rapidly at room temperature (25 ～ 35°C). Optimum culture conditions were obtained using this method to assay the glycolonitrile-hydrolyzing activity of Rhodococcus sp. CCZU10-1. The preferred carbon and nitrogen sources and ideal inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (5 g/L), and ?-caprolactam (2 mmol/L), respectively. The optimal growth temperature and initial medium pH for Rhodococcus sp. CCZU10-1 glycolonitrile-hydrolyzing activity were 30°C and pH 7.0. Modified ferric hydroxamate spectrophotometry could potentially be employed to assay other carboxylic acids.     

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.     

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.     

Carbonyl reductase SCRII from Candida parapsilosis catalyzes anti-Prelog reaction to (S)-1-phenyl-1,2-ethanediol with absolute stereochemical selectivity

An (S)-specific carbonyl reductase (SCRII) was purified to homogeneity from Candida parapsilosis by following an anti-Prelog reducing activity of 2-hydroxyacetophenone. Peptide mass fingerprinting analysis shows SCRII belongs to short-chain dehydrogenase/reductase family. Its coding gene was cloned and overexpressed in Escherichia coli. The recombinant SCRII displays the similar enzymatic characterization and catalytic properties to SCR. It catalyzes the enantioselective reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol with excellent optical purity of 100% in higher yield than SCR. Based on the sequence-structure alignment, several single-point mutations inside or adjacent to the substrate-binding loop or active site were designed. With respect to recombinant native SCRII, the A220 and E228 mutations almost lost enantioselectivity towards 2-hydroxyacetophenone reduction. The catalytic efficiencies (k_cat/K_m) for the A220 or E228 variants are <7% that of the unmutated enzyme. This work provides an excellent catalyst for enantiopure alcohol preparation and the lethal mutations of A220 and E228 suggest their importance in substrate-binding and/or catalysis.     

Isolation and lipid degradation profile of Raoultella planticola strain 232-2 capable of efficiently catabolizing edible oils under acidic conditions

The lipids (fats and oils) degradation capabilities of soil microorganisms were investigated for possible application in treatment of lipids-contaminated wastewater. We isolated a strain of the bacterium Raoultella planticola strain 232-2 that is capable of efficiently catabolizing lipids under acidic conditions such as in grease traps in restaurants and food processing plants. The strain 232-2 efficiently catabolized a mixture (mixed lipids) of commercial vegetable oil, lard, and beef tallow (1:1:1, w/w/w) at 20–35 °C, pH 3–9, and 1,000–5,000 ppm lipid content. Highly effective degradation rate was observed at 35 °C and pH 4.0, and the 24-h degradation rate was 62.5?±?10.5 % for 3,000 ppm mixed lipids. The 24-h degradation rate for 3,000 ppm commercial vegetable oil, lard, beef tallow, mixed lipids, and oleic acid was 71.8 %, 58.7 %, 56.1 %, 55.3?±?8.5 %, and 91.9 % at pH 4 and 30 °C, respectively. R. planticola NBRC14939 (type strain) was also able to efficiently catabolize the lipids after repeated subculturing. The composition of the culture medium strongly influenced the degradation efficiency, with yeast extract supporting more complete dissimilation than BactoPeptone or beef extract. The acid tolerance of strain 232-2 is proposed to result from neutralization of the culture medium by urease-mediated decomposition of urea to NH₃. The rate of lipids degradation increased with the rates of neutralization and cell growth. Efficient lipids degradation using strain 232-2 has been achieved in the batch treatment of a restaurant wastewater.     

Optimization of parameters for improvement of phenol degradation by Rhodococcus UKMP-5M using response surface methodology

Phenol is a toxic compound and is one of the major pollutants contained in the waste water from petroleum and its downstream industries. Response surface methodology (RSM) was used to optimize medium composition and culture condition for enhancement of growth of Rhodococcus UKMP-5M and phenol degradation rate in shake flask cultures. Phenol and (NH₄)₂SO₄ concentrations as well as temperature were the most significant factors that influenced growth and phenol degradation. Central composite design (CCD) was used for optimization of these parameters with growth, and degradation rates were used as the responses. Cultivation with 0.5 g/L phenol and 0.3 g/L (NH₄)₂SO₄ and incubation at 36 °C greatly enhanced growth of Rhodococcus UKMP-5M, where the final cell concentration increased from 0.117 g/L to 0.376 g/L. On the other hand, the degradation rate was greatly increased in cultivation with 0.7 g/L phenol and 0.4 g/L (NH₄)₂SO₄ and incubation at 37 °C. In this cultivation, the time taken to degrade 1 g/L phenol in the culture was reduced from 48 h to 27 h. The model for both responses was found significant and the predicted values were found to be in a good agreement with experimental values and subsequently validated. Increases in phenol degradation rate during Rhodococcus UKMP-5M cultivation corresponded well with increasing phenol hydroxylase activity.     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37°C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov., from a marine solar saltern and an aquaculture farm,respectively

Two halophilic archaea, strains GX21^T and R35^T, were isolated from a marine solar saltern and an aquaculture farm in China, respectively. Cells of the two strains were observed to be pleomorphic, flat, to contain gas vesicles, stain Gram-negative and produce red-pigmented colonies. Strain GX21^T was found to be able to grow at 25–50 °C (optimum 37 °C), at 2.6–4.8 M NaCl (optimum 3.4 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–8.5 (optimum pH 6.5) while strain R35^T was found to be able to grow at 25–45 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0–0.7 M MgCl₂ (optimum 0.03 M MgCl₂) and at pH 5.5–9.5 (optimum pH 6.5–7.0). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 15 % (w/v) for strain GX21^T and 12 % (w/v) for strain R35^T. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid and a minor lipid chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. 16S rRNA gene sequence analysis revealed that strains GX21^T and R35^T show 97.1 % sequence similarity to each other and are closely related to Haloplanus aerogenes TBN37^T (96.8 and 95.8 %), Haloplanus vescus RO5-8^T (96.7 and 96.1 %), Haloplanus salinus YGH66^T (96.4 and 95.8 %) and Haloplanus natans JCM 14081^T (96.3 and 95.4 %). The rpoB′ gene similarity between strains GX21^T and R35^T is 90.5 % and show 88.5–90.8 % similarity to the Haloplanus species with validly published names. The DNA G+C content of strain GX21^T and R35^T were determined to be 65.8 and 66.0 mol%, respectively. The DNA–DNA hybridization values between strain GX21^T and strain R35^T, and the two strains with the Haloplanus species with validly published names, showed less than 50 % DNA–DNA relatedness. It was concluded that strain GX21^T (=CGMCC 1.10456^T = JCM 17092^T) and strain R35^T (=CGMCC 1.10594 ^T = JCM 17271^T) represent two new species of Haloplanus, for which the names Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov. are proposed.     

Special Rhodococcus sp. CR-53 esterase Est4 contains a GGG(A)X-oxyanion hole conferring activity for the kinetic resolution of tertiary alcohols

Rhodococci are highly adaptable bacteria, capable to degrade or transform a large number of organic compounds, including recalcitrant or toxic products. However, little information is available on the lipases of the genus Rhodococcus, except for LipR, the first lipase isolated and described from strain Rhodococcus CR-53. Taking into consideration the interest raised by the enzymes produced by actinomycetes, a search for new putative lipases was performed in strain Rhodococcus CR-53. We describe here the isolation, cloning, and characterization of intracellular esterase Est4, a mesophilic enzyme showing preference for short-chain-length acyl groups, without interfacial activation. Est4 displays moderate thermal and pH stability and low tolerance to most tested ions, being inhibited by detergents like sodium dodecyl sulfate and Triton X-100®. Nevertheless, the enzyme shows good long-term stability when stored at 4–20 °C and neutral pH. Amino acid sequence analysis of Est4 revealed a protein of 313 amino acids without a signal peptide, bearing most of the conserved blocks that define bacterial lipase family IV, thus being assigned to this family. Detection of a GGG(A)X oxyanion hole in the enzyme motivated the evaluation of Est4 ability to convert tertiary alcohol esters. The newly discovered esterase Est4 from Rhodococcus CR-53 successfully hydrolyzed the tertiary alcohol esters linalyl acetate, terpinyl acetate, and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate.     

Separation of pure polychlorinated biphenyl isomers into two types of inducers on the basis of induction of cytochrome P-450 or P-448.

A number of isomerically pure polychlorinated biphenyls (PCBs) were tested as inducers of hepatic drug-metabolizing enzymes in the rat. The chlorinated biphenyl isomers can be categorized into two distinct groups of inducers, while commercial PCB mixtures have characteristics of both groups. Biphenyls chlorinated symmetrically in both the meta and para positions (3,4,3′,4′- and 3,4,5,3′,4′,5′-) increase the formation of cytochrome P-448, the ratio of the 455 to 430 peaks of the ethyl isocyanide difference spectrum, and aryl hydrocarbon hydroxylase and glucuronyl transferase activities, but decrease aminopyrine N-demethylase activity. These isomers are also the most toxic, as measured by weight loss. Biphenyl isomers chlorinated in both the para and ortho positions induce the formation of cytochrome P-450 rather than P-448, regardless of the chlorination of the meta position. These isomers, which include 2,4,2′,4′-tetra- and 2,4,5,2′,4′,5′-, 2,3,4,2′,3′,4′- and 2,4,6,2′,4′,6′-hexachlorobiphenyls, increase cytochrome P-450 and N-demethylase activity, but produce only a slight increase in aryl hydrocarbon hydroxylase activity, and do not alter the peak of the CO-difference spectrum or the ratio of the 455/430 peaks of the ethyl isocyanide difference spectrum. Isomers which are chlorinated in only one ring, or are chlorinated in both rings but not in the para positions, have very little activity as inducers of liver enzymes. Of the dichlorobiphenyls tested, 3,3′- and 4,4′-dichlorobiphenyls have very slight activity at extremely high doses.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

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.     

Purification and Characterization of an Extracellular Low Temperature-Active and Alkaline Stable Peptidase from Psychrotrophic Acinetobacter sp. MN 12 MTCC (10786)

An extracellular low temperature-active alkaline stable peptidase from Acinetobacter sp. MN 12 was purified to homogeneity with a purification fold of 9.8. The enzyme exhibited specific activity of 6,540 U/mg protein, with an apparent molecular weight of 35 kDa. The purified enzyme was active over broad range of temperature from 4 to 60 °C with optimum activity at 40 °C. The enzyme retained more than 75 % of activity over a broad range of pH (7.0–11.0) with optimum activity at pH 9.0. The purified peptidase was strongly inhibited by phenylmethylsulfonyl fluoride, giving an indication of serine type. The K _m and V _max for casein and gelatin were 0.3529, 2.03 mg/ml and 294.11, 384.61 μg/ml/min respectively. The peptidase was compatible with surfactants, oxidizing agents and commercial detergents, and effectively removed dried blood stains on cotton fabrics at low temperature ranging from 15 to 35 °C.     

Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta?(DE3)pLysS by induction with 1 mM of isopropyl-β-d-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0–8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH?6.5–7.5. The Vmax and the apparent Km value of the reductase were 67.6 μmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.     

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.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

Production of xylanase by an alkaline-tolerant marine-derived Streptomyces viridochromogenes strain and improvement by ribosome engineering

Xylanase is the enzyme complex that is responsible for the degradation of xylan; however, novel xylanase producers remain to be explored in marine environment. In this study, a Streptomyces strain M11 which exhibited xylanase activity was isolated from marine sediment. The 16S rDNA sequence of M11 showed the highest identity (99 %) to that of Streptomyces viridochromogenes. The xylanase produced from M11 exhibited optimum activity at pH 6.0, and the optimum temperature was 70 °C. M11 xylanase activity was stable in the pH range of 6.0–9.0 and at 60 °C for 60 min. Xylanase activity was observed to be stable in the presence of up to 5 M NaCl. Antibiotic-resistant mutants of M11 were isolated, and among the various antibiotics tested, streptomycin showed the best effect on obtaining xylanase overproducer. Mutant M11-1(10) isolated from 10 μg/ml streptomycin-containing plate showed 14 % higher xylanase activities than that of the wild-type strain. An analysis of gene rpsL (encoding ribosomal protein S12) showed that rpsL from M11-1(10) contains a K88R mutation. This is the first report to show that marine-derived S. viridochromogenes strain can be used as a xylanase producer, and utilization of ribosome engineering for the improvement of xylanase production in Streptomyces was also first successfully demonstrated.     

Didymosphaeria igniaria: a new microorganism useful for the enantioselective reduction of aryl-aliphatic ketones

Didymosphaeria igniaria is a promising biocatalyst in asymmetric reductions of prochiral aromatic-aliphatic ketones such as acetonaphthones, acetophenones, and acetylpyridines. The organism converted the substrates mainly to (S)-alcohols. Excellent results in terms of conversion and enantioselectivity (100% yield, >99% ee) were obtained with acetonaphthones. In case of acetyl pyridines, the optical purity of the product depended on the position of the carbonyl group on the pyridine ring and followed the order 2-acetyl ? 4-acetyl > 3-acetyl-pyridine. Transformation of o-methoxy-acetophenone gave optically pure (S)-(-)-1-(2-methoxyphenyl)-ethanol in 95% yield. The transformation of para-methyl ketone gave (R)-alcohol (81% ee), whereas para-bromo ketone gave (S)-alcohol (98% ee). Monitoring of the biotransformation of these substrates over time led to the conclusion that for both substrates, non-selective carbonyl group reduction occurred in the first step, followed by selective oxidation of the (R)-isomer of p-bromo-phenylethanol and selective oxidation of the (S)-isomer of p-methyl-phenylethanol. D. igniaria exhibited poor enantioselectivity in the reduction of bicyclic aryl-aliphatic ketones such as 1- and 2-tetralones. Only (S)-5-methoxy-1-tetralol was obtained in optically pure (>99% ee) form.