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1.
Chem Commun (Camb) ; 58(95): 13222-13225, 2022 Nov 29.
Article in English | MEDLINE | ID: mdl-36353944

ABSTRACT

An engineered (S)-selective imine reductase from Streptomyces sp. GF3546 (SIR46) showed twice the activity of the wild type and high thermostability at 50 °C. Using a biocatalyst based on SIR46, we investigated the activity toward high-concentration 2-methyl-1-pyrroline up to 500 mM, conversion rates of other imines, and reductive amination activity.


Subject(s)
Imines , Streptomyces , Oxidoreductases/metabolism , Amination , Streptomyces/metabolism , Amines , Stereoisomerism
2.
Biosci Biotechnol Biochem ; 83(4): 768-773, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30572801

ABSTRACT

Rhodococcus sp. 2N was found as a 1,3-propanediols-oxidizing strain from soil samples through enrichment culture using 2,2-diethyl-1,3-propanediol (DEPD) as the sole carbon source. The culture condition of the strain 2N was optimized, and the highest activity was observed when 0.3% (w/v) DEPD was added in the culture medium as an inducer. Chiral HPLC analysis of the hydroxyalkanoic acid converted from 2-ethyl-2-methyl-1,3-propanediol (EMPD) revealed that the strain 2N catalyzed the (R)-selective oxidation of EMPD. The reaction products and intermediates from DEPD and EMPD were identified by nuclear magnetic resonance analyses, and the results suggested that only one hydroxymethyl group of the propanediols was converted to carboxy group via two oxidation steps. Under optimized conditions and after a 72-h reaction time, the strain 2N produced 28 mM (4.1 g/L) of 2-(hydroxymethyl)-2-methylbutanoic acid from EMPD with a molar conversion yield of 47% and 65% ee (R).


Subject(s)
Butyrates/metabolism , Propylene Glycols/metabolism , Rhodococcus/metabolism , Biodegradation, Environmental , Butyrates/chemistry , Culture Media/chemistry , Culture Media/pharmacology , Kinetics , Magnetic Resonance Spectroscopy , Oxidation-Reduction , Propylene Glycols/chemistry , Rhodococcus/chemistry , Rhodococcus/drug effects
3.
J Biosci Bioeng ; 115(6): 651-3, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23290448

ABSTRACT

Verticillium sp. GF39, catalyzing the oxidation of 1-methylisoquinoline to 1-methylisoquinoline N-oxide, was found to be the highest N-oxide producer. Under the optimized reaction conditions, the whole cells of Verticillium sp. GF39 formed 5 mM 1-methylisoquinoline N-oxide from 1-methylisoquinoline with a molar conversion yield of 100% after a 10-h incubation at 20°C. The whole cells also acted on pyridine, 2-methylpyridine, quinoline and isoquinoline and formed the corresponding N-oxides.


Subject(s)
Cyclic N-Oxides/metabolism , Isoquinolines/metabolism , Verticillium/metabolism , Cyclic N-Oxides/chemistry , Isoquinolines/chemistry , Oxidation-Reduction , Picolines/metabolism , Pyridines/metabolism , Verticillium/chemistry
4.
Biotechnol Lett ; 35(5): 685-8, 2013 May.
Article in English | MEDLINE | ID: mdl-23288294

ABSTRACT

The nitrilase gene of Rhodococcus rhodochrous J1 was expressed in Escherichia coli using the expression vector, pKK223-3. The recombinant E. coli JM109 cells hydrolyzed enantioselectively 2-methyl-2-propylmalononitrile to form (S)-2-cyano-2-methylpentanoic acid (CMPA) with 96 % e.e. Under optimized conditions, 80 g (S)-CMPA l(-1) was produced with a molar yield of 97 % at 30 °C after a 24 h without any by-products.


Subject(s)
Aminohydrolases/metabolism , Bacterial Proteins/metabolism , Pentanoic Acids/metabolism , Aminohydrolases/genetics , Bacterial Proteins/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Nitriles/chemistry , Nitriles/metabolism , Pentanoic Acids/analysis , Pentanoic Acids/chemistry , Rhodococcus/enzymology , Rhodococcus/genetics , Stereoisomerism , Temperature
5.
Appl Microbiol Biotechnol ; 97(18): 8079-86, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23263364

ABSTRACT

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.


Subject(s)
Amines/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cloning, Molecular , Imines/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Streptomyces/enzymology , Amines/chemistry , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Gene Expression , Imines/chemistry , Molecular Sequence Data , NADP/metabolism , Oxidoreductases/genetics , Oxidoreductases/isolation & purification , Sequence Homology, Amino Acid , Soil Microbiology , Streptomyces/chemistry , Streptomyces/genetics , Streptomyces/isolation & purification , Substrate Specificity
6.
Biotechnol Lett ; 34(9): 1741-4, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22639090

ABSTRACT

Washed cells (62 mg) of Kitasatospora sp. GF12 in 4 ml buffer (pH 7) catalyzed the regioselective hydroxylation of 60 mM 1,3-adamantanediol [1,3-ad(OH)(2)] to 30.9 mM 1,3,5-adamantanetriol [1,3,5-ad(OH)(3)] over 120 h at 24 °C. Glycerol at 400 mM was added to the reaction mixture to recycle the intracellular NADH/NADPH. Whole cells of GF12, also catalyzed the hydroxylation of 10 mM 1-adamantanol (1-adOH), to 3.6 mM 1,3,5-ad(OH)(3).


Subject(s)
Adamantane/analogs & derivatives , Adamantane/metabolism , Biotechnology/methods , Streptomycetaceae/metabolism , Adamantane/chemistry , Glycerol/metabolism , Hydroxylation , NAD/metabolism , NADP/metabolism , Stereoisomerism , Temperature , Time Factors
7.
Biosci Biotechnol Biochem ; 76(4): 838-40, 2012.
Article in English | MEDLINE | ID: mdl-22484946

ABSTRACT

The 4-hydroxyacetophenone assimilating bacterium Arthrobacter sp. TGJ4 was isolated from a soil sample. The resting cell reaction suggested that the strain cleaved 4-hydroxyacetophenone and its 3-methoxy derivative to the corresponding carboxylic acids and formaldehyde. Some properties of the enzyme catalyzing the cleavage reaction were examined.


Subject(s)
Acetophenones/metabolism , Arthrobacter/metabolism , Soil Microbiology , Soil Pollutants/metabolism , Biodegradation, Environmental , Carboxylic Acids/metabolism , Formaldehyde/metabolism , Oxidation-Reduction , Vanillic Acid/metabolism
8.
Biosci Biotechnol Biochem ; 76(3): 567-74, 2012.
Article in English | MEDLINE | ID: mdl-22451401

ABSTRACT

The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 µM and 6.28 µmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.


Subject(s)
Biocatalysis , Burkholderia/enzymology , Dioxygenases/metabolism , Absorption , Amino Acid Sequence , Benzhydryl Compounds , Burkholderia/genetics , Burkholderia/isolation & purification , Cloning, Molecular , Dioxygenases/chemistry , Dioxygenases/genetics , Dioxygenases/isolation & purification , Enzyme Inhibitors/pharmacology , Escherichia coli/genetics , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Phenols/metabolism , Substrate Specificity , Temperature
9.
Biosci Biotechnol Biochem ; 75(9): 1778-82, 2011.
Article in English | MEDLINE | ID: mdl-21897027

ABSTRACT

The (R)-imine reductase (RIR) of Streptomyces sp. GF3587 was purified and characterized. It was found to be a NADPH-dependent enzyme, and was found to be a homodimer consisting of 32 kDa subunits. Enzymatic reduction of 10 mM 2-methyl-1-pyrroline (2-MPN) resulted in the formation of 9.8 mM (R)-2-methylpyrrolidine ((R)-2-MP) with 99% e.e. The enzyme showed not only reduction activity for 2-MPN at neutral pH (6.5-8.0), but also oxidation activity for (R)-2-MP under alkaline pH (10-11.5) conditions. It appeared to be a sulfhydryl enzyme based on the sensitivity to sulfhydryl specific inhibitors. It was very specific to 2-MPN as substrate.


Subject(s)
Imines/metabolism , Oxidoreductases/metabolism , Protein Subunits/chemistry , Pyrroles/metabolism , Pyrrolidines/metabolism , Streptomyces/enzymology , Chromatography, Gel , Dimerization , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Kinetics , Methylation , Molecular Weight , NADP/metabolism , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/chemistry , Oxidoreductases/isolation & purification , Streptomyces/chemistry , Substrate Specificity , Sulfhydryl Reagents/pharmacology , p-Chloromercuribenzoic Acid/pharmacology
10.
Org Biomol Chem ; 8(20): 4533-5, 2010 Oct 21.
Article in English | MEDLINE | ID: mdl-20820664

ABSTRACT

Streptomyces sp. GF3587 and 3546 were found to be imine-reducing strains with high R- and S-selectivity by screening using 2-methyl-1-pyrroline (2-MPN). Their whole-cell catalysts produced 91 mM R-2-methylpyrrolidine (R-2-MP) with 99.2%e.e. and 27.5 mM S-2-MP (92.3%e.e.) from 2-MPN at 91-92% conversion in the presence of glucose, respectively.


Subject(s)
Amines/chemical synthesis , Imines/chemistry , Pyrrolidines/metabolism , Catalysis , Glucose/metabolism , Oxidoreductases/chemistry , Pyrrolidines/chemistry , Stereoisomerism , Streptomyces/metabolism
11.
J Biosci Bioeng ; 109(6): 550-3, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20471592

ABSTRACT

To efficiently produce 1,3-adamantanediol (1,3-ad(OH)(2)) from 1-adamantanol (1-adOH), our stocks of culture strains and soil microorganisms were surveyed for hydroxylation activity towards 1-adOH. Among them, the soil actinomycete SA8 showing the highest hydroxylation activity was identified as Streptomyces sp. based on 16S ribosomal DNA sequence analysis. The reaction products were purified by silica gel column chromatography, and from NMR and MS analyses, they were identified as 1,3-ad(OH)(2) and 1,4-ad(OH)(2). Streptomyces sp. SA8 produced 5.9 g l(-1) 1,3-ad(OH)(2)from 6.2 g l(-1) 1-adOH in culture broth after 120 h at 25 degrees C. Using resting cells, 2.3 g l(-1) 1,3-ad(OH)(2) was produced after 96 h of incubation at a 69% conversion rate. In both cases, 1,4-ad(OH)(2) was formed as a byproduct at a rate of about 15%. Strain SA8 also hydroxylated 2-adamantanol and 2-methyl-2-adamantanol.


Subject(s)
Adamantane/analogs & derivatives , Streptomyces/metabolism , Adamantane/metabolism , Hydroxylation , Oxidation-Reduction , Sequence Analysis, DNA , Streptomyces/genetics , Substrate Specificity
12.
Appl Microbiol Biotechnol ; 71(4): 502-4, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16187097

ABSTRACT

Hydroxylation of adamantane using whole cells of bacteria, actinomyces, and molds was examined. The structure of the product was determined using gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectroscopy (MS). Among 470 strains tested, Streptomyces griseoplanus was highly regioselective to give 1-adamantanol (0.096 mmol) from adamantane (0.3 mmol) in a 32% molar conversion yield after 72-h cultivation in the presence of 3% (v/v) Tween 60. A P450 inhibitor such as 0.5 mM 1-aminobenzotriazole or menadione significantly inhibited the hydroxylation activity. These results suggested that a P450 oxidation system might be involved in this hydroxylation reaction.


Subject(s)
Adamantane/analogs & derivatives , Adamantane/metabolism , Alcohols/metabolism , Streptomyces/metabolism , Adamantane/chemistry , Alcohols/chemistry , Chromatography, Gas , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Enzyme Inhibitors/pharmacology , Hydroxylation , Magnetic Resonance Spectroscopy , Mass Spectrometry , Streptomyces/enzymology , Triazoles/pharmacology , Vitamin K 3/pharmacology
13.
Biotechnol Lett ; 27(17): 1305-10, 2005 Sep.
Article in English | MEDLINE | ID: mdl-16215830

ABSTRACT

Mucor circinelloides and Mortierella isabellina hydroxylated dehydroabietic acid (DehA). DehA was converted regio- and stereo-selectively by whole cells of Mr. circinelloides to give 2alpha-hydroxydehydroabietic acid in a 75% molar conversion yield (11 mM from 14.7 mM DehA) after 72 h in the cultivation medium containing 3% (v/v) Tween 80. With cells of Ma. isabellina, under the same conditions, 20.5 mM (6.5 g l(-1)) 2-hydroxydehydroabietic acid (alpha/beta=81/19) was formed from 26.4 mM DehA.


Subject(s)
Abietanes/metabolism , Mortierella/metabolism , Mucor/metabolism , Abietanes/analysis , Hydroxylation , Stereoisomerism
14.
Biotechnol Lett ; 26(21): 1643-8, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15604813

ABSTRACT

Conversion of heterocyclic and aromatic aldehydes to the corresponding carboxylic acids was carried out using Acetobacter rancens IFO3297, A. pasteurianus IFO13753 and Serratia liquefaciens LF14. IFO3297 produced 110 g 2-furoic acid l(-1) from furfural with a 95% molar yield. 5-Hydroxymethyl-2-furancarboxylic acid was produced from the corresponding aldehyde by using whole cells LF14. IFO13753 and LF14 both converted isophthalaldehyde, 2,5-furandicarbaldehyde, 2,5-thiophenedicarbaldehyde and 2,2' biphenyldicarbaldehyde to the corresponding formylcarboxylic acid with 86-91% molar yields.


Subject(s)
Acetobacter/metabolism , Aldehydes/metabolism , Carboxylic Acids/metabolism , Serratia/metabolism , Acetobacter/classification , Biotransformation , Heterocyclic Compounds/metabolism , Hydrocarbons, Aromatic/metabolism , Oxidation-Reduction , Serratia/classification , Species Specificity
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