Yasuhisa Asano

Find an error

Name:

Organization: Toyama Prefectural University

Department: 1 Biotechnology Research Center and Department of Biotechnology

Title:

Chemicals
References

Hydrogen peroxide as a new defensive compound in “benzoyl cyanide” producing polydesmid millipedes

Co-reporter:Yasumasa Kuwahara;Takuya Yamaguchi;Yayoi Ichiki;Tsutomu Tanabe

The Science of Nature 2017 Volume 104( Issue 3-4) pp:

Publication Date(Web):2017 April

DOI:10.1007/s00114-017-1435-y

Hydrogen peroxide was newly and simultaneously demonstrated with well-known hydrogen cyanide as a component of defensive secretions of “benzoyl cyanide” producing polydesmid millipedes. Presence of hydrogen peroxide was successively evidenced by Trinder reagent’s spray with colorless as well as oily smears of defensive secretions containing benzoyl cyanide and hydrogen cyanide by alkaline picrate paper treatment. Linear correlation was demonstrated between quantities of hydrogen peroxide and benzoyl cyanide. By qualitative assay, seven benzoyl cyanide containing polydesmidans (six species of adults and one species of a nymph at stadium I) tested positive to Trinder reagent, indicative of the presence of hydrogen peroxide (together with hydrogen cyanide), while two cyanogenic species without benzoyl cyanide exhibited negative responses to the reagent. Two types of millipedes were elucidated as species of cyanogenic Polydesmida.

New enzymatic methods for the synthesis of primary α-aminonitriles and unnatural α-amino acids by oxidative cyanation of primary amines with D-amino acid oxidase from porcine kidney

Co-reporter:Nobuhiro Kawahara;Kazuyuki Yasukawa

Green Chemistry (1999-Present) 2017 vol. 19(Issue 2) pp:418-424

Publication Date(Web):2017/01/23

DOI:10.1039/C6GC02003H

Oxidation of amino groups in amines or amino acids activates the sp3 Cα–H bond to form imines, making the alpha carbon atom a preferable target for nucleophilic reagents such as cyanide. Therefore, we focused on the oxidase reaction for the production of primary α-aminonitriles via imines. D-Amino acid oxidase from porcine kidney (pkDAO) and L-amino acid oxidase from Crotalus atrox catalyzed the synthesis of 2-amino-2-cyano-3-phenylpropanoic acid from phenylalanine and potassium cyanide (KCN). Mutant pkDAO (Y228L/R283G) catalyzed the synthesis of racemic-2-methyl-2-phenylglycinonitrile from (R)-α-methylbenzylamine and KCN. Based on these results, we developed a new cascade reaction for the synthesis of unnatural α-amino acids from primary amines using mutant pkDAO and nitrilase AY487533. This is the first report of the enzymatic synthesis of primary α-aminonitriles and unnatural α-amino acids. These methods will contribute widely to the synthesis of primary α-aminonitriles and unnatural α-amino acids in aqueous systems.

Translation-dependent bioassay for amino acid quantification using auxotrophic microbes as biocatalysts of protein synthesis

Co-reporter:Masafumi Kameya

Applied Microbiology and Biotechnology 2017 Volume 101( Issue 6) pp:2523-2531

Publication Date(Web):10 December 2016

DOI:10.1007/s00253-016-8027-1

Bioassay for amino acid quantification is an important technology for a variety of fields, which allows for easy, inexpensive, and high-throughput analyses. Here, we describe a novel translation-dependent bioassay for the quantification of amino acids. For this, the gene encoding firefly luciferase was introduced into Lactococcus lactis auxotrophic to Glu, His, Ile, Leu, Pro, Val, and Arg. After a preculture where luciferase expression was repressed, the cells were mixed with analytes, synthetic medium, and an inducer for luciferase expression. Luminescence response to the target amino acid appeared just after mixing, and linear standard curves for these amino acids were obtained during 15–60-min incubation periods. The rapid quantification of amino acids has neither been reported in previous works on bioassays nor is it theoretically feasible with conventional methods, which require incubation times of more than 4 h to allow for the growth of the microbe used. In contrast, our assay was shown to depend on protein translation, rather than on cell growth. Furthermore, replacement of the luciferase gene with that of the green fluorescent protein (GFP) or β-galactosidase allowed for fluorescent and colorimetric detection of the amino acids, respectively. Significantly, when a Gln-auxotrophic Escherichia coli mutant was created and transformed by a luciferase expression plasmid, a linear standard curve for Gln was observed in 15 min. These results demonstrate that this methodology can provide versatile bioassays by adopting various combinations of marker genes and host strains according to the analytes and experimental circumstances.

1-Phenyl-2-pentanone and methyl salicylate: new defense allomone components and their content shift during ontogenetic development of the millipede Nedyopus tambanus mangaesinus (Polydesmida: Paradoxosomatidae)

Co-reporter:Yasumasa Kuwahara;Masashi Morita;Yayoi Ichiki

Applied Entomology and Zoology 2017 Volume 52( Issue 3) pp:447-455

Publication Date(Web):20 May 2017

DOI:10.1007/s13355-017-0496-x

Twelve components were identified in hexane extracts of the polydesmid millipede Nedyopus tambanus mangaesinus (Attems, 1909) and their contents were examined at all stadia of ontogenetic development including two adult conditions (before and after hibernation). Two compounds, 1-phenyl-2-pentanone and methyl salicylate, were newly identified as components of the millipede together with six well-known polydesmoid compounds (benzaldehyde, benzyl alcohol, benzoic acid, benzoyl cyanide, mandelonitrile, and mandelonitrile benzoate) and four phenols (phenol, p-cresol, 2-methoxyphenol, and 2-methoxy-4-methylphenol). Benzaldehyde and benzoyl cyanide were distributed from nymphs at stadium I, and other components started to become detectable at more advanced stadia. The largest content (35.1%) of methyl salicylate was detected in nymphs at stadium I, together with benzaldehyde and benzoyl cyanide, implying the reinforcement of defensive functions during or after egg emergence. The content (18.8%) of 1-phenyl-2-pentanone reached a maximum in hibernated females among all stadia and conditions. Its female-biased distribution at the mating season (ca. 5.7-fold more than in males) implies its sexually related functions.

Effect of Glycosylation on the Biocatalytic Properties of Hydroxynitrile Lyase from the Passion Fruit, Passiflora edulis: A Comparison of Natural and Recombinant Enzymes

Co-reporter:Aem Nuylert;Dr. Yuko Ishida; Dr. Yasuhisa Asano

ChemBioChem 2017 Volume 18(Issue 3) pp:257-265

Publication Date(Web):2017/02/01

DOI:10.1002/cbic.201600447

AbstractA hydroxynitrile lyase from the passion fruit Passiflora edulis (PeHNL) was isolated from the leaves and showed high stability in biphasic co-organic solvent systems for cyanohydrin synthesis. Cyanohydrins are important building blocks for the production of fine chemicals and pharmaceuticals. Thus, to enhance production yields of PeHNL for industrial applications, we cloned and expressed recombinant PeHNL in Escherichia coli BL21(DE3) and Pichia pastoris GS115 cells without a signal peptide sequence. The aim of this study is to determine the effect of N-glycosylation on enzyme stability and catalytic properties in microbial expression systems. PeHNL from leaves (PeHNL-N) and that expressed in P. pastoris (PeHNL-P) were glycosylated, whereas that expressed in E. coli (PeHNL-E) was not. The enzymes PeHNL-N and PeHNL-P showed much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme PeHNL-E and the deglycosylated mutant N105Q from P. pastoris (PeHNL-P-N105Q). The glycosylated PeHNL-P also efficiently performed transcyanation of (R)-mandelonitrile with a 98 % enantiomeric excess in a biphasic system with diisopropyl ether. These data demonstrate the efficacy of these methods for improving enzyme expression and stability for industrial application through N-glycosylation.

Rücktitelbild: Cyanid-freie und breit anwendbare enantioselektive Syntheseplattform für chirale Nitrile durch einen biokatalytischen Zugang (Angew. Chem. 40/2017)

Co-reporter:Tobias Betke;Philipp Rommelmann;Keiko Oike; Dr. Yasuhisa Asano; Dr. Harald Gröger

Angewandte Chemie 2017 Volume 129(Issue 40) pp:12546-12546

Publication Date(Web):2017/09/25

DOI:10.1002/ange.201707179

Eine cyanidfreie Plattformtechnologie …… für die Synthese chiraler Nitrile durch biokatalytische Dehydratisierung einer großen Bandbreite an Aldoximen liefert die Produkte in vielen Fällen mit hoher Enantioselektivität. In der Zuschrift auf S. 12533 beschreiben Y. Asano, H. Gröger et al. zudem ein überraschendes Phänomen bezüglich der Enantiospezifität: In Abhängigkeit vom E- oder Z-Isomer des racemischen Substrats wird mit demselben Enzym das entgegengesetzte Enantiomer des Nitrils bevorzugt gebildet.

Cyanid-freie und breit anwendbare enantioselektive Syntheseplattform für chirale Nitrile durch einen biokatalytischen Zugang

Co-reporter:Tobias Betke;Philipp Rommelmann;Keiko Oike; Dr. Yasuhisa Asano; Dr. Harald Gröger

Angewandte Chemie 2017 Volume 129(Issue 40) pp:12533-12538

Publication Date(Web):2017/09/25

DOI:10.1002/ange.201702952

AbstractEine Cyanid-freie Plattformtechnologie zur Synthese von chiralen Nitrilen durch eine biokatalytische, enantioselektive Dehydratisierung zahlreicher Aldoxime wurde entwickelt. Die Nitrile wurden in vielen Fällen mit Enantiomerenüberschüssen von >90 % ee (bis zu 99 % ee) erhalten, und eine “privilegierte Substratstruktur” in Hinblick auf das Erzielen einer hohen Enantioselektivität wurde identifiziert. Des Weiteren wurde ein überraschendes Phänomen bei der Enantiospezifität beobachtet, das normalerweise in der Biokatalyse nicht auftritt: In Abhängigkeit vom E- und Z-Isomer des racemischen Aldoximsubstrats wurde mit demselben Enzym das gegensätzliche Enantiomer des gebildeten Nitrils als bevorzugtes Produkt erhalten.

Cyanide-Free and Broadly Applicable Enantioselective Synthetic Platform for Chiral Nitriles through a Biocatalytic Approach

Co-reporter:Tobias Betke;Philipp Rommelmann;Keiko Oike; Dr. Yasuhisa Asano; Dr. Harald Gröger

Angewandte Chemie International Edition 2017 Volume 56(Issue 40) pp:12361-12366

Publication Date(Web):2017/09/25

DOI:10.1002/anie.201702952

AbstractA cyanide-free platform technology for the synthesis of chiral nitriles by biocatalytic enantioselective dehydration of a wide range of aldoximes is reported. The nitriles were obtained with high enantiomeric excess of >90 % ee (and up to 99 % ee) in many cases, and a “privileged substrate structure” with respect to high enantioselectivity was identified. Furthermore, a surprising phenomenon was observed for the enantiospecificity that is usually not observed in enzyme catalysis. Depending on whether the E or Z isomer of the racemic aldoxime substrate was employed, one or the other enantiomer of the corresponding nitrile was formed preferentially with the same enzyme.

Back Cover: Cyanide-Free and Broadly Applicable Enantioselective Synthetic Platform for Chiral Nitriles through a Biocatalytic Approach (Angew. Chem. Int. Ed. 40/2017)

Co-reporter:Tobias Betke;Philipp Rommelmann;Keiko Oike; Dr. Yasuhisa Asano; Dr. Harald Gröger

Angewandte Chemie International Edition 2017 Volume 56(Issue 40) pp:12374-12374

Publication Date(Web):2017/09/25

DOI:10.1002/anie.201707179

A cyanide-free platform technology for the synthesis of chiral nitriles through biocatalytic enantioselective dehydration of a wide range of aldoximes leads to the nitrile products with high enantioselectivity in many cases. In their Communication on page 12361 ff., Y. Asano, H. Gröger, and co-workers also describe an interesting phenomenon with regard to the enantiospecificity: Depending on whether the E or Z isomer of the racemic substrate is used, the other enantiomer of the nitrile is obtained with the same enzyme.

Isolation and characterization of racemase from Ensifer sp. 23-3 that acts on α-aminolactams and α-amino acid amides

Co-reporter:Daisuke Matsui;Ken-ichi Fuhshuku

Journal of Industrial Microbiology & Biotechnology 2017 Volume 44( Issue 11) pp:1503-1510

Publication Date(Web):19 September 2017

DOI:10.1007/s10295-017-1981-5

Limited information is available on α-amino-ε-caprolactam (ACL) racemase (ACLR), a pyridoxal 5′-phosphate-dependent enzyme that acts on ACL and α-amino acid amides. In the present study, eight bacterial strains with the ability to racemize α-amino-ε-caprolactam were isolated and one of them was identified as Ensifer sp. strain 23-3. The gene for ACLR from Ensifer sp. 23-3 was cloned and expressed in Escherichia coli. The recombinant ACLR was then purified to homogeneity from the E. coli transformant harboring the ACLR gene from Ensifer sp. 23-3, and its properties were characterized. This enzyme acted not only on ACL but also on α-amino-δ-valerolactam, α-amino-ω-octalactam, α-aminobutyric acid amide, and alanine amide.

Cytochrome P450 CYP71AT96 catalyses the final step of herbivore-induced phenylacetonitrile biosynthesis in the giant knotweed, Fallopia sachalinensis

Co-reporter:Takuya Yamaguchi;Koji Noge

Plant Molecular Biology 2016 Volume 91( Issue 3) pp:229-239

Publication Date(Web):2016 June

DOI:10.1007/s11103-016-0459-6

The giant knotweed Fallopia sachalinensis (Polygonaceae) synthesizes phenylacetonitrile (PAN) from l-phenylalanine when infested by the Japanese beetle Popillia japonica or treated with methyl jasmonate (MeJA). Here we identified (E/Z)-phenylacetaldoxime (PAOx) as the biosynthetic precursor of PAN and identified a cytochrome P450 that catalysed the conversion of (E/Z)-PAOx to PAN. Incorporation of deuterium-labelled (E/Z)-PAOx into PAN emitted from the leaves of F. sachalinensis was detected using gas chromatography–mass spectrometry. Further, using liquid chromatography–tandem mass spectrometry, we detected the accumulation of (E/Z)-PAOx in MeJA-treated leaves. These results showed that (E/Z)-PAOx is the biosynthetic precursor of PAN. MeJA-induced mRNAs were analysed by differential expression analysis using a next-generation sequencer. Of the 74,329 contigs obtained from RNA-seq and de novo assembly, 252 contigs were induced by MeJA treatment. Full-length cDNAs encoding MeJA-induced cytochrome P450s CYP71AT96, CYP82AN1, CYP82D125 and CYP715A35 were cloned using 5′- and 3′-RACE and were expressed using a baculovirus expression system. Among these cytochrome P450s, CYP71AT96 catalysed the conversion of (E/Z)-PAOx to PAN in the presence of NADPH and a cytochrome P450 reductase. It also acted on (E/Z)-4-hydroxyphenylacetaldoxime and (E/Z)-indole-3-acetaldoxime. The broad substrate specificity of CYP71AT96 was similar to that of aldoxime metabolizing cytochrome P450s. Quantitative RT-PCR analysis showed that CYP71AT96 expression was highly induced because of treatment with MeJA as well as feeding by the Japanese beetle. These results indicate that CYP71AT96 likely contributes the herbivore-induced PAN biosynthesis in F. sachalinensis.

Origin of Stereoselectivity and Substrate/Ligand Recognition in an FAD-Dependent R-Selective Amine Oxidase

Co-reporter:Shogo Nakano, Kazuyuki Yasukawa, Takaki Tokiwa, Takeshi Ishikawa, Erika Ishitsubo, Naoya Matsuo, Sohei Ito, Hiroaki Tokiwa, and Yasuhisa Asano

The Journal of Physical Chemistry B 2016 Volume 120(Issue 41) pp:10736-10743

Publication Date(Web):September 20, 2016

DOI:10.1021/acs.jpcb.6b09328

Elucidation of the molecular mechanism of amine oxidases (AOx) will help to extend their reactivity by rational design and their application to deracemization of various amine compounds. To date, several studies have been performed on S-selective AOx, but relatively few have focused on R-selective AOx. In this study, we sought to elucidate the mechanism of pkAOx, an R-selective AOx that we designed by introducing the Y228L and R283G mutations into d-amino acid oxidase from pig kidney. Four crystal structures of the substrate-bound protein and first-principles calculations based on the correlated fragment molecular orbital (FMO) indicated that two aromatic residues, Tyr224 and Phe242, form stable π–π stacking interaction with substrates. Enzyme kinetics also supported the importance of Tyr224 in catalysis: the kcat/Km value of the Y224L mutant was reduced by 300-fold than that of wild-type (WT) when utilizing either (R)-methylbenzylamine [(R)-MBA] or (R)-1-(2-naphthyl)ethylamine [(R)-NEA] as the substrate. On the other hand, several Phe242 mutants exhibited higher reactivity toward (R)-NEA than the WT enzyme. In addition, FMO analysis indicated that pkAOx forms ∼13 kcal/mol more stable interaction with (R)-MBA than with (S)-MBA; this energy difference contributes to specific recognition of (R)-MBA in the racemate. Through the present study, we clarified three features of pkAOx: the roles of Tyr224 and Phe242 in catalysis, the origin of high stereoselectivity, and the potential to extend its reactivity toward amine compounds with bulky groups.

Inside Cover: Mutagenesis of an Asn156 Residue in a Surface Region of S-Selective Hydroxynitrile Lyase from Baliospermum montanum Enhances Catalytic Efficiency and Enantioselectivity (ChemBioChem 13/2015)

Co-reporter:Nobuhiro Kawahara; Dr. Yasuhisa Asano

ChemBioChem 2015 Volume 16( Issue 13) pp:

Publication Date(Web):

DOI:10.1002/cbic.201581302

In Silico Identification for α-Amino-ε-Caprolactam Racemases by Using Information on the Structure and Function Relationship

Co-reporter:Wisarut Payoungkiattikun;Seiji Okazaki

Applied Biochemistry and Biotechnology 2015 Volume 176( Issue 5) pp:1303-1314

Publication Date(Web):2015 July

DOI:10.1007/s12010-015-1647-6

In silico identification for enzymes having desired functions is attractive because there is a possibility that numerous desirable enzymes have been deposited in databases. In this study, α-amino-ε-caprolactam (ACL) racemases were searched from the NCBI protein database. Four hundred thirteen fold-type I pyridoxal 5′-phosphate-dependent enzymes which are considered to contain sequences of ACL racemase were firstly obtained by submitting the sequence of ACL racemase from Achromobacter obae to the database. By identifying Lys241 as a key amino acid residue, 13 candidates for ACL racemase were selected. Then, putative ACL racemase genes were synthesized as codon-optimized sequences for expression in Escherichia coli. They were subcloned and expressed in E. coli BL21 and underwent His-tag purification. ACL and amino acid amide racemizing activities were detected among ten of the candidates. The locus tags Oant_4493, Smed_5339, and CSE45_2055 derived from Ochrobactrum anthropi ATCC49188, Sinorhizobium medicae WSM 419, and Citreicella sp. SE45, respectively, showed higher racemization activity against d- and l-ACLs rather than that of ACL racemase from A. obae. Our results demonstrate that the newly discovered ACL racemases were unique from ACL racemase from A. obae and might be useful for applications in dynamic kinetic resolution for d- or l-amino acid production.

Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis

Co-reporter:Mohammad Dadashipour;Yuko Ishida;Kazunori Yamamoto

PNAS 2015 Volume 112 (Issue 34 ) pp:10605-10610

Publication Date(Web):2015-08-25

DOI:10.1073/pnas.1508311112

Hydroxynitrile lyase (HNL) catalyzes the degradation of cyanohydrins and causes the release of hydrogen cyanide (cyanogenesis). HNL can enantioselectively produce cyanohydrins, which are valuable building blocks for the synthesis of fine chemicals and pharmaceuticals, and is used as an important biocatalyst in industrial biotechnology. Currently, HNLs are isolated from plants and bacteria. Because industrial biotechnology requires more efficient and stable enzymes for sustainable development, we must continuously explore other potential enzyme sources for the desired HNLs. Despite the abundance of cyanogenic millipedes in the world, there has been no precise study of the HNLs from these arthropods. Here we report the isolation of HNL from the cyanide-emitting invasive millipede Chamberlinius hualienensis, along with its molecular properties and application in biocatalysis. The purified enzyme displays a very high specific activity in the synthesis of mandelonitrile. It is a glycosylated homodimer protein and shows no apparent sequence identity or homology with proteins in the known databases. It shows biocatalytic activity for the condensation of various aromatic aldehydes with potassium cyanide to produce cyanohydrins and has high stability over a wide range of temperatures and pH values. It catalyzes the synthesis of (R)-mandelonitrile from benzaldehyde with a 99% enantiomeric excess, without using any organic solvents. Arthropod fauna comprise 80% of terrestrial animals. We propose that these animals can be valuable resources for exploring not only HNLs but also diverse, efficient, and stable biocatalysts in industrial biotechnology.

Mutagenesis of an Asn156 Residue in a Surface Region of S-Selective Hydroxynitrile Lyase from Baliospermum montanum Enhances Catalytic Efficiency and Enantioselectivity

Co-reporter:Nobuhiro Kawahara; Dr. Yasuhisa Asano

ChemBioChem 2015 Volume 16( Issue 13) pp:1891-1895

Publication Date(Web):

DOI:10.1002/cbic.201500225

Abstract

The S-selective hydroxynitrile lyase from Baliospermum montanum (BmHNL) has broad substrate specificity toward aromatic substrates as well as high temperature stability, although with low enantioselectivity and specific activity. To expand the industrial application of this enzyme, we improved its enantioselectivity and specific activity toward (S)-mandelonitrile by mutagenesis. The specific activity of the BmHNL H103C/N156G mutant for (S)-mandelonitrile production was raised to 154 U mg⁻¹ (WT BmHNL: 52 U mg⁻¹). The enantiomeric excess was increased to 93 % (WT BmHNL: 55 %). The kinetic analysis revealed K_m for (R)-mandelonitrile and k_cat for (S)-mandelonitrile increased by the mutation at Asn156, thus contributing to the increase in enantiomeric excess. This is the first report on an improvement in catalytic efficiency and enantiomeric excess of BmHNL for (S)-mandelonitrile synthesis by random and site-directed mutagenesis.

Rapid and selective enzymatic assay for l-methionine based on a pyrophosphate detection system

Co-reporter:Masafumi Kameya, Mariko Himi, Yasuhisa Asano

Analytical Biochemistry 2014 Volume 447() pp:33-38

Publication Date(Web):15 February 2014

DOI:10.1016/j.ab.2013.11.002

Abstract

An enzymatic assay for l-methionine was developed by coupling adenosylmethionine synthetase (AdoMetS) to a pyrophosphate (PP_i) detection system, which was constructed using pyruvate, phosphate dikinase. To expand the use of this assay, the PP_i detection system was embodied as three different forms, which allowed PP_i to be measured by UV, visible, and fluorescent light detectors. The assay system was robust and could tolerate the addition of inorganic phosphate and ATP to the assay mixtures. l-Methionine could be accurately determined by coupling the PP_i detection system and AdoMetS. This AdoMetS coupling assay was highly selective to l-methionine and exhibited no significant activity to other proteinaceous amino acids, ammonia, or urea, unlike conventional enzymatic assays for l-methionine. Spike and recovery tests showed that the AdoMetS assay could accurately and reproducibly determine increases in l-methionine in human plasma samples without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. The high selectivity and robustness of the AdoMetS assay provide rapid and high-throughput analysis of l-methionine in various kinds of analytes.

Tailoring D-Amino Acid Oxidase from the Pig Kidney to R-Stereoselective Amine Oxidase and its Use in the Deracemization of -Methylbenzylamine

Co-reporter:Kazuyuki Yasukawa;Dr. Shogo Nakano;Dr. Yasuhisa Asano

Angewandte Chemie International Edition 2014 Volume 53( Issue 17) pp:

Publication Date(Web):

DOI:10.1002/anie.201308812

Abstract

The deracemization of racemic amines to yield enantioenriched amines using S-stereoselective amine oxidases (AOx) has recently been attracting attention. However, R-stereoselective AOx that are suitable for deracemization have not yet been identified. An R-stereoselective AOx was now evolved from porcine kidney D-amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards R amines. The mutant enzyme exhibited a high preference towards the substrate α-methylbenzylamine and was used to synthesize the S amine through deracemization. The findings of this study indicate that further investigations on the structure–activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis.

Tailoring D-Amino Acid Oxidase from the Pig Kidney to R-Stereoselective Amine Oxidase and its Use in the Deracemization of -Methylbenzylamine

Co-reporter:Kazuyuki Yasukawa;Dr. Shogo Nakano;Dr. Yasuhisa Asano

Angewandte Chemie 2014 Volume 126( Issue 17) pp:

Publication Date(Web):

DOI:10.1002/ange.201308812

Abstract

Identification and characterization of CYP79D16 and CYP71AN24 catalyzing the first and second steps in l-phenylalanine-derived cyanogenic glycoside biosynthesis in the Japanese apricot, Prunus mume Sieb. et Zucc.

Co-reporter:Takuya Yamaguchi;Kazunori Yamamoto

Plant Molecular Biology 2014 Volume 86( Issue 1-2) pp:215-223

Publication Date(Web):2014 September

DOI:10.1007/s11103-014-0225-6

Japanese apricot, Prunus mume Sieb. et Zucc., belonging to the Rosaceae family, produces as defensive agents the cyanogenic glycosides prunasin and amygdalin, which are presumably derived from l-phenylalanine. In this study, we identified and characterized cytochrome P450s catalyzing the conversion of l-phenylalanine into mandelonitrile via phenylacetaldoxime. Full-length cDNAs encoding CYP79D16, CYP79A68, CYP71AN24, CYP71AP13, CYP71AU50, and CYP736A117 were cloned from P. mume ‘Nanko’ using publicly available P. mume RNA-sequencing data, followed by 5′- and 3′-RACE. CYP79D16 was expressed in seedlings, whereas CYP71AN24 was expressed in seedlings and leaves. Enzyme activity of these cytochrome P450s expressed in Saccharomyces cerevisiae was evaluated by liquid and gas chromatography–mass spectrometry. CYP79D16, but not CYP79A68, catalyzed the conversion of l-phenylalanine into phenylacetaldoxime. CYP79D16 showed no activity toward other amino acids. CYP71AN24, but not CYP71AP13, CYP71AU50, and CYP736A117, catalyzed the conversion of phenylacetaldoxime into mandelonitrile. CYP71AN24 also showed lower conversions of various aromatic aldoximes and nitriles. The Km value and turnover rate of CYP71AN24 for phenylacetaldoxime were 3.9 µM and 46.3 min−1, respectively. The Km value and turnover of CYP71AN24 may cause the efficient metabolism of phenylacetaldoxime, avoiding the release of the toxic intermediate to the cytosol. These results suggest that cyanogenic glycoside biosynthesis in P. mume is regulated in concert with catalysis by CYP79D16 in the parental and sequential reaction of CYP71AN24 in the seedling.

Selective tryptophan determination using tryptophan oxidases involved in bis-indole antibiotic biosynthesis

Co-reporter:Masafumi Kameya, Hiroyasu Onaka, Yasuhisa Asano

Analytical Biochemistry 2013 Volume 438(Issue 2) pp:124-132

Publication Date(Web):15 July 2013

DOI:10.1016/j.ab.2013.03.024

A novel tryptophan assay was developed using tryptophan oxidases. Although many l-amino acid oxidases (LAAOs) have been reported to catalyze tryptophan oxidation, most of them have broad substrate specificity and oxidize multiple amino acids besides tryptophan. To obtain a tryptophan-specific LAAO, we focused on bis-indole antibiotic biosynthesis, a bacterial secondary metabolic pathway. A putative LAAO from Streptomyces sp. TP-A0274, StaO involved in staurosporine biosynthesis, was heterologously expressed, biochemically characterized, and shown to serve as a selective tryptophan oxidase for the first time. In addition, another LAAO, VioA involved in violacein biosynthesis in Chromobacterium violaceum, was characterized for comparison with StaO. Interestingly, StaO and VioA share similar properties, namely narrow substrate specificity and high affinity for l-tryptophan, despite the phylogenetic distance between these enzymes. Owing to these features, uncommon among known LAAOs, StaO and VioA assays can be used for selective and accurate quantification of l-tryptophan via a coupled colorimetric reaction. Indeed, StaO and VioA assays provided tryptophan concentrations in human plasma as accurately as those obtained by high-performance liquid chromatography. Therefore, these enzymes were clearly shown to offer an effective method for determining tryptophan in biological samples rapidly, inexpensively, and accurately. The results shown here also suggest the possibility of metabolism-oriented screening as a strategy to obtain enzymes highly selective for individual biomolecules.

Enzymatic Synthesis of Chiral Phenylalanine Derivatives by a Dynamic Kinetic Resolution of Corresponding Amide and Nitrile Substrates with a Multi-Enzyme System

Co-reporter:Kazuyuki Yasukawa

Advanced Synthesis & Catalysis 2012 Volume 354( Issue 17) pp:3327-3332

Publication Date(Web):

DOI:10.1002/adsc.201100923

Abstract

Mutant α-amino-ε-caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D-amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co-expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)-phenylalanine and non-natural (R)-phenylalanine derivatives (4-OH, 4-F, 3-F, and 2-F-Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)-phenylalanine with 84% yield and 99% ee from (RS)-phenylalaninamide (400 mM) in 22 h. (R)-Tyrosine and 4-fluoro-(R)-phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co-expresed two genes encoding mutant ACL racemase and L-amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)-phenylalanine derivatives. Moreover, 2-aminophenylpropionitrile was converted to (R)-phenylalanine by DKR using a combination of the non-stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes.

Determination of l-methionine using methionine-specific dehydrogenase for diagnosis of homocystinuria due to cystathionine β-synthase deficiency

Co-reporter:Shino Yamasaki-Yashiki, Shinjiro Tachibana, Yasuhisa Asano

Analytical Biochemistry 2012 Volume 428(Issue 2) pp:143-149

Publication Date(Web):15 September 2012

DOI:10.1016/j.ab.2012.06.019

To determine the l-methionine (l-Met) concentration in an extract from dried blood spots (DBSs) for newborn mass screening for homocystinuria (HCU) due to cystathionine β-synthase (CBS) deficiency, a new fluorometric microplate assay using a methionine-specific dehydrogenase (MetDH) and the diaphorase/reazusrin system was established. We created by directed mutagenesis an NAD+-dependent MetDH from phenylalanine dehydrogenase (PheDH) showing higher substrate specificity toward l-Met than l-phenylalanine (l-Phe). However, it also exhibited notable activity for branched-chain amino acids (BCAAs). BCAAs in blood clearly interfered with the determination of l-Met in the DBS specimens using a single application of MetDH. To measure l-Met selectively, we used a branched-chain amino acid transaminase (BCAT) to eliminate the BCAAs in the specimens and screened for a BCAT with low activity toward l-Met. In microplate assays using MetDH, pretreatment of specimens with the BCAT from Lactobacillus delbrueckii subsp. bulgaricus coupled with l-glutamate oxidase minimized the effects of BCAAs, and l-Met concentrations were determined with high accuracy even at elevated BCAA concentrations. This enzymatic end-point assay is suitable for determining l-Met concentrations in DBSs for neonatal screening for HCU due to CBS deficiency.

A simple assay of taurine concentrations in food and biological samples using taurine dioxygenase

Co-reporter:Motoki Matsuda, Yasuhisa Asano

Analytical Biochemistry 2012 Volume 427(Issue 2) pp:121-123

Publication Date(Web):15 August 2012

DOI:10.1016/j.ab.2012.04.030

Taurine demonstrates various physiological functions and pharmacological actions. A successful application of taurine dioxygenase (EC 1.14.11.17) for taurine determination is described. The gene encoding taurine dioxygenase was cloned from Escherichia coli strain K-12, and the enzyme was used to determine taurine in commercially available beverages and some biological samples. The measured values obtained using the current method are close to the declared values with the precolumn derivatization ultra-performance liquid chromatography (UPLC) procedure. Taurine dioxygenase can be used for taurine determination in food control, biological research, and diagnoses based on urinary taurine concentration.

Synthesis of optically active medium-sized α-aminolactams via ring-closing metathesis

Co-reporter:Ken-ichi Fuhshuku, Yasuhisa Asano

Tetrahedron 2012 68(33) pp: 6651-6655

Publication Date(Web):

DOI:10.1016/j.tet.2012.06.010

Hydroxynitrile Lyases: Insights into Biochemistry, Discovery, and Engineering

Co-reporter:Mohammad Dadashipour and Yasuhisa Asano

ACS Catalysis 2011 Volume 1(Issue 9) pp:1121

Publication Date(Web):August 1, 2011

DOI:10.1021/cs200325q

Hydroxynitrile lyases are valuable enzymes for asymmetric synthesis of cyanohydrins. These hydroxyl and nitrile–containing compounds are being used in production of very useful pharmaceuticals, agrochemicals, and other biologically active compounds using chemical or chemoenzymatic follow-up reactions in industry. Although a huge amount of information exists on the reaction parameters of these enzymes, including stability to pH and organic solvents, yield, reaction time, and valuable data on the enantiopurity of their products, cyanohydrins, there is a lack of update on the biochemistry, discovery, and engineering of the HNLs. Therefore, in the Introduction, we will have a look into these enzymes, cyanohydrins, and aldoxime-nitrile pathways. A brief view of functional groups and several examples of cyanohydrin-based chemicals and pharmaceuticals will also be described. Then we will present characteristics of many S- and R-selective HNLs with comparative tables for several enzymatic properties under biochemistry section. The methods of screening and discovery of these enzymes both from nature and a library of mutants will be described as well as their potential in the synthesis of chemicals. Cloning and expression of new HNLs will also be described under the discovery section. A pool of successful applications of protein engineering methods and the subsequent improvement in the properties of mutant HNLs will be reviewed in detail afterward.Keywords: cyanohydrins; directed evolution; HNL screening/discovery; hydroxynitrile lyase screening; protein/enzyme engineering;

Dynamic Kinetic Resolution of -Aminonitriles to Form Chiral -Amino Acids

Co-reporter:Kazuyuki Yasukawa;Ryuji Hasemi

Advanced Synthesis & Catalysis 2011 Volume 353( Issue 13) pp:2328-2332

Publication Date(Web):

DOI:10.1002/adsc.201100360

Abstract

We have succeeded in the enzymatic synthesis of (R)-α-aminobutyric acid from racemic α-aminobutyronitrile. This has been demonstrated by the use of non-stereoselective nitrile hydratase (NHase) from Rhodococcus opacus 71D, D-aminopeptidase from Ochrobactrum anthropi C1-38 and α-amino-ε-caprolactam (ACL) racemase from Achromobacter obae. Racemic α-aminobutyronitrile was completely converted in 6 h at 30 °C to (R)-α-aminobutyric acid whose optical purity was more than 99%. (S)-α-Aminobutyric acid was also synthesized from α-aminobutyronitrile by NHase, ACL racemase and L-amino acid amidase from Brevundimonas diminuta TPU 5720. In a similar manner, other (R)- or (S)-α-amino acids with more than 97.5% ee could be synthesized from the corresponding α-aminonitriles. This is the first report on the dynamic kinetic resolution (DKR) of α-aminonitriles to form chiral α-amino acids. The key enzyme in this DKR is non-stereoselective NHase, which had been newly screened from soil samples, and its gene cloned.

Highly selective l-threonine 3-dehydrogenase from Cupriavidus necator and its use in determination of l-threonine

Co-reporter:Techawaree Ueatrongchit, Yasuhisa Asano

Analytical Biochemistry 2011 Volume 410(Issue 1) pp:44-56

Publication Date(Web):1 March 2011

DOI:10.1016/j.ab.2010.11.003

l-Threonine level in blood plasma is a biomarker of some diseases and nitrogen imbalance in the body. The determination of l-threonine is interesting and is required for diagnosis and management of inherited metabolic disorder. This is the first report of the specific enzymatic determination of l-threonine by a newly discovered l-threonine 3-dehydrogenase (ThrDH, EC 1.1.1.103) from Cupriavidus necator NBRC 102504. ThrDH, a key enzyme in l-threonine catabolism in microorganisms and animals, catalyzes the NAD+-dependent oxidation of l-threonine to 2-amino-3-oxobutyrate. ThrDH from C. necator was purified to homogeneity and fully characterized. l-Threonine and dl-2-amino-3-hydroxyvalerate are the only substrates for ThrDH among other l-amino acids, alcohols, and amino alcohols. The primary amino acid structure of ThrDH belongs to the extended short-chain alcohol dehydrogenase superfamily and is related to GDP-mannose-3′,5′-epimerase (GME) from Arabidopsis thaliana. Both enzymes have a glycine-rich NAD+-binding domain at the N terminal and conserved catalytic triad of YxxxK residues, but substrate-binding residues of GME were not found in the ThrDH sequence. ThrDH significantly differs from known bacterial and archaea ThrDHs that belong to zinc-binding medium chain alcohol dehydrogenase because of low sequence similarity and the lack of a zinc-binding domain in the sequence. A specific, quantitative, and sensitive enzymatic endpoint method for l-threonine determination was developed by using a ThrDH microplate assay. The assay was successfully applied for determination of l-threonine in human serum and plasma. Our specific determination is simple, convenient, inexpensive, accurate, and suitable for mass screening determination of l-threonine in a number of samples.

Determination of plasma and serum l-lysine using l-lysine ε-oxidase from Marinomonas mediterranea NBRC 103028T

Co-reporter:Motoki Matsuda, Yasuhisa Asano

Analytical Biochemistry 2010 Volume 406(Issue 1) pp:19-23

Publication Date(Web):1 November 2010

DOI:10.1016/j.ab.2010.06.045

This article describes a successful application of l-lysine ε-oxidase (EC 1.4.3.20) for l-lysine determination. l-Lysine ε-oxidase was isolated from culture supernatant of Marinomonas mediterranea NBRC 103028T and was used for l-lysine determination. Comparison of the characteristics of l-lysine ε-oxidase with l-lysine α-oxidase, a commercial enzyme used for l-lysine determination, suggests that the use of l-lysine ε-oxidase would be more valuable for the determination of l-lysine because of its selectivity and sensitivity, especially in samples with low l-lysine concentration. The enzyme acted only on l-lysine and l-ornithine, to which the relative activity was only 3.4% of that on l-lysine. The value obtained by the colorimetric assay using l-lysine ε-oxidase and horseradish peroxidase was not affected by l-ornithine. The enzyme also shows a higher affinity for l-lysine (Km = 0.0018 mM). l-Lysine determination using l-lysine ε-oxidase in human plasma and serum was examined. The measured values were close to values determined by instrumental analyses using the precolumn AccQ·Tag Ultra Derivatization Kit. These results suggest that l-lysine ε-oxidase can be used for diagnosis based on plasma l-lysine concentration. This is the first report on the application of l-lysine ε-oxidase.

Purification and partial characterization of N-hydroxy-l-phenylalanine decarboxylase/oxidase from Bacillus sp. strain OxB-1, an enzyme involved in aldoxime biosynthesis in the “aldoxime–nitrile pathway”

Co-reporter:Yasuo Kato, Takashi Tsuda, Yasuhisa Asano

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2007 Volume 1774(Issue 7) pp:856-865

Publication Date(Web):July 2007

DOI:10.1016/j.bbapap.2007.04.010

An enzyme that catalyzes the conversion of N-hydroxy-l-phenylalanine to phenylacetaldoxime was shown to be present in the Z-phenylacetaldoxime-degrading bacterium, Bacillus sp. strain OxB-1. The aldoxime-forming enzyme, which is induced by l-phenylalanine, was purified 8,050-fold to apparent homogeneity with a yield of 15.2%. The enzyme has a subunit Mr of about 86,000. The enzyme converts N-hydroxy-l-phenylalanine (Km 0.99 mM) to only one geometrical isomer, namely Z-phenylacetaldoxime. Relatively large amounts of pyridoxal 5′-phosphate (PLP) are required to be present in the reaction mixture because PLP reacts non-enzymatically with the N-hydroxy amino acid substrate to form a nitrone. Several characteristics of the enzyme were compared with those of other PLP-dependent aromatic amino acid-converting enzymes described in the literature. The enzyme is tentatively named “N-hydroxy-l-phenylalanine decarboxylase/oxidase”. Finally, the possible biosynthesis and metabolism of phenylacetaldoxime in Bacillus sp. strain OxB-1 is discussed.

Spectroscopic and substrate binding properties of heme-containing aldoxime dehydratases, OxdB and OxdRE

Co-reporter:Katsuaki Kobayashi, Biswajit Pal, Shiro Yoshioka, Yasuo Kato, Yasuhisa Asano, Teizo Kitagawa, Shigetoshi Aono

Journal of Inorganic Biochemistry 2006 Volume 100(5–6) pp:1069-1074

Publication Date(Web):May 2006

DOI:10.1016/j.jinorgbio.2005.12.006

Aldoxime dehydratase (Oxd) is a novel hemeprotein that catalyzes the dehydration reaction of aldoxime to produce nitrile. In this study, we studied the spectroscopic and substrate binding properties of two Oxds, OxdB from Bacillus sp. strain OxB-1 and OxdRE from Rhodococcus sp. N-771, that show different quaternary structures and relatively low amino acid sequence identity. Electronic absorption and resonance Raman spectroscopy revealed that ferric OxdRE contained a six-coordinate low-spin heme, while ferric OxdB contained a six-coordinate high-spin heme. Both ferrous OxdRE and OxdB included a five-coordinate high-spin heme to which the substrate was bound via its nitrogen atom for the reaction to occur. Although the ferric Oxds were inactive for catalysis, the substrate was bound to the ferric heme via its oxygen atom in both OxdB and OxdRE. Electronic paramagnetic resonance (EPR) and rapid scanning spectroscopy revealed that the flexibility of the heme pocket was different between OxdB and OxdRE, which might affect their substrate specificity.

Application of an enzyme chip to the microquantification of l-phenylalanine

Co-reporter:Shinjiro Tachibana, Masayasu Suzuki, Yasuhisa Asano

Analytical Biochemistry 2006 Volume 359(Issue 1) pp:72-78

Publication Date(Web):1 December 2006

DOI:10.1016/j.ab.2006.09.006

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase–resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 × 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)6- and (His)9-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The Vmax and kcat values of the (His)6-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)9-PheDH variant, and the (His)6-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-μl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.

PmHNL catalyzed synthesis of (R)-cyanohydrins derived from aliphatic aldehydes

Co-reporter:Samik Nanda, Yasuo Kato, Yasuhisa Asano

Tetrahedron: Asymmetry 2006 Volume 17(Issue 5) pp:735-741

Publication Date(Web):6 March 2006

DOI:10.1016/j.tetasy.2006.02.003

Hydroxynitrile lyase from the Japanese apricot (Prunus mume) catalyzes the formation of several aliphatic cyanohydrins in an asymmetric fashion. By employing a biphasic reaction system, aliphatic aldehydes with various structural features can be converted to the corresponding (R)-cyanohydrins with good overall yield and enantiomeric excess.A series of saturated, unsaturated, and cyclic aliphatic aldehydes were subjected to (R)-HNL (Prunus mume) catalyzed asymmetric cyanohydrin synthesis.(2R,3E,5E)-2-Hydroxy-3,5-octadienenitrileC8H11ONEe = 97%[α]D25=-31.1 (c 1.4, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R)-2-Cyclobutyl-2-hydroxyacetonitrileC6H9ONEe = 92%[α]D25=+3.8 (c 1.1, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R)-2-Cyclopentyl-2-hydroxyacetonitrileC7H11ONEe = 94%[α]D25=+11.2 (c 1.0, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R)-2-Cyclohexenyl-2-hydroxyacetonitrileC8H11ONEe = 90%[α]D25=-16.7 (c 1.5, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R)-2-Hydroxy-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-3-yl)acetonitrileC11H15ONEe = 99%[α]D25=-21.1 (c 1.4, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R,3E)-3-Ethyl-2-hydroxy-3-pentenenitrileC7H11ONEe = 92%[α]D25=-32.1 (c 1.0, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R,3E)-2-Hydroxy-3-octenenitrileC8H13ONEe = 72%[α]D25=-16.3 (c 1.0, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R,3E)-2-Hydroxy-3-methyl-3-hexenenitrileC7H11ONEe = 96%[α]D25=-26.8 (c 1.2, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R(2R,3E)-3-Ethyl-2-hydroxy-3-heptenenitrileC9H15ONEe = 92%[α]D25=-36.8 (c 1.0, CHCl3)Source of chirality: asymmetric hydrocyanationAbsolute configuration: 2R

Asymmetric Synthesis of L-α-Methylcysteine with the Amidase from Xanthobacter flavus NR303

Co-reporter:Atsushi Inoue;Hidenobu Komeda

Advanced Synthesis & Catalysis 2005 Volume 347(Issue 7-8) pp:

Publication Date(Web):1 JUN 2005

DOI:10.1002/adsc.200505028

An amidase acting on d, l-α-methylcysteinamide was purified from Xanthobacter flavus NR303. The enzyme acted L-stereoselectively on d,l-α-methylcysteinamide to yield L-α-methylcysteine. Based on the N-terminal amino acid sequence of the amidase, the gene encoding the enzyme was cloned from the genomic DNA of X. flavus and sequenced. Analysis of 4840 bp of the genomic DNA revealed the presence of an open reading frame (mcaA) which encodes the amidase. This enzyme is composed of 355 amino acid residues (molecular mass, 38555 Da). The intact cells of the Escherichia coli transformant could be used for the L-stereoselective hydrolysis of racemic α-methylcysteinamide. It was activated in the presence of Mn²⁺, and had maximal activity at pH 7.0 and 55 °C. The E. coli transformant catalyzed the synthesis of L-α-methylcysteine from d, l-α-methylcysteinamide in a yield of 80% with high optical purity (>98% ee).

Lipase-mediated desymmetrization of glycerol with aromatic and aliphatic anhydrides

Co-reporter:Daniela I. Batovska, Shuichirou Tsubota, Yasuo Kato, Yasuhisa Asano, Makoto Ubukata

Tetrahedron: Asymmetry 2004 Volume 15(Issue 22) pp:3551-3559

Publication Date(Web):15 November 2004

DOI:10.1016/j.tetasy.2004.09.033

Chirazyme L-2 (Candida antarctica) catalyzed esterification of glycerol with aromatic and aliphatic anhydrides in 1,4-dioxane is described. All the aromatic monoacylglycerols (MAGs) were produced as (R)-enantiomers, while aliphatic MAGs were obtained either as racemic mixtures or the (S)-enantiomers. The influence of substituted aromatic rings, chain length, and presence of a conjugated double bond in the acyl donor moiety on the enantiotopic selectivity as well as the efficiency of the enzyme was studied.

Comparative expression of wild-type and highly soluble mutant His103Leu of hydroxynitrile lyase from Manihot esculenta in prokaryotic and eukaryotic expression systems

Co-reporter:Mohammad Dadashipour, Yasuhisa Fukuta, Yasuhisa Asano

Protein Expression and Purification (May 2011) Volume 77(Issue 1) pp:92-97

Publication Date(Web):1 May 2011

DOI:10.1016/j.pep.2010.12.010

Low protein solubility and inclusion body formation represent big challenges in production of recombinant proteins in Escherichia coli. We have recently reported functional expression of hydroxynitrile lyase from Manihot esculenta, MeHNL, in E. coli with high in vivo solubility and activity using directed evolution. As a part of attempts to clarify the mechanism of this phenomenon, we have described the possibility of expression of the highly active and soluble mutant MeHNL-His103Leu as well as wild-type enzyme in several expression systems. Methylotrophic yeast Pichia pastoris, protozoan host Leishmania tarentolae and two cell-free translations, including an E. coli lysate (WakoPURE system) and wheat germ translation system were used to compare expression profiles of the genes. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryotic-based systems. The wild-type and mutant enzyme showed high activity for both genes (up to 10 U/ml) in eukaryotic hosts P. pastoris and L. tarentolae, while those of E. coli exhibited about 1 and 15 U/ml, respectively. The different activity level in prokaryotic systems but the same level among the eukaryotic hosts indicate the phenomenon is specific to the E. coli system. Both the wild-type and mutant enzymes were functionally expressed in eukaryotic systems, probably using the folding assistants such as chaperones. Properties of expression systems used in this study were precisely compared, too.

Preparation of d-α-aminolactams by l-enantioselective degradation of α-aminolactam mediated by Mesorhizobium sp. L88

Co-reporter:Ken-ichi Fuhshuku, Momoko Takata, Hiroka Iwatsubo, Yasuhisa Asano

Biocatalysis and Agricultural Biotechnology (July 2014) Volume 3(Issue 3) pp:

Publication Date(Web):1 July 2014

DOI:10.1016/j.bcab.2014.01.003

By the screening of microorganisms that can assimilate α-amino-ε-caprolactam (ACL) as a sole source of carbon and nitrogen from 115 soil samples, two microorganisms that can hydrolyze ACL l-enantioselectively and degrade further have been isolated and identified as Mesorhizobium sp. L88 and Aneurinibacillus migulanus L168. The culture and reaction conditions of Mesorhizobium sp. L88 were optimized to apply the intact cells to the preparation of enantiomerically pure d-α-aminolactams. Under the optimized conditions, d-α-aminolactams with a five- to an eight-membered ring were prepared with an enantiomeric excess of >99.8%. Ring-opened α-amino-N'-alkylamides, lysine amide, ornithine amide, ε-caprolactam, and δ-valerolactam were not accepted as substrates.

l-Arginine oxidase from Pseudomonas sp. TPU 7192: Characterization, gene cloning, heterologous expression, and application to l-arginine determination

Co-reporter:Daisuke Matsui, Anna Terai, Yasuhisa Asano

Enzyme and Microbial Technology (January 2016) Volume 82() pp:151-157

Publication Date(Web):1 January 2016

DOI:10.1016/j.enzmictec.2015.10.002

•l-Arginine oxidase (AROD) was newly identified in Pseudomonas sp. TPU 7192.•AROD converted l-arginine to 2-ketoarginine and 4-guanidinobutyric acid.•The enzymatic properties of AROD were suitable for diagnostic applications.•Enzymatic assay for l-arginine using Pseudomonas AROD was developed.l-Arginine oxidase (AROD, EC 1.4.3.-) was discovered in newly discovered Pseudomonas sp. TPU 7192 and its characteristics were described. The molecular mass (MS) of the enzyme was estimated to be 528 kDa, which was accounted for by eight identical subunits with MS of 66 kDa each. AROD was identified as a flavin adenine dinucleotide (FAD)-dependent enzyme with 1 mol of FAD being contained in each subunit. It catalyzed the oxidative deamination of l-arginine and converted l-arginine to 2-ketoarginine, which was non-enzymatically converted into 4-guanidinobutyric acid when the hydrogen peroxide (H2O2) formed by l-arginine oxidation was not removed. In contrast, 2-ketoarginine was present when H2O2was decomposed. AROD was specific to l-arginine with a Km value of 149 μM. It exhibited maximal activity at 55 °C and pH 5.5. AROD was stable in the pH range 5.5–7.5 and >95% of its original activity was below 60 °C at pH 7.0. Since these enzymatic properties are considered suitable for the determination of l-arginine, the gene was cloned and expressed in a heterologous expression system. We herein successfully developed a new simple enzymatic method for the determination of l-arginine using Pseudomonas AROD.

Efficient preparation of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid catalyzed by Shinella sp. R-6 and Arthrobacter sp. S-2

Co-reporter:Ken-ichi Fuhshuku, Shunsuke Watanabe, Tetsuro Nishii, Akihiro Ishii, Yasuhisa Asano

Journal of Molecular Catalysis B: Enzymatic (April 2014) Volume 102() pp:115-119

Publication Date(Web):1 April 2014

DOI:10.1016/j.molcatb.2014.01.024

•Two bacterial strains that can enantioselectively hydrolyze 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide have been isolated from soil samples, and identified as Shinella sp. R-6 and Arthrobacter sp. S-2, respectively.•Shinella sp. R-6 was identified as a strain capable of exhibiting R-selective hydrolysis activity, while Arthrobacter sp. S-2 was capable of exhibiting S-selective hydrolysis activity.•The preparation of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid via the two-step whole-cell reaction was investigated using these two strains.Several microorganisms that can enantioselectively hydrolyze 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide have been isolated from soil samples. These strains were capable of growing in a medium containing 3,3,3-trifluoro-2-hydroxy-2-methylpropanamide as the sole nitrogen source. Among them, Shinella sp. R-6 was identified as a strain capable of exhibiting R-selective hydrolysis activity, while Arthrobacter sp. S-2 was capable of exhibiting S-selective hydrolysis activity. The preparation of both enantiomers of 3,3,3-trifluoro-2-hydroxy-2-methylpropanoic acid via the two-step whole-cell reaction was investigated using these two strains.Download full-size image

Parameters influencing asymmetric synthesis of (R)-mandelonitrile by a novel (R)-hydroxynitrile lyase from Eriobotrya japonica

Co-reporter:Techawaree Ueatrongchit, Hidenobu Komeda, Yasuhisa Asano, Aran H-Kittikun

Journal of Molecular Catalysis B: Enzymatic (April 2009) Volume 56(Issue 4) pp:208-214

Publication Date(Web):1 April 2009

DOI:10.1016/j.molcatb.2008.05.001

(R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a hydroxynitrile lyase from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. The effects of pH, temperature, organic solvent, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the enzyme were studied. Both pH and temperature had a large effect on the initial velocity and enantiomeric excess (e.e.) of the product, (R)-mandelonitrile. High enantiomeric purity of the product was observed at low pH and temperature because the non-enzymatic reaction producing racemates of mandelonitrile was almost suppressed. The optimum pH and temperature to obtain high e.e. were pH 4.0 and 10 °C, respectively. Surprisingly, the organic solvents had a significant influence on the initial velocity of the reaction but less influence on the enantiomeric purity of product. The EjHNL was very stable in ethyl acetate, diethyl ether, methyl-t-butyl ether, diisopropyl ether, dibutyl ether and hexane for 12 h. The best solvent for the highest initial velocity and e.e. was diethyl ether with an optimum aqueous phase content of 50% (v/v). The initial reaction rate increase as the aqueous phase content rose, but when the content was more than 50%, a reduction of e.e. was observed. Increasing the concentration of the substrates accelerated the initial velocity, but caused a slight decrease in the e.e. of the product. Under the optimized conditions, the conversion and e.e. of (R)-mandelonitrile for 3 h were 40 and 99%, respectively. The aqueous phase containing the enzyme also showed considerably efficient reusability for 4 batch reactions.

A novel d-stereoselective amino acid amidase from Brevibacterium iodinum: Gene cloning, expression and characterization

Co-reporter:Hidenobu Komeda, Yasuhisa Asano

Enzyme and Microbial Technology (5 September 2008) Volume 43(Issue 3) pp:276-283

Publication Date(Web):5 September 2008

DOI:10.1016/j.enzmictec.2008.03.008

The gene encoding d-stereoselective amino acid amide amidohydrolase (d-amino acid amidase, named DaaABi) was cloned from a chromosomal DNA library of Brevibacterium iodinum TPU 5850 and sequenced. The gene, daaABi, encoded a protein composed of 266 amino acids with a Mr of 30035. The deduced amino-acid sequence of the daaABi gene did not exhibit any similarity with any other previously reported d-amino acid amidases, but did show similarity with hypothetical class A β-lactamases. DaaABi protein was produced in Escherichia coli, purified to electrophoretic homogeneity, and characterized. The purified enzyme was about 290,000 based on gel filtration chromatography and about 30,000 based on SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is active as a decamer with identical subunits. DaaABi showed maximum activity at pH 7.2 and 35 °C. It exhibited strict d-stereoselective hydrolyzing activity towards a broad range of d-amino acid amides including d-methioninamide, d-lysinamide, d-glutaminamide, and d-phenylalaninamide, while l-amino acid amides, peptides composed of l- or d-amino acids, and β-lactam compounds could not serve as substrates for the enzyme. Almost complete hydrolysis of d-phenylalaninamide with highly strict d-stereoselectivity was achieved in 3 h from 180 mM of dl-phenylalaninamide using the purified DaaABi enzyme.

Hydroxynitrile lyase from Passiflora edulis: Purification, characteristics and application in asymmetric synthesis of (R)-mandelonitrile

Co-reporter:Techawaree Ueatrongchit, Ken’ichirou Tamura, Tohru Ohmiya, Aran H-Kittikun, Yasuhisa Asano

Enzyme and Microbial Technology (5 May 2010) Volume 46(Issue 6) pp:456-465

Publication Date(Web):5 May 2010

DOI:10.1016/j.enzmictec.2010.02.008

A hydroxynitrile lyase from leaves of Passiflora edulis (PeHNL) was purified and characterized for the first time. The enzyme is a monomer of 15 kDa and 18 kDa by SDS-PAGE, and gel filtration, respectively. Asymmetric synthesis of (R)-mandelonitrile from benzaldehyde and acetone cyanohydrin in a biphasic system employing the PeHNL from rinds of P. edulis was carried out. Several parameters influenced the enantiomeric purity of the product and initial velocity of the reaction. Both pH and temperature were important parameters controlling the enantiomeric purity of the product. The optimum pH and temperature were pH 4 and 10 °C, respectively. At the optimum pH and temperature, the spontaneous non-enzymatic reaction yielding the racemic mandelonitrile was almost completely suppressed. The PeHNL was stable (more than 80% residual activity after incubation for 12 h) in the system of methyl-t-butyl ether (MTBE), dibutyl ether (DBE), hexane (HEX), and diisopropyl ether (DIPE) while diethyl ether (DEE) and ethyl acetate (EA) were not suitable solvents. The initial velocity was markedly affected by the type of organic solvent in the biphasic system, while high enantiomeric purity was obtained when organic solvents having log P lower than 3.5 were used. The highest initial velocity of reaction and enantiomeric purity of (R)-mandelonitrile were obtained in the biphasic system of DBE with the aqueous phase content of 30% (v/v). The optimum substrate concentrations were 250 mM for benzaldehyde and 900 mM for acetone cyanohydrin, and the optimum enzyme concentration was 26.7 units/ml. The highest enantiomeric purity of (R)-mandelonitrile was successfully obtained with conversion and enantiomeric excess of 31.6% and 98.6%, respectively. The enzyme showed considerable reusability in batch reaction with high enantiomeric purity of product.Herein, we reported the characteristics of a unique (R)-PeHNL from leaves of P. edulis. The PeHNL from rinds had been isolated for the first time and the enzyme showed great ability in transcyanation of (R)-mandelonitrile with high e.e. in DBE as the co-organic solvent in a biphasic system.

A new aryl acylamidase from Rhodococcus sp. strain Oct1 acting on ω-lactams: Its characterization and gene expression in Escherichia coli

Co-reporter:Yasuhisa Fukuta, Shinpei Koizumi, Hidenobu Komeda, Yasuhisa Asano

Enzyme and Microbial Technology (5 March 2010) Volume 46(Issues 3–4) pp:237-245

Publication Date(Web):5 March 2010

DOI:10.1016/j.enzmictec.2009.09.010

Rhodococcus sp. strain Oct1 utilizing ω-octalactam as a sole source of carbon and nitrogen was isolated from soil. ω-Octalactam hydrolyzing enzyme was purified to homogeneity. The purified enzyme has a molecular weight of approximately 48,100 by SDS polyacrylamide gel electrophoresis and 99,100 by gel filtration, indicating that the enzyme consists of 2 subunits. The purified enzyme catalyzed the hydrolysis of ω-octalactam to form 8-aminooctanoic acid at a rate of 3.95 U/mg. The purified enzyme also acted on ω-heptalactam, ω-laurolactam, nitroacetoanilide substitutions, and various aliphatic amides. The most suitable substrate was o-nitroacetanilide for the enzyme (11.6 U/mg). The enzyme belongs to aryl acylamidase. The gene for the enzyme was cloned and the deduced amino acid sequence showed similarity to ω-laurolactam hydrolase from Rhodococcus sp. U224 (51%) and putative aryl acylamidase from Nocardia farcinica IFM 10152 (98%), and N-terminal amino acid sequence (28 residues) of aryl acylamidase from Nocardia globerula IFO 13510 (92%). Aryl acylamidases and 6-aminohexanoate-cyclic-dimer hydrolases are in the same phylogenic lineage. These enzymes were mostly active toward non-natural amides. From phylogenic analysis, these enzymes were classified into amidase signature family. The enzyme was produced in a soluble form as a fusion protein (extension of 13 amino acids at C-terminal) in Escherichia coli.

Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities

Co-reporter:Daisuke Matsui, Do-Hyun Im, Asami Sugawara, Yasuhisa Fukuta, ... Yasuhisa Asano

FEBS Open Bio (2014) Volume 4() pp:220-228

Publication Date(Web):1 January 2014

DOI:10.1016/j.fob.2014.02.002

•l-Amino acid oxidase/monooxygenase (l-AAO/MOG) from Pseudomonas sp. AIU813 was studied.•l-Lysine oxidase activity of l-AAO/MOG increased with p-chloromercuribenzoate concentration.•Variant l-AAO/MOG C254I showed 5-times higher oxidase and decreased monooxygenase activities.•3D structure of l-AAO/MOG provided a structural basis for its biochemical characteristics.In this study, it was shown for the first time that l-amino acid oxidase of Pseudomonas sp. AIU813, renamed as l-amino acid oxidase/monooxygenase (l-AAO/MOG), exhibits l-lysine 2-monooxygenase as well as oxidase activity. l-Lysine oxidase activity of l-AAO/MOG was increased in a p-chloromercuribenzoate (p-CMB) concentration-dependent manner to a final level that was five fold higher than that of the non-treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying l-AAO/MOG C254I mutant enzyme, which showed five-times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high-resolution three-dimensional structure of l-AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of l-AAO/MOG, Cys-254, is located near the aromatic cage (Trp-418, Phe-473, and Trp-516). Although the location of Cys-254 indicates that it is not directly involved in the substrate binding, the chemical modification by p-CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp-516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase.

Synthesis of (R)-β-nitro alcohols catalyzed by R-selective hydroxynitrile lyase from Arabidopsis thaliana in the aqueous–organic biphasic system

Co-reporter:Ken-ichi Fuhshuku, Yasuhisa Asano

Journal of Biotechnology (20 May 2011) Volume 153(Issues 3–4) pp:153-159

Publication Date(Web):20 May 2011

DOI:10.1016/j.jbiotec.2011.03.011

Both enantiomers of β-nitro alcohols are versatile chiral building blocks. However, their synthesis using enzymes as catalysts has received little attention, with the exception of (S)-β-nitro alcohols produced in a reaction catalyzed by an S-selective hydroxynitrile lyase (HNL) from Hevea brasiliensis (HbHNL). An R-selective HNL containing an α/β-hydrolase fold from the noncyanogenic plant Arabidopsis thaliana (AtHNL) accepts nitromethane (MeNO2) as a donor in a reaction with aromatic aldehydes to yield (R)-β-nitro alcohols (Henry reaction; nitro aldol reaction). This reaction proceeded in an aqueous–organic biphasic system. The organic solvent giving the highest enantioselectivity was n-butyl acetate (AcOBu) with an optimum aqueous phase content of 50% (v/v). This is the first example of the R-HNL-catalyzed synthesis of (R)-β-nitro alcohols.

Enhancement of stability of l-tryptophan dehydrogenase from Nostoc punctiforme ATCC29133 and its application to l-tryptophan assay

Co-reporter:Daisuke Matsui, Seiji Okazaki, Motoki Matsuda, Yasuhisa Asano

Journal of Biotechnology (20 February–10 March 2015) Volumes 196–197() pp:27-32

Publication Date(Web):20 February 2015

DOI:10.1016/j.jbiotec.2015.01.010

•TrpDH variant was obtained by random mutagenesis and complementation in a Trp auxotroph.•The specific activity and stability of the variant were higher than those of the wild type enzyme.•The variant was available for determination of Trp in human plasma.Microbial NAD+-dependent l-tryptophan dehydrogenase (TrpDH, EC1.4.1.19), which catalyzes the reversible oxidative deamination and the reductive amination between l-tryptophan and indole-3-pyruvic acid, was found in the scytonemin biosynthetic pathway of Nostoc punctiforme ATCC29133. The TrpDH exhibited high specificity toward l-tryptophan, but its instability was a drawback for l-tryptophan determination. The mutant enzyme TrpDH L59F/D168G/A234D/I296N with thermal stability was obtained by screening of Escherichia coli transformants harboring various mutant genes, which were generated by error-prone PCR using complementation in an l-tryptophan auxotroph of E. coli. The specific activity and stability of this mutant enzyme were higher than those of the wild type enzyme. We also revealed here that in these four mutation points, the two amino acid residues Asp168 and Ile296 contributed to increase the enzyme stability, and the Leu59, Ala234 residues to increase its specific activity. Growth of the strain harboring the gene of above 4 point mutated enzyme was accelerated by the enhanced performance. In the present study, we demonstrated that TrpDH L59F/D168G/A234D/I296N was available for determination of l-tryptophan in human plasma.

Rapid enzymatic assays for l-citrulline and l-arginine based on the platform of pyrophosphate detection

Co-reporter:Masafumi Kameya, Yasuhisa Asano

Enzyme and Microbial Technology (10 April 2014) Volume 57() pp:36-41

Publication Date(Web):10 April 2014

DOI:10.1016/j.enzmictec.2014.01.008

•Rapid enzymatic assays for l-citrulline and l-arginine were developed.•Higher selectivity than that of conventional enzymatic and colorimetric assays.•Accurate quantification even in the presence of a biological sample.•Usefulness of the pyrophosphate detection system as a platform of selective assays.Rapid determination of l-citrulline and l-arginine, physiologically important amino acids, is a beneficial technique from the scientific and medical viewpoints. In this study, enzymatic assays for l-citrulline and l-arginine were established and evaluated. l-Citrulline assay was constructed by coupling argininosuccinate synthetase to a pyrophosphate detection system, in which pyruvate phosphate dikinase was employed, so that the citrulline-dependent production of pyrophosphate could be determined. Furthermore, the l-arginine assay was developed by coupling arginine deiminase to the l-citrulline assay. Both assays exhibited high selectivity to l-citrulline and l-arginine without any significant reactivity to other proteinaceous amino acids. These assays were also resistant to various contaminants that interfered with the conventional l-citrulline and l-arginine assays. The high accuracy of these assays was demonstrated by measurements in the presence of human plasma. Because these assays can be conducted under the neutral pH without terminating the reaction progress, they allow not only measurements in static analyte solutions, but also real-time monitoring of l-citrulline and l-arginine synthesis in the reaction mixture. The features of these assays also demonstrated that the pyrophosphate detection system served as a useful platform to develop selective and robust enzymatic assays by being coupled to a pyrophosphate-producing enzyme.

(2R)-N-[(1R)-1-{5-[5-Chloro-3-fluoro-2-(5-methyl-1,2,4-oxadiazol- 3-yl)phenyl]-3-fluoro-2-pyridinyl}ethyl]-3,3,3-trifluoro-2-hydrox y-2-methylpropanamide

CAS:858412-39-2

(2R)-N-[(1R)-1-{5-[5-Chloro-3-fluoro-2-(5-methyl-1,2,4-oxadiazol- 3-yl)phenyl]-3-fluoro-2-pyridinyl}ethyl]-3,3,3-trifluoro-2-hydrox y-2-methylpropanamide