Newly discovered enzymes and cascades for the determination of amino acids

The high stereo- and substrate specificities of enzymes have been utilized for micro-determination of amino acids. With the successful practical use of oxidoreductases such as the NAD+-dependent L-Phe dehydrogenase for phenylketonuria among more than 5,400,000 of neonates in Japan [1], many enzymes with higher substrate specificities have been screened and discovered from Nature [2]. Please click Additional Files below to see the full abstract

Continuity and change in screening for industrial enzymes and protein engineering- A tribute to the late Professor Hideaki Yamada

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Soluble expression of genes for enzymes in Escherichia coli

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In silico methods in enzyme screening and gene expression

INTMSAlign is a software to assign consensus residues of target protein utilizing large amount of their family sequences. We generated three protein sequences with S-selective hydroxynitrile lyase (S-HNL) activity, which we call designed S-HNLs; these proteins folded as efficiently as the native S-HNL (1). a-Amino-e-caprolactam (ACL) racemase from Achromobacter obae has been shown to be an effective catalyst for the dynamic kinetic resolution of amino acid amide and a-aminonitriles to form chiral amino acids. We searched for ACL racemase in silico with INTMSAlign software. By fixing Lys 241 as one of the key residues, we discovered thirteen ACL racemase genes from 413 fold type-I PLP genes (2). Insolubility of proteins expressed in Escherichia coli expression hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). We discovered a phenomenon of soluble expression of HNL from Manihot esculenta, in E. coli. By random mutagenesis, we found that a single point mutation H103L, and mutation with alterations at three positions (Lys-Pro mutations at positions 176, 199 and 224) cause total solubility in E. coli even when grown at 37°C (3). If a relationship between soluble expression and mutation points could be established, it will become very easy to generate a mutant for correctly folded expression in E. coli. Using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules of thumb to help identify hotspots: one focuses on the hydrophobicity of amino acids in the a-helix structure, and another one focuses the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. Using these two relationships together, we succeeded in developing methods to improve the solubility of expressed proteins in E. coli (4). References: (1) S. Nakano and Y. Asano, Sci. Rep., 5, 8193 (2015). (2) W. Payoungkiattikun, S. Okazaki, S. Nakano, A. Ina, A. H-Kittikun, and Y. Asano, Appl. Biochem. Biotechnol., 176 (5), 1303-1314 (2015). (3) Y. Asano, M. Dadashipour, M. Yamazaki, N. Doi, and H. Komeda. Prot. Eng. Des. Sel., 24 (8), 607-616 (2011). (4) D. Matsui, S. Nakano, M. Dadashipour, and Y. Asano, submitted

Aldoxime Dehydratase Mutants as Improved Biocatalysts for a Sustainable Synthesis of Biorenewables-Based 2-Furonitrile

Choi J-E, Shinoda S, Asano Y, Gröger H. Aldoxime Dehydratase Mutants as Improved Biocatalysts for a Sustainable Synthesis of Biorenewables-Based 2-Furonitrile. Catalysts. 2020;10(4): 362.2-Furonitrile is an interesting nitrile product for the chemical industry due to its use as intermediate in the field of fine chemicals and pharmaceuticals or as a potential sweetener, as well as due to its access from biorenewables. As an alternative to current processes based on, e.g., the ammoxidation of furfural with ammonia as a gas phase reaction running at > 400 °C, we recently reported an enzymatic dehydration of 2-furfuryl aldoxime being obtained easily from furfural and hydroxylamine. However, improving the catalytic properties of the aldoxime dehydratase biocatalyst from Rhodococcus sp. YH3-3 (OxdYH3-3) in terms of activity and stability remained a challenge. In this contribution, the successful development of aldoxime dehydratase OxdYH3-3 mutants that were generated by directed evolution and its enhanced activity toward 2-furfuryl aldoxime is reported. The mutant OxdYH3-3 N266S showed an improved activity of up to six times higher than the wild type when utilizing a substrate concentration of 50–100 mM of 2-furfuryl aldoxime

Reconstruction of ancestral L-amino acid oxidases to broaden substrate selectivity

Characteristic functions of enzymes, such as high thermal stability and substrate specificity, are attained during the evolutionary process. Ancestral sequence reconstruction (ASR) is applied to infer the process by designing artificial enzymes which are located on ancestral node of phylogenetic tree; here, the inferred enzymes called ancestral enzymes. Ancestral enzymes often exhibit substrate promiscuity and high thermal stability of which functions are suitable to perform enzyme engineering. In addition, applicability of the ASR is high because the method requires only sequence data to design ancestral enzymes. Thus, we believe that artificial enzymes contributing to progress in enzyme engineering can be designed by ASR. Please click Additional Files below to see the full abstract

A Simple Enzymatic Method for Production of a Wide Variety of D-Amino Acids Using L-Amino Acid Oxidase from Rhodococcus sp. AIU Z-35-1

A simple enzymatic method for production of a wide variety of D-amino acids was developed by kinetic resolution of DL-amino acids using L-amino acid oxidase (L-AAO) with broad substrate specificity from Rhodococcus sp. AIU Z-35-1. The optimum pH of the L-AAO reaction was classified into three groups depending on the L-amino acids as substrate, and their respective activities between pH 5.5 and 8.5 accounted for more than 60% of the optimum activity. The enzyme was stable in the range from pH 6.0 to 8.0, and approximately 80% of the enzyme activity remained after incubation at 40°C for 60 min at pH 7.0. D-Amino acids such as D-citrulline, D-glutamine, D-homoserine or D-arginine, which are not produced by D-aminoacylases or D-hydantoinases, were produced from the racemic mixture within a 24-hr reaction at 30°C and pH 7.0. Thus, the present method using L-AAO was versatile for production of a wide variety of D-amino acids

Deduced catalytic mechanism of d-amino acid amidase from Ochrobactrum anthropi SV3

The catalytic mechanism of d-amino acid amidase from Ochrobactrum anthropi SV3 has been deduced

An Optimal Design Method for CMOS Even-Stage Ring Oscillators Containing Latches

This paper describes a design method for complementary metal oxide semiconductor (CMOS) ring oscillators composed of even-stage inverters. First, we propose a quantitative method to evaluate oscillation stability for an even-stage ring oscillator with a CMOS latch. The method uses static noise margin analysis to evaluate the static random access memory (SRAM) cell\u27s data storage stability, by observing the similarity between the oscillator and SRAM cell circuitry. Next, the method is extended to oscillators with multiple latches. Finally, by analyzing oscillation stability using this method, we find that the range of stable oscillation conditions can be drastically widened by adding multiple single-channel latch circuits, and also by an appropriate design of their polarities and insertion positions. We also clarify through Monte Carlo simulations, that the optimized oscillator circuit is robust under process, voltage and temperature fluctuations and device characteristic variations