Ichiro Watanabe

Bio: Ichiro Watanabe is an academic researcher from Mitsubishi. The author has contributed to research in topics: Acrylonitrile & Amide. The author has an hindex of 8, co-authored 22 publications receiving 280 citations.

Optimal Conditions for Cultivation of Rhodococcus sp. N-774 and for Conversion of Acrylonitrile to Acrylamide by Resting Cells

Abstract: The culture conditions for Rhodococcus sp. N-774 cells showing high nitrile hydratase activity and the reaction conditions for acrylamide production by the resting cells were optimized. Thiamine was essential for the growth of the strain. Yeast extract and Fe2 + or Fe3 + remarkably promoted the formation of nitrile hydratase of the cells. The reaction proceeded optimally at temperatures below 30°C. Incubation for 1 hr at above 40°C resulted in inactivation of the enzyme. Through reaction at a temperature as low as 0°C, the inhibition and inactivation of the enzyme activity by the substrate, acrylonitrile, and the product, acrylamide, were remarkably reduced, and higher accumulation of acrylamide could be attained. Under the optimal conditions, a more than 20% (w/v) acrylamide solution was obtained with a conversion yield of nearly 100%. Thus, the aqueous acrylamide solution obtained showed a high enough quality for use for the commercial preparation of polyacrylamide.

High-level secretory production of phospholipase A1 by Saccharomyces cerevisiae and Aspergillus oryzae.

Abstract: Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of lecithin to form lysolecithin. The PLA1 gene, which had been cloned from Aspergillus oryzae, was expressed in Saccharomyces cerevisiae and A. oryzae. Through the modification of the medium composition and the feeding conditions of substrate, the production level of PLA1 by S. cerevisiae was increased to a level fivefold higher than that indicated in a previous report. In the case of A. oryzae, introduction of multicopies of PLA1 expression units, and the morphological change from the pellet form to the filamentous form were effective for the enhancement of PLA1 production. We succeeded in producing 3,500 U/ml of PLA1 using an industrial-scale fermentor.

Development of a transformation system for the filamentous, ML-236B (compactin) — producing fungus Penicillium citrinum

Abstract: We present here the first report of a transformation system developed for the filamentous, ML-236B (compactin)-producing fungus Penicillium citrinum. Hygromycin B-resistant colonies were obtained after treatment of protoplasts with a vector containing an Escherichia coli hygromycin B phosphotransferase gene fused to a 3-phosphoglycerate kinase promoter from Aspergillus nidulans. The transformation rate was 194 transformants per microgram circular DNA per 4 x 10(5) viable protoplasts under optimized transformation conditions. Transformation took place via the integration of plasmid DNA into the fungal chromosomal DNA. Most of the integration events appeared to produce tandemly iterated arrays of plasmid molecules at different sites in the chromosome. The transformed, drug-resistant, phenotype and the integrated plasmids were mitotically stable with or without selection in a majority of cases. The demonstration of such a transformation system is an essential first step in the application of recombinant DNA technology to strain improvement and for the production of novel ML-236B derivatives.

Screening of Microorganisms Producing D-β-Acetylthioisobutyric Acid from Methyl DL-β-Acetylthioisobutyrate

Abstract: Microorganisms producing D-β-acetylthioisobutyric acid from methyl D-β-acetylthioisobutyrate were screened from stock cultures. The D-β-acetylthioisobutyric acid-producing ability was found in 15 strains belonging to the genera Pseudomonas, Agrobacterium, Enterobacter, Cellulomonas, Rhodococcus, Brevibacterium, and Torulopsis. A strain of Pseudomonas fluorescens, IFO 3081, was selected as the best microorganism. The cells having activity (558 units/g of dry cells) could be easily prepared by cultivation at 25°C at pH 6.6 for 24 hr in a glucose-containing medium. The D-form of methyl DL-β-acetylthioisobutyrate was selectively hydrolyzed with the cells so that D-β-acetylthioisobutyric acid (97.2% enantiomeric excess) was produced in a high yield.

Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations.

Abstract: Cytochromes P450 (CYPs) belong to the superfamily of heme b containing monooxygenases with currently more than 21,000 members. These enzymes accept a vast range of organic molecules and catalyze diverse reactions. These extraordinary capabilities of CYP systems that are unmet by other enzymes make them attractive for biotechnology. However, the complexity of these systems due to the need of electron transfer from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) via redox partner proteins for the initial hydroxylation step limits a broader technical implementation of CYP enzymes. There have been several reviews during the past years tackling the potential CYPs for synthetic application. The aim of this review is to give a critical overview about possibilities and chances for application of these interesting catalysts as well as to discuss drawbacks and problems related to their use. Solutions to overcome these limitations will be demonstrated, and several selected examples of successful CYP applications under industrial conditions will be reviewed.

Roles of key active-site residues in flavocytochrome P450 BM3

Abstract: The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 microM, kcat 3960 min-1; Y51F mutant, Km 432 microM, kcat 6140 min-1; wild-type, Km 288 microM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (DeltaG) resulting from a smaller DeltaG of substrate binding. The side chain of Phe-42 acts as a phenyl 'cap' over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2. 08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 microM, kcat 14620 min-1; compared with values of 4.7 microM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a 'fast' to a 'slow' rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat 'fast', 760 (1620) min-1; kcat 'slow', 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole >10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid >30-fold more tightly than wild-type.

Phospholipases and their industrial applications.

Abstract: Phospholipids are present in all living organisms. They are a major component of all biological membranes, along with glycolipids and cholesterol. Enzymes aimed at modifying phospholipids, namely, phospholipases, are consequently widespread in nature, playing very diverse roles from aggression in snake venom to signal transduction and digestion in humans. In this review, we give a general overview of phospholipases A1, A2, C and D from a sequence and structural perspective and their industrial application. The use of phospholipases in industrial processes has grown hand-in-hand with our ability to clone and express the genes in microbial hosts with commercially attractive amounts. Further, the use in industrial processes is increasing by optimizing the enzymes by protein engineering. Here, we give a perspective on the work done to date to express phospholipases in heterologous hosts and the efforts to optimize them by protein engineering. We will draw attention to the industrial processes where phospholipases play a key role and show how the use of a phospholipase for oil degumming leads to substantial environmental benefits. This illustrates a very general trend: the use of enzymes as an alternative to chemical processes to make products often provides a cleaner solution for the industrial processes. In a world with great demands on non-polluting, energy saving technical solutions—white biotechnology is a strong alternative.

Role of cultivation media in the development of yeast strains for large scale industrial use

Abstract: The composition of cultivation media in relation to strain development for industrial application is reviewed. Heterologous protein production and pentose utilization by Saccharomyces cerevisiae are used to illustrate the influence of media composition at different stages of strain construction and strain development. The effects of complex, defined and industrial media are compared. Auxotrophic strains and strain stability are discussed. Media for heterologous protein production and for bulk bio-commodity production are summarized.