Hirosuke Okada

Physiology of ?-amylase production by immobilized Bacillus amyloliquefaciens

SummaryBacillus amyloliquefaciens 321S cells were immobilized with 3.4% κ-carrageenan gel in bead form, and α-amylase production by the immobilized cells was studied. Cells in the gel, after the population reached maximum were restricted to a layer of 50 μm thickness, from the surface of the gel, suggesting that oxygen diffusion is the growth limiting factor. The specific respiratory activity and the growth rate of the entrapped cells under such conditions were 1/2 and 1/5 ∼1/10, respectively, that of free cells. In spite of the repressed respiration and growth, the specific rate of α-amylase production of the entrapped cells reached the maximum value of free cells or higher.In continuous culture, in an aerated vessel with a volume ratio of gel beads to medium of 1:2, the maximum production rate of α-amylase was obtained at a dilution rate of 1.0 h−1, which was double the maximum specific growth rate of the strain.These results showed that bacterial α-amylase production, which is a nongrowth-associated type of synthesis was achieved with the use of immobilized cells.

The complete nucleotide sequence of the xylanase gene (xynA) of Bacillus pumilus

The complete nucleotide sequence of the xylanase (EC 3.2.1.8) gene (xynA and its flanking regions of Bacillus pumilus IPO, a hyperproducer of xylanase, was determined. A 684 bp open reading frame for xylanase gene was observed. The amino acid sequence of the N‐terminal region of xylanase was determined to be Arg‐Thr‐Ile‐Thr‐, suggesting the processing at Ala27 of pre‐xylanase. The amino acid composition and M r (22384) of xylanase deduced from DNA sequence agreed with the results obtained with the purified enzyme. The signal sequence consisted of 27 amino acids, of which 3 were basic amino acid residues in the region near the N‐terminus and 15 were hydrophobic amino acid residues. The ribosome binding sequence complementary to the 3′ ‐end of 16 S rRNA of B, subtilis was found 7 bp upstream of the initiation codon, ATG.

Stabilization of xylanase by random mutagenesis.

Four heat‐resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser‐26 to Trp, Gly‐38 to Asp and Thr‐126 to Ser; N102, Gly‐38 to Asp; N104, Gly‐38 to Ser and Arg‐48 to Lys; F1, Ser‐12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.

Reaction of glutaraldehyde with amino and thiol compounds

Abstract Stoichiometry, pH dependence, and reversibility of the reaction of glutaraldehyde with various amino and thiol compounds were investigated to elucidate the chemical nature of glutaraldehyde. For glutaraldehyde, three commercial samples were examined. They have different spectral characteristics probably due to the difference in the content of α, β-unsaturated aldehyde polymers formed by aldol condensations of glutaraldehyde, but the amount of such unsaturated structures is very small, and the chemical reactivity of these samples are almost the same. Therefore, the chemical reactivity characteristic of glutaraldehyde is not due to the α, β-unsaturated aldehydes. Glutaraldehyde reacts with the amino group in a wide pH range ( ≧ pH 3 ). The reactions at pH 7 and 9 are almost irreversible, though a little reversibility is observed. The reaction rate becomes very slow after the initial rapid phase. The average molar ratio of the amino and aldehyde groups consumed during the reaction of glutaraldehyde with the amino group is in a range of 0.3-0.2. Glutaraldehyde reacts with cysteine with a stoichiometric relationship of one mol of the thiol and amino groups of cysteine per mol of glutaraldehyde. Glutaraldehyde does not react with the thiol group without the presence of the primary amino group; the average stoichiometric relationship is 0.5–0.6 mol of the thiol group, about 0.4 mol of the amino group, and 1 mol of glutaraldehyde, under the conditions of excess in the thiol and amino groups. These results indicate the complex nature of the glutaraldehyde reaction.

Isolation of xylose reductase gene of Pichia stipitis and its expression in Saccharomyces cerevisiae.

A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast,Pichia stipitis. DNA sequence analysis showed that the gene consists of 951 bp. The gene introduced inSaccharomyces cerevisiae was transcribed to mRNA, and a considerable amount of enzyme activity was observed constitutively, whereas transcription and translation inP stipitis were inducible.S. cerevisiae carrying the xylose reductase gene could not, however, grow on xylose medium, and could not produce ethanol from xylose. Since xylose uptake and accumulation of xylitol byS. cerevisiae were observed, the conversion of xylitol to xylulose seemed to be limited.

Structure of patulolide a, a new macrolide from penicillium urticae mutants

Abstract A new macrolide named patulolide A was isolated from the culture broth of Penicillium urticae mutants. The structure and absolute configuration of it was determined.

Preparation of polyethylene glycol‐bound NAD and its application in a model enzyme reactor

Recently many kinds of water-soluble macromolecular NAD derivatives have been prepared and their coenzymatic properties and applications in biochemical reactors have been reported [l-4]. However, there have been no reports of systematic investigations about the structure and cofactor activity of macromolecular NAD derivatives. We have synthesized a NAD derivative carrying a vinyl group [5]. The NAD derivative was copolymerized with acrylamide or methacrylamide and various kinds of water-soluble macromolecular NAD derivatives (polymeric NAD derivatives) were obtained [5-71. Based on the results of the investigation into the coenzymatic properties of the polymeric NAD derivatives, it was suggested that an NAD derivative with a smaller molecular size and a lower NAD content in the polymer chain would have higher cofactor activity. Indeed, a polymeric NAD derivative prepared by copolymeri~ation with me~ac~l~ide had good cofactor activity [6], but the polymeric NAD derivative was not suitable for application in enzyme reactors because of its low NAD content in the total polymer and the wide distribution of its molecular size. Therefore, we planned to make a new watersoluble macromolecular NAD derivative with a fvted NAD content and an appropriate molecular size. From among soluble polymers suitable for binding of NAD, we used polyethylene glycol, which was commercially available in a variety of molecular weights (1500-20 000) and had only two functional groups at its ends. Here, polyethylene glycol-bound NAD (PEG-NAD) was prepared by coupling of Nd-(2-car-

Genomic DNA structure of two new horseradish-peroxidase-encoding genes.

Genomic DNAs encoding the horseradish peroxidase (HRP) isozymes, prxC2 and prxC3, were cloned and sequenced. By comparing the sequences of the HRP isozyme-encoding genes, prxC1a and prxC1b and their cDNA [Fujiyama et al., Eur. J. Biochem. 173 (1988) 681-687], , it was concluded that prxC2 and prxC3 consisted of four exons and three introns as in the prxC1 gene family. The position of introns in coding regions were the same in all four prx genes. Genes prxC2 and prxC3 coded for 347 and 349 amino acid (aa) residues, respectively, including putative signal sequences at the N termini. In the flanking regions of both genes, putative promoters and polyadenylation signals were found. Nucleotide sequence homology in the coding region was 71% between prxC1a and prxC2, and 66% between prxC1a and prxC3. The aa sequence homologies in plant and microbial peroxidases were compared.

Cloning and sequence analysis of a cDNA for cellulase (FI-CMCase) from Aspergillus aculeatus

SummaryWa have cloned and characterized the cDNA coding for a major component of cellulase, endoglucanase (FI-CMCase), produced by Aspergillus aculeatus. The cDNA was isolated from a A. aculeatus cDNA library using synthetic oligonuceotide mixtures that correspond to the internal amino acid sequence of the mature FI-CMCase protein. Nucleotide sequence analysis of the cloned cDNA insert revealed a 711 bp open reading frame that encoded a protein of 237 amino acid residues. The primary structure of FI-CMCase deduced from the nucleotide sequence of cDNA agreed with that found by amino acid sequencing of peptide fragments obtained by digestion with several proteinases and cyanogen bromide cleavage. There may be a signal peptide sequence of 16 amino acid residues at the N-terminus. The molecular mass of the mature protein calculated from the cDNA is 24002 daltons, which compares favorably with molecular mass estimates of purified FI-CMCase obtained from SDS-PAGE (25000 Da). No distinct homology was found between the amino acid sequence of FI-CMCase and known cellulase sequences of other microorganisms. This study is the first example of cDNA cloning of an endoglucanase from the genus Aspergillus.

Effect of acetylation of Bacillus subtilis α-amylase on the kinetics of heat inactivation

Abstract Bacillus subtilis α-amylase (α-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) was acetylated with p-nitrophenyl acetate and the effect of acetylation on the thermostability of the enzyme was investigated. The thermostability of the α-amylase was increased by acetylation at temperatures higher than 70 °C and decreased at temperatures lower than 67 °C. The compensation effect was also observed for the heat inactivation of acetyl α-amylases and the temperature of compensation Tc was approximately 68 °C. This effect seems to be due to the conformational change of the enzyme caused by acetylation. The significance of the compensation effect and Tc in the study of the denaturation and stability of proteins was discussed.