Tadashi Kamikubo

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.

Elucidation of adsorption processes of cellulases during hydrolysis of crystalline cellulose

To elucidate the effect of adsorption of cellulases during hydrolysis of crystalline cellulose, the relationship between the rate of hydrolysis and the adsorption of crude cellulases onto crystalline cellulose was investigated under various experimental conditions. Several phases of adsorption have been proposed to explain the process of cellulose hydrolysis by these enzymes. The process of hydrolysis calculated on the basis of these phases fitted well with that obtained experimentally.

Light regulates the gene expression of ribulosebisphosphate carboxylase at the levels of transcription and gene dosage in greening pea leaves

Ribulosebisphosphate carboxylase, composed of large and small subunits, is induced by light in pea leaves. During induction, the synthesis rate of the two mRNAs and the gene dosage were measured. The relative rates of synthesis of the two mRNAs changed with the time of illumination, while the relative gene dosage changed only for the large subunit. The increase in the synthesis rate of the large subunit mRNA was shown to be at least partly due to an increase in gene dosage. These results indicate that the light induction of ribulosebisphosphate carboxylase in the pea is controlled at the levels of transcription and, for the large subunit, also of gene dosage.

Evaluation of chemical pretreatment for enzymatic solubilization of rice straw

SummaryRice straw was treated with NaOH, peracetic acid (PA), and sodium chlorite (NaClO2). Quantitative changes in the composition of the treated straw, crystallinity of the treated straw and extracted cellulose, and susceptibility of the treated straw to Trichoderma reesei cellulase were studied. The alkali treatment resulted in a remarkable decrease in hemicellulose as well as lignin. Consequently, the recovery of residual straw after NaOH treatment was lowest among the three chemical reagents evaluated. The treatment with PA or NaCIO2 resulted in a slight loss in hemicellulose and cellulose in the straw. The three chemical treatments caused little or no breakdown of the crystalline structure of cellulose in the straw. The treated straw was solubilized with the culture filtrate of T. reesei. The degree of enzymatic solubilization relative to the amount of residual straw was 69% after treatment with 0.25 N NaOH, 42% after treatment with 20% PA, and 50% after treatments with NaClO2 (twice). The degree of enzymatic solubilization relative to the amount of the untreated straw, however, was 30% after treatment with 0.25 N NaOH, 32% after treatment with 20% PA, and 37% after treatments with NaClO2 (twice).

Production of single-cell protein from enzymatic hydrolyzate of rice straw

SummaryThe components of rice straw, pretreated with sodium chlorite, cellulose and hemicellulose were solubilized with culture filtrate of Pellicularia filamentosa or Trichoderma reesei. The ratio of glucose to total sugar in the solution obtained from the cellulose component with the culture filtrate of Pellicularia filamentosa was approximately twice that of Trichoderma reesei.Ten yeast strains (Candida utilis, C. tropicalis, C. guilliermondii, C. parapsilosis, Torulopsis xylinus, Trichosporon cutaneum, Debaryomyces hansenii, Rhodotorula glutinis, Saccharomyces fragilis and Saccharomyces cerevisiae) were cultivated as test organisms for single-cell protein (SCP) production on sugar solutions obtained from the straw, cellulose and hemicellulose components, pretreated with the culture filtrate of Pellicularia filamentosa. Sugar consumption, in terms of total sugar and cell yield, of the culture with the sugar solution obtained from pretreated straw were; 70% and 6.8 g/l for Candida tropicalis, 56% and 6.4 g/l for Torulopsis xylinus, 76% and 10.1 g/l for Trichosporon cutaneum, and 74% and 7.6 g/l for Candida guilliermondii. In addition, the highest consumption with respect to total sugar (87%) and the best dry cell yield (15.6 g/l) were observed with the culture of Trichosporon cutaneum using the sugar solution obtained from the hemicellulose component.

Long term continuous ATP regeneration by enzymes of the alcohol fermentation pathway and kinases of yeast

SummaryContinuous ATP regeneration from adenosine using enzymes of the alcohol fermentation pathway, adenosine kinase and adenylate kinase of bakers yeast was investigated using a reactor equipped with a semipermeable membrane. The addition of DTT, which protected the thiol groups of some enzymes against oxidation, increased the duration of the period of ATP formation from 42 h (previously the longest period) to 100 h. With the addition of a yeast extract containing intermediates of alcohol fermentation, a stable steady state was attained in which a yield of more than 75% ATP continued for 2 weeks. These results suggest that the long term continuous ATP formation attained might be due to the protection and stabilization of enzymes by the yeast extract which was added to prevent inactivation.

Large-scale purification and subunit structure of DNA-dependent RNA polymerase II from cauliflower inflorescence

DNA-dependent RNA polymerase II (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) from cauliflower inflorescence (Brassica oleracae, var. botrytis) was highly purified by polyethyleneimine treatment on a large scale. The solubilized enzyme was partially purified by polyethyleneimine fractionation and subjected to chromatography on DEAE-Sephadex and phosphocellulose, and subsequently to sedimentation in a glycerol gradient. The specific activity (231 nmol/mg per 10 min) of this enzyme was comparable to that reported for other purified eukaryotic RNA polymerases. Analysis of the purified RNA polymerase II by polyacrylamide gel electrophoresis under nondenaturing conditions revealed a single band. The subunit composition of the enzyme was analyzed by electrophoresis under denaturing conditions. The RNA polymerase II contained subunits with molecular weights and molar ratios (in parentheses) of 180 000(1), 130 000(2), 48 000(2), 25 000(4), and 19 500(4).

Optimal conditions for pretreatment of rice straw with n-butylamine for enzymatic solubilization

SummaryThree different pretreatment methods with n-butylamine (n-BA) were used to obtain fermentable sugars in a high yield from rice straw. The optimal conditions of each method were as follows: treating at boiling point for 1 h under refluxing in 10 w/w% n-BA with the weight ratio of n-BA to original rice straw more than 1.0, autoclaving at 120°C for 1 h in 1 w/w% n-BA with the weight ratio more than 0.1, and wetting for 2 h with the circulating condensate of the vapour evaporated from 2.5 w/w% n-BA with the weight ratio more than 0.8. Soaking rice straw with n-BA before the above pretreatments was not needed. For the circulation pretreatment, the overall cumulative yield of total sugars (70% of cellulose and hemicellulose in original rice straw) was best for both pretreatment and enzymatic solubilization steps, because there was no decomposition of monosaccharides during the pretreatment. Furthermore, the optimal degree of delignification for enzymatic solubilization of the pretreated rice straw was approximately 60% of lignin in the original.

DNA-dependent RNA polymerase III from cauliflower. Characterization and template specificity.

Class III DNA-dependent RNA polymerase (EC 2.7.7.6) was highly purified from cauliflower (Brassica oleracea, var. bortytis) by using polyethyleneimine precipitation. The specific activity of the enzyme was comparable to that reported for mammalian enzymes. Glycerol gradient sedimentation analysis indicated that the sedimantation coefficient (23 S) was slightly higher than that of enzyme II from cauliflower. The class III enzyme was inhibited by alpha-amanitin at high concentrations (50% inhibition at 200 microgram/ml). The Km value for nucleoside triphosphate was determined. Template specificities for single synthetic polymers showed that the enzyme read pyrimidine homopolymers as templates and preferred poly(dT) to poly(dC). The enzyme transcribed both strands of homopolymer pairs of poly(dI). poly(dC) and poly(dA).poly(dT). The synthetic polyribonucleotides were not effectively read. Competition experiments with these synthetic polymers indicated that the enzyme had different binding specificities which were not the same as their template specificities. The different binding affinities and template specificites for synthetic templates of the three classes of enzyme suggest that the enzyme can discriminate among different template sequences.

Cellulases from Eupenicillium javanicum

Publisher Summary Extracellular cellulases that a fungus produces usually consist of several components with different substrate specificities. These cellulase components act synergistically in the hydrolysis of crystalline cellulose. Eupenicillium javanicum produces several cellulase components in growth medium. This chapter describes cellulases from Eupenicillium javanicum . The cellulase activity from E. javanicum is comparable to that from Trichoderma reesei QM 9414.