Erythritol production by Moniliella megachiliensis using nonrefined glycerol waste as carbon source.

UNLABELLED: The number of naphtha plants is being reduced due to a worldwide shift in energy sources. Consequently, a shortage of chemical materials heavily dependent on naphtha-oil, especially C4 compounds such as butene and butane-diol, is an urgent issue in chemical manufacturing. Erythritol is a rare C4 compound produced by fermentation processes using glucose as the carbon source. Since erythritol is considerably more expensive than hydrocarbons derived from naphtha-oil, a reduction in its cost is critical. We found that Moniliella megachiliensis, a highly osmotolerant yeast strain, can utilize nonrefined glycerol waste derived from palm oil or beef tallow and convert it to erythritol. Cell growth on glycerol was almost the same as on glucose, and the cells could grow in up to 300 mg ml(-1) glycerol. When 200 mg ml(-1) nonrefined glycerol was supplied, the yield of erythritol from the glycerol was approx. 60%, which is slightly higher than that obtained using glucose. The cost of glycerol waste is considerably lower than that of glucose. Thus, the conversion of glycerol waste into valuable erythritol, proposed here, is attractive and promising from the viewpoint of ensuring a supply of C4 hydrocarbons and utilizing a waste natural resource. SIGNIFICANCE AND IMPACT OF THE STUDY: A shortage of C4 hydrocarbon depending much on naptha-oil has become urgent problem due to rapid reduction of naphtha plants together with global energy revolution. Erythritol, obtained by fermentation, is a rare C4 polyol that can be converted to C4 hydrocarbons. Erythritol is considerably expensive than hydrocarbons, a reduction in cost is critical issue. To meet this, we proposed to utilize low-cost glycerol waste from bio-diesel fuel as a carbon source. Moniliella megachiliensis successfully converted glycerol waste to erythritol. This proposal is promising to obtain C4 hydrocarbon substitute, and concomitantly to dispose a large amount of glycerol waste discharged.

Cloning and nucleotide sequence of the inulin fructotransferase (DFA I-producing) gene of Arthrobacter globiformis S14-3.

A gene encoding an inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3 was cloned and the nucleotides sequenced, for the first time. The sequence indicated that the native enzyme protein is composed of 392 amino acid residues. The native enzyme is an extracellar enzyme produced in the culture supernatant of A. globiformis S14-3, but the nucleotide sequence of the gene lacks a sequence for signal peptide for secretion. The 1.5-kb DNA fragment encoding the gene was found to produce the active enzyme in the culture supernatant of an E. coli clone, under the control of the lac promoter of pUC119.

Monitoring tripeptidase activity using capillary electrophoresis. Comparison with the ninhydrin assay.

Capillary electrophoresis (CE) was used to assay the activity of a tripeptidase from a crude extract of Lactococcus lactis subsp. lactis NCDO 712 against the substrate, Gly-Gly-Phe and a comparison with a standard ninhydrin assay was made. Standard curves of the substrates and products showed a significantly variable colorimetric reaction to ninhydrin making accurate quantification of the tripeptidase problematic. The CE assay further demonstrated that the presence of contaminating enzymes in crude cell-free extracts can cause secondary reactions that are not apparent from the ninhydrin assay data. The CE assay was also able to generate enzyme kinetics data and monitor, during purification, the presence of co-eluting contaminating activities. The speed and sensitivity with CE allows routine analysis of the tripeptidase activity without any derivatization normally required for this enzyme.

Steric course of the hydrolysis of alpha,alpha-trehalose and alpha-D-glucosyl fluoride catalyzed by pig kidney trehalase.

We are unable to confirm the report of Labat et al.3 that pig kidney trehalase hydrolyzes alpha,alpha-trehalose to form solely alpha-D-glucose. Highly purified trehalase from pig renal cortex was found, in reactions monitored by 1H-n.m.r. spectra, to hydrolyze alpha,alpha-trehalose with the formation of both alpha- and beta-D-glucose. That the beta anomer constitutes the enzymically mobilized glucosyl residue is indicated by the further finding that beta-D-glucose is the product formed on hydrolysis of alpha-D-glucosyl fluoride by the enzyme. Present results show the stereochemical behavior of pig kidney trehalase in hydrolyzing alpha,alpha-trehalose to be indistinguishable from that reported by ourselves and others for trehalase preparations from a range of biological sources including rabbit renal cortex.

Catalytic versatility of Bacillus pumilus beta-xylosidase: glycosyl transfer and hydrolysis promoted with alpha- and beta-D-xylosyl fluoride.

Bacillus pumilus beta-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of beta-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with alpha- and beta-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of beta-D-xylopyranosyl fluoride at a high rate, V = 6.25 mumol min-1 mg-1 at 0 degrees C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon alpha-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, 1H NMR spectra of a digest of alpha-D-xylosyl fluoride showed the substrate to be specifically converted to alpha-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this "inverting" enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from alpha-D-xylosyl fluoride to form a beta-D-xylosidic product, followed by hydrolysis of the latter to produce alpha-D-xylose. The transient intermediate product formed enzymically from alpha-D-xylosyl fluoride in the presence of [14C]xylose was isolated and shown by its specific radioactivity and 1H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-beta-D-xylopyranosyl-D-xylopyranose containing one [14C]xylose residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Catalytic versatility of trehalase: synthesis of alpha-D-glucopyranosyl alpha-D-xylopyranoside from beta-D-glucosyl fluoride and alpha-D-xylose.

Trehalase was previously shown (see ref. 5) to hydrolyze alpha-D-glucosyl fluoride, forming beta-D-glucose, and to synthesize alpha, alpha-trehalose from beta-D-glucosyl fluoride plus alpha-D-glucose. Present observations further define the enzyme's separate cosubstrate requirements in utilizing these nonglycosidic substrates. alpha-D-Glucopyranose and alpha-D-xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with beta-D-glucosyl fluoride. As little as 0.2mM added alpha-D-glucose (0.4mM alpha-D-xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25mM beta-D-glucosyl fluoride at 0 degrees. Digests of beta-D-glucosyl fluoride plus alpha-D-xylose yielded the alpha, alpha-trehalose analog, alpha-D-glucopyranosyl alpha-D-xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when beta-D-glucosyl fluoride is the donor, and for water when alpha-D-glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.

Isolation of a membrane protein from R rubrum chromatophores and its abnormal behavior in SDS-polyacrylamide gel electrophoresis due to a high binding capacity for SDS.

A membrane protein insoluble in water was isolated by gel chromatography in the presence of 0.1% sodium dodecyl sulfate (SDS) from chromatophores of a photosynthetic bacterium, Rhodospirillum rubrum. This is one of the major membrane proteins of the chromatophore. The protein was found to bind about four grams of SDS per gram, a value which is more than twice the amount generally observed with protein polypeptides derived from water-soluble globular proteins. The electrophoretic behavior of the complex between the membrane protein and SDS is abnormal due to this high capacity for binding SDS. Estimation of the molecular weight of this protein by SDS-polyacrylamide gel electrophoresis was thus impossible. Such an anomaly in SDS binding is unlikely to be restricted to the particular membrane protein described in this paper. The possibility of such a deviation from standard behavior in the interaction with SDS should be taken into consideration in studies of other membrane proteins, since SDS is often used both in analytical and preparative procedures.