Yoshiki Tani

Production and Properties of a Lipopeptide Biosurfactant from Bacillus subtilis C9

Abstract A lipopeptide biosurfactant, C9-BS produced by Bacillus (B.) subtilis C9, emulsified hydrocarbons, vegetable oil and crude oil. A high yield of C9-BS was obtained from a culture of B. subtilis C9 using a carbohydrate substrate, while a hydrocarbon substrate inhibited the production of the biosurfactant. The optimum medium for the production of C9-BS was as follows (g/l): glucose, 40; NH4HCO3, 13.5; K2HPO4, 10.5; NaH2PO4, 1.5; MgSO4·7H2O, 0.5; MnSO4·4H2O, 0.05; yeast extract, 0.5. The initial pH was 8.0 and the culture temperature was 30°C. The biosurfactant C9-BS was obtained by collection of the foam that overflowed in the fermentor culture. The production of the biosurfactant by B. subtilis C9 was growth-associated. Under O2-limited conditions, C9-BS was produced in a 3-fold higher yield compared to that under O2-sufficient conditions. C9-BS is soluble in ethanol, acetone, methanol, butanol, chloroform, dichloromethane and alkaline water. The biosurfactant C9-BS lowered the surface tension of water to 28.5 dyne/cm, and the CMC (critical micelle concentration) was 40 μM. C9-BS was stable from pH 5.0 to pH 9.5, in incubation at 100°C for 1 h, and at a salt concentration of 1,000 mM for NaCl and 10 mM for CaCl2.

Degradation of polyethylene by a fungus, Penicillium simplicissimum YK

Abstract We isolated a strain of Penicillium simplicissimum , YK, for use in the biodegradation of polyethylene, characterizing the fungus and examining how to treat the polyethylene before cultivation to make degradation more complete. Degradation was monitored by high-temperature gel-permeation chromatography of the molecular weight distribution of polyethylene before and after the fungus was cultivated with it. Polyethylene with starting molecular weights of 4000 to 28,000 had lower molecular weights after 3 months of liquid cultivation with hyphae of the fungus. UV irradiation of polyethylene or its incubation with nitric acid at 80°C for 6 days before cultivation caused functional groups to be inserted into the polyethylene. The strain grew better on a solid medium with 0.5% polyethylene when it was irradiated for 500 h than when it was not irradiated. Polyethylene with a molecular weight of 100,000 or higher after nitric acid treatment had lower molecular weight after 3 months of liquid cultivation with hyphae of the fungus. The efficiency of polyethylene degradation depended on the growth phase in pure cultivation of the fungus. Functional groups inserted into polyethylene aided biodegradation. Bioremediation of polyethylene may become possible.

Characterization of a biosurfactant, mannosylerythritol lipid produced from Candida sp. SY16.

Abstract One yeast strain, SY16, was selected as a potential producer of a biosurfactant, and identified as a Candida species. A biosurfactant produced from Candida sp. SY16 was purified and confirmed to be a glycolipid. This glycolipid-type biosurfactant lowered the surface tension of water to 29 dyne/cm at critical micelle concentration of 10 mg/l (1.5 × 10−5 M), and the minimum interfacial tension was 0.1 dyne/cm against kerosene. Thin-layer and high-pressure liquid chromatography studies demonstrated that the glycolipid contained mannosylerythritol as a hydrophilic moiety. The hydrophilic sugar moiety of the biosurfactant was determined to be β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by nuclear magnetic resonance (NMR) and fast atom bombardment mass–spectroscopy analyses. The hydrophobic moiety, fatty acids, of the biosurfactant was determined to be hexanoic, dodecanoic, tetradecanoic, and tetradecenoic acid by gas chromatography–mass spectroscopy. The structure of the native biosurfactant was determined to be 6-O-acetyl-2,3- di-O-alkanoyl-β-d-mannopyranosyl-(1 → 4)-O-meso-erythritol by NMR analyses. We newly determined that an acetyl group was linked to the C-6 position of the d-mannose unit in the hydrophilic sugar moiety.

Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting.

Flow cytometry used in combination with single-cell sorting is a powerful technique for the identification and isolation of microbial cells with particular characteristics, especially when such cells grow more slowly than other cells in a large heterogeneous population. Many applications of flow cytometry with cell sorting, originally used by specialists studying mammalian cells, have been modified so that microorganisms also can be evaluated. The methods can now be used more widely because of the increasing availability of the expensive equipment. There are means for the fluorescence detection of a wide variety of properties, such as amounts of various cell components, specific sequences of peptides and nucleotides, cell functions, and enzyme activities. From the extensive literature, representative reports of an assortment of uses of flow cytometry with cell sorting are reviewed in this article, intended to introduce the technique and its many advantages to microbiologists.

yaaD and yaaE are involved in vitamin B6 biosynthesis in Bacillus subtilis.

We show that yaaD and yaaE are involved in vitamin B6 (B6) biosynthesis in Bacillus subtilis. This is the first report which identifies genes involved in B(6) biosynthesis in B. subtilis. Based on homology, yaaD and yaaE belong to the highly conserved SNZ and SNO families, respectively. Disruptants of yaaD and yaaE required pyridoxal (PL) or pyridoxine (PN), and grew in the same way as the wild type in a minimal medium supplemented with 0.05 mM PL. The SNZ family is considered to be involved in singlet-oxygen resistance. Singlet-oxygen quenchers, L(+)-ascorbic acid and reduced glutathione, did not support the growth of these disruptants. Both yaaD and yaaE were transcribed at the highest level during the middle- to late-exponential phase and at a much lower level during the stationary phase. Neither PL nor PN affected the transcriptional rates of yaaD and yaaE. It is concluded that yaaD and yaaE are involved in B6 biosynthesis in B. subtilis, and are transcribed at the highest level during the middle- to late-exponential phase.

Efficient screening for astaxanthin-overproducing mutants of the yeast Xanthophyllomyces dendrorhous by flow cytometry

Astaxanthin possesses higher antioxidant activity than other carotenoids and, for this and other reasons, has great commercial potential for use in the aquaculture, pharmaceutical, and food industries. The basidiomycetous yeast Xanthophyllomyces dendrorhous is one of the best natural producers of astaxanthin, but wild-type cells accumulate only a small amount of astaxanthin. In this study, we developed an efficient flow cytometry method to screen for astaxanthin-overproducing mutants of X. dendrorhous. We first examined the relationship between cellular astaxanthin content and the intensity of fluorescence emitted from the cell. Although the fluorescence emission maximum of astaxanthin dissolved in acetone occurred at 570 nm, intracellular astaxanthin content correlated better with emission at around 675 nm in different X. dendrorhous strains. Using this emission wavelength, we screened cells mutagenized with ethyl methanesulfonate and successfully isolated mutants that produced 1.5-3.8-fold more astaxanthin than parent cells. This method enabled us to obtain overproducers five times more efficient than conventional screening from plate culture.

Polyol production by culture of methanol-utilizing yeast.

Four methanol-utilizing yeasts, Candida boidinii, Hansenula polymorpha, Hansenula ofunaensis, and Pichia pinus, produced polyols from corresponding sugars in a methanol medium. H. polymorpha produced larger amounts of xylitol than the other yeasts. Productivity was the highest at pH 8 when 5 g (dry)/l cultured cells were incubated with 2.5 g/l urea as the nitrogen source in a medium containing 1% (v/v) methanol and 1 g/l MgSO4.7H2O. Under these conditions, 57 g/l xylitol was obtained from 110 g/l D-xylose after 3 d of cultivation. The largest amount of xylitol (58 g/l; yield, 0.62 g/g) was produced from 125 g/l, D-xylose and 5% (w/v) glycerol instead of methanol after 4 d of cultivation.

A non-conventional dissimilation pathway for long chain n-alkanes in Acinetobacter sp. M-1 that starts with a dioxygenase reaction

Abstract n-Alkane oxidation in a long chain n-alkane utilizer, Acinetobacter sp. M-1, was investigated. In Acinetobacter, n-alkanes have been postulated to be converted to the acid via a non-conventional oxidation pathway: n-alkane→n-alkyl hydroperoxide→aldehyde→acid (Finnerty, W.R.: Lipids of Acinetobacter. In Proceedings of the World Conference on Biotechnology for the Fats and Oils Industry, 184–188, 1988). However, there is little biochemical information on the enzymes involved in the postulated pathway, particularly the enzyme catalyzing the first step. In this study, we purified and n-alkane-oxidizing enzyme to apparent homogeneity by SDS-PAGE. The enzyme was a flavoprotein, and required molecular oxygen and Cu2+ for its activity, but did not require a reduced coenzyme such as NAD(P)H. A hydroperoxide was detected as a product of the enzyme reaction. We assume that the n-alkane-oxidizing enzyme is a dioxygenase. In addition, as a fatty alcohol does not appear to be an intermediate, fatty alcohol dehydrogenase is assumed not to participate in the n-alkane oxidation. The validity of the postulated pathway is supported by the following observations: (i) n-alkane monooxygenase activity was not detected, (ii) fatty alcohol dehydrogenase activities were low, and (iii) NAD(P)H-dependent long chain fatty aldehyde dehydrogenase activities were strongly induced in n-alkane-grown cells. NAD(P)H-dependent fatty aldehyde reductase activity was also found in n-alkane-grown cells, which may contribute to the formation of waxes that are cell reserve substances in n-alkane-utilizing Acinetobacter.

Degradation of dibenzothiophene by sulfate-reducing bacteria cultured in the presence of only nitrogen gas.

To remove sulfur compounds in petroleum, we isolated sulfate-reducing bacteria that could degrade dibenzothiophene in the presence of only nitrogen gas. Among the 19 strains isolated, some could grow in the presence of 10% (v/v) kerosene and of which two strains were identified as Desulfomicrobium escambium and Desulfovibrio longreachii. Gas chromatography of the ethyl-acetate extract of bacterial cultures, in which 10% or more of the dibenzothiophene initially present was degraded, gave five unknown peaks as the presumable degradation products. Thus, desulfurization of dibenzothiophene could be carried out without oxygen or hydrogen in a pathway different from the anaerobic one already reported, in which biphenyl is detected as the main product.

Functional analysis of fructosyl-amino acid oxidases of Aspergillus oryzae

ABSTRACT Three active fractions of fructosyl-amino acid oxidase (FAOD-Ao1, -Ao2a, and -Ao2b) were isolated from Aspergillus oryzae strain RIB40. N-terminal and internal amino acid sequences of FAOD-Ao2a corresponded to those of FAOD-Ao2b, suggesting that these two isozymes were derived from the same protein. FAOD-Ao1 and -Ao2 were different in substrate specificity and subunit assembly; FAOD-Ao2 was active toward Nε-fructosyl Nα-Z-lysine and fructosyl valine (Fru-Val), whereas FAOD-Ao1 was not active toward Fru-Val. The genes encoding the FAOD isozymes (i.e., FAOAo1 and FAOAo2) were cloned by PCR with an FAOD-specific primer set. The deduced amino acid sequences revealed that FAOD-Ao1 was 50% identical to FAOD-Ao2, and each isozyme had a peroxisome-targeting signal-1, indicating their localization in peroxisomes. The genes was expressed in Escherichia coli and rFaoAo2 showed the same characteristics as FAOD-Ao2, whereas rFaoAo1 was not active. FAOAo2 disruptant was obtained by using ptrA as a selective marker. Wild-type strain grew on the medium containing Fru-Val as the sole carbon and nitrogen sources, but strain ΔfaoAo2 did not grow. Addition of glucose or (NH4)2SO4 to the Fru-Val medium did not affect the assimilation of Fru-Val by wild-type, indicating glucose and ammonium repressions did not occur in the expression of the FAOAo2 gene. Furthermore, conidia of the wild-type strain did not germinate on the medium containing Fru-Val and NaNO2 as the sole carbon and nitrogen sources, respectively, suggesting that Fru-Val may also repress gene expression of nitrite reductase. These results indicated that FAOD is needed for utilization of fructosyl-amino acids as nitrogen sources in A. oryzae.