微生物转化方法生产香草酸与香草醛的初步研究

从实验室保藏的菌种中筛选到一株黑曲霉（Aspergillus niger）SW-33,能够将1g/L的阿魏酸底物转化为0.23g/L的香草酸,相应的摩尔转化率为29.35％;流加四次底物阿魏酸后,产物浓度达到1.11g/L,相应的摩尔转化率为44.9％。为了提高产物浓度,对培养基和发酵条件进行优化,使得该菌株能够将1g/l的阿魏酸底物转化为0.46g/L的香草酸,相应的摩尔转化率为57.81％。提取得到的香草酸,经HPLC测定,纯度为85.9％;提取收率为75.2％。用含香草酸的转化液,或者用提取的结晶香草酸,加入朱红密孔菌（Prcnporus cinnabarnus）SW-0203发酵培养液,可得到转化产物香草醛。     

Decomposition of vanillin by soil microorganisms

In chernozem soil, vanillin was decomposed via vanillic and protocatechuic acid before the aromatic ring opened. The rate curves of oxygen consumption for the oxidation of vanillin were seen to have more than one maximum. During incubation of the soil with vanillin, the number of bacteria increased, especially those capable of utilizing vanillin as the sole carbon source. Of the 21 such strains isolated, 15 were identified asPseudomonas sp., five asCellulomonas sp. and one asAchromobacter sp. It was found that the course of the oxidation of vanillin varied at different p.H values and in different strains was found that the course of the oxidation of vanillin varied at different p.H values and in different strains of bacteria. In some cases, the phase of the oxidation of vanillin to vanillic acid was clearly differentiated from the subsequent decomposition of vanillic acid.     

Degradation of veratric acid and other lignin-related aromatic compounds by the white-rot fungus Pycnoporus cinnabarinus

Metabolism of veratric acid and other aromatic compounds has been studied in two strains of Pycnoporus cinnabarinus. In non-agitated cultures which contained cellulose as an additional carbon source, veratric acid was demeth(ox)ylated to vanillic acid which accumulated in the medium. Under these conditions, ¹⁴CO₂ evolution from [4-O¹⁴CH₃]-veratric acid preceded that from [3-O¹⁴CH₃]-veratric acid in the case of both strains. ¹⁴CO₂ evolution was markedly accelerated and increased when 100% oxygen was employed instead of air. Oxygen had not so strong effect on the decarboxylation of ¹⁴COOH-labelled vanillic and p-hydroxybenzoic acid but it did increase decarboxylation of ¹⁴COOH-labelled veratric acid, indicating the effect of oxygen on the preceding demeth(ox)ylation. There were indications, for example rapid demethylation of veratric acid in early stages of growth when apparent phenol oxidase (laccase) activity was zero, for an existence of a separate demethylase enzyme. However, the participation of phenol oxidases in demeth(ox)ylation cannot be ruled out. Degradation pattern of vanillic acid was basically similar in P. cinnabarinus compared to Sporotrichum pulverulentum (Phanerochaete chrysosporium). Also the effect of carbon source was similar: cellulose as a carbon source enhanced degradation of vanillic acid through methoxyhydroquinone whereas in glucose medium, vanillic acid was reduced to the respective aldehyde and alcohol.Non-standard abbreviations CBQ cellobiose: quinone oxidoreductase - MHQ methoxyhydroquinone     

Isolation of Enterobacteria able to degrade simple aromatic compounds from the wastewater from olive oil extraction

Summary Enterobacteria growing on wastewater from olive oil extraction were selected. Among this microflora, strains of Klebsiella oxytoca and Citrobacter diversus able to degrade simple monomeric aromatic compounds were isolated by enrichment culture of the effluent lacking simple sugars. In this preliminary investigation, the phenolic acids tested on solid and liquid media were gentisic, protocatechuic, p-hydroxybenzoic, benzoic, vanillic and ferulic. It was shown that the biodegradation of an aromatic acid is tightly dependent on both the type and the position of the radical substituted on the aromatic ring. Citrobacter was the most efficient strain in metabolizing ferulic acid in liquid medium at a concentration of 1.5 g/l. The substrate biodegradation yield achieved exceeded 86%.     

Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.     

Biotransformation of 2-acetylthiophene by micromycetes

Summary The biotransformation of 2-acetylthiophene by 800 strains of micromycetes has been investigated. Among them, 460 strains have been selected on solid media and cultivated in a liquid synthetic medium. Of these, 106 strains were able to completely deplete 2-acetylthiophene with or without production of intermediary metabolites. 2-Thienylglyoxylic acid was not detected but 72 strains produced 2-thiophenecarboxylic acid. Among them, eight strains have been selected for further optimization of this bioconversion.     

Chitin-glucan complex production by Schizophyllum commune submerged cultivation

Chitin-glucan complex is a fungal origin copolymer that finds application in medicine and cosmetics. Traditionally, the mycelium of Micromycetes is considered as an industrial chitin-glucan complex source. Basidiomycete Schizophyllum commune submerged cultivation for chitin-glucan complex production was studied. In different S. commune strains chitin-glucan complex composed 15.2 +/- 0.4 to 30.2 +/- 0.2% of mycelium dry weight. Optimized conditions for chitin-glucan complex production (nutrient medium composition in g/l: sucrose - 35, yeast extract - 4, Na2HPO4*12H2O - 2.5, MgSO4*7 H2O - 0.5; medium initial pH 6.5; aeration intensity 21 of air per 11 of medium; 144 hours of cultivation) resulted in 3.5 +/- 0.3 g/l complex yield. Redirection of fungal metabolism from exopolysaccharide synthesis to chitin-glucan complex accumulation was achieved most efficiently by aeration intensity increase. Chitin-glucan complex from S. commune had the structure of microfibers with diameter 1-2 microm, had water-swelling capacity of 18 g/g, and was composed of 16.63% chitin and 83.37% glucan with a degree of chitin deacetylation of 26.9%. S. commune submerged cultivation is a potent alternative to Micromycetes for industrial-scale chitin-glucan complex production.     

An attempt to channel the transformation of vanillic acid into vanillin by controlling methoxyhydroquinone formation in Pycnoporus cinnabarinus with cellobiose

The effects of adding cellobiose on the transformation of vanillic acid to vanillin by two strains of Pycnoporus cinnabarinus MUCL39532 and MUCL38467 were studied. When maltose was used as the carbon source in the culture medium, very high levels of methoxyhydroquinone were formed from vanillic acid. When cellobiose was used as the carbon source and/or added to the culture medium of P. cinnabarinus strains on day 3 just before vanillic acid was added, it channelled the vanillic acid metabolism via the reductive route leading to vanillin. Adding 3.5 g l⁻¹ cellobiose to 3-day-old maltose cultures of P. cinnabarinus MUCL39532 and 2.5 g l⁻¹ cellobiose to 3-day-old cellobiose cultures of P. cinnabarinus MUCL38467, yielded 510 mg l⁻¹ and 560 mg l⁻¹ vanillin with a molar yield of 50.2 % and 51.7 % respectively. Cellobiose may either have acted as an easily metabolizable carbon source, required for the reductive pathway to occur, or as an inducer of cellobiose:quinone oxidoreductase, which is known to inhibit vanillic acid decarboxylation. Received: 24 July 1996 / Received revision: 29 November 1996 / Accepted: 29 November 1996     

Isolation and characterization of bacteria capable of metabolizing lignin-derived low molecular weight compounds

Lignin, a major component of biomass, composed of homogeneous phenolic monomers and functions as a synthetic precursor in the production of specialty chemicals or polymers. In this study, bacterial strains that metabolize lignin-derived low molecular weight compounds (LLCs) were cultured which are capable of LLC bioconversion. We used an LLC mixture primarily composed of vanillin (VL), syringaldehyde (SA), vanillic acid (VA) and p-hydroxybenzoic acid which were prepared from a commercial alkaline lignin product. Enrichment culture was repeated twice in a medium containing the soil sample, the LLCs and inorganic salts. Three bacterial strains belonging to the genera Pseudomonas, Ochrobactrum, and Klebsiella were isolated. We found that only VL, SA, and VA were metabolized by the Pseudomonas strain, which was then found to grow in a medium with VL or VA as the sole source of carbon and energy. The VL isomers, namely, ovanillin and isovanillin were converted to the corresponding carboxylic acids but were not utilized as carbon sources by Pseudomonas. VL and VA are intermediates in the pathway of bacterial degradation of eugenol via ferulic acid. Several bacterial strains that metabolize VL, eugenol, and ferulic acid have been reported but such strains are rarely isolated from enrichment culture medium containing LLCs, due to insufficient induction by the precursors in the LLC medium. In this study, we demonstrated that the microorganisms involved in the bioconversion of LLCs can be isolated from simple enrichment culture.     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Production of patulin by Micromycetes

In the context of the screening for antifungal and antibacterial molecules among the Micromycetes, we investigated the ability of 850 strains grown in liquid medium to produce patulin. The compound was produced by 58 fungi, most of which had not been mentioned in the literature, especially Mucorales and Fungi Imperfecti. Biological tests showed that dermatophytes are the most sensitive to this molecule. It also appears that the origin of the producing strains influences their toxin producing activity.     

木质纤维素酸解副产物对D-乳酸生产菌Sporolactobacillus sp.Y2-8生长及发酵的影响

选择乙酸根、糠醛、5-羟甲基糠醛、苯酚、香草酸和丁香醛等6种典型木质纤维素酸解副产物,考察它们对D-乳酸生产菌Sporolactobacillus sp.Y2-8生长及发酵的影响。实验结果表明:酚类物质抑制作用最强烈,0.25 g/L丁香醛已经完全抑制了菌体的生长和D-乳酸的发酵;苯酚和香草酸在低浓度(≤1.0 g/L)时抑制作用较小,但质量浓度达到3 g/L时对D-乳酸产量的抑制率分别为99%和70%;3 g/L糠醛和5-羟甲基糠醛对产物的抑制率分别为60%与20%,抑制作用小于酚类;乙酸根的影响最小,10 g/L的乙酸钠对菌体的生长和发酵几乎无抑制作用;当抑制物混合时,存在着相互促进作用,抑制作用更强烈。     

Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of bacillus coagulans

A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and ¹H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.     

Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [14C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.     

Polysaccharide-protein complexes isolated from different strains of Neisseria meningitidis serogroup B

Fractionation of the biomass of 3 serogroup B N. meningitidis strains, obtained from solid serum-free and liquid synthetic media, by increasing concentrations of cetavlone revealed that the formation of natural polysaccharide-protein complexes with the ratio of their components approaching 1:1 was possible under the conditions ensuring the intensive synthesis of capsular polysaccharide. Two strains, 125 and 1642, grown on a solid amino peptide-containing medium regularly produced two polysaccharide-protein complexes with the protein/polysaccharide ratio approaching 1:1. One of these complexes passed easily into the supernatant fluid and could be precipitated with 0.1% cetavlone. The second complex was more firmly bound to the outer membrane of the cell and could be precipitated with 1% cetavlone. In most experiments an additional fraction with high protein content in relation to sialic acid was isolated from the biomass.     

Screening of microorganisms for biodegradation of poly(lactic-acid) and lactic acid-containing polymers.

The ability of some microorganisms to use lactic acid stereocopolymers and copolymers with glycolic acid as sole carbon and energy sources was studied under controlled or natural conditions. First, 14 filamentous fungal strains were tested in liquid cultures, adopting total lactic acid consumption, nitrogen source exhaustion, and maximal biomass production as selection criteria. Two strains of Fusarium moniliforme and one strain of Penicillium roqueforti were able to totally assimilate DL-lactic acid, partially soluble racemic oligomers (MW = 1,000), and the nitrogen source. Only one strain of F. moniliforme was able to grow on a poly(lactic acid)-glycolic acid copolymer (MW = 150,000) after 2 months of incubation at 28 degrees C on synthetic agar medium. Mycelium development was examined by scanning electron microscopy. F. moniliforme filaments were observed to grow not only at the copolymer surface but also through the bulk of the copolymer. In a second approach, plates made of a racemic poly(lactic acid) were buried in the soil before being incubated in petri dishes containing mineral agar medium under controlled conditions. Five strains of different filamentous fungi were isolated, and their ability to assimilate racemic poly(lactic acid) oligomers was tested in liquid cultures.     

Design of a low-cost culture medium based in whey permeate for biomass production of enological Lactobacillus plantarum strains

The production of malolactic starter cultures requires the obtention of suitably large biomass at low-cost. In this work it was possible to obtain a good amount of biomass, at laboratory scale, of two enological strains of Lb. plantarum, by formulating a culture medium based on whey permeate (WP), a by-product of the cheese industry usually disposed as waste, when this was supplemented with yeast extract (Y), salts (S) and Tween 80 (T) (WPYST). Bacteria grown in WPYST medium exhibited good tolerance to stress conditions of synthetic wine (pH 3.5, ethanol 13% vol/vol). However, when WPYST was added with 8% vol/vol ethanol, cultures inoculated in synthetic wine, showed a lower viability and capacity to consume L-malic acid than when they were cultured in WPYST without ethanol. Subsequently, strains grown in WPYST were inoculated in sterile wine samples (final stage of alcoholic fermentation) of the red varietals Merlot and Pinot noir, and incubated at laboratory scale. Cultures from WPYST, inoculated in Pinot noir wine, showed a better performance than bacteria grown in MRS broth, and exhibited a consumption of L-malic acid higher than 90%. However, cultures from WPYST or from MRS broth, inoculated in sterile Merlot wine, showed a lower survival. This study allowed the formulation of a low-cost culture medium, based on a by-product of the food industry, which showed to be adequate for the growth of two enological strains of Lb. plantarum, suggesting their potentiality for application in the elaboration of malolactic starter cultures.     

Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [¹⁴C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.     

Effect of different K+ concentrations on Cryptococcus neoformans phenoloxidase activity

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe3+ and Cu2+ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K+ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomly chosen, were tested for phenoloxidase activity, according to the modified protocols of Polacheck et al., Torres-Guerrero et al. and Rhodes. According to the results obtained, it has been observed that K+ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.     

Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose–xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L⁻¹ acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L⁻¹) in all cultures. Xylose fermentation in acetic –acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.