Biodegradation of tetracycline by the yeast strain Trichosporon mycotoxinivorans XPY-10

We investigated the behavior of tetracycline degradation and its degradation products upon treatment of isolated yeast that we termed “XPY-10.” XPY-10 was isolated from wastewater and identified as Trichosporon mycotoxinivorans by morphological and physiological tests and 5.8S rRNA ITS sequencing. In our experiments, 78.28 ± 0.8% of tetracycline was removed within 7 days with XPY-10. The degradation of tetracycline fitted well with the first-order kinetic model. We also speculated upon the biodegradation products formed during biodegradation. The possible structures of five products were determined using liquid chromatography–tandem mass spectrometry. During practical application, XPY-10 was shown to have an obvious influence on biodegradation, and 89.61% of tetracycline was removed in feedlot sewage after 7 days of reaction. The chemical oxygen demand removal reached 73.47%.     

Bioconversion of biodiesel refinery waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2

ABSTRACT: BACKGROUND: The microbial bioemulsifiers was surface active compounds, are more effective in stabilizing oil-in-water emulsions. The yeasts have been isolated to produce bioemulsifiers from vegetable oils and industrial wastes. RESULTS: Trichosporon mycotoxinivorans CLA2 is bioemulsifier-producing yeast strain isolated from effluents of the dairy industry, with ability to emulsify different hydrophobic substrates. Bioemulsifier production (mg/L) and the emulsifying activity (E24) of this strain were optimized by response surface methodology using mineral minimal medium containing refinery waste as the carbon source, which consisted of diatomaceous earth impregnated with esters from filters used in biodiesel purification. The highest bioemulsifier production occurred in mineral minimal medium containing 75 g/L biodiesel residue and 5 g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58 g/L biodiesel refinery residue and 4.6 g/L ammonium sulfate, and under these conditions, the model estimated an emulsifying activity of 85%. Gas chromatography and mass spectrometry analysis suggested a bioemulsifier molecule consisting of monosaccharides, predominantly xylose and mannose, and a long chain aliphatic groups composed of octadecanoic acid and hexadecanoic acid at concentrations of 48.01% and 43.16%, respectively. The carbohydrate composition as determined by GC-MS of their alditol acetate derivatives showed a larger ratio of xylose (49.27%), mannose (39.91%), and glucose (10.81%). 1 H NMR spectra confirmed by COSY suggested high molecular weight, polymeric pattern, presence of monosaccharide's and long chain aliphatic groups in the bioemulsifier molecule. CONCLUSIONS: The biodiesel residue is an economical substrate, therefore seems to be very promising for the low-cost production of active emulsifiers in the emulsification of aromatics, aliphatic hydrocarbons, and kerosene.     

Novel Ring Cleavage Products in the Biotransformation of Biphenyl by the Yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4′-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-Trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Comparative Utilization of Paraffins by a Trichosporon Species

Utilization of normal and isoparaffins, separately and in mixtures, by a Trichosporon sp. was investigated. From a mixture of normal paraffins and isoparaffins, the organism consumed straight-chain paraffins, leaving the branched paraffins relatively unchanged. When offered separately, the highest utilization of n-alkanes by the organism was obtained in the range of undecane to octadecane; n-pentadecane was poorly utilized. From a mixture of n-alkanes, the rate of consumption of shorter-chain alkanes, n-decane to n-dodecane, was found to be relatively faster and more uniform than that of longer-chain alkanes.     

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Ornatipolide,a Novel Phenolic Metabolite from the Basidiomycete,Boletus ornatipes

A novel phenolic metabolite, ornatipolide, was isolated from the Boletus ornatipes fungus. Its structure was established by a combination of spectroscopic and chemical methods, and by an X-ray crystallographic analysis.     

Cleavage of BSA by a dipeptide seryl-histidine

A dipeptide seryl-histidine (Ser-His) was found to have theprotein cleavage activity. BSA was cleaved into smear at aroundpH 5.0–6.0, with a half-life around 15 hr at 60 °C.Phosphate could accelerate the reaction. This is a brand newprotein cleavage system. Since Ser and His are well-knowncatalytic residues at the active sites of many serine proteases,this results might provide clues to the possible roles of shortoligopeptides in the origins of modern enzymes.     

Cleavage of BSA by a dipeptide seryl-histidine

Summary A dipeptide seryl-histidine (Ser-His) was found to have the protein cleavage activity. BSA was cleaved into smear at around pH 5.0–6.0, with a half-life around 15 hr at 60 °C. Phosphate could accelerate the reaction. This is a brand new protein cleavage system. Since Ser and His are well-known catalytic residues at the active sites of many serine proteases, this results might provide clues to the possible roles of short oligopeptides in the origins of modern enzymes.     

Cleavage of Leishmania Mini-exon Sequence by Oligonucleotides Conjugated to a Dimidazole Construction

Abstract

RNA sequences derived from the Leishmania amazonensis mini-exon and pre-mini-exon sequences have been targeted with complementary oligonucleotides bearing a diimidazole construction mimicking active center of ribonuclease A. The conjugates were shown to cleave the target RNAs at specific positions.     

Cleavage of a tail-anchored protein by signal peptidase

Tail-anchored proteins are post-translationally targeted and inserted into the endoplasmic reticulum membrane. They do not use the co-translational signal-recognition particle (SRP)-dependent pathway, but rather utilize an ill-defined, ATP-dependent mechanism. Here, we show that a tail-anchored protein can be cleaved by signal peptidase and that the sequence requirements for efficient cleavage seem to be the same as for cleavage of co-translationally targeted SRP-dependent proteins.     

Inhibition of Hydroxyquinol Auto-Oxidation by a Cell Extract of Trichosporon cutaneum

A cell extract from acetate-grown Trichosporon cutaneum WY2-2inhibited auto-oxidation of phenolics, especially that of hydroxyquinol.It prevented auto-oxidation of hydroxyquinol without directinteraction with hydroxyquinol. Bovine erythrocyte superoxidedismutase had similar characteristics as the cell extract, andthe elution patterns of superoxide dismutase activity and ofthe inhibitory activity to hydroxyquinol auto-oxidation froma Sephadex G-150 column coincided. These results indicate thatthe inhibitory activity in the cell extract is mainly due tosuperoxide dismutase. High activity of superoxide dismutase(20–30 unit/mg protein) and its isozyme profiles suggestan intimate relation between the regulation of superoxide dismutaseand catabolism of phenolics via hydroxyquinol. ¹Present address: Biological Institute, Faculty of Science,Nagoya University, Nagoya 464, Japan.²Present address: Shin Nihon Chemical Co. Ltd., 19-10, Showa-cho,Anjoh, Aichi 446, Japan. (Received November 15, 1985; Accepted July 3, 1986)     

发酵性丝孢酵母(Trichosporon fermentans)发酵产油脂的研究

通过摇瓶发酵,考察了碳源浓度、氮源种类和浓度对发酵性丝孢酵母(Trichosporonfermentans)发酵产油脂的影响,对发酵产油脂条件的初步优化结果为:在葡萄糖100 g/L、蛋白胨1.8 g/L、初始pH 7.0的培养基中,以10%的接种量,于33℃、190 r/min的摇床上发酵120 h,可得菌体生物量为18.2 g/L,干细胞的油脂含量为68.5%。     

发酵性丝孢酵母蛋白酶稳定性的研究

研究了发酵性丝孢酵母所产蛋白酶的最适作用条件及稳定性。结果表明,发酵性丝孢酵母蛋白酶的最适反应温度为55℃,最适反应p H为7.0;有良好的热稳定性,50℃保温30 min,仍保留78%以上的酶活力;贮存稳定性也较好,10℃贮存60 h,仍保留80%左右的酶活力;5 mmol/L的金属离子Mn2+和Ca2+对蛋白酶有激活作用,其中Ca2+激活作用最显著,使酶活提高了0.87倍;1%(w/w)的表面活性剂吐温-100可以促进酶活力,使酶活提高了24.6%,1%(w/w)的SDS对酶活力有抑制作用,将残余酶活降低为原酶液的67.1%;2.5%(w/w)的葡萄糖、半乳糖和海藻糖等能抑制该蛋白酶活力,其中海藻糖抑制作用最显著,使残余蛋白酶活力降低到原酶液的23.6%;甘油可作为酶保护剂保护蛋白酶稳定性,且浓度为7.5%(w/w)的甘油能明显激活酶活力。     

Identification and Structural Analysis of a Novel Carboxysome Shell Protein with Implications for Metabolite Transport

Bacterial microcompartments (BMCs) are polyhedral bodies, composed entirely of proteins, that function as organelles in bacteria; they promote subcellular processes by encapsulating and co-localizing targeted enzymes with their substrates. The best-characterized BMC is the carboxysome, a central part of the carbon-concentrating mechanism that greatly enhances carbon fixation in cyanobacteria and some chemoautotrophs. Here we report the first structural insights into the carboxysome of Prochlorococcus, the numerically dominant cyanobacterium in the world's oligotrophic oceans. Bioinformatic methods, substantiated by analysis of gene expression data, were used to identify a new carboxysome shell component, CsoS1D, in the genome of Prochlorococcus strain MED4; orthologs were subsequently found in all cyanobacteria. Two independent crystal structures of Prochlorococcus MED4 CsoS1D reveal three features not seen in any BMC-domain protein structure solved to date. First, CsoS1D is composed of a fused pair of BMC domains. Second, this double-domain protein trimerizes to form a novel pseudohexameric building block for incorporation into the carboxysome shell, and the trimers further dimerize, forming a two-tiered shell building block. Third, and most strikingly, the large pore formed at the 3-fold axis of symmetry appears to be gated. Each dimer of trimers contains one trimer with an open pore and one whose pore is obstructed due to side-chain conformations of two residues that are invariant among all CsoS1D orthologs. This is the first evidence of the potential for gated transport across the carboxysome shell and reveals a new type of building block for BMC shells.     

Uptake of phenol by Trichosporon cutaneum.

The soil yeast Trichosporon cutaneum, which is distinguished by having a strictly oxidative metabolism, can be induced to utilize phenol as a sole carbon source. The present paper shows that such phenol-induced cells contain a specific, energy-dependent uptake system for phenol. Phenol uptake is not directly linked to its o-hydroxylation inside the cell, the first step of phenol metabolism. The Km for uptake is 235 +/- 30 microM, that for hydroxylation only 4.5 +/- 0.5 microM. Further, the phenol analog 2,6-dimethylphenol, which can not be hydroxylated, competes with phenol for the uptake system. The pH dependence of uptake indicates that phenolate is an essential form during the uptake process. The energy requirement for phenol uptake is indicated by effects of various inhibitors of energy generation, including proton-conducting uncouplers. Direct monitoring of proton movements in a pH-stat during phenol uptake indicates a phenol-proton symport. One proton is cotransported with every phenol molecule. Phenol competes with the uptake of sucrose and glycerol by cells grown on these substrates. Under such conditions the uptake of phenol seems to proceed through a different system, with lower affinity for phenol than in phenol-grown cells.     

藤黄微球菌降解真菌毒素玉米赤霉烯酮的研究

目的研究并优化藤黄微球菌降解真菌毒素玉米赤霉烯酮(ZEN)的因素条件。方法采用HPLC的检测方法对藤黄微球菌降解真菌毒素玉米赤霉烯酮的影响因素(培养基、温度、pH、摇床转速、培养时间和金属离子等)进行优化研究。结果藤黄微球菌在0.05 mol/LMnCl2、初始pH为7.0的LB培养基中,37℃,180 r/min,连续培养120 h,能降解99%的ZEN毒素(初始浓度为2μg/ml)。结论藤黄微球菌降解真菌毒素ZEN的能力与培养基成分、pH和添加的金属离子种类密切相关。     

Use of a Novel Fluorinated Organosulfur Compound To Isolate Bacteria Capable of Carbon-Sulfur Bond Cleavage

The vacuum residue fraction of heavy crudes contributes to the viscosity of these oils. Specific microbial cleavage of C—S bonds in alkylsulfide bridges that form linkages in this fraction may result in dramatic viscosity reduction. To date, no bacterial strains have been shown conclusively to cleave C—S bonds within alkyl chains. Screening for microbes that can perform this activity was greatly facilitated by the use of a newly synthesized compound, bis-(3-pentafluorophenylpropyl)-sulfide (PFPS), as a novel sulfur source. The terminal pentafluorinated aromatic rings of PFPS preclude growth of aromatic ring-degrading bacteria but allow for selective enrichment of strains capable of cleaving C—S bonds. A unique bacterial strain, Rhodococcus sp. strain JVH1, that used PFPS as a sole sulfur source was isolated from an oil-contaminated environment. Gas chromatography-mass spectrometry analysis revealed that JVH1 oxidized PFPS to a sulfoxide and then a sulfone prior to cleaving the C—S bond to form an alcohol and, presumably, a sulfinate from which sulfur could be extracted for growth. Four known dibenzothiophene-desulfurizing strains, including Rhodococcus sp. strain IGTS8, were all unable to cleave the C—S bond in PFPS but could oxidize PFPS to the sulfone via the sulfoxide. Conversely, JVH1 was unable to oxidize dibenzothiophene but was able to use a variety of alkyl sulfides, in addition to PFPS, as sole sulfur sources. Overall, PFPS is an excellent tool for isolating bacteria capable of cleaving subterminal C—S bonds within alkyl chains. The type of desulfurization displayed by JVH1 differs significantly from previously described reaction results.     

Cleavage of a viral polyprotein by a cellular proteolytic activity.

The 200,000-dalton polyprotein encoded by the bottom component RNA of cowpea mosaic virus was synthesized in rabbit reticulocyte lysates, and this in vitro-synthesized protein was isolated from the lysate reaction mixture by sucrose density gradient centrifugation. Incubation of the isolated polyprotein with buffer caused no change in the protein, but incubation with reticulocyte lysates or with fractionated lysate proteins resulted in cleavage of the protein into the expected cleavage products (32,000- and 170,000-dalton proteins). This finding indicated that reticulocytes contain a proteolytic activity that is needed for the primary cleavage reaction. A cleavage assay in which we used partially purified preparations showed that cleavage was an ATP-dependent reaction.     

Cleavage of a model DNA replication fork by a methyl-specific endonuclease

Epigenetic DNA methylation is involved in many biological processes. An epigenetic status can be altered by gain or loss of a DNA methyltransferase gene or its activity. Repair of DNA damage can also remove DNA methylation. In response to such alterations, DNA endonucleases that sense DNA methylation can act and may cause cell death. Here, we explored the possibility that McrBC, a methylation-dependent DNase of Escherichia coli, cleaves DNA at a replication fork. First, we found that in vivo restriction by McrBC of bacteriophage carrying a foreign DNA methyltransferase gene is increased in the absence of homologous recombination. This suggests that some cleavage events are repaired by recombination and must take place during or after replication. Next, we demonstrated that the enzyme can cleave a model DNA replication fork in vitro. Cleavage of a fork required methylation on both arms and removed one, the other or both of the arms. Most cleavage events removed the methylated sites from the fork. This result suggests that acquisition of even rarely occurring modification patterns will be recognized and rejected efficiently by modification-dependent restriction systems that recognize two sites. This process might serve to maintain an epigenetic status along the genome through programmed cell death.