厌氧微生物菌群XH-1对2,4,6-三氯苯酚的降解特性

有机污染物2,4,6-三氯苯酚(2,4,6-TCP)普遍存在于地下水和河流底泥等厌氧环境中。为了探究厌氧微生物菌群XH-1对2,4,6-TCP的降解能力,本研究以2,4,6-TCP为底物,接种XH-1建立微宇宙培养体系,并以中间产物4-氯苯酚(4-CP)和苯酚为底物分别进行分段富集培养,利用高效液相色谱分析底物的降解转化,同时基于16S rRNA基因高通量测序分析微生物群落结构变化。结果表明: 2,4,6-TCP(122 μmol·L^-1)以0.15 μmol·d^-1的速率在80 d内被完全降解转化,降解中间产物分别为2,4-二氯苯酚(2,4-DCP)、4-氯苯酚和苯酚,所有中间产物最终在325 d被完全降解。高通量测序结果表明,脱卤杆菌和脱卤球菌可能驱动2,4,6-TCP还原脱氯,其中,脱卤球菌可能在4-CP的脱氯转化中发挥重要作用,并与丁酸互营菌和产甲烷菌联合作用彻底降解2,4,6-TCP。     

Biohydroxylation of N,N-dialkylarylamines by the isolated topsoil bacterium Bacillus megaterium

Topsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and was identified as Bacillus megaterium by 16S rDNA analysis and DNA/DNA-hybridization. In contrast to the well-known C-hydroxylation by liver microsomes, leading to p-hydroxylation, B. megaterium formed o- and p-monohydroxylated products, i.e. N,N-dimethyl-2-aminophenol and N,N-dimethyl-4-aminophenol, both identified by gas chromatography–mass spectrometry (GC–MS) using synthesized reference compounds. The observed hydroxylation showed slight regioselectivity in favour of the o-hydroxylated product. Two further substrates, N,N-diethylaniline and N-ethyl-N-methylaniline, were also successfully biohydroxylated by B. megaterium with corresponding regioselectivity. Interestingly, aniline, known to be transformed easily by cytochrome P-450meg into p-aminophenol, was not accepted as substrate.     

UBA domain protein SUF1 interacts with NatA-complex subunit NAA15 to regulate thermotolerance in Arabidopsis

During recovery from heat stress, plants clear away the heat-stress-induced misfolded proteins through the ubiquitin-proteasome system(UPS). In the UPS, the recognition of substrate proteins by E3 ligase can be regulated by the N-terminal acetyltransferase A(NatA) complex. Here, we determined that Arabidopsis STRESS-RELATED UBIQUITIN-ASSOCIATEDDOMAIN PROTEIN FACTOR 1(SUF1) interacts with the NatA complex core subunit NAA15 and positively regulates NAA15. The suf1 and naa15 mutants are sensitive ...     

Chemo-enzymatic synthesis of (R)-(+)-aminoglutethimide by kinetic resolution of (±)-4-cyano-4-phenyl-1-hexanol

Chemo-enzymatic approaches for the synthesis of the family of aromatase inhibitory drug via lipase-catalyzed kinetic resolution of (±)-4-cyano-4-phenyl-1-hexanol (2) as appropriate precursors were described. Enzymatic transesterification of primary alcohol (±)-2 using Pseudomonas cepacia (Amano PS, PCL) provided the enantiopure alcohol (R)-(−)-2 with 99% ee at conversion of 86%, while that of (±)-2 using Pseudomonas fluorescens (Amano AK, LAK) provided the (S)-(+)-2 with 96% ee at conversion of 86%. Chemical transformation of substrate (R)-(−)-2 gave (R)-(+)-aminoglutethimide (1) in enantioselectively high yield.     

An Escherichia coli system for assay of Flp site-specific recombination on substrate plasmids

We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the Flp site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40. pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying Flp-recognition targets (FRT) are transformed into BL-FLP, and the consequences of Flp-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect Flp-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used Flp substrate plasmid, pNEOβGAL (O'Gorman et al. (1991) Science 251, 1351–1355).     

桦褐孔菌膜二肽酶在巴斯德毕赤酵母中的分泌表达

本研究利用巴斯德毕赤酵母Pichia pastoris蛋白表达体系表达了药用担子菌桦褐孔菌的一个二肽酶基因。该二肽酶基因编码区全长1814bp,包含6个内含子,编码465个氨基酸。生物信息学分析发现,二肽酶基因编码的蛋白中不含信号肽序列,但在第55–77位氨基酸之间存在一个跨膜结构。将含跨膜结构和去跨膜结构蛋白的cDNA序列分别克隆到酵母分泌型表达载体pPICZαA上,电转化至巴斯德毕赤酵母X-33中,用1%(V/V)甲醇诱导重组菌株表达目标蛋白,采用SDS-PAGE和Western-blot检测表达蛋白。结果显示,巴斯德毕赤酵母可表达含跨膜结构的完整基因,但目标蛋白不能分泌到胞外,存在于破碎细胞的沉淀中,且没有催化活性;而去跨膜结构的蛋白则可分泌表达到胞外,并具有催化活性。Ni-NTA纯化去跨膜结构的桦褐孔菌二肽酶浓度可达0.12mg/mL,并发现其在pH 7.3、反应温度50℃、反应时间2h的条件下,以Gly-Gly为底物时,其比活为433U/mg。同时检测到其对Ile-Leu、Trp-Trp和Phe-Phe具有较高的水解活性。     

热带假丝酵母高效利用甘油研究

目的:热带假丝酵母以油脂为底物发酵时会产生副产物甘油,研究对热带假丝酵母gk基因进行过表达,将副产物甘油转化为能量,提高油脂转化利用效率。方法:以热带假丝酵母Candida tropicalis 1798中的甘油激酶(gk)为研究对象,利用PCR技术获得同源臂基因gkpR,通过一步法无缝克隆将同源臂和G418抗性基因(kanr)连接至pPICzαA载体,同时将解脂假丝酵母Candida lipolytica 1457中的启动子基因pGAP无缝连接至载体中的gkpR,构成质粒pPICzαA-gkp,并电转化至C.tropicalis 1798感受态细胞中,通过一次同源单交换,将启动子pGK替换为pGAP。结果:经过G418抗性筛选和PCR鉴定,成功获得pGAP基因替换菌株C.tropicalis 1798-gkPr;发酵验证结果显示,启动子基因替换C.tropicalis 1798在以甘油为底物培养时重组菌OD600值比野生型菌株高46.4%,重组菌培养基中甘油剩余量比野生菌降低56.1%,表明启动子替换能促进C.tropicalis1798对甘油的吸收利用。此外,以油脂为底物进行发酵实验时还发现重组菌产长链二元酸的量比野生菌提高32.7%。结论:通过启动子替换手段构建的重组菌C.tropicalis 1798-gkPr,提高了热带假丝酵母对油脂组分中甘油成分的利用效率。     

Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)

Peroxidase (POD) and superoxide dismutase (SOD) enzyme activities were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias), or hygromycin phosphotransferase (hpt) genes. All of the transformed lines analyzed had elevated levels of all POD isoforms. Three POD isoforms with pI values of approximately 4.5, 4.8, and 8.4, and one additional pair of isoforms with a pI value of approximately 8.8 were separated from tissue extracts of all transgenic lines. Isoelectrofocusing patterns revealed the induction of one isoform of SOD with a pI of about 5.6 in all transgenic lines compared with non-transformed embryogenic tissue. These results indicate that the process of transformation may disrupt redox homeostasis in alflalfa tissues.     

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal

Synthesis of (R)-2-trimethylsilyl-2-hydroxyl-propionitrile via asymmetric transcyanation of acetyltrimethylsilane with acetone cyanohydrin in an aqueous/organic biphasic system catalyzed by (R)-hydroxynitrile lyase from Prunus japonica seed meal was successfully carried out for the first time. The optimal volume ratio of aqueous to organic phase, buffer pH value and reaction temperature were 15% (v/v), 5.0 and 30°C, respectively, under which both substrate conversion and product enantiomeric excess (ee) were 99%. Silicon atom in the substrate showed great effect on the reaction. Acetyltrimethylsilane was a much better substrate for (R)-hydroxynitrile lyase from Prunus japonica than its carbon analogue.     

Acetylcholine-activated currents in quail myotubes expressing viral oncogenes

Acetylcholine-activated currents were recorded in cultured myotubes arising from embryonic quail myoblasts transformed by the v-src and v-ras oncogenes. In src-myotubes, the whole cell inward current decayed more slowly than in non-transformed controls. In ras-myotubes, the current had a faster decay and smaller amplitude than in the controls. The single-channel conductance and mean open times recorded from cell-attached patches were similar in transformed and control cells. However, in ras-myotubes the frequency of channel openings strongly decreased with time.

It is concluded that oncogenic tyrosine-specific protein kinase (v-src product) and G-like p21 protein (v-ras product) can induce differential changes in the function of nicotinic ACh receptor, perhaps related to specific biochemical events elicited in the establishment of the transformed state.     

Steroid 11ß-hydroxylation by a fungal microsomal cytochrome P450

The steroid 11ß-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4–5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011ß system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011 from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11ß-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M_r 60 kD) and a NADPH-cytochrome P450 reductase (M_r 79 kD).     

Microhabitat selection in intertidal gobiid fishes: Species- and size-associated variation

Patterns of space use and the individual-based behaviour of microhabitat selection were investigated in three intertidal gobiid fishes, Bathygobius fuscus, Chaenogobius annularis and C. gulosus, from Kyushu, southern Japan. While the three species tended to occupy slightly different types of tidepool, their patterns of distribution largely overlapped in the field. Laboratory experiments involving choice of shelter (i.e. underneath a stone plate) and four different substrate types were conducted to examine size- and time-related variation in habitat selection. The shelter area was preferred by small- and large-sized C. gulosus (day and night), large C. annularis (day and night) and small C. annularis (daytime only), while no preference was evident in small B. fuscus (day and night) and small C. annularis (night). Patterns of substrate choice also differed among species, size groups and between day and night. Size differences in substrate use were evident in B. fuscus and C. gulosus but not in C. annularis, while diel differences were shown by all species groups except large B. fuscus. The gravel and sand substrates tended to be used more frequently than the bare rock substrate, but the strength of preference of a particular substrate type varied among individuals/species. Our results demonstrate that habitat selection by the three gobiid species is variable depending on species, body size and time of day, which must ultimately bear upon mitigating intra-/interspecific interactions in tidepool environments.     

Biotransformation of (S)-(−)- and (R)-(+)-limonene using Solanum aviculare and Dioscorea deltoidea plant cells

(S)-(−)- and (R)-(+)-limonene was transformed to carvone via corresponding cis- and trans-carveol using Solanum aviculare and Dioscorea deltoidea plant cells. Both carveols and carvone formed were racemic in all biotransformations.     

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: beta-galactosidase from Kluyveromyces lactis

Three recombinant DNA vectors carrying the β-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the β-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded β-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However, S. cerevisiae transformed with     

Ferredoxin reductase enhances heterologously expressed cytochrome CYP105D1 in Escherichia coli and Streptomyces lividans

The ability of two different ferredoxin reductases from Streptomyces coelicolor, to enhance the amount of active recombinant Streptomyces griseus soyC (CYP105D1) was investigated in both Escherichia coli and Streptomyces lividans. In E. coli a two-plasmid system and a single operon construct were used for expression of the CYP105D1 and the ferredoxin reductase(s) under the control of T7 promoters. Expression levels of CYP105D1 were found to range between 85 and 280 nmol l⁻¹ cell culture after prolonged growth. In S. lividans the CYP105D1 and its ferredoxin were cloned downstream of the Pact1 promoter in the E. coli/Streptomyces shuttle vector pBW160. The recombinant E. coli and S. lividans cells converted 7-ethoxycoumarin into 7-hydroxycoumarin efficiently. Expression of a ferredoxin reductase as an operon with CYP105D1 and its ferredoxin enhances the o-dealkylation of 7-ethoxycoumarin. Ferredoxin NADPH reductase was found to enhance the level of the active form of CYP105D1 monooxygenase when no substrate was present.     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

Characterization of transgenic root cultures of Trifolium repens, Trifolium pratense and Lotus corniculatus and transgenic plants of Lotus corniculatus

Hairy root cultures of three species of legume, Lotus corniculatus, Trifolium repens and T. pratense were established using a wild-type strain (C58C1 with pRi 15834) of Agrobacterium rhizogenes. Southern hybridization analysis confirmed that the lines were genetically transformed. Copy numbers of TL-DNA in different lines varied from one to eight. Examination of the transformed root cultures revealed changes in anatomy, morphology and cytology. Plants that had regenerated from hairy roots of L. corniculatus showed changes in morphology, physiology and cytology but no change in several parameters of nitrogen fixation activity.     

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFLl, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS : (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.     

Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete

A thermostable laccase was isolated from an unidentified fungal isolate [Enz. Microb. Technol. 33 (2003) 212], and tentatively named UD4. This work indicates that the enzyme has unique properties other than its thermostability. Investigation into the kinetic parameters of the thermostable laccase yielded an unusually high affinity for ABTS as a substrate (low K_m) when compared with available published data for other laccase isozymes. The specificity constant (k_cat/K_m) was found to be considerably higher than laccase from other sources and is comparable to “white” laccase from Pleurotus ostreatus (POXA1). However, POXA1 isozyme exhibits a large turnover number (k_cat) that contributes to its high specificity constant whereas the high specificity constant for UD4 laccase is achieved by having a high substrate affinity. The UD4 thermostable laccase, like most other laccases, is able to utilize guaiacol as a substrate, whereas POXA1 is unable to oxidize guaiacol, indicating a broader substrate range for the thermostable laccase from UD4. The thermostable laccase is inhibited by sodium azide through non-competitive inhibition, and by thioglycolic acid and hydroxylamine through competitive inhibition. The high specificity constant, substrate affinity and broader substrate range of the thermostable laccase from UD4 indicates that it is a highly favourable candidate enzyme for industrial application.     

Regiospecific hydroxylation of 3-(methylaminomethyl) pyridine to 5-(methylaminomethyl) -2 (1H) -pyridinone byArthrobacter ureafaciens

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1H)-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells ofArthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1H)-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.