Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

Several genes that may be involved in embryogenesis have been isolated from somatic embryos of carrot by many workers. However, the function of these genes has not been discovered yet. As the first step toward finding the function of these genes, we established a rapid and efficient method for transformation of carrot by using direct embryogenesis from hypocotyl segments treated with 2,4-dichlorophenoxyacetic acid (2,4-D) for a short period.     

Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

7-Keto-8-aminopelargonic acid synthetase (KAPA synthetase) which catalyzes the formation of KAPA from pimelyl CoA and l-alanine, and is involved in biotin biosynthesis, was partially purified from a cell-free extract of Bacillus sphaericus by a procedure involving ammonium sulfate fraction ation, protamine treatment, and DEAE-cellulose column chromatography. The reaction product was bioautographically confirmed to be KAPA. Some properties of the enzyme were also investigated. Among the amino acids, only l-alanine was active as a substrate, condensing with pimelyl CoA, The reaction required pyridoxal phosphate but the other vitamin B₆ compounds were inert. Typical inhibitors of pyridoxal phosphate enzymes showed marked inhibition to the reaction. Various amino acids such as l-cysteine, glycine, d-alanine, l-serine, l-histidine, and d-histidine were also found to be inhibitory.     

The Pimelyl-CoA Synthetase Responsible for the First Step in Biotin Biosynthesis by Microorganisms

Cell-free extracts of various bacteria were active in catalyzing the synthesis of pimelyl-CoA from pimelic acid and CoA. The pimelyl-CoA formed was determined in a reaction coupled with the 7-keto-8-aminopelargonic acid (KAPA) synthetase system, which is required to convert pimely-CoA to KAPA, and by microbiologically assaying the amount of KAPA formed. The enzyme synthesizing pimelyl-CoA was named pimely-CoA synthetase and should belong to EC 6.2.1. The pimelyl-CoA synthetase reaction required pimelic acid, CoA, ATP and Mg²⁺. The enzyme was partially purified from a cell-free extract of Bacillus megaterium. Using purified enzyme, characterization of the enzyme was performed. The enzyme reaction was remarkably inhibited by typical metal-chelating agents. Mn²⁺ and ADP could replace Mg²⁺ and ATP, respectively. No feedback repression was observed even with the addition of 1.0 µg per ml of biotin to the culture medium.     

Enzyme System Involved in the Decomposition of Phenyl Mercuric Acetate by Mercury-resistant Pseudomonas

The enzymatic decomposition of phenyl mercuric acetate (PMA) to metallic mercury by a mercury-resistant Pseudomonas has been studied. The formation of the system involved in the decomposition was found to be inducible. Glucose dehydrogenase (D-glucose: NAD oxidoreductase), arabinose dehydrogenase (L-arabinose: NADP oxidoreductase), cytochrome c and the “decomposing enzyme” which catalyzes the splitting of the C–Hg linkage, were separated from cell free extract by gel filtration on a column of Sephadex G–150. The cytochrome fraction was further separated into two types, c-I and c-II, by chromatography on a column of CM-Sephadex. Each of these cytochromes showed absorption peaks at 416, 519 and 547 mμ in the reduced form, but they were different in the molecular weight; cytochrome c-I was estimated to be about 26,000, and cytochrome c-II about 14,000. The reconstruction of these enzymes demonstrated that a reduced NAD(P) generating system, glucose dehydrogenase or arabinose dehydrogenase, cytochrome c-I and the “decomposing enzyme” were required for the decomposition of PMA. A hypothetical scheme for the decomposition of PMA was proposed and the decomposition mechanism was discussed.     

泛蛋白连接酶Ubc9参与氧还蛋白Ref—1的降解

氧还蛋白Ｒｅｆ－１是一种双功能蛋白质,在细胞氧还调控和ＤＮＡ无嘌呤／无嘧啶损伤修复中起重要作用。发寻找与它相互作用的蛋白Ｒｉｐｓ（Ｒｅｒ－１ ｉｎｔｅｒａｃｔｉｎｇ ｐｒｏｔｅｒｎｓ）,用Ｒｅｆ－１氧还功能域进行了酵母双杂交库的筛选,得到了５种阳性克隆。其中Ｒｉｐ３经测序证实为泛蛋白连接酶Ｕｂｃ９。Ｈｅｌａ细胞中共过表达Ｕｂｃ９可以明显抑制Ｒｅｆ－１对ＡＰ－１报告系统的增强作用。Ｗｅｓｔｅｒｎ印迹     

Microbial Degradation of Bergenia,a Phenolic C-Glucoside

A strain of soil bacteria was isolated by elective culture with bergenia, a C-glucoside having dihydroisocoumarin structure, as a sole carbon source, and was identified as Erwinia herbicola. In growth or replacement medium, the bacterium degraded bergenin to yield at least two major degradation products, one of them being identified as 4-O-methylgallic acid (compound I), an aglycone of bergenin. The bacterium seemed to utilize the sugar moiety of bergenin preferentially as carbon and energy sources, since the rate of further transformation of compound I by the bacterium was slow. In replacement culture with compound I, gallic acid was detected as one of the metabolites. A possible pathway for microbial degradation of bergenin is proposed.     

Bacillus Species Proteins Involved in Spore Formation and Degradation: From Identification in the Genome,to Sequence Analysis,and Determination of Function and Structure

The members of Bacillus species are Gram-positive, ubiquitous spore-forming bacilli. Several genomic sequences have been made available during recent years, including Bacillus subtilis, a model organism among this genus, Bacillus anthracis, and their analyses provided a wealth of information about spore-forming bacteria. Some members of this species can cause serious diseases in livestock and humans. An important pathogen in this group of organisms is B. anthracis, which is the causative agent of anthrax. A summary of the B. subtilis genome information, based on the publicly released sequence, that allowed for the identification and characterization of new and novel proteins of this organism as well as similar proteins from other members of Bacillus species is provided. The primary goal for this work is to present a review of the genome sequence-identified genes that encode proteins involved in the sporulation, germination, and outgrowth processes. These three processes are essential for spore development and later its transformation into a vegetative cell. Additionally, for a few selected examples of the protein products of the identified genes, the application of bioinformatics and modeling tools is illustrated in order to determine their likely structure and function. Two three-dimensional models of the structures of such proteins, PrfA endonuclease and phosphatase PhoE, are presented together with the structure-based functional conclusions. The review of such studies provides an example of how the genomic sequence can be utilized in order to elucidate the structure and function of proteins, in particular proteins of the Bacillus species. Because only a limited number of proteins of Bacillus species organisms are involved in the synthesis and degradation of spores and have been characterized to date, this genome-based analysis may provide new insights into the developmental processes of bacterial organism.     

Stimulation of Pectolytic Enzyme Formation of Erwinia aroideae by an Active Factor in Carrot Extracts

Formation of pectinase system in Erwinia aroideae was stimulated to a considerable extent when the cells were incubated in a pectin medium containing carrot extracts. The active factor in the extract was purified about 30 fold by ethanol precipitation, and further purification was achieved by ninhydrin treatment, charcoal adsorption, dialysis and gel filtration with Sephadex G-10. Although crude carrot extract preparation also stimulated protease formation in this organism, no stimulating activity for protease formation was found in the purified factor. Acetate and butyrate which had been shown to stimulate pectinase formation, were found to stimulated protease formation as well. Pectinase formation by this organism was also stimulated by polyamines and inorganic phosphate to a considerable extent.     

Aspergillomarasmine A and B,Potent Microbial Inhibitors of Endothelin-converting Enzyme

When screening for inhibitors of endothelin-converting enzyme (ECE), we isolated and identified Aspergillomarasmine A and B (AM-A and B) as potent inhibitors of ECE from the culture broth of Paecilomyces sp. N877. Both AM-A and AM-B had apparent activity in an in vivo experiment with big ET-1 induced sudden death, although the inhibitory activities of these compounds would be mainly due to the chelating effect.     

生物素对地衣芽孢杆菌生长和乳酸形成的影响

生物素对地衣芽孢杆菌生长和乳酸形成的影响胡尚勤（重庆师范学院生物系重庆６３００４７）地衣芽孢杆菌（Ｂａｃｉｌｕｓｌｉｃｇｅｎｉｆｏｒｍｉｓ）是我国９０年代首次从正常待产妇阴道拭子分离出来的一个无毒新菌株。现在国内已经用它来生产“整肠生”活菌剂。它是一...     

碳酸酐酶1对骨形成作用的研究

目的:为了研究碳酸酐酶1(carbonic anhydrase I,CA1)在生物矿化和骨形成过程中的作用。方法:选择HeLa细胞为研究对象,用地塞米松(dexamethasone,DEX)、β-磷酸甘油(β-glycerophosphate,β-GP)和维他命C(ascorbic acid,AA)诱导其钙化,茜素红染色法观察矿化结节的形成,荧光定量PCR检测细胞骨化标志蛋白Runx2/cbfa1的表达。CA1表达水平的变化采用Western blot和荧光定量PCR的方法检测。同时用碳酸酐酶抑制剂乙酰唑胺处理HeLa细胞,观察CA1表达被抑制后对HeLa细胞钙化的影响。结果:HeLa细胞在诱导后形成大量矿化结节,Runx2/cbfa1转录水平明显升高,显示DEX、β-GP和AA可诱导HeLa细胞钙化和骨化,HeLa细胞钙化后CA1表达明显增高。HeLa细胞经乙酰唑胺处理后,CA1的表达量减少,矿化结节形成明显减少,说明抑制CA1表达可以抑制HeLa细胞矿化和骨化过程。结论:CA1在生物矿化和新骨形成过程中起着重要作用。     

Irgm 1参与动脉粥样硬化斑块的形成

目的:探讨免疫相关GTP酶1(Irgm 1)对小鼠血管动脉粥样硬化(AS)斑块形成的影响。方法:高脂饲料喂养野生型(WT)、ApoE~(-/-)Irgm 1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠3个月,建立AS模型;取小鼠主动脉弓,免疫荧光染色方法观察WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1的表达情况及部位;Western blot方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1蛋白表达情况;Q-PCR方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1 m RNA表达情况;油红O染色观察ApoE~(-/-)Irgm1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠血管AS斑块形成情况;结果:与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠主动脉弓AS斑块中Irgm 1+细胞明显增多,Irgm 1+细胞主要位于血管AS斑块的表面;与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠血管AS斑块中Irgm 1蛋白表达显著增多(P0.001),Irgm 1 m RNA表达显著增多(P0.01);与ApoE~(-/-)Irgm1~(+/-)组相比,ApoE~(-/-)Irgm1~(+/+)组小鼠主动脉弓AS斑块面积显著增大(P0.01);结论:Irgm 1能够促进血管AS斑块的形成。     

Microbial Biomass and Community Composition Involved in Cycling of Organic Phosphorus in Sediments of Lake Dianchi,Southwest China

Organic phosphorus (P_o) was a major fraction of phosphorus (P) in sediments of lakes, and microbes were involved in most of its relevant biogeochemical cycling. Forms and quantification of P_o were investigated by sequential fractionation in 18 sediments of Lake Dianchi, Southwest China. Microbial biomass and community structure in these sediments were determined by phospholipid fatty acids (PLFAs). Distribution of P_o fractions were in the rank order that humic P_o > nucleic acid and polyphosphate > residual P > Ca-Al-P_o > Fe-P_o > sugar P_o > acid soluble P_o > H₂O-P_o. The recoveries of P_o and P_i in these detailed sequential fractions including residual P shows that the total contents of P_o in sediments of lakes were overestimated by the Standards, Measurements and Testing (SMT) protocol (ignition method). Microbial biomass including Gram-positive bacteria (14.4–20.0%), Gram-negative bacteria (32.7–38.4%), microeukaryotes (14.9–24.4%), aerobic bacteria (43.6–55.8%), anaerobic bacteria (0–2.9%) and type ? methanotrophs (17.6–24.4%) were assigned. Microbial mass and their composition were strongly correlated with H₂O-P_o, Fe-P_o, nucleic acid and polyphosphate, and humic P_o, though residual P was likely inert for microbes in sediments. The formation and degradation of P_o was closely related with microbial activities in sediments. These findings have implications for understanding the role of microbes on cycling of P_o and organic matter in sediments of lakes.     

PGU1 gene natural deletion is responsible for the absence of endo-polygalacturonase activity in some wine strains of Saccharomyces cerevisiae

The PGU1 gene encodes an endo-polygalacturonase enzyme in Saccharomyces cerevisiae. The literature reports that most S. cerevisiae strains possess this gene, despite a wide range of enzyme activity levels. Nevertheless, a few wine strains lack the PGU1 gene. We investigated the PGU1 locus sequence in these strains. The results indicated that the gene had been replaced by a partial Ty mobile element, whereas the gene promoter was still at the expected location. As all the strains lacking the PGU1 gene experienced the same phenomenon, it was tempting to hypothesize a common phylogenetic origin. However, fingerprints only allowed grouping of a few of them within one cluster.     

Conversion of Capsular Polysaccharide,Involved in Pellicle Formation,to Extracellular Polysaccharide by galE Deletion in Acetobacter tropicalis

Acetobacter tropicalis SKU1100 produces a pellicle-forming capsular polysaccharide (CPS), consisting of galactose, glucose, and rhamnose. We cloned the galE gene, a UDP-galactose synthesis gene, from A. tropicalis SKU1100 by PCR. A galE-disruptant was prepared and found not to produce CPS and thus not to form a pellicle under the static condition. Instead, the ΔgalE mutant secreted an extracellular polysaccharide (EPS), which was purified and found to have a unique character, different from the original CPS.     

Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous,Xenobiotic-Metabolizing Enzyme in Vertebrates

The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a ¹⁰T₅₀ (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.     

Linalool Dehydratase-Isomerase,a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg⁻¹ for the linalool dehydratase and 410 nanokatals mg⁻¹ for the geraniol isomerase, with apparent K_m values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).     

Cloning and Overexpression of a Tagged CMP-N-Acetylneuraminic Acid Synthetase from E. coli Using a Lambda Phage System and Application of the Enzyme to the Synthesis of CMP-N-Acetylneuraminic Acid

The gene coding from CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase (Ec 2.7.7.43) was amplified from total DNA of E. coli strain K-235 through a primer-directed polymerase chain reaction. The gene was fused with a modified ribosome binding site of the original CMP-NeuAc synthetase gene and a decapeptide tag sequence which served as a marker for screening of expressed proteins. The gene was cloned into lambda ZAP vector at EcoRI and XbaI sites and overexpressed in E. coli Sure at a level approximately 1000 times that of the wild type. The decapeptide-containing enzyme retained almost the same specificity as indicated by the V_max and K_m values using CTP and NeuAc as substrates. A preparative synthesis of CMP-NeuAc based on the recombinant enzyme was demonstrated.     

有机磷农药的微生物降解研究进展

微生物因种类和代谢多样性在有机磷农药降解中表现出独特的优势。对微生物降解有机磷农药的机制、微生物的获得、基因工程菌的构建及研究展望进行了综述。     

有机磷农药微生物降解研究进展

微生物降解是有机磷农药在环境中去毒降解的主要方式,是治理环境污染的一项有效手段。该文综述了有机磷农药降解菌的分离鉴定、降解机理与代谢途径、降解基因的克隆及表达、降解菌制剂和酶制剂的应用、以及有机磷农药微生物降解研究趋势五个方面的研究现状。