Factors affecting 2-hydroxypropiophenone formation by benzoylformate decarboxylase from Pseudomonas putida

Benzoylformate (100 mM) was quantitatively converted to the acyloin compound, 2-hydroxypropiophenone (61.76 mM) and benzaldehyde (38.2 mM) by an enzyme extract from Pseudomonas putida ATCC 12633 in the presence of 1.6M acetaldehyde. Biotransformations were carried out at pH 6.0 and 30 degrees C with an incubation time of 60 min. Activity of the acyloin forming enzyme, benzoylformate decarboxylase, was 1.23 units/mL in the biotransformation mixture. Acyloin formation increased dramatically with pH in the range 4-5 and had a broad activity plateau in the pH range 5-8. A broad temperature optimum for acyloin formation was also observed in the range 20-40 degrees C.     

Carboligation Reactions Mediated by Benzoylformate Decarboxylase Immobilized on a Magnetic Solid Support

In this study, magnetic nanoparticles (Fe₃O₄, magnetite) with immobilized metal affinity ligands (MSS) were prepared and characterized by X‐ray diffraction (XRD), transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FTIR), and vibrating‐sample magnetometer (VSM) methods for purification and immobilization of the histidine‐tagged recombinant benzoylformate decarboxylase (BFD). The MSS support was shown to be eligible for selective binding of HIS‐tagged BFD by SDS‐page analysis. Loading capacity of the MSS support was determined as 43.6 ± 1.1 mg/g. The regeneration ability for protein binding was also studied. An immobilized BFD was tested to catalyze benzoin condensation and representative cross acyloin reaction. Conversion and enantiomeric excess values were comparable with that of free enzyme catalyzed reactions. Chirality 25:415–421, 2013. © 2013 Wiley Periodicals, Inc.     

Novel carbenicillin-hydrolyzing β-lactamase (CARB-5) from Acinetobacter calcoaceticus var. anitratus

A strain of Acinetobacter calcoaceticus var. anitratus highly resistant to ticarcillin but susceptible to ticarcillin in combination with clavulanic acid (2 mg/l) was found to produce a constitutive beta-lactamase. This enzyme was periplasmic with a characteristic substrate profile of a carbenicillin-hydrolyzing enzyme. Enzyme inhibition was detected with antiserum (anti-CARB-3), pCMB, cloxacillin, clavulanic acid and sulbactam. This novel enzyme with a molecular mass of 28,000 resembles other plasmid-mediated carbenicillinases (CARB) but differs in its apparent isoelectric point estimated as 6.3 and has been designated CARB-5 on this basis.     

Enzymatic production of (R)-phenylacetylcarbinol by pyruvate decarboxylase from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

Herein, we synthesized (R)-phenylacetylcarbinol (PAC), a pharmaceutical intermediate for ephedrine and pseudoephedrine, from benzaldehyde and pyruvate using a recombinant pyruvate decarboxylase (PDC) from Zymomonas mobilis. A whole cell reaction consisting of 30 mM benzaldehyde, 60 mM pyruvate, and a mutant PDC enzyme (PDC W329M; 1.6 mg DCW/mL) produced 12.4 mM (R)-PAC and less than 0.3 mM benzyl alchohol in 15 h at 20°C, outperforming the crude enzyme extract reaction (1.2 mM (R)-PAC) and minimizing formation of benzyl alchohol, the major by-product of S. cerevisiae whole cell reaction. These observations suggested that recombinant E. coli whole cell reactions are more efficient than crude enzyme extract or yeast-based reactions. We also demonstrated that the E. coli whole cell reaction performed effectively without expensive thiamin diphosphate cofactor. Finally, whole cell reaction (8 mg DCW/mL) was carried out with 200 mM benzaldehyde, 400 mM pyruvate in 10 mL of 500 mM phosphate buffer (pH 6.5), and 72 mM (R)-PAC was produced with 36% conversion for 15 h. © KSBB     

Optimisation of reaction conditions for production ofS-(−)-2-hydroxypropiophenone byAcinetobacter calcoaceticus

Summary Whole cells and cell-free extracts ofAcinetobacter calcoaceticus containing benzoylformate decarboxylase efficiently condensed benzoylformate and acetaldehyde to produce the acyloin compoundS-(–)-2-hydroxypropiophenone. Optimal concentrations of acetaldehyde cosubstrate for this reaction were found to be 1600 and 800 mM when whole cells and cell-free extracts were used respectively as biocatalysts. In both cases, optimal benzoylformate concentration was 100 mM. Temperature and pH optima for the biotransformation reaction were 30°C and 6.0 respectively. Under optimised conditions, maximum production of 2-hydroxypropiophenone, amounting to 8.4 g L^–1, occurred after a 2-h incubation. Product formation equivalent to 6.95 g in 1 h corresponded to a productivity of 267 mg acyloin per g dry cells per h.     

Metabolic Consequences of Inhibition of Cerebral Adenosylmethionine Decarboxylase by Methylglyoxal bis(Guanylhydrazone)

Abstract: Effects of intracerebroventricularly injected methylglyoxal bis(guanylhydrazone) on polyamine metabolism in mouse brain were recorded during 180 h after a single dose of 3.4 μmol/kg body weight. Cerebral concentrations of 31 other amino compounds were also asayed during the experiment. The drug caused a significant inhibition of adenosylmethionine decarboxylase that lasted for 50 h, with the maximal decrease, about 70%, occurring between 5 and 10 h after injection. Significant decreases of brain spermidine and spermine concentrations were observed in three phases. Two transient decreases occurred at 10 and at 35 h, and a longer-lasting one between 60 and 100 h. Ornithine decarboxylase was stimulated within 5 h after the injection, reaching a maximal level about 30-fold above normal at 60 h, and returned to control level at 140 h. This stimulation was accompanied by significant accumulation of the reaction product, putrescine, in the brain. It was maximally > 10-fold above normal at 160 h, and was still significantly above control at the end of the observation period. The time course of changes in the parameters of polyamine metabolism was regarded as support of a previously presented hypothesis that limiting putrescine concentration may play a role in the regulation of cerebral polyamine metabolism. In addition, the present results emphasize the possibility that changes in the activities of catabolic reactions may also contribute to the regulation of cerebral polyamine concentrations. Of the 31 amino compounds analyzed, only the concentrations of ornithine, urea, glutamine, and glutamate showed significant changes from normal. Ornithine concentration first was significantly increased at 25 h, whereafter it decreased and was somewhat below normal for most of the period between 60 and 180 h. The urea concentration showed a tendency to increase throughout the experiment, being significantly elevated at the end. These changes were regarded as suggesting that the increased need for ornithine in putrescine synthesis is satisfied mainly by increased arginine uptake and degradation. The magnitude of urea accumulation suggested that metabolism of ornithine to glutamate was also accelerated. An unexpected shift toward glutamine in the glutamine/glutamate relationship was observed during the first 100 h. However, the total concentration of these two compounds was quite constant throughout the experiment. Inhibition of ornithine decarboxylase by intraventricular injection of 2-difluoromethylornithine was tried during the study, but sufficient doses could not be used without induction of acute side effects.     

Enantiospecific synthesis of carbocyclic (+)-(E)-5-(2-bromovinyl)-2'-deoxyuridine

(+)-1-[(1R, 3S, 4R)-3-hydroxy-4-hydroxymethylcyclopentyl]-5-[(E)-2- bromovinyl]-1H,3H-pyrimidin-2,4-dione 10 was synthesized starting from (+)-endo-5-norbornen-2-yl acetate. This chiral educt was obtained by enzymatic hydrolysis of racemic esters of endo-5-norbornen-2-ol.     

Continuous production of (R)-phenylacetylcarbinol in an enzyme-membrane reactor using a potent mutant of pyruvate decarboxylase from Zymomonas mobilis.

The optimization of a continuous enzymatic reaction yielding (R)-phenylacetylcarbinol (PAC), an intermediate of the L-ephedrine synthesis, is presented. We compare the suitability of three pyruvate decarboxylases (PDC), PDC from Saccharomyces cerevisiae, PDC from Zymomonas mobilis, and a potent mutant of the latter, PDCW392M, with respect to their application in the biotransformation using acetaldehyde and benzaldehyde as substrates. Among these, the mutant enzyme was the most active and most stable one. The reaction conditions of the carboligation reaction were investigated by determining initial rate velocities with varying substrate concentrations of both aldehydes. From the resulting data a kinetic model was inferred which fits the experimental data with sufficient reliability to deduce the optimal concentrations of both substrates for the enzymatic process. The results demonstrate that the carboligation is most efficiently performed using a continuous reaction system and feeding both aldehydes in equimolar concentration. Initial studies using a continuously operated enzyme-membrane reactor gave (R)-PAC with a space-time yield of 81 g L(-1). d(-1) using a substrate concentration of 50 mM of both aldehydes. The yield was easily increased by cascadation of enzyme-membrane reactors. The new strategy allows the synthesis of (R)-PAC from cheap substrates in an aqueous reaction system. It thereby overcomes the limitation of by-product formation that severely limits the current fermentative process.     

刚毛柽柳腺苷甲硫氨酸脱羧酶（ThSAMDC）基因的克隆与胁迫下的表达分析

S-腺苷甲硫氨酸脱羧酶（S-adenosyl-L-methionine decarboxylase,SAMDC）是一种通过参与多胺的代谢途径来调节植物生理生化过程的限速酶。通过分析刚毛柽柳（Tamarix hispida）的转录组数据,获得并克隆了SAMDC基因的cDNA序列,将其命名为ThSAMDC。该cDNA序列全长2085bp,包含tiny ORF（tORF）、upstream ORF（uORF）和main ORF（mORF）3个植物SAMDC基因特征ORF。主开放读码框（mORF）长1107bp,编码369个氨基酸多肽,相对分子质量为40.34kD,理论等电点（PI）为4.72。ThSAMDC编码蛋白具有多个较强的亲水性区域,无明显跨膜区。通过与其他多个物种的氨基酸多序列比对结果表明,ThSAMDC具有两个典型的高度保守的结构域：酶原剪切位点（LSESSLF）与蛋白快速降解有关的PEST（TIHVTPEDGFSYAS）结构域。系统发育树结果表明ThSAMDC与菠菜（SoSAMDC）氨基酸序列一致性最高,为77%。实时荧光定量RT-PCR分析显示,ThSAMDC在NaCl、PEG、ABA、CdCl₂诱导表达均上调,预示着ThSAMDC可能在刚毛柽柳非生物胁迫应答过程中发挥重要作用。     

Production of a new terpenoid from biotransformation of (-)-isolongifolanol by Aspergillus niger and suppression of SOS-inducing activity

Abstract

The stereoselective oxidation of (—)-isolongifolanol (1) with a longifolene skeleton by Aspergillus niger (NBRC 4414) as a biocatalyst and suppressive effect on umuC gene expression by chemical mutagens furylfuramid and AFB1 of the SOS response in Salmonella typhimurium TA1535/pSK1002 were investigated. Compound 1 was converted to a new terpenoid, (-)-(2S,8R)-8,12-dihydroxy-isolongifolanol (2). Its structure was determined by NMR, IR, specific rotation and mass spectrometry. The metabolites suppressed the SOS-inducing activity of furylfuramid and AFB₁ in the umu test. Compound 1 suppressed 51% of the SOS-inducing activity against furylfuramid at < 1.0 mM. Compound 2 suppressed 15% and 24% of the SOS-inducing activity against furylfuramid and AFB1 at < 1.0 mM respectively.     

Stereoselective bioreduction of 1-(5-phenylfuran-2-yl)-ethanones mediated by baker's yeast

Abstract

Baker's yeast mediated reduction of various phenylfuran-2-yl-ethanones has been studied. The influence of the reaction conditions, the type and position of the substituents, as well the presence of various additives on the enantiomeric composition of the products and the reaction yield are discussed. The absolute configuration of the reaction products was established using a retrosynthetic procedure.     

Production of 2-Phenylethanol in Roses as the Dominant Floral Scent Compound from L-Phenylalanine by Two Key Enzymes,a PLP-Dependent Decarboxylase and a Phenylacetaldehyde Reductase

We investigated the biosynthetic pathway for 2-phenylethanol, the dominant floral scent compound in roses, using enzyme assays. L-[²H₈] Phenylalanine was converted to [²H₈] phenylacetaldehyde and [²H₈]-2-phenylethanol by two enzymes derived from the flower petals of R. ‘Hoh-Jun,’ these being identified as pyridoxal-5′-phosphate-dependent L-aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). The activity of rose petal AADC to yield phenylacetaldehyde was nine times higher toward L-phenylalanine than toward its D-isomer, and this conversion was not inhibited by iproniazid, a specific inhibitor of monoamine oxidase. Under aerobic conditions, rose petal AADC stoichiometrically produced NH₃ together with phenylacetaldehyde during the course of decarboxylation and oxidation, followed by the hydrolysis of L-phenylalanine. Phenylacetaldehyde was subsequently converted to 2-phenylethanol by the action of PAR. PAR showed specificity toward several volatile aldehydes.     

Production of a new compound, 7,10-dihydroxy-8-(E)-octadecenoic acid from oleic acid byPseudomonas sp. PR3

Summary Sixty-two cultures from the ARS Culture Collection and 10 cultures isolated from soil and water samples collected in Illinois were screened for their ability to convert agricultural oils to value-added industrial chemicals. A new compound, 7,10-dihydroxy-8-(E)-octadecenoic acid (DOD) was produced from oleic acid by a new strain,Pseudomonas sp. PR3 isolated from a water sample in Morton, IL. Strain PR3 is a motile, small rod-shaped, Gram-negative bacterium. It has multiple polar flagellae and is oxidase-positive. Strain PR3 grows aerobically and cannot grow anaerobically. The strain produces white, smooth colonies on agar plate and no water-soluble pigment. The yield of the product was greater than 60%. The optimum time, pH and temperature for the production of DOD were: 2 days, 7.0, and 30°C, respectively. Glycerol and dextrose support the growth of strain PR3, but the cells grown from the former failed to catalyse the conversion of oleic acid to DOD. The production of DOD is unique in that it involves a hydroxylation at two positions and a rearrangement of the double bond of the substrate molecule.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

石鲽病原琼氏不动杆菌形态型Ⅰ的鉴定

从由杀鲑气单胞菌(Aeromonas salmonicida)所引起的石鲽(Stone flounder,Kareius bicoloratus L)细菌性败血感染症病(死)鱼肝、脾、肾及肠内容物中,同时检出了作为继发感染的病原菌。经对6株纯培养菌在形态特征、理化特性等方面较系统的表观分类学指征鉴定及代表菌株DNA中G C mol％的测定,表明为琼氏不动杆菌的一个新形态型并定名为琼氏不动杆菌形态型Ⅰ(Acinetobacter junii morphovar Ⅰ);同时,对该菌进行了血清型、对抗菌类药物的敏感性及致病作用等方面的试验,初步表明此6株菌具有同种的表面(K)及同种的菌体(O)抗原,对供试37种抗菌类药物在不同菌株间的敏感及耐药无明显差异,对供试石鲽及牙鲆均具有较强的致病作用。     

鲍曼不动杆菌IRS-PCR基因分型研究

目的 用低频限制性位点聚合酶链反应(IRS-PCR)对鲍曼不动杆菌进行基因分型,分析基因型与鲍曼不动杆菌耐药谱的关系,并初步探讨其在分子流行病学中的作用.方法 随机收集2008年8月至2009年8月临床分离的73株鲍曼不动杆菌,采用K-B法进行药物敏感试验确定鲍曼不动杆菌耐药谱;同时利用IRS-PCR对此73株鲍曼不动杆菌进行基因分型;并分析IRS-PCR分型与鲍曼不动耐药谱的关系;结合IRS-PCR分型结果与73株鲍曼不动杆菌感染病例的临床资料,分析在此时间段鲍曼不动杆菌在我院流行感染的情况.结果 药物敏感试验将73株鲍曼不动杆菌菌株分为A1(19株全耐药型)和A2 ～ A31(54株耐药谱型)31个药敏谱.IRS-PCR法将其分为A～W共23个基因型,其中A、C、B、D和E型为5种优势菌株,分别为14、11、10、8和6株.对比研究发现A1型菌株(15/19)主要集中在基因型A、C、D内,而基因型B包含A15型耐药菌株9株(69.2％),基因型E包含A3型耐药菌株3株(42.9％).A基因型在院内特别是ICU中心引起2次爆发流行,而C和D型主要在呼吸内科引起感染.结论 IRS-PCR基因分型与药敏分型有较高的一致性,且IRS-PCR基因分型在早期发现和预防感染暴发流行方面优于药敏分型.     

Production of S-(+)-ibuprofen from a nitrile compound by Acinetobacter sp. strain AK226.

S-(+)-2-(4'-Isobutylphenyl)propionic acid [S-(+)-ibuprofen] was produced from racemic 2-(4'-isobutylphenyl)propionitrile (Ibu-CN) by an isolated bacterial strain, Acinetobacter sp. strain AK226. Ammonium acetate, acetonitrile, or n-butyronitrile as a carbon source in the culture medium was effective for bacterial growth and induction of this activity. The optimum pH of the reaction was around 8.0. S-(+)-Ibuprofen formed from Ibu-CN by resting cells was present in a 95% enantiomeric excess. Acinetobacter sp. strain AK226 appeared to possess a nitrilase for Ibu-CN because 2-(4'-isobutylphenyl)propionamide was not detected in the reaction mixture and 2-(4'-isobutylphenyl)propionamide was not hydrolyzed to S-(+)-ibuprofen. Since S-(+)-ibuprofen was preferentially produced while the R enantiomer of Ibu-CN was left almost intact over the time course of the reaction, the putative nitrilase appeared to be highly specific for the S enantiomer of Ibu-CN.     

Formation of S-[2-(N7-guanyl)ethyl] adducts by the postulated S-(2-chloroethyl)cysteinyl and S-(2-chloroethyl)glutathionyl conjugates of 1,2-dichloroethane

The formation of S-[2-(N7-guanyl)ethyl]glutathione (GEG) from dihaloethanes is postulated to occur through two intermediates: the S-(2-haloethyl)glutathione conjugate and the corresponding episulfonium ion. We report the formation of GEG when deoxyguanosine (dG) was incubated with chemically synthesized S-(2-chloroethyl)glutathione (CEG). The depurination of GEG was shown to be first order with a half-life of 7.4 +/- 0.4 h at 27 degrees C. Evidence is also presented for the formation of S-[2-(N7-guanyl)ethyl]-L-cysteine (GEC) in incubation mixtures containing dG and S-(2-chloroethyl)-L-cysteine (CEC), the corresponding cysteine conjugate of CEG. This finding demonstrates that this (haloethyl)cysteine conjugate does not require activation by enzymatic action of cysteine conjugate beta-lyase but, instead, can directly alkylate DNA. The half-life of the depurination of GEC was 6.5 +/- 0.9 h, which is no different from that of GEG. Of the two conjugates, CEC is a somewhat more active alkylating agent toward dG than CEG as N7-guanylic adduct was detected in reaction mixtures with lower concentrations of CEC than with CEG.     

水-有机溶剂介质中固定化不动杆菌细胞催化拆分(RS)-环戊烯酮乙酸酯的研究

不动杆菌CGMCC 0789的海藻酸凝胶包埋固定化细胞可高对映选择性地水解拆分环戊烯酮(简称HMPC)乙酸酯.异丙醇对固定化细胞的活力和对映选择性有显著提高.反应体系中异丙醇浓度为10%(体积分数,全文同)时,固定化细胞的活力最高,为6.22 mmol/(L·min·g)细胞干重,是未添加异丙醇的对照组的150%.此时E值为94±6,是对照组的1.7倍.以光学纯环戊烯酮乙酸酯为底物,对部分纯化的不动杆菌酯酶进行了动力学考察.通过动力学参数推算,10%异丙醇存在时,酯酶的对映选择性(E值)为36.5,是空白的2.3倍.10%异丙醇存在下,固定化细胞仍具有良好的操作稳定性.连续反应10批,固定化细胞的活力保持良好.     

Preparation of (R)-3-hydroxy-3-alkylbutanolides by biocatalytic hydration of 3-alkyl-2-butenolides using Rhodococcus rhodochrous

Biocatalytic hydration of 3-methyl- or 3-ethyl-2-butenolide with resting cells of Rhodococcus rhodochrous ATCC 17895 gave the corresponding (R)-3-hydroxy-3-alkylbutanolide in moderate yield and with 95% e.e. © Rapid Science Ltd. 1998     

Resolution and Synthesis of (S)-1-(2-Naphthyl)ethanol with Immobilized Pea Protein: As a New Biocatalyst

(S)-1-(2-Naphthyl)ethanol was yielded by immobilized pea (Pisum sativum L.) protein (IPP) from (R, S) 2-naphthyl ethanol (>99% ee, yield; about 50%), in which the (R)-enantiomer was selectively oxidized to 2-acetonaphthone. IPP could be reused consecutively at least three times without any decrease of yield and optical purity.