S-2-氯丙酸脱卤酶的定向进化及其应用

为提高S-2-氯丙酸脱卤酶的活力,通过易错PCR的方法将源于假单胞菌(Pseudomonas sp.CGMCC 3267)的脱卤酶(DehII)进行定向进化,进化酶DehII-B2的比活提高3.9倍。同源模建两者的三维结构,并与底物进行分子对接。结果表明,突变位点为A7I,进化酶DehII-B2的结合能比原始酶降低了1.4kcal/mol,活性中心Asp10与底物α碳原子的距离缩短了0.321 6nm,因而加快了酶反应速率、提高了酶比活。目前,该酶的比活高于实验室已得酶。与原始酶相比,其最适温度和热稳定性略有增加,最适pH和pH稳定性没有明显变化。对该酶的应用做了初步的探索,结果表明在40℃,60mmol/L底物浓度下反应10h,转化率达到49.6%,ees>99.9%,因此该酶有一定的应用价值。     

Biodegradation of polyethylene glycol by symbiotic mixed culture (obligate mutualism)

Neither Flavobacterium sp. nor Pseudomonas sp. grew on a polyethylene glycol (PEG) 6000 medium containing the culture filtrate of their mixed culture on PEG 6000. The two bacteria did not grow with a dialysis culture on a PEG 6000 medium. Flavobacterium sp. grew well on a dialysis culture containing a tetraethylene glycol medium supplemented with a small amount of PEG 6000 as an inducer, while poor growth of Pseudomonas sp. was observed. Three enzymes involved in the metabolism of PEG, PEG dehydrogenase, PEG-aldehyde dehydrogenase and PEG-carboxylate dehydrogenase (ether-cleaving) were present in the cells of Flavobacterium sp. The first two enzymes were not found in the cells of Pseudomonas sp. PEG 6000 was degraded neither by intact cells of Flavobacterium sp. nor by those of Pseudomonas sp., but it was degraded by their mixture. Glyoxylate, a metabolite liberated by the ether-cleaving enzyme, inhibited the growth of the mixed culture. The ether-cleaving enzyme was remarkably inhibited by glyoxylate. Glyoxylate was metabolized faster by Pseudomonas sp. than by Flavobacterium sp., and seemed to be a key material for the symbiosis.     

Degradation of 2-chloroethanol by wild type and mutants of Pseudomonas putida US2

A strain of Pseudomonas putida was isolated that was able to degrade 2-chloroethanol. The degradation proceeded via 2-chloroacetaldehyde and chloroacetate to glycolate. In crude extracts the enzymes for this degradation pathway could be detected. All enzymes proved to be inducible. The dehalogenase that catalyzed the dehalogenation of chloroacetate to glycolate was further characterized. It consisted of a single polypeptide chain with a molecular mass of 28 kDa. After induction the dehalogenase was expressed at a high level. In a mutant resistant to high concentrations of 2-chloroethanol the dehalogenase was no longer expressed. The mechanism of resistance seemed to be due to the inability to convert chloroacetate and export of this compound out of the cell.Non-standard abbreviations CEO 2-chloroethanol - DCPIP 2,6-dichlorophenolindophenol - FPLC fast protein liquid chromatography - PAGE polyacrylamide gelelectrophoresis - PES phenazine ethosulfate - PMS phenazine methosulfate - PQQ pyrroloquinoline quinone     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Biodegradation and metabolic pathway of β-chlorinated aliphatic acid in Bacillus sp. CGMCC no. 4196

In this study, a bacterial Bacillus sp. CGMCC no. 4196 was isolated from mud. This strain exhibited the ability to degrade high concentration of 3-chloropropionate (3-CPA, 120 mM) or 3-chlorobutyrate (30 mM), but not chloroacetate or 2-chloropropionate (2-CPA). The growing cells, resting cells, and cell-free extracts from this bacterium had the capability of 3-CPA degradation. The results indicated that the optimum biocatalyst for 3-CPA biodegradation was the resting cells. The 3-CPA biodegradation pathway was further studied through the metabolites and critical enzymes analysis by HPLC, LC-MS, and colorimetric method. The results demonstrated that the metabolites of 3-CPA were 3-hydroxypropionic acid (3-HP) and malonic acid semialdehyde, and the critical enzymes were 3-CPA dehalogenase and 3-HP dehydroxygenase. Thus, the mechanism of the dehalogenase-catalyzed reaction was inferred as hydrolytic dehalogenation which was coenzyme A-independent and oxygen-independent. Finally, the pathway of β-chlorinated aliphatic acid biodegradation could be concluded as follows: the β-chlorinated acid is first hydrolytically dehalogenated to the β-hydroxyl aliphatic acid, and the hydroxyl aliphatic acid is oxidized to β-carbonyl aliphatic acid by β-hydroxy aliphatic acid dehydroxygenase. It is the first report that 3-HP was produced from 3-CPA by β-chlorinated aliphatic acid dehalogenase.     

Identification of 4-chlorobenzoyl-coenzyme A as intermediate in the dehalogenation catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3

The intermediate in the reaction catalysed by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4-chlorobenzoyl-CoA. One component of 4-chlorobenzoate debalogenase worked as a a 4-chlorobenzoyl-CoA ligase catalysing the formation of 4-chlorobenzoyl-CoA from 4-chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4-chlorobenzoyl-CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4-hydroxybenzoate with concomitant release or coenzyme A.     

Purification, characterization, and gene cloning of a novel fluoroacetate dehalogenase from Burkholderia sp. FA1

Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of the highly stable carbon–fluorine bond in an aliphatic compound. The bacterial isolate FA1, which was identified as Burkholderia, grew on fluoroacetate as the sole carbon source to produce fluoroacetate dehalogenase (FAc-DEX FA1). The enzyme was purified to homogeneity and characterized. The molecular weights were estimated to be 79,000 and 34,000 by gel filtration and SDS-polyacrylamide gel electrophoresis (PAGE), respectively, suggesting that the enzyme is a dimer. The purified enzyme was specific to haloacetates, and fluoroacetate was the best substrate. The activities toward chloroacetate and bromoacetate were less than 5% of the activity toward fluoroacetate. The K_m and V_max values for the hydrolysis of fluoroacetate were 5.1 mM and 11 μmol per minute milligram, respectively. The gene coding for the enzyme was isolated, and the nucleotide sequence was determined. The open reading frame consisted of 912 nucleotides, corresponding to 304 amino acid residues. Although FAc-DEX FA1 showed high sequence similarity to fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX H1) (61% identity), the substrate specificity of FAc-DEX FA1 was significantly different from that of FAc-DEX H1: FAc-DEX FA1 was more specific to fluoroacetate than FAc-DEX H1.     

Nitrile-hydrolyzing enzyme from Meyerozyma guilliermondii and its potential in biosynthesis of 3-hydroxypropionic acid

3-Hydroxypropionic acid (3-HP) is an important platform chemical in organic synthesis. Traditionally, 3-HP was produced by chemical methods and fermentation process. In this work, a novel enzymatic method was developed for green synthesis of 3-HP. A yeast strain harboring nitrile-hydrolyzing enzyme was newly isolated from environmental samples using 3-hydroxypropionitrile (3-HPN) as the sole nitrogen source. It was identified to be Meyerozyma guilliermondii CGMCC12935 by sequencing of the 18S ribosomal DNA and internal transcribed spacer, together with analysis of the morphology characteristics. The catalytic properties of M. guilliermondii CGMCC12935 resting cells were determined, and the optimum activity was achieved at 55 °C and pH 7.5. The enzyme showed broad substrate specificity towards nitriles, especially 3-HPN, aminoacetonitrile and 3-cyanopyridine. The presence of Ag⁺, Pb²⁺ and excess substrate inhibited the enzyme activity, whereas 5% (v/v) ethyl acetate had a positive effect on the enzyme activity. M. guilliermondii CGMCC12935 resting cells by addition of 3% glucose could thoroughly hydrolyze 500 mM 3-HPN into 3-HP within 100 h and the maximal accumulative production of 3-HP reached 216.33 mM, which was over twofolds than the control group with no additional glucose. And this work would lay the foundation for biological production of 3-HP in industry.

     

Characteristics and DNA-Sequence of a Cryptic Haloalkanoic Acid Dehalogenase from Agrobacterium tumefaciens RS5

Agrobacterium tumefaciens RS5 harbors two different hydrolytic haloalkanoic acid dehalogenase genes, one coding for a nonstereospecific enzyme (DhlS5II) and a second for a cryptic L-isomer-specific dehalogenase (DhlS5I). The latter gene was cloned and expressed in Escherichia coli. Biochemical characterization and sequence analysis of dhlS5I shows its membership to the class of the L-isomer-specific hydrolytic dehalogenases. Highest homology of 72% was found to the dehalogenase LdexYL from Pseudomonas sp. YL. Both enzymes share an unusual high temperature optimum of 65°C. Controlled by a vector promoter, high specific dehalogenase activities up to 32 U mg⁻¹ protein were obtained in E. coli, and putatively, owing to its own σ⁷⁰-dependent promoter, a constitutive low-level expression of dhlS5I of 0.4 U mg⁻¹ protein was measured. Received: 27 June 1997 / Accepted: 4 August 1997     

Purification and characterization of cholesterol oxidase from Pseudomonas sp. and taxonomic study of the strain

Summary A cholesterol-oxidase-producing microorganism, strain COX629, isolated from soil was identified as Pseudomonas sp. The cholesterol oxidase produced by Pseudomonas sp. strain COX629 was purified 2400-fold to homogeneity in an overall yield of 60% from culture broth. The enzyme was a monomer with a molecular weight of 56 000, as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Sephadex G-150 gel column chromatography. The enzyme showed optimum activity at pH 7.0 and was stable over a rather wide pH range of 4.0 to 11.0. The enzyme showed a high substrate specificity for 3-hydroxysteroids and the K _m value for the oxidation of cholesterol by this enzyme was about 0.2 mM. A characteristic of the enzyme is marked stability at high temperature.     

Enzymatic dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 in organic solvents

Summary 4-Chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 showed dehalogenating activity in various organic solvents. In alcohols like methanol (150%) or ethanol (120%) higher activities than in water (100%) were obtained. In apolar solvents like petroleum ether (5%) and nhexane (5%) only trace activities were observed. The solvents did not increase the stability of the enzyme. 4-Chlorobenzoic acid methylester, a substance not soluble in water, was not dehalogenated in organic solvents.     

Dichloromethane dehalogenase of Hyphomicrobium sp. strain DM2.

Dichloromethane dehalogenase, a highly inducible glutathione-dependent enzyme catalyzing the conversion of dichloromethane into formaldehyde and inorganic chloride, was purified fivefold with 60% yield from Hyphomicrobium sp. strain DM2. The electrophoretically homogeneous purified enzyme exhibited a specific activity of 17.3 mkat/kg of protein. Its pH optimum was 8.5. The enzyme was stable at -20 degrees C for at least 6 months. A subunit molecular weight of 33,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration of native dichloromethane dehalogenase yielded a molecular weight of 195,000. Subunit cross-linking with dimethyl suberimidate confirmed the hexameric tertiary structure of the enzyme. Dichloromethane dehalogenase was highly specific for dihalomethanes. Its apparent Km values were 30 microM for CH2Cl2, 15 microM for CH2BrCl, 13 microM for CH2Br2, 5 microM for CH2I2, and 320 microM for glutathione. Several chlorinated aliphatic compounds inhibited the dichloromethane dehalogenase activity of the pure enzyme. The Ki values of the competitive inhibitors 1,2-dichloroethane and 1-chloropropane were 3 and 56 microM, respectively.     

Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL

The gene encoding thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL was isolated, and its over-expression system was constructed. Gene library was prepared fromSau3AI fragments of total DNA fromPs. sp. YL, pUC118 as a vector andEscherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.Abbreviations IPTG isopropyl -D-thiogalactopyranoside - KPB potassium phosphate buffer - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside     

Mass spectrometric analysis of the reactions catalyzed by -2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of -2-haloalkanoic acids to produce the corresponding -2-hydroxyalkanoic acids. Asp10 of -2-haloacid dehalogenase from Pseudomonas sp. YL nucleophilically attacks the α-carbon atom of the substrate to form an ester intermediate, which is subsequently hydrolyzed by an activated water molecule. We previously showed that the replacement of Thr14, Arg41, Ser118, Lys151, Tyr157, Ser175, Asn177, and Asp180 causes significant loss in the enzyme activity, indicating the involvement of these residues in catalysis. In the present study, we tried to determine which process these residues are involved in by monitoring the formation of the ester intermediate by measuring the molecular masses of the mutant enzymes using ionspray mass spectrometry. When the wild-type enzyme and the T14A, S118D, K151R, Y157F, S175A, and N177D mutant enzymes were mixed with the substrate, the ester intermediate was immediately produced. In contrast, the R41K, D180N, and D180A mutants formed the intermediate much more slowly than the wild-type enzyme, indicating that Arg41 and Asp180 participate in the formation of the ester intermediate. This study presents a new method to analyze the roles of amino acid residues in catalysis.     

Optimization of metallo-keratinase production by Pseudomonas sp. LM19 as a potential enzyme for feather waste conversion

Locally isolated bacterium Pseudomonas sp. LM19, a metallo-keratinase producer was used to hydrolyze the highly rigid keratin recalcitrant in this study. The production of crude keratinase by Pseudomonas sp. LM19 is influenced by both physical and nutritional parameters. The highest keratinase activity of 127?U/ml (2.15-fold) was observed in feather meal medium supplemented with fructose and peptone at a C/N ratio of 40. The optimum pH and temperature for keratinase production were found to be pH 8 and 30?°C, using 1% (w/v) feather as substrate. The degradation rate of the feathers was increased 2.4-fold at optimized physical and nutritional conditions. Feather degradation by Pseudomonas sp. LM19 led to the production of free amino acids such as arginine, glycine, leucine, and serine. The information on the production of keratinase by Pseudomonas sp. LM19 obtained from this study warrants further research for possible commercial application.     

Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k _cat/K _m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate. Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998     

Microbial hydrolysis of 7-xylosyl-10-deacetyltaxol to 10-deacetyltaxol

Enterobacter sp. CGMCC 2487, a bacterial strain isolated from the soil around a Taxus cuspidata Sieb. et Zucc. plant, was able to remove the xylosyl group from 7-xylosyltaxanes. The xylosidase of this strain was an inducible enzyme. In the bioconversion of 7-xylosyl-10-deacetyltaxol (7-XDT) to 10-deacetyltaxol (10-DT), for the purpose of enhancing the conversion efficiency, the effects of NH₄⁺, oat xylan, temperature, pH value, cell density and substrate concentration on the bioconversion have been systematically investigated. 3.0 mM NH₄⁺, 0.6% oat xylan in the media could enhance the yield of 10-DT; the optimum biocatalytic temperature was 26 °C and optimum pH value was 6.0. The highest conversion rate and yield of 10-DT from 7-XDT reached 92% and 764 mg/L, respectively. In addition, the biocatalytic capacity of the cell cultures remained 66.1% after continuous three batches. These results indicate that converting 7-XDT to 10-DT, a useful intermediate for the semisynthesis of paclitaxel or other taxane-based anticancer drugs by a novel bacterial strain, Enterobacter sp. CGMCC 2487, would be an alternative for the practical application in the future.     

Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.     

Biochemical and molecular characterisation of the 2,3-dichloro-1-propanol dehalogenase and stereospecific haloalkanoic dehalogenases from a versatile <Emphasis Type="Italic">Agrobacterium</Emphasis> sp.

We previously reported the presence of both haloalcohol and haloalkanoate dehalogenase activity in the Agrobacterium sp. strain NHG3. The versatile nature of the organism led us to further characterise the genetic basis of these dehalogenation activities. Cloning and sequencing of the haloalcohol dehalogenase and subsequent analysis suggested that it was part of a highly conserved catabolic gene cluster. Characterisation of the haloalkanoate dehalogenase enzyme revealed the presence of two stereospecific enzymes with a narrow substrate range which acted on d -2-chloropropionic and I-2-chloropropionoic acid, respectively. Cloning and sequencing indicated that the two genes were separated by 87 bp of non-coding DNA and were preceded by a putative transporter gene 66 bp upstream of the d-specific enzyme.     

Purification and properties of a high-affinity L-2-haloacid dehalogenase from Azotabacter sp. strain RC26

A monomeric 29 kDa protein showing dehalogenase activity on several halogenated carboxylic acids has been purified from Azotobacter sp. strain RC26. The purified enzyme is specific for the L isomer of optically active 2-haloacids leading to the inversion of the product configuration. The dehalogenase is active at temperatures ranging from 30 to 60C and shows a relatively high affinity for the substrate. The combined thermal stability, high substrate affinity and resistance to enzyme inhibitors found for the RC26 dehalogenase may be relevant for its use as catalyst in biotransformation processes.