Characterization of a HMT2-like enzyme for sulfide oxidation from Pseudomonas putida

The open reading frame pp0053, which has a high homology with the sequence of mitochondrial sulfide dehydrogenase (HMT2) conferring cadmium tolerance in fission yeast, was amplified from Pseudomonas putida KT2440 and expressed in Escherichia coli JM109(DE3). The isolated and purified PP0053-His showed absorption spectra typical of a flavin adenine dinucleotide (FAD)--binding protein. The PP0053-His catalyzed a transfer of sulfide-sulfur to the thiophilic acceptor, cyanide, which decreased the Km value of the enzyme for sulfide oxidation and elevated the sulfide-dependent quinone reduction. Reaction of the enzyme with cyanide elicited a dose-dependent formation of a charge transfer band, and the FAD-cyanide adduct was supposed to work for a sulfur transfer. The pp0053 deletion from P. putida KT2440 led to activity declines of the intracellular catalase and ubiquinone-H2 oxidase. The sulfide-quinone oxidoreductase activity in P. putida KT2440 was attributable to the presence of pp0053, and the activity showed a close relevance to enzymatic activities related to sulfur assimilation.     

A whole cell biocatalyst for double oxidation of cyclooctane

A novel whole cell cascade for double oxidation of cyclooctane to cyclooctanone was developed. The one-pot oxidation cascade requires only a minimum of reaction components: resting E. coli cells in aqueous buffered medium (=catalyst), the target substrate and oxygen as environmental friendly oxidant. Conversion of cyclooctane was catalysed with high efficiency (50% yield) and excellent selectivity (>94%) to cyclooctanone. The reported oxidation cascade represents a novel whole cell system for double oxidation of non-activated alkanes including an integrated cofactor regeneration. Notably, two alcohol dehydrogenases from Lactobacillus brevis and from Rhodococcus erythropolis with opposite cofactor selectivities and one monooxygenase P450 BM3 were produced in a coexpression system in one single host. The system represents the most efficient route with a TTN of up to 24363 being a promising process in terms of sustainability as well.     

Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93–97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7–12) and temperature (5–85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6–12, thermoactivity (5–85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.     

Use of isolated cyclohexanone monooxygenase from recombinant Escherichia coli as a biocatalyst for Baeyer-Villiger and sulfide oxidations

The performance, in Baeyer-Villiger and heteroatom oxidations, of a partially purified preparation of cyclohexanone monooxygenase obtained from an Escherichia coli strain in which the gene of the enzyme was cloned and overexpressed was investigated. As model reactions, the oxidations of racemic bicyclo[3.2.0]hept-2-en-6-one into two regioisomeric lactones and of methyl phenyl sulphide into the corresponding (R)-sulphoxide were used. Enzyme stability and reuse, substrate and product inhibition, product removal, and cofactor recycling were evaluated. Of the various NADPH regeneration systems tested, 2-propanol/alcohol dehydrogenase from Thermoanerobium brockii appeared the most suitable because of the low cost of the second substrate and the high regeneration rate. Concerning enzyme stability, kosmotropic salts were the only additives able to improve it (e.g., half-life from 1 day in diluted buffer to 1 week in 1 M sodium sulphate) but only under storage conditions. Instead, significant stabilization under working conditions was obtained by immobilization on Eupergit C (half-life approximately 2.5 days), a procedure that made it possible to reuse the catalyst up to 16 times with complete substrate (5 g x L(-1)) conversion at each cycle. Reuse of free enzyme was also achieved in a membrane reactor but with lower efficiency. Water-organic solvent biphasic systems, which would overcome substrate inhibition and remove from the aqueous phase, where reaction takes place, the formed product, were unsuccessful because of their destabilizing effect on cyclohexanone monooxygenase. More satisfactory was continuous substrate feeding, which shortened reaction times and, very importantly, yielded in the case of bicyclo[3.2.0]hept-2-en-6-one (10 g x L(-1)) both lactone products with high optical purity (enantiomeric excess > or = 96%), which was not the case when all of the substrate was added in a single batch.     

One-pot bio-synthesis of propyl gallate by a novel whole-cell biocatalyst

Propyl gallate has an excellent antioxidative capacity and some pharmaceutical potentials. In order to examine the feasibility for one-pot bio-synthesis of propyl gallate catalyzed by a whole-cell biocatalyst in organic media, a whole-cell biocatalyst of Aspergillus niger was prepared and utilized to catalyze the transesterification with tannic acid as a raw material. Furthermore, both the catalytic system and the reaction mode were optimized to further improve the conversion rate of substrate. The result shows that a promising conversion rate, 43%, was achieved by the pH-tuned mycelium-bound tannase. The rate is over than or very close to that achieved by isolated tannase. The study on reaction mode indicates that the simulated continuous catalysis is the most suitable to the transesterification as compared to batch catalysis and batch catalysis coupled with product separation. Accordingly, the one-pot bio-synthesis of propyl gallate by the novel whole-cell biocatalyst has such three advantages as easy operability of the biocatalyst, high efficiency of reaction mode, and the abundance of the natural raw material, which will contribute to constructing an efficient and eco-friendly method for one-pot synthesis of propyl gallate in an economical and ecological manner.     

一株异养型细菌对无机硫化物的降解特性和培养条件优化

畜禽屠宰加工、鱼粉饲料加工等一些食品工业生产过程中会释放出大量的硫化氢恶臭气体,导致周边环境的严重污染。为实现以培养异养型细菌脱除硫化氢气体的目的,取分离到的异养脱硫细菌XJ-2,通过诱变筛选得到一株高效脱硫菌株ZJNB-B3,其脱硫率达97%。基于形态学研究、API 50 CHB生理生化鉴定及16S r RNA基因测序,鉴定该菌为蜡状芽胞杆菌Bacillus cereus ZJNB-B3。该菌Gen Bank登录号为MF679650。降解特性研究表明,ZJNB-B3菌株对有毒的硫化物有较高耐受性,耐受上限高达300 mg/L。采用响应面法优化环境因素对菌株降解硫化物效率的影响,得到在最适培养温度30℃下,初始S~2–浓度为211.8 mg/L、初始p H值6.72、接种量为5.04%时,菌株氧化脱硫效果最显著,经过实测在48 h产生的硫酸盐浓度为63.8 mg/L,脱硫率达97.3%。菌株在氧化硫化物时不会产生硫酸抑制菌株的生长,可以在p H值温和的环境条件下脱硫,因此,该菌有较高的工业应用价值。本研究为异养型细菌应用于工业反应器脱除硫化氢恶臭气体提供了小试研究基础。     

A mathematical model for the bacterial oxidation of a sulfide ore concentrate

The effect of dilution rate and feed solids concentration on the bacterial leaching of a pyrite/arsenopyrite ore concentrate was studied. A mathematical model was developed for the process based on the steady-state data collected over the range of dilution rates (20 to 110 h) and feed solids concentrations (6 to 18% w/v) studied. A modified Monod model with inhibition by arsenic was used to model bacterial ferrous ion oxidation rates. The model assumes that (i) pyrite and arsenopyrite leaching occurs solely by the action of ferric iron produced from the bacterial oxidation of ferrous iron and (ii) bacterial growth rates are proportional to ferrous ion oxidation rate. The equilibrium among the various ionic species present in the leach solution that are likely to have a significant effect on the bioleach process were included in the model. (c) 1994 John Wiley & Sons, Inc.     

Simultaneous oxidation of ammonium, p-cresol and sulfide using a nitrifying sludge in a multipurpose bioreactor: a novel alternative

A nitrifying continuous stirred tank reactor was used as multipurpose bioreactor and it was operated for 325 days at 220 mg NH(4)(+)-N/Ld, 89 mg p-cresol-C /Ld and 36-76 mg S(2-)/Ld. The bioreactor was fed in sequential way, firstly with ammonium, achieving a consumption efficiency of 89%, with a nitrate yield of 0.99. Afterward, p-cresol was fed, achieving ammonium and p-cresol consumption efficiencies of 95% and 100%, respectively. The nitrate yield was higher and no aromatic intermediaries from p-cresol were detected. Finally sulfide was fed and the consumption efficiencies for all substrates were of 100%, being nitrate, HCO(3)(-) and sulfate the end products. The kinetic results showed that biological sulfide consumption was 13-fold faster than the chemical oxidation. This is the first time that a nitrifying reactor can be used for multiple purposes and also for the simultaneous removal of ammonium, sulfide and p-cresol in one step.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

A fission yeast gene for mitochondrial sulfide oxidation

A cadmium-hypersensitive mutant of the fission yeast Schizosaccharomyces pombe was found to accumulate abnormally high levels of sulfide. The gene required for normal regulation of sulfide levels, hmt2(+), was cloned by complementation of the cadmium-hypersensitive phenotype of the mutant. Cell fractionation and immunocytochemistry indicated that HMT2 protein is localized to mitochondria. Sequence analysis revealed homology between HMT2 and sulfide dehydrogenases from photosynthetic bacteria. HMT2 protein, produced in and purified from Escherichia coli, was soluble, bound FAD, and catalyzed the reduction of quinone (coenzyme Q2) by sulfide. HMT2 activity was also detected in isolated fission yeast mitochondria. We propose that HMT2 functions as a sulfide:quinone oxidoreductase. Homologous enzymes may be widespread in higher organisms, as sulfide-oxidizing activities have been described previously in animal mitochondria, and genes of unknown function, but with similarity to hmt2(+), are present in the genomes of flies, worms, rats, mice, and humans.     

Established and novel tools to investigate biocatalyst stability

We report on novel developments regarding the influence of temperature and salt on protein biocatalysts. The influence of temperature on the activation, unfolding, and deactivation of enzymes can now be described quantitatively with simple, analytical models. We demonstrate that enzyme deactivation phenomena can be determined via T-ramping and observation of instantaneous rates. We calculate the total turnover number analytically on the basis of the deactivation mechanism. We also report on the latest efforts to quantify the influence of salts on protein biocatalyst stability. While effects cannot yet be rationalized completely, we nevertheless found novel correlations between protein unfolding and deactivation and ion hydration.     

Established and novel tools to investigate biocatalyst stability

We report on novel developments regarding the influence of temperature and salt on protein biocatalysts. The influence of temperature on the activation, unfolding, and deactivation of enzymes can now be described quantitatively with simple, analytical models. We demonstrate that enzyme deactivation phenomena can be determined via T-ramping and observation of instantaneous rates. We calculate the total turnover number analytically on the basis of the deactivation mechanism. We also report on the latest efforts to quantify the influence of salts on protein biocatalyst stability. While effects cannot yet be rationalized completely, we nevertheless found novel correlations between protein unfolding and deactivation and ion hydration.     

Breaking the mirror: l-Amino acid deaminase,a novel stereoselective biocatalyst

Enantiomerically pure amino acids are of increasing interest for the fine chemical, agrochemicals and pharmaceutical industries. During past years l-amino acids have been produced from deracemization of dl-solution employing the stereoselective flavoenzyme d-amino acid oxidase. On the other hand, the isolation of corresponding d-isomer was hampered by the scarce availability of a suitable l-amino acid oxidase activity. On this side, l-amino acid deaminase (LAAD), only present in the Proteus bacteria, represents a suitable alternative. This FAD-containing enzyme catalyzes the deamination of l-amino acids to the corresponding α-keto acids and ammonia, with no hydrogen peroxide production (a potentially dangerous oxidizing species) since the electrons of the reduced cofactor are transferred to a membrane-bound cytochrome. Very recently the structure of LAAD has been solved: in addition to a FAD-binding domain and to a substrate-binding domain, it also possesses an N-terminal putative transmembrane α-helix (residues 8–27, not present in the crystallized protein variant) and a small α + β subdomain (50–67 amino acids long, named “insertion module”) strictly interconnected to the substrate binding domain. Structural comparison showed that LAAD resembles the structure of several soluble amino acid oxidases, such as l-proline dehydrogenase, glycine oxidase or sarcosine oxidase, while only a limited structural similarity with d- or l-amino acid oxidase is apparent. In this review, we present an overview of the structural and biochemical properties of known LAADs and describe the advances that have been made in their biotechnological application also taking advantage from improved variants generated by protein engineering studies.     

Organosoluble enzyme-polymer complexes: A novel type of biocatalyst for nonaqueous media

Summary A novel type of organosoluble biocatalyst, representing a non-covalent complex of enzyme with a sugar-based amphiphilic polymer is described. The subtilisin Carlsbergpalmitoyl poly(sucrose acrylate) complex was found to be soluble and catalytically active in a number of organic solvents of different nature.     

Preparation and characterization of urea-formaldehyde-pepsin bioconjugate: a new biocatalyst system

This study describes the synthesis of urea formaldehyde (UF) microspheres by a dispersion polycondensation polymerization method. These microspheres with proper F/U molar ratio can provide highly reactive groups, capable of further condensation with the amino acid residues of enzyme/proteins. Presence of methylols groups in UF microspheres was confirmed by 13C NMR study. Pepsin, a proteolytic enzyme, was immobilized on the UF microspheres to form bioconjugate system. As compared to the free enzyme in solution, the pepsin in the bioconjugate system exhibited significantly enhanced pH and temperature stability. The urea-formaldehyde-pepsin bioconjugate system also exhibited excellent proteolytic activity over eight successive reuse cycles with more than 50% of initial activity. A highlight of this new biocatalyst is the ease with which separation of this biocatalyst from the reaction medium may be achieved by mild centrifugation.     

Bubble-free oxygenation of a bi-enzymatic system: effect on biocatalyst stability

The effect of bubble-free oxygenation on the stability of a bi-enzymatic system with redox mediator regeneration for the conversion of lactose to lactobionic acid was investigated in a miniaturized reactor with bubbleless oxygenation. Earlier investigations of this biocatalytic oxidation have shown that the dispersive addition of oxygen can cause significant enzyme inactivation. In the process studied, the enzyme cellobiose dehydrogenase (CDH) oxidizes lactose at the C-1 position of the reducing sugar moiety to lactobionolactone, which spontaneously hydrolyzes to lactobionic acid. 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was used as electron acceptor for CDH and was continuously regenerated (reoxidized) by laccase, a blue multi-copper oxidase. Oxygen served as the terminal electron acceptor of the reaction and was fully reduced to water by laccase. The overall mass transfer coefficient of the miniaturized reactor was determined at 30 and 45 degrees C; conversions were conducted both in the reaction-limited and diffusion-limited regime to study catalyst inactivation. The bubbleless oxygenation was successful in avoiding gas/liquid interface inactivation. It was also shown that the oxidized redox mediator plays a key role in the inactivation mechanism of the biocatalysts unobserved during previous studies.     

Characterization of a novel L-serine transport system in Escherichia coli.

A novel transport system for L-serine was found in Escherichia coli cells grown on medium containing amino acid mixture. This novel system is distinguishable from the known three transport systems for L-serine, namely, the serine-threonine system, one of the leucine-isoleucine-valine systems, and the glycine-alanine system. Uptake of L-serine via this novel system was inhibited by none of the amino acids tested, indicating that it is highly specific for L-serine. This system was induced by L-leucine, but not by L-serine. The Km for L-serine was 50 microM, and the Vmax was 23 nmol/min per mg of cell protein. Transport of L-serine via this system was strongly inhibited by KCN, an inhibitor of the respiratory chain, or by carbonyl cyanide m-chlorophenylhydrazone, an H+ conductor. Uptake of H+ was induced by L-serine influx. These results indicate that an H+-serine cotransport mechanism is operative in this novel L-serine transport system.