Mutation of PEB4 alters the outer membrane protein profile of Campylobacter jejuni

Although anaerobic bioremediation of chlorinated organic contaminants in the environment often requires exogenous supply of hydrogen as an electron donor, little is known about the ability of hydrogen-producing bacteria to grow in the presence of chlorinated solvents. In this study, 18 Clostridium strains including nine uncharacterized isolates originating from chlorinated solvent contaminated groundwater were tested to determine their ability to fermentatively produce hydrogen in the presence of three common chlorinated aliphatic groundwater contaminants: 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. Some strains produced hydrogen in media containing concentrations as high as 29.7 mM DCA, 9.8 mM TCA, and 1.1 mM PCE. None of the strains biotransformed chlorinated solvents under the conditions tested. Results demonstrate that many Clostridium species are chlorinated solvent tolerant, producing hydrogen even in the presence of high concentrations of DCA, TCA, and PCE. These findings have important implications for bioremediation of contaminated soil and groundwater.     

Stabilization of water-in-oil emulsion by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 and its application to biotransformation

Rhodococcus opacus B-4, which has recently been isolated as an organic solvent-tolerant bacterium, stabilized water-in-oil (w/o) emulsions by inhibition of droplet coalescence when the cells were dispersed in 90% (v/v) organic solvents. Confocal microscopy revealed that many bacterial cells assembled at the interface between oil and water droplets, though free cells were also detectable at the inside of water droplets. Bacterial cells in the w/o emulsion were capable of utilizing both a water-soluble (glucose) and an oil-soluble substrate (oleic acid) as an energy source. Availability of the w/o emulsion as an immobilized cell system in organic solvents was demonstrated using production of indigo from indole and production of o-cresol from toluene as model conversions. When glucose and oleic acid were simultaneously supplied as energy sources, the w/o emulsion culture of R. opacus B-4 produced indigo and o-cresol at levels of 0.217 and 2.12 mg ml⁻¹, respectively, by 12 h.     

非水酶学和非水相生物催化研究进展

近年来工业生物技术飞速发展,酶学和生物催化领域也取得突破性进展,特别在酶在非水相中活性及稳定性研究,耐溶剂生物催化剂的筛选、构建、修饰和改造,生物相容性和环境相容性好的绿色介质等方面取得了较大的进展。最近的研究热点和未来几年的研究方向主要为:基于基因组信息的耐溶剂酶的虚拟筛选和构建;基于自然界筛选新酶基因的耐溶剂酶重构和改造;离子液体等环境友好的绿色介质系统等几个方面。     

Solvent tolerant Pseudomonads as a source of novel lipases for applications in non-aqueous systems

Abstract

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are ubiquitous biocatalysts known to catalyze the hydrolysis of water insoluble triglycerides in aqueous medium and carry out the reverse reaction (synthesis) under organic solvent rich medium. Microbial lipases have received a great deal of attention in the field of food technology, pharmaceutical sciences, chemical and detergent industries due to their stability, selectivity, mild operation conditions and broad substrate specificity. Despite these advantages, low activity and stability displayed in organic medium has restricted their commercial application in organic synthesis. Researchers have explored alternative ways to modify the enzymes making them suitable for use in non-conventional media. In this context, harvesting lipases from “Solvent Tolerant Microbes” has recently become an attractive approach. These microbes are able to grow in the presence of high concentrations of organic solvents, generally known to have detrimental effect on microorganisms. Such microbes survive through novel adaptation mechanisms and secretion of solvent stable enzymes having efficient functionality in solvent-rich media. These enzymes could be useful for bioconversion in non-conventional media. In the current review, this approach is described with an emphasis on characteristics, applications and genetic aspect of lipases from the genus Pseudomonas.     

芽胞杆菌(Bacillus sp.)YX-1耐有机溶剂葡萄糖脱氢酶的基因克隆与酶学性质

[目的] 从芽胞杆菌(Bacillus sp.)YX-1基因组中克隆出一种有机溶剂耐受型的葡萄糖脱氢酶基因,实现了该基因在大肠杆菌中的高效表达,研究了重组蛋白的酶学性质.[方法] 依据芽胞杆菌属中葡萄糖脱氢酶氨基酸序列的保守性,设计合理引物,钓取来源于Bacillus sp.YX-1的葡萄糖脱氢酶基因,构建诱导型表达载体pET28a-gdh,于大肠杆菌中进行表达.镍柱亲和层析法纯化重组蛋白,考察了重组蛋白的酶学性质.[结果] 葡萄糖脱氢酶基因全长为786 bp,编码261个氨基酸.酶学研究结果表明:该酶最适反应温度为45℃,最适pH值为8.0;具有良好的有机溶剂耐受性,于50％的辛烷、环己烷、癸烷中室温放置1h后,酶活仍能保持90％以上;具有较宽的底物谱,对多种糖均具有一定的催化活性,其中催化D-葡萄糖的活力最高,产生还原型辅酶因子;对辅酶NADH和NADPH具有相似的依赖性,对NAD+和NADP+的催化比活分别为8.37 U/mg和8.62 U/mg.[结论]利用生物信息学成功地挖掘出Bacillus sp.YX-1一种耐有机溶剂的葡萄糖脱氢酶,为氧化还原酶在有机相反应中的的辅酶再生循环提供了新型的生物催化剂.     

Utilization of hydrophobic bacterium <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 as whole-cell catalyst in anhydrous organic solvents

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 μg ml⁻¹ by 24 h.     

Isolation and characterization of Deinococcus geothermalis T27, a slightly thermophilic and organic solvent-tolerant bacterium able to survive in the presence of high concentrations of ethyl acetate

A solvent-tolerant, slightly thermophilic bacterium was isolated at 45 degrees C in the presence of toluene vapor provided as the sole carbon source. Strain T27 was identified as Deinococcus geothermalis T27. It could tolerate high concentrations of solvent provided as a nonaqueous layer (5% and 20%, v/v) to a cell suspension and had a remarkable ability to tolerate a broad range of solvents having log P(ow) values ranging from 5.6 of n-decane to as low as 0.7 of ethyl acetate. It was also able to utilize some of the solvents tested as a growth substrate at 45 degrees C. The addition of Ca(2+) ion, glucose and fructose partially promoted solvent tolerance. Cells exposed to ethyl acetate appeared to have a smaller size; however, the cell structure was not altered and was apparently well defined even after solvent shock. The tolerance of D. geothermalis T27 in the presence of high levels of toxic solvent stress at a comparatively high temperature indicated its potential use in biotechnological applications as well as bioremediation of xenobiotics.     

Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn²⁺ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M²⁺) cofactors, except Ca²⁺, for catalysis. We found that Zn²⁺ yielded the highest enzyme complex thermostability (T_m of 87 °C) and solvent tolerance. All AbHpaI•M²⁺ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn²⁺ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M²⁺ cofactors). For the aldol condensation reaction, AbHpaI•M²⁺ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M²⁺ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M²⁺ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn²⁺-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca²⁺ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn²⁺ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.     

An organic solvent-stable alkaline protease from Pseudomonas aeruginosa strain K: Enzyme purification and characterization

The organic solvent-tolerant strain K protease was purified to homogeneity by ammonium sulphate precipitation and anion exchange chromatography with 124-fold increase in specific activity. The molecular mass of the purified enzyme as revealed by SDS-PAGE electrophoresis is 51,000 Da. The strain K protease was an alkaline metalloprotease with an optimum pH and temperature of 10 and 70 °C, respectively. The enzyme showed stability and activation in the presence of organic solvents with log P_a/w values equal or more than 4.0. After 14 days of incubation, the purified protease was activated 1.11, 1.82, 1.50, 1.75 and 1.80 times in 1-decanol, isooctane, decane, dodecane and hexadecane, respectively.     

Preparation of dodecanol-tolerant strains of Yarrowia lipolytica

Dodecanol (1% v/v) and dodecanoic acid (1% w/v) inhibited growth of Yarrowia lipolytica in complex media supplemented with glucose but dodecanedioic acid (1% w/v) was not toxic. Dodecanol-tolerant strains were prepared from the wild type strain H222 as well as the acyl-CoA oxidase deleted (deltaPOX2, POX3, POX5) strain MTLY35. These strains grew in rich media containing up to 10% (v/v) dodecanol. Dodecanol-tolerant strains remained dodecanol tolerant after they had been cultured in rich media without dodecanol. No significant amount of dodecanedioic acid was accumulated by the dodecanol-tolerant strains when grown on glucose in the presence of dodecanol.