Organic Solvent Tolerance Research Articles

Purification and characterization of an organic solvent-tolerant and detergent-stable lipase from Haloferax mediterranei CNCMM 50101

The present study investigates the purification and biochemical characterization of an extracellular lipase (HML) from Haloarchaea Haloferax mediterranei strain ATS1, isolated from the Sebkha (Medea, Algeria). The pure protein was obtained with ammonium sulfate precipitation (40–70%)-dialysis and UNO Q-6 FPLC, and characterized biochemically. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer, with a molecular mass of 45,011.09 Da. It showed optimum lipase activity at pH 7 and 60 °C. HML showed a higher specific activity on triacylglycerols with long-chains fatty acids, indicating that HML is a true lipase. This enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggested its belonging to the serine lipase family. The Km and Vmax for HML toward olive oil were 1.01 mM and 1195 U/mg, respectively. Compared to Lipolase, HML displayed an elevated organic solvent tolerance, an outstanding stability to surfactants, oxidizing, and auxiliary agents, a considerable compatibility with various commercialized laundry detergents, and wash performance analysis revealed that it could remove oil-stains effectively. Overall, HML has a number of attractive properties that make it a potential promising candidate for the synthesis of non-aqueous peptides and detergent formulations.

IrrE Improves Organic Solvent Tolerance and Δ1-Dehydrogenation Productivity of Arthrobacter simplex.

During steroid bioconversion, organic solvents are widely used for facilitating hydrophobic substrate dissolution in industry. Thus, strains that tolerate organic solvents are highly desirable. IrrE, a global transcriptional factor, was introduced into Arthrobacter simplex with Δ1-dehydrogenation ability. The results evidenced that IrrE did not affect cell biological traits and biotransformation performance under non-stress conditions. However, the recombinant strain achieved a productivity higher than that of the control strain in systems containing more ethanol and substrate, which coincided with cell viability under ethanol stress, the major stress factor during biotransformation. It also demonstrated that IrrE caused genome-wide transcriptional perturbation, and several defense proteins or systems were linked with higher organic solvent tolerance. IrrE simultaneously enhanced cell resistance to various stresses, and its horizontal impacts showed strain and stress dependence. In conclusion, the introduction of exogenous global regulators is an efficient approach to enhance organic solvent tolerance in steroid-transforming strains, resulting in higher productivity.

A cold tolerant lipase develops enhanced activity, thermal tolerance and solvent stability in the presence of calcium nanoparticles: An alternative approach to genetic modulation

Abstract An extracellular cold active lipase producing bacterium was isolated from soil. It was identified as Pseudomonas aeruginosa KC1 strain (GenBank accession number KT334371 ). There are no previous reports on purification of cold active lipase protein from Pseudomonas aeruginosa. The lipase of molecular weight 54 kDa was purified to 33 fold with 8% recovery. The enzyme was active within the range 10–40 °C with maximum activity at 15 °C; pH 7.0–8.5 with 4-nitrophenyl butyrate substrate. Substrate utilization by lipase showed better affinity for short chain fatty acid esters. The enzyme activity was enhanced by Ca2+, Ba2+, Fe2+ and Mg2+; inhibited by Hg2 +, Ni2+, Zn2+ and Co2+. Nano-calcium enabled KC1 lipase (NP-lip) showed enhanced activity for both short and long chain fatty acid esters (NP: 8.8 µg/mL) compared to CaCl2 (1 mM). The activity of the NP-lip system increased 72% at 15 °C and 7 fold at 55 °C while retaining activity for 4 h. Lowering of Km (55% at 15 °C; 45% at 55 °C) and increased Vmax (7 fold at 15 °C; 3.5 fold at 55 °C) was observed for NP-lip system. Heat deactivation kinetics for NP-lip showed drastic improvement in half-life at higher temperatures and entropy-enthalpy compensation. Furthermore the NP-lip system was stable in organic solvents and was effective in the esterification of butyl butyrate and trans-esterification of sunflower oil in n-hexane. This remarkable simultaneous enhancement of activity, temperature and organic solvent tolerance of the NP-lip has potential for industrial usage.

Expression and characterization of a 9-cis-epoxycarotenoid dioxygenase from Serratia sp. ATCC 39006 capable of biotransforming isoeugenol and 4-vinylguaiacol to vanillin

A 9-cis-epoxycarotenoid dioxygenase gene from Serratia sp. ATCC 39,006 (SeNCED) was overexpressed in soluble form in E.coli. SeNCED showed the maximum activity at 30 °C and pH 8.0, and it was stable relatively at range of pH 5–10 and temperature of 20 °C to 30 °C. SeNCED effectively catalyzes the side chain double bond cleavage of isoeugenol and 4-vinylguaiacol to vanillin. The kinetic constant Km values toward isoeugenol and 4-vinylguaiacol were 18.92 mM and 6.31 mM and Vmax values were 50.73 IU/g and 4.77 IU/g, respectively. Moreover, the SeNCED exhibited an excellent organic solvent tolerance and the enzyme activity was substantially improved at presence of 10% of trichloromethane. The produced vanillin was achieved at an around 0.53 g/L (3.47 mM) and 0.33 g/L (2.17 mM) after 8 h reaction at 4 mM of isoeugenol and 4-vinylguaiacol, respectively, using transformed Escherichia coli cells harboring SeNCED in the presence of trichloromethane.

Improving Catalytic Performance of Burkholderiacepacia Lipase by Chemical Modification with Functional Ionic Liquids

Various imidazolium and choline-based functional ionic liquids(ILs) comprising different cations and anions were grafted onto Burkholderiacepacia lipase(BCL) through surface amino acids coupling. The catalytic ac-tivity, thermostability, organic solvent tolerance and adaptability to temperature and pH changes of the modified BCL were then evaluated in olive oil hydrolysis reaction. The results showed that different combinations of cations and anions in ILs had important influence on the catalytic performance of the modified lipases. BCL modified with IL [Choline][H2PO4] was the most improved lipase, in which increases by 1.2 folds in relative activity, 2.5 folds in typi-cal proton solvent(10% methanol, volume fraction), and 1.4 folds in thermostability(after incubation at 70 °C for 2 h) were achieved in relative toits native form. BCL modified with [HOOCEPEG350IM][BF4] had higher optimal tempe- rature and pH, and better thermosability compared with the native and other modified BCLs. The conformational changes of BCLs were also confirmed by fluorescence spectroscopy and circular dichroism spectroscopy.

Characterization of a novel alkaline esterase from Altererythrobacter epoxidivorans CGMCC 1.7731T

ABSTRACTA novel esterase gene (e25) was identified from Altererythrobacter epoxidivorans CGMCC 1.7731T by genome sequence screening. The e25 gene is 948 nucleotides in length and encodes a 315 amino acid protein (E25) with a predicted molecular mass of 33,683 Da. A phylogenetic tree revealed that E25 belongs to the hormone-sensitive lipase (HSL) family of lipolytic enzymes. An activity assay of E25 showed that it exhibited the highest catalytic efficiency when using p-nitrophenyl caproate (C6) as a substrate. The optimum pH and temperature were determined to be approximately pH 9 and 45°C, and the Km and Vmax values were 0.12 mM and 1,772 µmol/min/mg, respectively. After an incubation at 40°C for 80 min, E25 retained 75% of its basal activity. The enzyme exhibited good tolerance to metal cations, such as Ba2+, Ca2+, and Cu2+ (10 mM), but its activity was strongly inhibited by Co2+, Ni2+, Mn2+, and Zn2+. The E25 enzyme was stimulated by glycerol and retained over 60% of its basal activity in the presence of 1% Tween-80 and Triton X-100. Overall, the activity of E25 under alkaline conditions and its organic solvent and detergent tolerance indicate that E25 could be useful as a novel industrial catalyst in biotechnological applications.

Purification and characterization of a novel organic solvent-tolerant and cold-adapted lipase from Psychrobacter sp. ZY124.

By screening 25 different psychrophilic strains isolated from the Arctic habitat, we isolated a strain capable of producing lipase. We identified this strain as Psychrobacter sp. ZY124 based on the amplified 16S rDNA sequence. The lipase, named as Lipase ZC12, produced from the supernatant of Psychrobacter sp. ZY124 cultured at 15°C was purified to homogeneity by ammonium sulfate precipitation followed by Phenyl Sepharose FF gel hydrophobic chromatography. Based on the obtained amino acid sequence, Lipase ZC12 is classified as a member of the Proteus/psychrophilic subfamily of lipase family I.1; it has a molecular weight of 37.9kDa. We also determined that the apparent optimum temperature for Lipase ZC12 activity is 40°C. Lipase ZC12 shows remarkable organic solvent tolerance by remaining more 50% after incubated with 10-90% different organic solvents. In addition, acyl chain esters with C12 or longer were confirmed to be preferable substrates for Lipase ZC12. Lipase ZC12 also shows better stereoselectivity for (R, S)-1-phenylethanol chiral resolution in n-hexane solvent with (S)-1-phenylethanol (eep 92%) and conversion rate (39%) by transesterification reactions. These properties may provide potential applications in biocatalysis and biotransformation in non-aqueous media, such as in detergent, transesterification or esterification and chiral resolution.

Highly efficient synthesis of arbutin esters catalyzed by whole cells of Candida parapsilosis.

Acylation modification of phenol glycosides is currently of great interest due to the improved bioavailability and multiple functions. In this work, mono- or diesters of arbutin, an important phenol glycoside derivative, can be controllably synthesized by using whole-cell biocatalytic systems. Among fourteen microbial strains selected, Candida parapsilosis cells showed the best catalytic activity and high organic solvent tolerance. Compared with the best pure solvent tetrahydrofuran, the use of a binary solvent pyridine-isooctane gave a slightly lower conversion (98.3% vs. 97.2%) and selectivity (85.3% vs. 80.5%) and much higher substrate solubility (37.1 vs. 214.0 mg mL−1), in a 24 h bioconversion of arbutin with a VP-arbutin molar ratio of 15 and whole cell dosage of 30 mg mL−1. The production of various arbutin esters with different fatty acid chain lengths can be realized by using this whole-cell strategy, with the substrate conversion and 6′-regioselectivity of 54.1–98.3% and 83.2–99.0%, respectively.

Purification and biochemical characterization of a thermostable and acid-stable alpha-amylase from Bacillus licheniformis B4-423

Novel thermostable amylase need to be continuously explored with the improvement of industrial requirements. A new acidophilic and thermostable amylase producing bacterium isolated from spring was identified as Bacillus strain on the basis of 16S rDNA. The amylase was purified by ammonium sulphate precipitation, gel chromatography and anion exchange chromatography. SDS-PAGE revealed that the enzyme was monomeric with a molecular weight of 58 kDa. The amylase exhibited optimal activity at pH 5.0 and temperature 100 °C. Then the enzyme showed high stability in pH ranges 4.0-10.0 and more than 90% of maximal activity was found from 20 °C to 80 °C. Apart from good stability toward SDS and non-ionic detergent, the purified enzyme exhibited high compatibility with some inhibitors such as urea and EDTA. The results demonstrated the stability of the enzyme in different organic solvents. Moreover, we determined the amylase gene, compared the structure with α-amylase BAA and BLA and found some thermostability determinants in our enzyme. Overall, presenting various properties were including high thermostability, Ca2+-independency, broad temperature and pH profiles, organic-solvent tolerance as well as excellent stability with detergents. Such characteristics have not been reported for this type of enzyme, and the α-amylase will be a suitable candidate in industrial fields.

Micelle-mediated transport disturbance providing extracellular strategy for alleviating n-butanol stress on Escherichia coli.

One barrier inhibiting further progress in biofuel production is the toxicity of biofuels towards their producers. It is promising to apply gene-based intracellular techniques to engineer better strains with higher organic solvent tolerance. These methods are, however, complex. In the present study, we developed a simple, manageable, and commercial extracellular prototypal strategy to alleviate n-butanol (n-BuOH) stress on Escherichia coli via a micelle-mediated transport disturbance. When the concentration of sodium dodecyl sulfate, a typical anionic surfactant, is high enough to form micelles, n-BuOH will be trapped into/onto the micelles, and the negative charge prevents the n-BuOH from approaching the cells. Our study provides an extracellular strategy to relieve the stress from n-BuOH, and it also exhibits a new angle to advance microbial factories through extracellular routines.

Multiple Roles for Two Efflux Pumps in the Polycyclic Aromatic Hydrocarbon-Degrading Pseudomonas putida Strain B6-2 (DSM 28064).

Microbial bioremediation is a promising approach for the removal of polycyclic aromatic hydrocarbon (PAH) contaminants. Many degraders of PAHs possess efflux pump genes in their genomes; however, their specific roles in the degradation of PAHs have not been clearly elucidated. In this study, two efflux pumps, TtgABC and SrpABC, were systematically investigated to determine their functions in a PAH-degrading Pseudomonas putida strain B6-2 (DSM 28064). The disruption of genes ttgABC or srpABC resulted in a defect in organic solvent tolerance. TtgABC was found to contribute to antibiotic resistance; SrpABC only contributed to antibiotic resistance under an artificial overproduced condition. Moreover, a mutant strain without srpABC did not maintain its activity in long-term biphenyl (BP) degradation, which correlated with the loss of cell viability. The expression of SrpABC was significantly upregulated in the course of BP degradation. BP, 2-hydroxybiphenyl, 3-hydroxybiphenyl, and 2,3-dihydroxybiphenyl (2,3-DHBP) were revealed to be the inducers of srpABC 2,3-DHBP was verified to be a substrate of pump SrpABC; SrpABC can enhance the tolerance to 2,3-DHBP by pumping it out. The mutant strain B6-2ΔsrpS prolonged BP degradation with the increase of srpABC expression. These results suggest that the pump SrpABC of strain B6-2 plays a positive role in BP biodegradation by pumping out metabolized toxic substances such as 2,3-DHBP. This study provides insights into the versatile physiological functions of the widely distributed efflux pumps in the biodegradation of PAHs.IMPORTANCE Polycyclic aromatic hydrocarbons (PAHs) are notorious for their recalcitrance to degradation in the environment. A high frequency of the occurrence of the efflux pump genes was observed in the genomes of effective PAH degraders; however, their specific roles in the degradation of PAHs are still obscure. The significance of our study is in the identification of the function and mechanism of the efflux pump SrpABC of Pseudomonas putida strain B6-2 (DSM 28064) in the biphenyl degradation process. SrpABC is crucial for releasing the toxicity caused by intermediates that are unavoidably produced in PAH degradation, which enables an understanding of how cells maintain the intracellular balance of materials. The findings from this study provide a new perspective on PAH recalcitrance and shed light on enhancing PAH degradation by genetic engineering.

Biochemical properties of free and immobilized Candida viswanathii lipase on octyl-agarose support: Hydrolysis of triacylglycerol and soy lecithin

Abstract Microbial lipases are important enzymes in food and pharmaceutical industries. In this work, a Candida viswanathii lipase was purified by hydrophobic interaction chromatography on octyl Sepharose. The purification presented 78.4% yield and the enzyme was 8.7-fold purified, with specific activity 700.4 U/mg protein and 69 kDa molecular weight. Immobilization of the enzyme on the same support presented 72.5% yield and a derivative with 101% expressed activity (109.2 U/g support), indicating hyperactivation of the enzyme. Optimal activity for both free and immobilized lipase was observed at pH 4.0 and 45 °C. The free and immobilized lipase showed broad-range stability from acid to neutral pH, and apparent activation and stability on organic solvents. The derivative was 60-fold thermostabilized in relation to the free enzyme and fully retained its activity after four cycles of p-nitrophenyl palmitate hydrolysis. Slight activation was observed with dithiothreitol and β-mercaptoethanol. The free and immobilized lipase efficiently hydrolyzed monoesters, simple and mixed long chain triacylglycerols, as well as soy lecithin. The activity and stability in acid pH, the organic solvent tolerance and the lecithin hydrolysis indicate high potential application of the enzyme and its derivative in textile, food and pharma industries and for chemical synthesis.

Immobilization of Candida antarctica Lipase B Onto ECR1030 Resin and its Application in the Synthesis of n‐3 PUFA‐Rich Triacylglycerols

In this study, Candida antarctica lipase B (CALB) is immobilized onto ECR1030 resin and the obtained immobilized preparation is used for the synthesis of n‐3 polyunsaturated fatty acids (PUFA)‐rich triacylglycerols (TAG). The immobilization process is systematically studied. Under the optimized conditions, the immobilized preparation of ECR1030‐CALB with an esterification activity of 10 058 U g−1 is obtained, which is comparable with the commercially available Novozym 435. Confocal microscopy images showed that CALB diffused from the surface to the center of carrier during immobilization. The basic properties of ECR1030‐CALB is also investigated and it is found that the thermostability, acidic and alkaline stability, and organic solvent tolerance of ECR1030‐CALB are comparable with Novozym 435. Interestingly, ECR1030‐CALB showed significantly higher specificity toward EPA and DHA compared with Novozym 435, which made it suitable for the synthesis of n‐3 PUFA‐rich TAG. Overall, the prepared ECR1030‐CALB with excellent esterification activity, basic properties, and catalytic performance might be a promising alternative to commercial Novozym 435. Practical Applications: A previous study found that ECR1030 resin was a robust and promising carrier for the immobilization of CALB. However, the detailed immobilization conditions, the basic properties, and catalytic performance of the immobilized preparations using ECR1030 resin as carrier are still unknown. Consequently, knowledge of the above unknown information for the immobilization of CALB using ECR1030 resin as carrier is of great importance for their further practical applications in lipid chemistry.

Expression of TolC and Organic Solvent Tolerance of Escherichia Coli Ciprofloxacin Resistant Mutants.

AcrAB-TolC is a major efflux pump in Escherichia coli. It was reported that tolC is overexpressed and involves in improving the organic solvent tolerance level in Escherichia colimarR mutants that are resistant to several antibiotics, such as ciprofloxacin. Low and intermediate levels resistance did not improve organic solvent tolerance. Thus, it was decided to measure tolC expression and organic solvent tolerance in high level ciprofloxacin resistant mutants. tolC expression was measured by real time PCR and organic solvent tolerance assay was conducted by counting bacterial colonies on LBGMg agar. Results showed that tolC expression was increased significantly (P<0.05) and organic solvent tolerance was slightly improved in high resistant mutants. It was concluded that high organic solvent tolerance may need higher expression of tolC.

The significance of proline and glutamate on butanol chaotropic stress in Bacillus subtilis 168

BackgroundButanol is an intensively used industrial solvent and an attractive alternative biofuel, but the bioproduction suffers from its high toxicity. Among the native butanol producers and heterologous butanol-producing hosts, Bacillus subtilis 168 exhibited relatively higher butanol tolerance. Nevertheless, organic solvent tolerance mechanisms in Bacilli and Gram-positive bacteria have relatively less information. Thus, this study aimed to elucidate butanol stress responses that may involve in unique tolerance of B. subtilis 168 to butanol and other alcohol biocommodities.ResultsUsing comparative proteomics approach and molecular analysis of butanol-challenged B. subtilis 168, 108 butanol-responsive proteins were revealed, and classified into seven groups according to their biological functions. While parts of them may be similar to the proteins reportedly involved in solvent stress response in other Gram-positive bacteria, significant role of proline in the proline–glutamate–arginine metabolism was substantiated. Detection of intracellular proline and glutamate accumulation, as well as glutamate transient conversion during butanol exposure confirmed their necessity, especially proline, for cellular butanol tolerance. Disruption of the particular genes in proline biosynthesis pathways clarified the essential role of the anabolic ProB-ProA-ProI system over the osmoadaptive ProH-ProA-ProJ system for cellular protection in response to butanol exposure. Molecular modifications to increase gene dosage for proline biosynthesis as well as for glutamate acquisition enhanced butanol tolerance of B. subtilis 168 up to 1.8% (vol/vol) under the conditions tested.ConclusionThis work revealed the important role of proline as an effective compatible solute that is required to protect cells against butanol chaotropic effect and to maintain cellular functions in B. subtilis 168 during butanol exposure. Nevertheless, the accumulation of intracellular proline against butanol stress required a metabolic conversion of glutamate through the specific biosynthetic ProB-ProA-ProI route. Thus, exogenous addition of glutamate, but not proline, enhanced butanol tolerance. These findings serve as a practical knowledge to enhance B. subtilis 168 butanol tolerance, and demonstrate means to engineer the bacterial host to promote higher butanol/alcohol tolerance of B. subtilis 168 for the production of butanol and other alcohol biocommodities.

Effect of Glycosylation on the Biocatalytic Properties of Hydroxynitrile Lyase from the Passion Fruit, Passiflora edulis: A Comparison of Natural and Recombinant Enzymes.

A hydroxynitrile lyase from the passion fruit Passiflora edulis (PeHNL) was isolated from the leaves and showed high stability in biphasic co-organic solvent systems for cyanohydrin synthesis. Cyanohydrins are important building blocks for the production of fine chemicals and pharmaceuticals. Thus, to enhance production yields of PeHNL for industrial applications, we cloned and expressed recombinant PeHNL in Escherichia coli BL21(DE3) and Pichia pastoris GS115 cells without a signal peptide sequence. The aim of this study is to determine the effect of N-glycosylation on enzyme stability and catalytic properties in microbial expression systems. PeHNL from leaves (PeHNL-N) and that expressed in P. pastoris (PeHNL-P) were glycosylated, whereas that expressed in E. coli (PeHNL-E) was not. The enzymes PeHNL-N and PeHNL-P showed much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme PeHNL-E and the deglycosylated mutant N105Q from P. pastoris (PeHNL-P-N105Q). The glycosylated PeHNL-P also efficiently performed transcyanation of (R)-mandelonitrile with a 98 % enantiomeric excess in a biphasic system with diisopropyl ether. These data demonstrate the efficacy of these methods for improving enzyme expression and stability for industrial application through N-glycosylation.

Biodegradation and detoxification of azo solvent dye by ethylene glycol tolerant ligninolytic ascomycete strain of Pseudocochliobolus verruculosus NFCCI 3818

Abstract Ligninolytic microorganisms or enzymes mediated biodegradation of toxic pollutants was studied extensively; but hydrophobic waste management is a major environmental concern. For this reason, 80 fungal strains were isolated from various ligninocellulosic containing microhabitats and around 27.91% strains, demonstrated the ligninolytic activity. Screened isolates were further examined for organic solvent tolerance and the fungal strain LSF9 displayed better growth along with laccase (157 UL −1 ), lignin peroxidase (LiP; 12 UL −1 ) and manganese peroxidase (MnP; 8.6 UL −1 ) activity in the presence of water miscible ethylene glycol. The strain LSF9 was further identified as Pseudocochliobolus verruculosus based on the morphotaxonomic and molecular approach. The P. verruculosus was able to degrade water insoluble Solvent yellow 2 and display 98% decolorization with maximum laccase activity (52.74 UL −1 ). The degradation profile and metabolic fate of fungus treated dye was revealed by HPLC, FTIR and GC-MS analysis. The phytotoxicity study of fungus treated dye on Triticum aestivum (wheat) and Vigna radiata (mong bean) confirmed the nontoxic nature as compare to the phytotoxic Solvent yellow 2 dye. Thus, the present study suggests the potential of ethylene glycol tolerant P. verruculosus for environmental decontamination of solvent soluble azo dyes.

Mechanisms of quinolone resistance in Escherichia coli isolated from companion animals, pet-owners, and non-pet-owners.

The present study investigated the prevalence and mechanisms of fluoroquinolone (FQ)/quinolone (Q) resistance in Escherichia (E.) coli isolates from companion animals, pet-owners, and non-pet-owners. A total of 63 E. coli isolates were collected from 104 anal swab samples, and 27 nalidixic acid (NA)-resistant isolates were identified. Of those, 10 showed ciprofloxacin (CIP) resistance. A plasmid-mediated Q resistance gene was detected in one isolate. Increased efflux pump activity, as measured by organic solvent tolerance assay, was detected in 18 NA-resistant isolates (66.7%), but was not correlated with an increase in minimum inhibitory concentration (MIC). Target gene mutations in Q resistance-determining regions (QRDRs) were the main cause of (FQ)Q resistance in E. coli. Point mutations in QRDRs were detected in all NA-resistant isolates, and the number of mutations was strongly correlated with increased MIC (R = 0.878 for NA and 0.954 for CIP). All CIP-resistant isolates (n = 10) had double mutations in the gyrA gene, with additional mutations in parC and parE. Interestingly, (FQ)Q resistance mechanisms in isolates from companion animals were the same as those in humans. Therefore, prudent use of (FQ)Q in veterinary medicine is warranted to prevent the dissemination of (FQ)Q-resistant bacteria from animals to humans.

Wickerhamomyces subpelliculosus as whole-cell biocatalyst for stereoselective bioreduction of ketones

Newly isolated strains of Wickerhamomyces subpelliculosus were recognized as excellent whole-cell biocatalyst for bioreduction of various ketones. The biocatalytic properties of the new strains were demonstrated in this study by stereoselective bioreduction of acetophenone 1a, 2-heptanone 1b, phenylacetone 1c, 3,4-dimethoxyphenylacetone 1d and 1-cyclopropyl-2-(2-methoxy-4-nitrophenoxy)ethanone 1e. Our study is the first report on application of W. subpelliculosus as whole-cell biocatalyst for stereoselective bioreduction of prochiral ketones. In these processes, both the freshly harvested cell paste and the lyophilized cell powder were tested as biocatalyst using glucose or 2-propanol at various concentrations as cosubstrates for cofactor regeneration. The newly isolated strains of W. subpelliculosus showed diverse characteristics, including optimal pH, temperature and organic solvent tolerance. Bioreductions of phenylacetone 1c applying glucose as cosubstrate under various mild conditions resulted (S)-1-phenylpropanol [(S)-2c] in good to excellent conversion (c=63.4%–99.9%) with excellent enantiomeric excess [ee(S)-2c=98.7%–99.8%].

Gene cloning and characterization of a psychrophilic phthalate esterase with organic solvent tolerance from an Arctic bacterium Sphingomonas glacialis PAMC 26605

Phthalate esters (PEs) are commonly used as plasticizers to soften household and industrial plastic products. The wide use of PEs has become a public health concern as they are considered toxic to human health and PE contamination of the environment is increasing. In this study, we cloned a gene encoding the psychrophilic phthalate esterase (hereafter referred to as EstSP1) from an Arctic bacterium, Sphingomonas glacialis PAMC 26605, and characterized the properties of recombinant EstSP1 protein. EstSP1 belongs to the α/β hydrolase family and its catalytic triad consists of Ser 161, Asp 253, and His 283 residues. Thermal stability and protein folding of EstSP1 were reduced under ambient temperatures when compared with those at 4°C, despite EstSP1 maintaining its secondary structure at 4°C–50°C. EstSP1 is unique among cold-adapted esterases in that it not only has substrate preferences for phthalates with shorter ester chains (C2–C6), but also tolerance to polar organic solvents, including alcohols and DMSO. Furthermore, EstSP1 exhibited a larger change in conformational flexibility in ethanol, isopropanol, and DMSO when evaluated by acrylamide-induced fluorescence quenching. The results indicate that EstSP1 is a novel organic solvent-tolerant, psychrophilic phthalate esterase.