Characterization of a thermostable protease from Bacillus subtilis BSP strain.

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Expression and characterization of an organic solvent tolerant recombinant lipase from Staphylococcus capitis SH6 for food wastewater treatment.

The study illustrated here aims on an organic solvent tolerant lipase from Staphylococcus capitis (SCL). The gene part, encoding the mature lipase, was cloned and sequenced. The concluded polypeptide sequence, equivalent to the protein, consist of 388 amino acid residues with a molecular mass of about 45 kDa. A structure-based alignment of the SCL amino acid sequence shows high identities with those many staphylococcal lipases. From this alignment of sequences, the catalytic triad (Ser 117, Asp 308 and His 347) of SCL could be identified. The mature part of the SCL was expressed in Escherichia coli and the recombinant lipase (r-SCL) was purified to homogeneity. The purified r-SCL presented a quite interesting stability at low temperatures (< 30 °C) and the enzyme was found to be highly stable in polar organic solvent and at a pH ranging from 3 to 12. After that, we have demonstrated that the recombinant enzyme may be implicated in the biodegradability of oily wastewater from effluents of fast-food restaurants; the maximum conversion yield into fatty acids obtained at 30 °C, was 65%.

Simultaneous purification and characterization of detergent-stable, solvent-tolerant haloextremozymes protease and lipase from Haloferax sp. strain GUBF 2.

Industrial important proteases and lipases are in increasing demand for various biotechnological applications. In the present study, the concomitantly produced protease and lipase by Haloferax sp. strain GUBF 2 were simultaneously purified as a heterogeneous lipase (45 and 66 kDa) and homogeneous protease (180 kDa); with 28.3 and 31.36 fold purity, respectively using Sephadex G-200. The aforementioned extremozymes were active at pH 3-13, 20-80 °C, 1-5 M NaCl, with optimal activity at pH 6, 70 °C, and 3 M NaCl, thus exhibiting attributes of true haloextremozymes. The Km and Vmax of purified lipase were 3.47 mM and 16.2 U/mL, while protease were 3.29 mg/mL and 28.5 U/mL, respectively. FTIR bands corresponding to the vibrations of amide II and amide III were detected in haloextremozymes which could perhaps be used to determine the secondary structure of the purified proteins. Furthermore, the activity of both enzymes was stimulated by Ca2+ and inhibited by 10 mM Hg2+ and phenylmethyl sulphonyl fluoride (PMSF). Additionally, these haloextremozymes are stable in the presence of detergent additives and organic solvents. In addition, purified protease displayed 74.3 ± 4.85% in-vitro blood clot dissolution activity. Conclusively this study revealed the key features, unusual properties, and possible biomedical applications of detergent-stable and organic solvent-tolerant haloextremozymes from Haloferax sp. strain GUBF 2 to date unexplored.

Expression and characterization of a novel organic solvent tolerant protease from Bacillus sphaericus DS11.

Organic solvent-tolerant proteases have many applications in the synthesis of peptides. In this study, we have developed a low-cost and convenient method to produce highly concentrated organic solvent-tolerant protease. Organic solvent tolerant protease (OSP) gene from Bacillus sphaericus DS11 was cloned and expressed in Bacillus subtilis WB800. The optimum pH of the recombinant protease was 9.0. The optimum temperature of the recombinant protease was 40 °C. The recombinant protease was purified by ethanol with the yield of (87.33%). The yield of OSP enriched by ethanol was higher than that of by Ni-chelating affinity chromatography, which indicated that precipitation of the recombinant OSP with ethanol is a relatively low-cost and fast method for organic solvent -tolerant protease preparation. These results showed that this enzyme could be very useful in different industrial applications.

New Recombinant Cold-Adapted and Organic Solvent Tolerant Lipase from Psychrophilic Pseudomonas sp. LSK25, Isolated from Signy Island Antarctica.

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5⁻30 °C and at pH 6⁻8. A significant enhancement of lipolytic activity was observed in the presence of Ca2+ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents.

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 37.9 kDa. 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.

Changes in the membrane fatty acid composition in Anoxybacillus flavithermus subsp. yunnanensis E13T as response to solvent stress.

Anoxybacillus flavithermus subsp. yunnanensis is currently the first species of strictly thermophilic bacteria that is able to tolerate a broad range of solvents. Unlike most of solvent-tolerant mesophilic bacteria, the bacterium does not synthesize unsaturated fatty acids. Our results revealed that in growing cells of A. flavithermus subsp. yunnanensis E13T, ethanol and toluene resulted in an increase in straight-chain fatty acids, mainly C16:0, leading to a more rigid membrane. Moreover, the increase in straight-chain fatty acids caused by ethanol was much higher than that of toluene. High temperature had little effect on the fatty acid composition by itself, whereas the combined conditions of high temperature and ethanol caused the dramatic increase in straight-chain fatty acids (mainly C16:0), that was balanced by decreasing branched fatty acids. The increase was also temperature dependent. The proportion of C16:0 further increased above 60 °C. No similar evidence was found in four other species of Anoxybacillus. The results suggested that A. flavithermus subsp. yunnanesis seems to develop a different response to solvents compared to its mesophilic counterparts, which consist of an increase in the saturated straight/branched ratio.

Assessment of alkaline cholesterol oxidase purified from Rhodococcus sp. PKPD-CL for its halo tolerance, detergent and organic solvent stability.

The novel bacterium, Rhodococcus sp. PKPD-CL was isolated and identified from the 'Chilika Lake' located at Odisha state of India, which is a largest brackish water habitat in Asia. Rhodococcus sp. PKPD-CL produces extracellular halo tolerant, detergent and organic solvent stable alkaline cholesterol oxidase. It has apparent molecular weight of 60 kDa and was purified 59 fold by using 60% saturated ammonium sulfate fractionation, anion exchange followed by size exclusion chromatographic techniques with 37% recovery. It showed substrate specificity for 3ß-hydroxysteroids with Km of 1.1 × 10(-4)M for cholesterol. The pH, 8.0 and the temperature, 37 °C were required for its optimum activity. Enzyme is considerably stable at pH 6.0-8.5 and temperature up to 50 °C. At 4 and 30 °C it maintained its 100% activity up to 60 days. The isoelectric point of the enzyme was 9.5. It showed 80% residual activity with 20% NaCl (3.42 M) and 83% relative activity with 12% NaCl (2.05 M) concentration. The metal ions like Zn(2+), Cu(2+), Ag+, Fe(3+), Ba(2+) inhibited the enzyme activity >60% while Hg(2+) served a potent inhibitor whereas Mg(2+) found to be a good enhancer for it. The enzyme was stable in presence of chemical reagents (NaN3, EDTA), detergents (Tween-80, Tween-20, Triton X-100, sodium cholate) and various organic solvents (isopropanol, ethanol, benzene, chloroform, methanol, toluene, ethyl acetate, butanol and dimethylsulfoxide). Such a multi stress tolerant and versatile enzyme produced by Rhodococcus sp. PKPD-CL may serve as a good choice for industrial applications.

Role of α-helical structure in organic solvent-activated homodimer of elastase strain K.

Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.

High-level synthesis and secretion of laccase, a metalloenzyme biocatalyst, by the halophilic archaeon Haloferax volcanii.

Haloarchaea and their enzymes have extremophilic properties desirable for use as platform organisms and biocatalysts in the bioindustry. These GRAS (generally regarded as safe) designated microbes thrive in hypersaline environments and use a salt-in strategy to maintain osmotic homeostasis. This unusual strategy has resulted in the evolution of most of the intracellular and extracellular enzymes of haloarchaea to be active and stable not only in high salt (2-5M) but also in low salt (0.2M). This salt tolerance is correlated with a resilience to low water activity, thus, rendering the haloarchaeal enzymes active and stable in organic solvent and temperatures of 50-60°C used in the enzymatic biodelignification and saccharification of lignocellulosic materials. High-level secretion of haloarchaeal enzymes to the extracellular milieu is useful for many applications, including enzymes that deconstruct biomass to allow for lignin depolymerization and simultaneous fermentation of sugars released from hemicellulose and cellulose fractions of lignocellulosics. Here we detail strategies and methods useful for high-level secretion of a laccase, HvLccA, that mediates oxidation of various phenolics by engineering a recombinant strain of the haloarchaeon Haloferax volcanii.

Characterization of an organic solvent-tolerant polysaccharide lyase from Microbulbifer thermotolerans DAU221.

Alginate and its derivatives are annually produced approximately 30,000 tons or more and are applied to various industries as they are natural polymers. The global market for alginate and its derivatives has been growing steadily. There is little research compared to other enzymes produced through biomass degradation or modification. An alginate lyase, MtAl138, from Microbulbifer thermotolerans DAU221 was cloned and identified in Escherichia coli BL21 (DE3). MtAl138 contains a highly conserved motif (R538TELR, Q607IH609, and YFKAGVY716NQ), which indicates that it belongs to the polysaccharide lyase family 7 (PL7). MtAl138, with a molecular weight of 77 kDa worked optimally at 45 °C and pH 7.4. MtAl138 showed twice as much activity as when there was no NaCl when there was between 100 and 600 mM NaCl. Moreover, its activity increased in organic solvents such as benzene, hexane, methanol, and toluene. Based on the thin layer chromatography analyses, MtAl38 is an endo-type enzyme that produces di-, tri-, or tetrasaccharides from polyG and polyM. This study provided that MtAl138 is an endoenzyme that showed outstanding enzymatic activity at concentrated salt solutions and organic solvents, which makes it a reasonably attractive enzyme for use in various industries.

Coenzyme Binding Site Analysis of an Isopropanol Dehydrogenase with Wide Substrate Spectrum and Excellent Organic Solvent Tolerance.

NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for the regeneration of coenzyme are expected as costly of the coenzymes. Herein, a solvent-tolerant isopropanol dehydrogenase (IDH) showing lower similarity (37%) with other proteins was obtained and characterized. The enzyme exhibits high catalysis ability of its substrates methanol, ethanol, ethylene glycol, glycerol, isopropanol, n-butanol, isobutanol, and acetone. And it has good adaptability in organic solvents (isopropanol, acetonitrile, acetone, and acetophenone). Interaction force and the corresponding amino acid residues between IDH and NAD+ or NADP+ were parsed by docking. The wide substrate spectrum, excellent organic solvent tolerance, and good biocatalytic activity make the excavated enzyme a promising biocatalyst for the production of chiral compounds industrially and the construction of coenzyme regeneration systems in aqueous organic phase or organic phase.

Unravelling protein -organic solvent interaction of organic solvent tolerant elastase from Pseudomonas aeruginosa strain K crystal structure.

The utilization of organic solvents as reaction media for enzymatic reactions provides numerous industrially attractive advantages. However, an adaptation of enzyme towards organic solvent is unpredictable and not fully understood because of limited information on the organic solvent tolerant enzymes. To understand how the enzyme can adapt to the organic solvent environment, structural and computational approaches were employed. A recombinant elastase from Pseudomonas aeruginosa strain K was an organic solvent tolerant zinc metalloprotease was successfully crystallized and diffracted up to 1.39 Å. Crystal structure of elastase from strain K showed the typical, canonical alpha-beta hydrolase fold consisting of 10-helices (118 residues), 10- ß-strands (38 residues) and 142 residues were formed other secondary structure such as loop and coil to whole structure. The elastase from Pseusomonas aeruginosa strain K possess His-140, His-144 and Glu-164 served as a ligand for zinc ion. The conserved catalytic triad was composed of Glu-141, Tyr-155 and His-223. Three-dimensional structure features such as calcium-binding and presence of disulphide-bridge contribute to the stabilizing the elastase structure. Molecular dynamic (MD) simulation of elastase revealed that, amino acid residues located at the surface area and disulphide bridge in Cys-30 to Cys-58 were responsible for enzyme stability in organic solvents.

Characterization of Organic Solvent-Tolerant Lipolytic Enzyme from Marinobacter lipolyticus Isolated from the Antarctic Ocean.

The Antarctic marine environment provides a good source of novel lipolytic enzymes that possess beneficial properties, i.e., resistance to extreme physical and chemical conditions. We found a lipolytic Escherichia coli colony that was transformed using genomic DNA from Marinobacter lipolyticus 27-A9 isolated from the Antarctic Ross Sea. DNA sequence analysis revealed an open reading frame of lipolytic enzyme gene. The gene translates a protein (LipA9) of 404 amino acids with molecular mass of 45,247 Da. Recombinant LipA9 was expressed in E. coli BL21 (DE3) cells and purified by anion exchange and gel filtration chromatography. The kcat/Km of LipA9 was 175 s-1 µM-1, and the optimum temperature and pH were 70 °C and pH 8.0, respectively. LipA9 had quite high organic solvent stability; it was stable toward several common organic solvents up to 50% concentration. Substrate specificity studies showed that LipA9 preferred a short acyl chain length of p-nitrophenyl ester and triglyceride. Sequence analysis showed that LipA9 contained catalytic Ser72 and Lys75 in S-x-x-K motif, like family VIII esterases. Homology modeling and site-directed mutagenesis studies revealed that Tyr141 and Tyr188 residues were located near the conserved motif and played an important role in catalytic activity.

An alternative genome-integrated method for undomesticated Bacillus subtilis and related species.

Given their applicability in genetic engineering, undomesticated Bacillus strains are extensively used as non-natural hosts for chemical production due to their high tolerance of toxic substrates or products. However, they are difficult to genomically modify due to their low transformation efficiencies. In this study, the Bacillus-E. coli shuttle vector pHY300PLK, which is widely used in gram-positive bacteria, was adopted for genome integration in organic solvent-tolerant Bacillus isolates. The Bacillus-replicative vector was used to deliver homologous recombinant DNA and propagate itself inside the host cell, increasing the likelihood of genome integration of the recombinant DNA. Then, the unintegrated vectors were cured by cell cultivation in antibiotic-free medium with facilitation of nickel ions. The developed protocol was successfully demonstrated and validated by the disruption of amyE gene in B. subtilis 168. With an improved clonal selection protocol, the probability of clonal selection of the amyE::cat genome-integrated mutants was increased up to 42.0 ± 10.2%. Genome integration in undomesticated, organic solvent tolerant Bacillus strains was also successfully demonstrated with amyE as well as proB gene creating the gene-disrupted mutants with the corresponding phenotype and genotype. Not only was this technique effectively applied to several strains of undomesticated B. subtilis, but it was also successfully applied to B. cereus. This study validates the possibility of the application of Bacillus-replicative vector as well as the developed protocol in a variety of genome modification of undomesticated Bacillus species.

Cloning, purification, and characterization of an organic solvent-tolerant chitinase, MtCh509, from Microbulbifer thermotolerans DAU221.

BACKGROUND: The ability to use organic solvents in enzyme reactions offers a number of industrially useful advantages. However, most enzymes are almost completely inactive in the presence of organic solvents. Thus, organic solvent-tolerant enzymes have potential applications in industrial processes. RESULTS: A chitinase gene from Microbulbifer thermotolerans DAU221 (mtch509) was cloned and expressed in Escherichia coli BL21 (DE3). The molecular weight of the expressed MtCh509 protein was approximately 60 kDa, and it was purified by His-tag affinity chromatography. Enzymatic assays showed that the optimum temperature for MtCh509 chitinase activity was 55 °C, and the enzyme was stable for 2 h at up to 50 °C. The optimum pH for MtCh509 activity was in the sub-acidic range, at pH 4.6 and 5.0. The activity of MtCh509 was maintained in presence of 1 M salt, gradually decreasing at higher concentrations, with residual activity (20%) detected after incubation in 5 M salt. Some organic solvents (benzene, DMSO, hexane, isoamyl alcohol, isopropyl alcohol, and toluene; 10-20%, v/v) increased the reactivity of MtCh509 relative to the aqueous system. When using NAG3, as a substrate, MtCh509 produced NAG2 as the major product, as well as NAG4, demonstrating that MtCh509 has transglycosylation activity. The K m and V max values for MtCh509 using colloidal chitin as a substrate were 9.275 mg/mL and 20.4 U/mg, respectively. Thus, MtCh509 could be used in extreme industrial conditions. CONCLUSION: The results of the hydrolysate analysis and the observed increase in enzyme activity in the presence of organic solvents show that MtCh509 has industrially attractive advantages. This is the first report on an organic solvent-tolerant and transglycosylating chitinase from Microbulbifer species.

Organic Solvent-Tolerant Esterase from Sphingomonas glacialis Based on Amino Acid CompositionAnalysis: Cloning and Characterization of EstSP2.

Organic solvent-tolerant (OST) enzymes are widely applied in various industries due to their activity and stability in organic solvents, higher substrate solubility, and increased stereo-selectivity. However, the criteria for identifying OST enzymes largely remain unresolved. In this study, we compared the amino acid composition of 19 OST esterases and 19 non-OST esterases. OST esterases have increased ratio of Ala and Arg residues and decreased ratio of Asn, Ile, Tyr, and Ser residues. Based on the amino acid composition analysis, we cloned acarboxylesterase (EstSP2) from a psychrophilic bacterium, Sphingomonas glacialis PAMC 26605, and characterized its recombinant protein. EstSP2 is substrate specific to p-nitrophenyl acetate and hydrolyzed aspirin, with optimal activityat 40°C; at 4°C, the activity is approximately 50% of its maximum. As expected, EstSP2showstolerance in up to 40% concentration of polar organic solvents, including dimethyl sulfoxide, methanol, and ethanol. The results of this study suggest that selection of OST esterases based on their amino acid composition analysis could be a novel approach to identify OST esterases produced from bacterial genomes.

Crystal Structure and Biochemical Characterization of an Aminopeptidase LapB from Legionella pneumophila.

Aminopeptidases are a group of exopeptidases that catalyze the removal of a wide range of N-terminal amino acid residues from peptides and proteins. They have many important commercial applications in the food industry. We determined the crystal structure of an aminopeptidase LapB from Legionella pneumophila. The overall structure reveals that the N-terminal protease-associated (PA) domain presents a new fold and shields the active site cavity of the conserved C-terminal peptidase domain. The steady-state kinetic analysis of LapB and the PA domain deletion mutant indicate that the PA domain inhibited enzyme activity of the peptidase domain. Interestingly, the activity of LapB was largely increased by various organic solvents such as ethanol, propanol, and methanol at the concentration of 60% (v/v). CD analysis provided evidence that organic solvents induce the PA domain conformational changes that eliminate the inhibition role. The unique properties indicate the application potential of LapB in the food processing industry.

Optimization of thermostable organic solvent-tolerant lipase production by thermotolerant Rhizopus sp. using solid-state fermentation of palm kernel cake.

This study enhanced the production of thermostable organic solvent-tolerant (TS-OST) lipase by locally isolated thermotolerant Rhizopus sp. strain using solid-state fermentation (SSF) of palm kernel cake (PKC). The optimum conditions were achieved using a series of statistical approaches. The cultivation parameters, which include fermentation time, moisture content, temperature, pH, inoculum size, various carbon and nitrogen sources, as well as other supplements, were initially screened by the definitive screening design, and one-factor-at-a-time using PKC as the basal medium. Three significant factors (olive oil concentration, pH, and inoculum size) were further optimized using face-centred central composite design. The results indicated a successful and significant improvement of lipase activity by almost two-fold compared to the initial screening production. The findings showed that the optimal conditions were 2% (v/w) inoculum size, 2% (v/w) olive oil, 0.6% (w/w) peptone, 2% (v/w) ethanol, 70% moisture content at initial pH 10.0 and 45 °C within 72 h of fermentation. Process optimization resulted in maximum lipase activity of 58.63 U/gram dry solids (gds). The analysis of variance showed that the statistical model was significant (p value <0.0001) and reliable with a high value of R2 (0.98) and adjusted R2 (0.96). This indicates a better correlation between the actual and predicted responses of lipase production. By considering this study, the low-cost PKC through SSF appears to be promising in the utilization of agro-industrial waste for TS-OST lipase production. This is because satisfactory enzyme activity could be attained that promises industrial applications.

Development of a Novel Escherichia coli-Kocuria Shuttle Vector Using the Cryptic pKPAL3 Plasmid from K. palustris IPUFS-1 and Its Utilization in Producing Enantiopure (S)-Styrene Oxide.

The novel cryptic pKPAL3 plasmid was isolated from the Gram-positive microorganism Kocuria palustris IPUFS-1 and characterized in detail. pKPAL3 is a circular plasmid that is 4,443 bp in length. Open reading frame (ORF) and homology search analyses indicated that pKPAL3 possesses four ORFs; however, there were no replication protein coding genes predicted in the plasmid. Instead, there were two nucleotide sequence regions that showed significant identities with untranslated regions of K. rhizophila DC2201 (NBRC 103217) genomic sequences, and these sequences were essential for autonomous replication of pKPAL3 in Kocuria cells. Based on these findings, we constructed the novel Escherichia coli-Kocuria shuttle vectors pKITE301 (kanamycin resistant) and pKITE303 (thiostrepton resistant) from pKPAL3. The copy numbers of the constructed shuttle vectors were estimated to be 20 per cell, and they exhibited low segregation stability in Kocuria transformant cells in the absence of antibiotics. Moreover, constructed vectors showed compatibility with the other K. rhizophila shuttle vector pKITE103. We successfully expressed multiple heterologous genes, including the styrene monooxygenase gene from Rhodococcus sp. ST-10 (rhsmo) and alcohol dehydrogenase gene from Leifsonia sp. S749 (lsadh), in K. rhizophila DC2201 using the pKITE301P and pKITE103P vectors under the control of the glyceraldehyde 3-phosphate dehydrogenase (gapdh) promotor. The RhSMO-LSADH co-expressing K. rhizophila was used as a biocatalyst in an organic solvent-water biphasic reaction system to efficiently convert styrene into (S)-styrene oxide with 99% ee in the presence of 2-propanol as a hydrogen donor. The product concentration of the reaction in the organic solvent reached 235 mM after 30 h under optimum conditions. Thus, we demonstrated that this novel shuttle vector is useful for developing biocatalysts based on organic solvent-tolerant Kocuria cells.