Leifsonia Sp Research Articles

Selective transformation of crocin-1 to crocetin-glucosyl esters by β-glucosidase (Lf18920) from Leifsonia sp. ZF2019: Insights from molecular docking and point mutations

Crocetin di/mono-glucosyl esters (crocin-4 and crocin-5) are rarely distributed in nature, limiting their potential applications in the food and pharmaceutical industries. In the present study, a novel GH3 family β-glucosidase Lf18920 was identified from Leifsonia sp. ZF2019, which selectively hydrolyzed crocin-1 (crocetin di-gentiobiosyl ester) to crocin-5 and crocin-4, but not to its aglycone, crocetin. Under the optimal condition of 40 °C and pH 6.0 for 120 min, Lf18920 almost completely hydrolyzed crocin-1, yielding 73.50±5.66 % crocin-4 and 16.19±1.38 % crocin-5. Molecular docking and point mutation studies revealed that Lf18920 formed a narrow binding channel that facilitated crocin-1 binding. Five single amino acid variants (D50A, D53A, W274A, G420A, and Q421A) were constructed, all of which showed reduced hydrolytic activity. Mutations at D50 and D53, located distal to the active site, increased binding energy and decreased hydrolytic activity, while mutations at W274, G420, and Q421, proximal to the active site, disrupted hydrolytic function. These findings suggest that the narrow binding channel and specific enzyme-substrate interactions are crucial for Lf18920’s selective hydrolytic activity. Overall, this study is the first to report a β-glucosidase capable of selectively transforming crocin-1 to crocetin di/mono-glucosyl esters, offering potential for synthesizing crocin-4 and crocin-5.

Utilization of nickel-graphite electrode as an electron donor for high-efficient microbial removal of solved U(VI) mediated by Leifsonia sp.

Enzymatically catalyzed reduction of metals by bacteria has potential application value to uranium-mine wastewater. However, its practical implementation has long been restricted by its intrinsic drawbacks such as low efficiency and long treatment-time. This study aims to explore the effect of electrodes on U (VI) removal efficiency by a purified indigenous bacteria isolated from a uranium mining waste pile in China. The effects of current intensity, pH, initial U (Ⅵ) concentration, initial dosage of bacteria and contact time on U (Ⅵ) removal efficiency were investigated via static experiments. The results show that U(VI) removal rate was stabilized above 90% and the contact time sharply shortened within 1 h when utilized nickel-graphite electrode as an electron donor. Over the treatment ranges investigated maximum removal of U (Ⅵ) was 96.04% when the direct current was 10 mA, pH was 5, initial U (Ⅵ) concentration was 10 mg/L, and dosage of Leifsonia sp. was 0.25 g/L. In addition, it is demonstrated that U (VI) adsorption by Leifsonia sp. is mainly chemisorption and/or reduction as the quasi-secondary kinetics is more suitable for fitting the process. FTIR results indicated that amino, amide, aldehyde and phosphate -containing groups played a role in the immobilization of U (VI) more or less. SEM and EDS measurements revealed that U appeared to be more obviously aggregated on the surface of cells. A plausible explanation for this, supported by XPS, is that U (VI) was partially reduced to U (IV) by direct current then precipitated on the cells surface. These observations reveal that Nickel-graphite electrode exhibited good electro-chemical properties and synergistic capacity with Leifsonia sp. which potentially provides a new avenue for uranium enhanced removal/immobilization by indigenous bacteria.

Metabolomics Reveals Lysinibacillus capsici TT41-Induced Metabolic Shifts Enhancing Drought Stress Tolerance in Kimchi Cabbage (Brassica rapa L. subsp. pekinensis).

Climate change has increased variable weather patterns that affect plants. To address these issues, we developed a microbial biocontrol agent against drought stress in kimchi cabbage (Brassica rapa L. subsp. pekinensis). We selected three bacterial strains (Leifsonia sp. CS9, Bacillus toyonensis TSJ7, and Lysinibacillus capsici TT41) because they showed a survival rate of up to 50% and good growth rate when treated with 30% PEG 6000. The three strains were treated with kimchi cabbage to confirm their enhanced drought stress resistance under non-watering conditions. Among the three strains, the TT41 treated group showed a significant increase in various plant parameters compared with the negative control on the 7th day. We performed extensive profiling of primary and secondary metabolites from kimchi cabbage and the TT41 strain. Multivariate and pathway analyses revealed that only the TT41 group clustered with the well-watered group and showed almost the same metabolome on the 7th day. When treated with TT41, lactic acid was identified as an indicator metabolite that significantly improved drought stress tolerance. Furthermore, lactic acid treatment effectively induced drought stress tolerance in kimchi cabbage, similar to that achieved with the TT41 strain.

Transformation of Gardenia Yellow Waste Production into Gardenia Blue Pigment by Leifsonia sp. ZF2019 and Evaluation of Its Physicochemical Stability

To produce gardenia blue pigment (GBP) by natural fermentation, a novel bacteria, Leifsonia sp. ZF2019, was isolated from the larvae of a pest of Gardenia jasminoides Ellis fruits and shown to be able to convert gardenia yellow waste production (GYWP) into GBP. The fermentation medium and growth conditions were screened, and the GBP yield, color value, and stability were compared to those of Aspergillus niger fermentation. Leifsonia sp. ZF2019 was shown to effectively produce GBP in medium containing GYWP and leucine (Leu). A temperature of 35 °C, 0.8% leu content, a fermentation period of 72 h, and a pH value of 7.0 were shown to be optimal culture conditions. GBP produced by Leifsonia sp. ZF2019 was of higher yield and quality than that produced by Aspergillus niger fermentation. GBP was purified by D4020 macroporous adsorbent resin, and the effects of pH, temperature, light, oxidant and reductant, conventional food additives, and metal ions on its stability were evaluated. GBP produced by Leifsonia sp. ZF2019 fermentation was as stable as GBP generated directly by pure genipin. LC-MS revealed that the molecular formula of GBP was C37H45N2O8. Our results provide a reference for the preparation of efficient, stable, reliable, and high-quality gardenia blue pigments.

Biochemical characterization of a novel glucose-tolerant GH3 β-glucosidase (Bgl1973) from Leifsonia sp. ZF2019.

Beta-glucosidase (Bgl) is an enzyme with considerable food, beverage, and biofuel processing potential. However, as many Bgls are inhibited by their reaction end product glucose, their industrial applications are greatly limited. In this study, a novel Bgl gene (Bgl1973) was cloned from Leifsonia sp. ZF2019 and heterologously expressed in E. coli. Sequence analysis and structure modeling revealed that Bgl1973 was 748 aa, giving it a molecular weight of 78kDa, and it showed high similarity with the glycoside hydrolase 3 (GH3) family Bgls with which its active site residues were conserved. By using pNPGlc (p-nitrophenyl-β-D-glucopyranoside) as substrate, the optimum temperature and pH of Bgl1973 were shown to be 50°C and 7.0, respectively. Bgl1973 was insensitive to most metal ions (12.5mM), 1% urea, and even 0.1% Tween-80. This enzyme maintained 60% of its original activity in the presence of 20% NaCl, demonstrating its excellent salt tolerance. Furthermore, it still had 83% residual activity in 1M of glucose, displaying its outstanding glucose tolerance. The Km, Vmax, and kcat of Bgl1973 were 0.22mM, 44.44μmol/min mg, and 57.78s-1, respectively. Bgl1973 had a high specific activity for pNPGlc (19.10 ± 0.59 U/mg) and salicin (20.43 ± 0.92 U/mg). Furthermore, molecular docking indicated that the glucose binding location and the narrow and deep active channel geometry might contribute to the glucose tolerance of Bgl1973. Our results lay a foundation for the studying of this glucose-tolerant β-glucosidase and its applications in many industrial settings. KEY POINTS: • A novel β-glucosidase from GH3 was obtained from Leifsonia sp. ZF2019. • Bgl1973 demonstrated excellent glucose tolerance. • The glucose tolerance of Bgl1973 was explained using molecular docking analysis.

Comparative Genomic Understanding of Gram-Positive Plant Growth-Promoting Leifsonia

Root-associated bacteria play critical roles in shaping plant health for abiotic and biotic stress tolerance. The Leifsonia genus includes pathogenic and beneficial bacteria but limited research is available comparatively across species regarding their association with plants. We recently identified the plant growth-promoting Leifsonia sp. C5G2 isolated from the rhizosphere of Plectranthus scutellarioides (coleus) that increased stress tolerance and quality of water-stressed greenhouse ornamentals. Using this beneficial strain as a model, we performed comparative genomics with beneficial, commensal, and pathogenic Leifsonia spp. to better understand plant association and growth promotion under abiotic stress. Comparative phylogenetic analyses showed that beneficial and commensal Leifsonia spp. clustered separately from pathogenic Leifsonia xyli subsp. xyli. In addition, the beneficial or commensal strains share biosynthetic gene and orthologous protein clusters that are not shared with known phytopathogens in our analysis. The phytopathogenic Leifsonia strains also share noticeably higher percent homology with plant-associated virulence factors in closely related Clavibacter spp. than the beneficial or commensal strains. Genome analysis supplemented with in vitro assays validated the findings that our model growth-promoting Leifsonia sp. strain C5G2 was highly prolific on diverse carbon sources. This is unlike known fastidious growth behaviors of phytopathogenic Leifsonia spp. Beneficial Leifsonia sp. C5G2 imparts water stress tolerance in plants, and multiple genes were identified in the genomes of beneficial Leifsonia strains that are potentially involved in bacterial osmotic stress and the production of osmoprotectants and vitamin B9. This work sheds light on the diversity of the genus Leifsonia, with a better understanding of potential mechanisms employed by poorly understood beneficial bacteria.

Bioaugmentation of Soil Contaminated with Zinc

An experiment described in this work aimed to establish the role of bioaugmentation in minimizing adverse outcomes of loamy sand contamination with zinc. The bioaugmentation was conducted with the use of microbial strains being most resistant to the action of zinc, which were isolated from the soil contaminated with 1250 mg Zn2+ kg−1 dry matter (DM) of soil after 12-month incubation. The soil was inoculated with 4 strains of bacteria (Bacillus licheniformis KT986159.1, Bacillus sp. KF956639.1, Gordonia amicalis KM113029.1, Leifsonia sp. KJ191763.1) and 4 strains of fungi (Penicillium raperi KC797647.1, Penicillium janthinellum AY373921.1, Penicillium glabrum LT558918.1, Trichoderma harzianum LN714612.1). In the case of the non-contaminated soil, a metal dose of 250 mg Zn2+ kg−1 DM of soil contributed to enhanced proliferation and microorganisms and enzymatic activity. In turn, zinc in its highest dose (1250 mg Zn2+ kg−1 DM of soil) evoked adverse effects, which were manifested in reduced numbers and diversity of microorganisms and suppressed activity of soil enzymes. This contamination rate stimulated only the proliferation of fungi, but their ecophysiological diversity was reduced either. The bioaugmentation treatment minimized adverse effects of zinc. Unfortunately, the use of autochthonous microorganisms failed to reduce zinc bioavailability in the soil.

Effect of Leifsonia sp. on retardation of uranium in natural soil and its potential mechanisms

Uranium mining and milling activities for many years resulted in release of uranium into the adjoining soil in varying degrees. Bioremediation approaches (i.e., immobilization via the action of bacteria) resulting in uranium bearing solid is supposed as an economic and clean in-situ approach for the treatment of uranium contaminated sites. This study purposes to determine the immobilization efficiency of uranium in soil by Leifsonia sp. The results demonstrated that cells have a good proliferation ability under the stress of uranium and play a role in retaining uranium in soil. Residual uranium in active Leifsonia-medium group (66%) was higher than that in the controls, which was 31% in the deionised water control, 46% in the Leifsonia group, and 47% in the medium group, respectively. This indicated that Leifsonia sp. facilitates the immobilization efficiency of uranium in soil by converting part of the reducible and oxidizable fraction of uranium into the residual fraction. X-ray photoelectron fitting results showed that tetravalent states uranium existed in the soil samples, which indicated that the hexavalent uranium was converted into tetravalent by cells. This is the first report of effect of Leifsonia sp. on uranium immobilization in soil. The findings implied that Leifsonia sp. could, to some extent, prevent the migration and diffusion of uranium in soil by changing the chemical states into less toxicity and less risky forms.

Influence of Leifsonia sp. on U(VI) removal efficiency and the Fe-U precipitates by zero-valent iron.

Zero-valent iron (ZVI) has been widely applied to the remediation of uranium (U)-contaminated water. Notably, indigenous bacteria may possess potential positive or unfavorable influence on the mechanism and stability of Fe-U precipitates. However, the focus of the researches in this field has mainly been on physical and/or chemical aspects. In this study, batch experiments were conducted to explore the effects of an indigenous bacterium (Leifsonia sp.) on Fe-U precipitates and the corresponding removal efficiency by ZVI under different environmental factors. The results showed that the removal rate and capacity of U(VI) was significantly inhibited and decreased by ZVI when the pH increased to near-neutral level (pH = 6~8). However, in the ZVI + Leifsonia sp. coexistence system, the U(VI) removal efficiency were maintained at high levels (over 90%) within the experimental scope (pH = 3~8). This revealed that Leifsonia sp. had a synergistic effect on U(VI) remove by ZVI. According to scanning electron microscope and energy dispersive X-ray detector (SEM-EDX) analysis, dense scaly uranium-phosphate precipitation was observed on ZVI + Leifsonia sp. surface. The X-photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) analysis indicated that Leifsonia sp. facilitated the generation of U(VI)-phosphates precipitates. The X-ray diffraction (XRD) analyses further revealed that new substances, such as (Fe(II)Fe(III)2(PO4)2(OH)2), Fe(II)(UO2)2(PO4)2·8H2O, Fe(II)Fe(III)5(PO4)4(OH)2·4H2O, etc., were produced in the coexisting system of ZVI and Leifsonia sp. This study provides new insights on the feasibility and validity of site application of ZVI to U(VI)-contaminated subsurface water in situ. Graphical abstract.

Production and characterization of cellulose from Leifsonia sp.

Abstract Bacterial cellulose is gaining popularity due to its purity and promising applications in several industries like biomedical, food, packaging industries etc. In the present study, a newly isolated bacterium, Leifsonia sp. has been isolated from inexpensive fruit wastes. The synthesized cellulose from this bacterium has been identified and characterized. To optimize the synthesis of cellulose, various process parameters such as pH, temperature, inoculum concentration, incubation period and additional carbon and nitrogen sources were chosen. Different additives were also selected for the present study to evaluate its impact on cellulose yield. Amongst all the additives used, maltose, soy whey and calcium chloride had profound influence in increasing the production of bacterial cellulose. The isolated strain yielded 5.97 g/L of cellulose at optimal growth conditions of pH 6.5, 30 °C under 7 days of incubation period. Further, characterization of bacterial cellulose was carried out using X-ray diffraction, field emission scanning electron microscopy and Fourier-transform infrared spectroscopy studies to evaluate its properties for suitable applications.

Development of an Efficient and Cost-Effective Enzymatic Process for Production of (R)-[3,5-bis(trifluoromethyl)phenyl] Ethanol Using Carbonyl Reductase Derived from Leifsonia sp. S749.

(R)-[3,5-bis(trifluoromethyl) phenyl] ethanol [(R)-3,5-BTPE] is a crucial chiral intermediate for the synthesis of the NK-1 receptor antagonists aprepitant, rolapitant and fosaprepitant. The carbonyl reductase KR01 from Leifsonia sp. S749, discovered by protein sequence alignment, could convert 3',5'-bis(trifluoromethyl) acetophenone (3,5-BTAP) into (R)-3,5-BTPE with excellent activity and enantioselectivity. In order to enhance the conversion efficiency at high substrate concentrations, the reaction conditions were optimized by response surface analysis. The results showed that 600g/L 3,5-BTAP was bioreduced to (R)-3,5-BTPE (> 99.9% enantiomeric excess) by the recombinant Escherichia coli/pET-28a (+)-KR01 whole cells, with a 98.3% conversion and 59g/L/h productivity under the optimized reaction conditions. In addition, the recombinant E. coli cells could be repeatedly used up to seven times in the reaction mixture containing 90% isopropanol (IPA). This is the highest substrate loading and productivity for the bioreduction of 3,5-BTAP by carbonyl reductase ever reported, and this method represents an efficient and cost-effective process for production of (R)-3,5-BTPE.

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.

Characterization of two cryptic plasmids from Kocuria palustris IPUFS-1 and construction of novel Escherichia coli–Kocuria shuttle vector for biocatalysis

Two cryptic plasmids, designated pKPAL1 and pKPAL2, were identified from the gram-positive bacterium Kocuria palustris IPUFS-1, which was isolated from a fish source. The 2251-bp and 2488-bp circular genomes of pKPAL1 and pKPAL2, respectively, were sequenced. Subsequent open reading frame (ORF) and homology search analyses suggested that pKPAL1 and pKPAL2 possess two and three ORFs, respectively, and encode the putative replication proteins, RepA and RepB, like the genomes of several plasmids in gram-positive bacteria. Thus, pKPAL1 and pKPAL2 were inferred to belong to the ColE2 plasmid family. We constructed novel Escherichia coli-Kocuria shuttle vectors pKITE101-103 based on pKPAL1. The constructed shuttle vector was stably maintained in Kocuria transformant cells, and vector copy number was estimated to be about 60 per cell. Leifsonia sp. S749 alcohol dehydrogenase (LSADH) was efficiently expressed in Kocuria rhizophila DC2201 using the pKITE103P vector under the control of the promoter of glyceraldehyde 3-phosphate dehydrogenase (gapdh). Herein, we demonstrate that the novel shuttle vector is a useful tool for developing biocatalysts based on organic solvent-tolerant Kocuria cells.

Studies of action of heavy metals on caffeine degradation by immobilised <i>Leifsonia sp.</i> strain SIU

Caffeine is an important naturally occurring compound that can be degraded by bacteria. Excessive caffeine consumption is known to have some adverse problems. Previously, Leifsonia sp. strain SIU capable of degrading caffeine was isolated from agricultural soil. The bacterium was tested for its ability to degrade caffeine as the sole carbon and nitrogen source. The isolate was encapsulated in gellan gum and its ability to degrade caffeine in the presence of heavy metals was determined. Out of the nine heavy metals tested, Copper (Cu), Mercury (Hg), and Silver (Ag) had significant effects on caffeine degradation at 1mg/L. Therefore, the concentration of these heavy metals was varied from 0 - 1 mg/L to see at what concentration each metal it has effect. Ag and Hg showed effect at 0.1 mg/L with caffeine degradation of 64.05 and 52.17% respectively, while Cu showed effect at 0.8 mg/L with caffeine degradation of 64.74%. These bacterium features make it an ultimate means for caffeine bioremediation. This is the first report of effect of heavy metals on caffeine degradation by immobilised Leifsonia sp. strain SIU. Keywords : Caffeine, Degradation, Heavy metals, Immobilisation, Leifsonia sp.

Production and characterization of bacterial cellulose by Leifsonia sp. CBNU-EW3 isolated from the earthworm, Eisenia fetida

A total of five bacterial strains were isolated from earthworm, Eisenia fetida and examined for bacterial cellulose (BC) production in Hestrin-Schramm medium (HS). Among the five strains tested, CBNU-EW3 exhibited excellent BC production and was identified as Leifsonia sp. by 16S rDNA sequence analysis. BC production by Leifsonia sp. CBNU-EW3 was optimum at pH 5, 30°C, and with glucose and yeast extract as carbon and nitrogen sources, respectively, according to 15 day-long experiments. (XRD) analysis of the dried pellicle indicated that the BC was partially crystalline type I. Fourier transform infrared spectroscopy (FT-IR) analysis showed that the obtained pellicle contained the same functional groups as typical BC. Field emission scanning electron microscopy (FESEM) images showed that the BC micro-fibril matrix consisted of a flat surface with large pore size and cellulose aggregation.

Enzymatical and microbial degradation of cyclic dipeptides (diketopiperazines)

Diketopiperazines (DKPs) are cyclic dipeptides, representing an abundant class of biologically active natural compounds. Despite their widespread occurrence in nature, little is known about their degradation. In this study, the enzymatical and microbial cleavage of DKPs was investigated. Peptidase catalyzed hydrolysis of certain DKPs was formerly reported, but could not be confirmed in this study. While testing additional peptidases and DKPs no degradation was detected, indicating peptidase stability of the peptide bond in cyclic dipeptides. Besides confirmation of the reported degradation of cyclo(l-Asp-l-Phe) by Paenibacillus chibensis (DSM 329) and Streptomyces flavovirens (DSM 40062), cleavage of cyclo(l-Asp-l-Asp) by DSM 329 was detected. Other DKPs were not hydrolyzed by both strains, demonstrating high substrate specificity. The degradation of cyclo(l-Asp-l-Phe) by DSM 40062 was shown to be inducible. Three strains, which are able to hydrolyze hydantoins and dihydropyrimidines, were identified for the degradation of DKPs: Leifsonia sp. K3 (DSM 27212) and Bacillus sp. A16 (DSM 25052) cleaved cyclo(dl-Ala-dl-Ala) and cyclo(l-Gly-l-Phe), and Rhizobium sp. NA04-01 (DSM 24917) degraded cyclo(l-Asp-l-Phe), cyclo(l-Gly-l-Phe) and cyclo(l-Asp-l-Asp). The first enantioselective cleavage of cyclo(dl-Ala-dl-Ala) was detected with the newly isolated strains Paenibacillus sp. 32A (DSM 27214) and Microbacterium sp. 40A (DSM 27211). Cyclo(l-Ala-d-Ala) and cyclo(l-Ala-l-Ala) were completely degraded, whereas the enantiomer cyclo(d-Ala-d-Ala) was not attacked. Altogether, five bacterial strains were newly identified for the cleavage of DKPs. These bacteria may be of value for industrial purposes, such as degradation of undesirable DKPs in food and drugs and production of (enantiopure) dipeptides and amino acids.

Efficient biocatalysis for the production of enantiopure (S)-epoxides using a styrene monooxygenase (SMO) and Leifsonia alcohol dehydrogenase (LSADH) system

Herein we report the production of enantiopure epoxides through biocatalysis using recombinant Escherichia coli cells expressing Rhodococcus sp. ST-10 styrene monooxygenase (SMO) and Leifsonia sp. S749 alcohol dehydrogenase (LSADH) genes are described. Rhodococcus sp. ST-10 SMO catalyzed the epoxidation of various alkenes, including styrene derivatives, vinyl pyridines, and linear alkenes, to give (S)-epoxides. NADH was regenerated by the reduction of NAD+ by LSADH with 2-propanol. The E. coli biocatalyst was used in an aqueous/organic biphasic reaction system and the reaction conditions were optimized. Under the optimized conditions, 170mM of (S)-styrene oxide was obtained from styrene in the organic phase with excellent enantiomeric excess (99.8%). This biocatalytic process was used to synthesize various (S)-epoxides.

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol–water medium

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)-water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K(m) and V(max) values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA-water medium.

Inulin fructotransferase (DFA III-producing) from Leifsonia sp. T88-4

An inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Leifsonia sp. T88-4 was purified and characterized, for the first time. The enzyme was purified from culture supernatant of the microorganism 32.4-fold with a yield of 11.3% using a DEAE–Toyopearl chromatography and two times of Super Q-Toyopearl chromatography. The enzyme showed maximum activity at pH 5.0 and 65 °C. This maximum temperature was highest in the inulin fructotransferase (DFA III-producing) reported to date. The enzyme activity was stable up to 60 °C after 30 min heat treatment. The molecular mass of the enzyme was estimated to be 44 kDa by SDS–PAGE and 74 kDa by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (16 amino acid residues) was determined as METNVYDVTDVGVPGR. The K m value of the reaction at pH 5.0 and 60 °C was estimated to be 1.0 mM.

Gene Cloning and Expression ofLeifsoniaAlcohol Dehydrogenase (LSADH) Involved in Asymmetric Hydrogen-Transfer Bioreduction to Produce (R)-Form Chiral Alcohols

The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30-50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).