Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine.

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.

Fibroblast growth factor 9 reduces TBHP-induced oxidative stress in chondrocytes and diminishes mouse osteoarthritis by activating ERK/Nrf2 signaling pathway.

Osteoarthritis (OA) is a degenerative and progressive disease that affects joints. Pathologically, it is characterized by oxidative stress-mediated excessive chondrocyte apoptosis and mitochondrial dysfunction. Fibroblast growth factor 9 (FGF9) has been shown to exert antioxidant effects and prevent degenerative diseases by activating ERK-related signaling pathways. However, the mechanism of FGF9 in the pathogenesis of OA and its relationship with anti-oxidative stress and related pathways are unclear. In this study, mice with medial meniscus instability (DMM) were used as the in vivo model whereas TBHP-induced chondrocytes served as the in vitro model to explore the mechanism underlying the effects of FGF9 in OA and its association with anti-oxidative stress. Results showed that FGF9 reduced oxidative stress, apoptosis, and mitochondrial dysfunction in TBHP-treated chondrocytes and promoted the nuclear translocation of Nrf2 to activate the Nrf2/HO1 signaling pathway. Interestingly, silencing the Nrf2 gene or blocking the ERK signaling pathway abolished the antioxidant effects of FGF9. FGF9 treatment reduced joint space narrowing, cartilage ossification, and synovial thickening in the DMM model mice. In conclusion, the present findings demonstrate that FGF9 can inhibit TBHP-induced oxidative stress in chondrocytes through the ERK and Nrf2-HO1 signaling pathways and prevent the progression of OA in vivo.

Oral Prevalence of Candida Species in Patients Undergoing Systemic Glucocorticoid Therapy and the Antifungal Sensitivity of the Isolates.

BACKGROUND: Candida species are commonly detected as colonizers of the oral cavity; candidiasis or candidemia can develop in patients who are immunocompromised. Use of topical or inhaled glucocorticoids can alter the spectrum of Candida species and can promote oral candidiasis. The present study aims to evaluate the diversity of Candida species in the oral cavity and their susceptibility to antifungal agents in patients undergoing treatment with systemic glucocorticoids (SGCs) compared with non-users. METHODS: We conducted a descriptive, analytical, cross-sectional study that enrolled 120 patients with oral problems who were undergoing treatment with SGCs and who were admitted to the hospital of the First Affiliated Hospital, College of Medicine, Zhejiang University and Zhejiang Hospital, Hangzhou, China, between February 2019 and September 2019. One hundred and twenty age-and sex-matched patients were recruited as the SGC non-user control group. Demographic data included oral complaints and underlying diseases; symptoms of oral candidiasis were identified on physical examination. Candida species were collected using a concentrated oral rinse. Identification of fungal isolates was based on conventional phenotypic methods assisted by DNA sequence analysis of the internal transcribed spacer (ITS) rDNA gene region. Antifungal activities of anidulafungin, amphotericin B, micafungin, caspofungin, 5-flucytosine, posaconazole, voriconazole, itraconazole, and fluconazole were evaluated using the Sensititre YeastOneTM YO10 panel supplemented by the CLSI-M27-A3 protocol. RESULTS: Fifty-two (43.33%) out of the 120 patients undergoing with SGCs were diagnosed with oral candidiasis, compared with 14 (11.67%) of the non-users (P < 0.05). Likewise, we collected 88 strains from 73.33% of the SGC users compared with only 48 (40%) from non-users (P < 0.05). Candida albicans was detected most frequently in both groups (45.45% vs 66.67%, respectively; P = 0.033); the overall frequency of non-Candida albicans (NCA) strains isolated from patients treated with SGCs were significantly higher than that identified among non-users (51.14% vs 33.33%, respectively; P = 0.046), although there were no significant differences concerning any single species of NCA. Resistance of C. albicans to itraconazole (P = 0.004) and fluconazole (P = 0.001) was significantly higher in patients treated with SGCs than in non-users; however, echinocandins, amphotericin B, voriconazole, and posaconazole were all active against strains from both participant groups with no significant differences detected. CONCLUSION: Taken together, our findings indicate that SGC therapy may result in an increased prevalence of oral candidiasis as reflected by the clinical presentations and strains isolated; these findings were also associated with an increased frequency of NCA strains. SGC therapy was also associated with an increased frequency of C. albicans strains that were resistant to both itraconazole and fluconazole. The impact of SGC therapy on Candida species in the oral cavity requires further study.

Composite coal fly ash solid acid catalyst in synergy with chloride for biphasic preparation of furfural from corn stover hydrolysate.

A solid acid catalyst SO42-/SnO2-Al2O3-CFA was synthesized based on industrial waste coal fly ash (CFA) as carrier and applied in the conversion of oxalic acid pretreated corn stover hydrolysate to produce furfural. Physical properties of the solid acid catalyst were characterized by SEM, FTIR, XRD, BET, EDAX, and NH3-TPD. Highly wrinkled structure of SO42-/SnO2-Al2O3-CFA could provide more specific surface area for the covalent linkage between SiO2 and SnO2. Factors influencing the efficacy of SO42-/SnO2-Al2O3-CFA were systematically explored. The highest furfural yield of 84.7% was reached in NH4Cl-toluene biphasic system at 180 °C for 30 min. The recyclability of SO42-/SnO2-Al2O3-CFA and toluene could be achieved for five batches with stable performance in transformation of xylose-rich corn stover hydrolysate. This study provided a novel solid acid catalyst with promising potential in the synthesis of furfural from corn stover.

Detoxification of furfural residues hydrolysate for butanol fermentation by Clostridium saccharobutylicum DSM 13864.

The toxicity of furfural residues (FRs) hydrolysate is a major obstacle in its application. This work focused on the detoxification of FRs hydrolysate and its application in butanol fermentation. Combination of activated carbon and resin 717 was appropriate for the detoxification of hydrolysate. Mixed sterilization of FRs hydrolysate and corn steep liquor (CSL) was better than the separate ones, since proteins in CSL could adsorb and remove toxic components during sterilization. The results further confirmed that simultaneous sterilization of activated carbon + resin and fermentation medium was more efficient for detoxification and butanol production, in which 76.4% of phenolic compounds and 99.3% of Maillard reaction products were removed, 8.48 g/L butanol and 12.61 g/L total solvent were obtained. This study provides feasible and economic approaches for the detoxification of FRs hydrolysate and its application in butanol production.

Enhanced curdlan production with nitrogen feeding during polysaccharide synthesis by Rhizobium radiobacter.

Curdlan is a secondary metabolite synthesized by Agrobacterium sp. and some other bacteria. A newly isolated exopolysaccharide-producing strain was identified to be Rhizobium radiobacter CGMCC 12099. The polysaccharide product was confirmed to be curdlan with a molecule weight of 1.4×10(5)Da, and its molecular structure was determined by HPLC and infrared spectrum. Although nitrogen source is necessary for cell reproduction, curdlan production is largely dependent on nitrogen limitation, as well as cell vitality. Here, a nitrogen feeding strategy was investigated to elevate the curdlan production by R. radiobacter. The optimal concentration and addition time of (NH4)2HPO4 were investigated. The results showed that the enhanced cell density was correlated to the amount of (NH4)2HPO4 added. Also, nitrogen addition in earlier fermentation stage was beneficial to the cell growth and curdlan production. Furthermore, continuously feeding strategy was employed by feeding (NH4)2HPO4 at a constant rate of 1.24g/h at 35(th)h of fermentation for 9h, achieving a final curdlan production of 65.27g/L, productivity of 0.544g/L/h and glucose conversion rate of 38.89%. The curdlan production was improved by 2.1 times compared with that without nitrogen addition. This study provides a feasible and cheap nitrogen feeding strategy to enhance curdlan production.

DNA microarray of global transcription factor mutant reveals membrane-related proteins involved in n-butanol tolerance in Escherichia coli.

BACKGROUND: Escherichia coli has been explored as a platform host strain for biofuels production such as butanol. However, the severe toxicity of butanol is considered to be one major limitation for butanol production from E. coli. The goal of this study is therefore to construct butanol-tolerant E. coli strains and clarify the tolerance mechanisms. RESULTS: A recombinant E. coli strain harboring σ(70) mutation capable of tolerating 2 % (v/v) butanol was isolated by the global transcription machinery engineering (gTME) approach. DNA microarrays were employed to assess the transcriptome profile of butanol-tolerant strain B8. Compared with the wild-type strain, 329 differentially expressed genes (197 up-regulated and 132 down-regulated) (p < 0.05; FC ≥ 2) were identified. These genes are involved in carbohydrate metabolism, energy metabolism, two-component signal transduction system, oxidative stress response, lipid and cell envelope biogenesis and efflux pump. CONCLUSIONS: Several membrane-related proteins were proved to be involved in butanol tolerance of E. coli. Two down-regulated genes, yibT and yghW, were identified to be capable of affecting butanol tolerance by regulating membrane fatty acid composition. Another down-regulated gene ybjC encodes a predicted inner membrane protein. In addition, a number of up-regulated genes, such as gcl and glcF, contribute to supplement metabolic intermediates for glyoxylate and TCA cycles to enhance energy supply. Our results could serve as a practical strategy for the construction of platform E. coli strains as biofuel producer.

Standardization of Type 2 Diabetes Outpatient Expenditure with Bundled Payment Method in China.

BACKGROUND: In recent years, the prevalence of type 2 diabetes among Chinese population has been increasing by years, directly leading to an average annual growth rate of 19.90% of medical expenditure. Therefore, it is urgent to work on strategies to control the growth of medical expenditure on type 2 diabetes on the basis of the reality of China. Therefore, in this study, we explored the feasibility of implementing bundled payment in China through analyzing bundled payment standards of type 2 diabetes outpatient services. METHODS: This study analyzed the outpatient expenditure on type 2 diabetes with Beijing Urban Employee's Basic Medical Insurance from 2010 to 2012. Based on the analysis of outpatient expenditure and its influential factors, we adopted decision tree approach to conduct a case-mix analysis. In the end, we built a case-mix model to calculate the standard expenditure and the upper limit of each combination. RESULTS: We found that age, job status, and whether with complication were significant factors that influence outpatient expenditure for type 2 diabetes. Through the analysis of the decision tree, we used six variables (complication, age, diabetic foot, diabetic nephropathy, cardiac-cerebrovascular disease, and neuropathy) to group the cases, and obtained 11 case-mix groups. CONCLUSIONS: We argued that it is feasible to implement bundled payment on type 2 diabetes outpatient services. Bundled payment is effective to control the increase of outpatient expenditure. Further improvements are needed for the implementation of bundled payment reimbursement standards, together with relevant policies and measures.

Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

Characterization and Soluble Expression of D-Hydantoinase from Pseudomonas fluorescens for the Synthesis of D-Amino Acids.

An active D-hydantoinase from Pseudomonas fluorescens was heterogeneously overexpressed in Escherichia coli BL21(DE3) and designated as D-PfHYD. Sequence and consensus analysis suggests that D-PfHYD belongs to the dihydropyrimidinase/hydantoinase family and possesses catalytic residues for metal ion and hydantoin binding. D-PfHYD was purified to homogeneity by nickel affinity chromatography for characterization. D-PfHYD is a homotetramer with molecular weight of 215 kDa and specific activity of 20.9 U mg(-1). D-PfHYD showed the highest activity at pH 9.0 and 60 °C. Metal ions such as Mn(2+), Fe(2+), and Fe(3+) could activate D-PfHYD with 20 % improvement. Substrate specificity analysis revealed that purified D-PfHYD preferred aliphatic to aromatic 5'-monosubstituted hydantoins. Among various strategies tested, chaperone GroES-GroEL was efficient in improving the soluble expression of D-PfHYD. Employing 1.0 g L(-1) recombinant E. coli BL21(DE3)-pET28-hyd/pGRO7 dry cells, 100 mM isobutyl hydantoin was converted into D-isoleucine with 98.7 % enantiomeric excess (ee), isolation yield of 78.3 %, and substrate to biocatalyst ratio of 15.6. Our results suggest that recombinant D-PfHYD could be potentially applied in the synthesis of D-amino acids.

Simultaneous saccharification and fermentation of dilute alkaline-pretreated corn stover for enhanced butanol production by Clostridium saccharobutylicum DSM 13864.

Simultaneous saccharification and fermentation (SSF) process was applied for biobutanol production by Clostridium saccharobutylicum DSM 13864 from corn stover (CS). The key influential factors in SSF process, including corn steep liquor concentration, dry biomass and enzyme loading, SSF temperature, inoculation size and pre-hydrolysis time were optimized. In 5-L bioreactor with SSF process, butanol titer and productivity of 12.3 g/L and 0.257 g/L/h were achieved at 48 h, which were 20.6% and 21.2% higher than those in separate hydrolysis and fermentation (SHF), respectively. The butanol yield reached 0.175 g/g pretreated CS in SSF, representing 50.9% increase than that in SHF (0.116 g/g pretreated CS). This study proves the feasibility of efficient and economic production of biobutanol from CS by SSF.

Biobutanol production from corn stover hydrolysate pretreated with recycled ionic liquid by Clostridium saccharobutylicum DSM 13864.

In this study, corn stover (CS) hydrolysates, pretreated by fresh and recycled ionic liquid (IL) [Bmim][Cl], were utilized in butanol fermentation by Clostridium saccharobutylicum DSM 13864. An efficient CS pretreatment procedure using [Bmim][Cl] was developed, giving a glucose concentration of 18.7 g L(-1) using ten times recycled [Bmim][Cl], representing about 77% of that produced with fresh IL (24.2 g L(-1)). Fermentation of hydrolysate I (pretreated by fresh IL) resulted in 7.4 g L(-1) butanol with a yield of 0.21 g g total-sugar(-1) and a productivity of 0.11 g L(-1)h(-1), while 7.9 g L(-1) butanol was achieved in fermentation using hydrolysate II (pretreated by ten times reused IL) with similar levels of acetone and ethanol, as well as yield and productivity. This study provides evidence for the efficient utilization of IL in CS pretreatment for biobutanol fermentation.

Enzymatic preparation of D-phenyllactic acid at high space-time yield with a novel phenylpyruvate reductase identified from Lactobacillus sp. CGMCC 9967.

An NADH-dependent phenylpyruvate reductase (LaPPR) was identified through screening the shotgun library of Lactobacillus sp. CGMCC 9967. It belongs to D-3-phosphoglycerate dehydrogenase (PGDH) subfamily of 2-hydroxy acid dehydrogenase superfamily. LaPPR was stable at pH 6.5 and 30 °C, with a half-life of 152 h. LaPPR has a substrate preference towards aromatic to aliphatic keto acids, and various keto acids could be reduced into D-hydroxy acids with excellent enantioselectivity (>99%). By construction the coexpression system with glucose dehydrogenase, as much as 100 g L(-1) phenylpyruvic acid was asymmetrically reduced into D-phenyllactic acid with 91.3% isolation yield and 243 g L(-1) d(-1) productivity. The results suggest that LaPPR is a promising biocatalyst for the efficient synthesis of optically pure D-phenyllactic acid.

Enhancing cellulose accessibility of corn stover by deep eutectic solvent pretreatment for butanol fermentation.

In this study, an effective corn stover (CS) pretreatment method was developed for biobutanol fermentation. Deep eutectic solvents (DESs), consisted of quaternary ammonium salts and hydrogen donors, display similar properties to room temperature ionic liquid. Seven DESs with different hydrogen donors were facilely synthesized. Choline chloride:formic acid (ChCl:formic acid), an acidic DES, displayed excellent performance in the pretreatment of corn stover by removal of hemicellulose and lignin as confirmed by SEM, FTIR and XRD analysis. After optimization, glucose released from pretreated CS reached 17.0 g L(-1) and yield of 99%. The CS hydrolysate was successfully utilized in butanol fermentation by Clostridium saccharobutylicum DSM 13864, achieving butanol titer of 5.63 g L(-1) with a yield of 0.17 g g(-1) total sugar and productivity of 0.12 g L(-1)h(-1). This study demonstrates DES could be used as a promising and biocompatible pretreatment method for the conversion of lignocellulosic biomass into biofuel.

Cloning, Expression, and Characterization of budC Gene Encoding meso-2,3-Butanediol Dehydrogenase from Bacillus licheniformis.

The budC gene encoding a meso-2,3-butanediol dehydrogenase (BlBDH) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli BL21(DE3). Sequence analysis reveals that this BlBDH belongs to short-chain dehydrogenase/reductase (SDR) superfamily. In the presence of NADH, BlBDH catalyzes the reduction of diacetyl to (3S)-acetoin (97.3% ee), and further to (2S,3S)-2,3-butanediol (97.3% ee and 96.5% de). Similar to other meso-2,3-BDHs, it shows oxidative activity to racemic 2,3-butanediol whereas no activity toward racemic acetoin in the presence of NAD(+). For diacetyl reduction and 2,3-butanediol oxidation, the pH optimum of BlBDH is 5.0 and 10.0, respectively. Unusually, it shows relatively high activity over a wide pH range from 5.0 to 8.0 for racemic acetoin reduction. BlBDH shows lower K m and higher catalytic efficiency toward racemic acetoin (K m = 0.47 mM, k cat /K m = 432 s(-1)·mM(-1)) when compared with 2,3-butanediol (K m = 7.25 mM, k cat /K m = 81.5 s(-1)·mM(-1)), indicating its physiological role in favor of reducing racemic acetoin into 2,3-butanediol. The enzymatic characterization of BlBDH provides evidence for the directed engineering of B. licheniformis for producing enantiopure 2,3-butanediol.

Comparative Study of Three Commonly Used Methods for Hospital Efficiency Analysis in Beijing Tertiary Public Hospitals, China.

BACKGROUND: Tertiary hospitals serve as the medical service center within the region and play an important role in the medical and health service system. They are also the key targets of public hospital reform in the new era in China. Through the reform of health system, the public hospital efficiency has changed remarkably. Therefore, this study aimed to provide some advice for efficiency assessment of public hospitals in China by comparing and analyzing the consistency of results obtained by three commonly used methods for examining hospital efficiency, that is, ratio analysis (RA), stochastic frontier analysis (SFA), and data envelopment analysis (DEA). METHODS: The theoretical basis, operational processes, and the application status of RA, SFA, and DEA were learned through literature analysis. Then, the empirical analysis was conducted based on measured data from 51 tertiary public hospitals in Beijing from 2009 to 2011. RESULTS: The average values of hospital efficiency calculated by SFA with index screening and principal component analysis (PCA) results and those calculated by DEA with index screening results were relatively stable. The efficiency of specialized hospitals was higher than that of general hospitals and that of traditional Chinese medicine hospitals. The results obtained by SFA with index screening results and the results obtained by SFA with PCA results showed a relatively high correlation (r-value in 2009, 2010, and 2011 were 0.869, 0.753, and 0.842, respectively, P < 0.01). The correlation between results obtained by DEA with index screening results and PCA results and results obtained by other methods showed statistical significance, but the correlation between results obtained by DEA with index screening results and PCA results was lower than that between results obtained by SFA with index screening results and PCA results. CONCLUSIONS: RA is not suitable for multi-index evaluation of hospital efficiency. In the given conditions, SFA is a stable efficiency analysis method. In the evaluation of hospital efficiency, DEA combined with PCA should be adopted with caution due to its poor stability.

Identification of key residues in Debaryomyces hansenii carbonyl reductase for highly productive preparation of (S)-aryl halohydrins.

Key residues of Debaryomyces hansenii carbonyl reductase in the determination of the reducing activity towards aryl haloketones were identified through combinatorial mutation of conserved residues. This study provides a green and efficient biocatalyst for the synthesis of (S)-aryl halohydrins.

Hydrophobic Mutagenesis and Semi-rational Engineering of Arginine Deiminase for Markedly Enhanced Stability and Catalytic Efficiency.

Due to its systemic arginine degradation, arginine deiminase (ADI) has attracted attentions as an anti-tumor drug. Its low activity at physiological conditions among other limitations has necessitated its engineering for improved properties. The present study describes the hydrophobic mutagenesis and semi-rational engineering of ADI from Pseudomonas plecoglossicida (PpADI). Using an improved ADI variant M13 (D38H/A128T/E296K/H404R/I410L) as parent, site saturation mutagenesis at position 162 resulted in an over 20 % increase in protein solubility. Compared with M13 (15.23 U/mg), mutants M13-2 (M13+S245D) and M13-5 (M13+R243L) exhibited enhanced specific activity of 21.19 and 31.20 U/mg at physiological conditions. M13-5 displayed enhanced substrate specificity with a dramatic reduction in its K m value (from 0.52 to 0.16 mM). It is speculated that the improvements in M13-5 could mainly be attributed to the enhanced structural stability due to an R243L substitution. The hydrophobic contribution of Leu 243 was supported by mutant M13-9 (M13+A276W) generated based on the hydrophobic mutagenesis concept. M13-9 showed a specific activity of 18.68 U/mg, as well as remarkable thermal and pH stability. It retained over 90 % activity over pH range from 4.5 to 8.5. At 60 °C, the half-life of M13-9 was enhanced from 4 to 17.5 min in comparison with M13, and its specific activity at 62 °C (93.0 U/mg) was approximately fivefold of that determined at 37 °C. Our results suggest that the increased hydrophobicity around the active regions of PpADI might be crucial in improving its structural stability and ultimately catalytic efficiency.

Soluble Expression of (+)-γ-Lactamase in Bacillus subtilis for the Enantioselective Preparation of Abacavir Precursor.

Chiral Vince lactam (γ-lactam) is an important precursor of many carbocyclic nucleoside analogues and pharmaceuticals. Here, a (+)-γ-lactamase encoding gene delm from Delftia sp. CGMCC 5755 was identified through genome hunting. To achieve its soluble and functional expression, Escherichia coli and Bacillus subtilis expression systems were introduced. Compared with E. coli system, recombinant (+)-γ-lactamase showed improved protein solubility and catalytic activity in B. subtilis 168. Reaction conditions for enantioselective resolution of γ-lactam were optimized to be at 30 °C, pH 9.0, and 300 rpm when employing the recombinant B. subtilis 168/pMA5-delm whole cells. Kinetic analysis showed that the apparent V max and K m were 0.595 mmol/(min · gDCW) and 378 mmol/L, respectively. No obvious substrate inhibition was observed. In a 500-mL reaction system, enantioselective resolution of 100 g/L γ-lactam was achieved with 10 g/L dry cells, resulting in 55.2 % conversion and 99 % ee of (-)-γ-lactam. All above suggested that recombinant B. subtilis 168/pMA5-delm could potentially be applied in the preparation of optically pure (-)-γ-lactam.

Cloning and Characterization of an Enantioselective l-Menthyl Benzoate Hydrolase from Acinetobacter sp. ECU2040.

A new esterase gene abmbh, encoding a benzoate hydrolase which can enantioselectively hydrolyze l-menthyl benzoate to l-menthol, was recently identified from the genomic library of a soil isolate Acinetobacter sp. ECU2040. The abmbh gene contains a 1080-bp open reading frame encoding a protein of 360 amino acids with a calculated molecular mass of 40.7 kDa. The corresponding enzyme AbMBH was functionally expressed in Escherichia coli BL21 (DE3), purified, and characterized. The AbMBH displayed the maximum activity towards p-nitrophenyl butyrate at 50 °C, and an optimum pH of 8.5. A K M of 2.6 mM and a k cat of 0.26 s(-1) were observed towards dl-menthyl benzoate. The AbMBH exhibited a moderate enantioselectivity (E = 27.5) towards dl-menthyl benzoate. It can also catalyze the enantioselective hydrolysis of a variety of racemic menthyl esters, including dl-menthyl acetate, dl-menthyl chloroacetate, and dl-menthyl butyrate.