Improvement and characterization of a hyperthermophilic glucose isomerase from <Emphasis Type="Italic">Thermoanaerobacter</Emphasis><Emphasis Type="Italic">ethanolicus</Emphasis> and its application in production of high fructose corn syrup

High fructose corn syrup (HFCS) is an alternative of liquid sweetener to sucrose that is isomerized by commercial glucose isomerase (GI). One-step production of 55 % HFCS by thermostable GI has been drawn more and more attentions. In this study, a new hyperthermophilic GI from Thermoanaerobacter ethanolicus CCSD1 (TEGI) was identified by genome mining, and then a 1317 bp fragment encoding the TEGI was synthesized and expressed in Escherichia coli BL21(DE3). To improve the activity of TEGI, two amino acid residues, Trp139 and Val186, around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected for site-directed mutagenesis. The specific activity of mutant TEGI-W139F/V186T was 2.3-fold and the value of k _cat/K _m was 1.86-fold as compared to the wild type TEGI, respectively. Thermostability of mutant TEGI-W139F/V186T at 90 °C for 24 h showed 1.21-fold extension than that of wild type TEGI. During the isomerization of glucose to fructose, the yield of fructose could maintain above 55.4 % by mutant TEGI-W139F/V186T as compared to 53.8 % by wild type TEGI at 90 °C. This study paved foundation for the production of 55 % HFCS using the thermostable TEGI.     

Identification of halohydrin dehalogenase mutants that resist COBE inhibition

The biocatalytic cascade conversion of ethyl 4-chloroacetoacetate (COBE) to ethyl (R)-4-cyano-3-hydroxybutyrate ((R)-HN) for the preparation of atorvastatin represents significant economic and environmental benefits, and is catalyzed by alcohol dehydrogenase and halohydrin dehalogenase (HHDH). However, as the activity of HHDH is inhibited by COBE, the cascade reaction is an inefficient one-pot reaction. In this study, substrate inhibition kinetics analysis was performed and the inhibition by COBE was found to be competitive reversible inhibition. Molecular simulation analysis was used to determine the inhibition mechanism by COBE. The results showed that COBE bound to the active center of HHDH via the formation of hydrogen bonds with the OH groups of S132 and Y145. Site saturation mutagenesis of residues around the active site and at the entrance of the access tunnel was performed, and two target mutant residues were identified, F136 and W249. Small focused mutagenesis on these two residues was performed and the F136V/W249F mutant was successfully found to relieve the activity inhibition of HHDH by COBE. The half inhibiting concentration of mutant F136V/W249F was found to be 20-fold higher than wild-type HHDH. The efficiency of the multi-enzymatic one-pot system for the synthesis of (R)-HN from COBE using mutant F136V/W249F was improved significantly.     

Characterization and immobilization of a novel SGNH hydrolase (Est24) from Sinorhizobium meliloti

A novel oligomeric SGNH hydrolase (Est24) from Sinorhizobium meliloti was identified, actively expressed in Escherichia coli, characterized, and immobilized for industrial application. Sequence analysis of Est24 revealed a putative catalytic triad (Ser¹³-Asp¹⁶³-His¹⁶⁹), with moderate homology to other SGNH hydrolases. Est24 was more active toward short-chain esters, such as p-nitrophenyl acetate, butyrate, and valerate, while the S13A mutant completely lost its activity. Moreover, the activity of Est24 toward α- and β-naphthyl acetate, and enantioselectivity on (R)- and (S)-methyl-3-hydroxy-2-methylpropionate were tested. Est24 exhibited optimum activity at mesophilic temperature ranges (45–55 °C), and slightly alkaline pH (8.0). Structural and mutagenesis studies revealed critical residues involved in the formation of a catalytic triad and substrate-binding pocket. Cross-linked enzyme aggregates (CLEAs) of Est24 with and without amyloid fibrils were prepared, and amyloid fibril-linked Est24 with amyloid fibrils retained 83 % of its initial activity after 1 h of incubation at 60 °C. The high thermal stability of immobilized Est24 highlights its potential in the pharmaceutical and chemical industries.     

Cloning,expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency

(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) is a valuable chiral intermediate for the synthesis of antiemetic drug Aprepitant and Fosaprepitant. A Leifsonia xyli HS0904-derived carbonyl reductase (LXCAR), an effective biocatalyst for the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to (R)-BTPE, was overexpressed in Escherichia coli BL21 (DE3). Bioinformatics analysis indicated that the amino acid sequence of recombinant LXCAR showed 89 % similarity to short-chain dehydrogenase/reductase. E. coli recombinant carbonyl reductase crude extract showed a specific activity of 1.54 U/mg, which was 62 times higher than that of L. xyli HS0904 crude extract. By using error-prone polymerase chain reaction and site-directed mutagenesis, the engineered LXCAR demonstrated superior catalytic activity toward BTAP, and the obtained mutant LXCAR-S154Y exhibited nearly 13-fold, 5.4-fold, and 2.3-fold increase in k _cat/K _m value, k _cat value, and specific activity toward BTAP, respectively, compared to the recombinant LXCAR. Additionally, the reduction of BTAP by whole cells of mutant LXCAR-S154Y afforded a best yield of 99.6 % for (R)-BTPE within 2 h at 200 mM BTAP, which was shortened by 28 and 2 h compared to those catalyzed by L. xyli HS0904 cells and recombinant E. coli cells expressing LXCAR, respectively. Moreover, a yield of 82.5 % for (R)-BTPE was achieved within 12 h at an increased BTAP concentration of up to 1,000 mM (256 g/l), representing a 1.9-fold increase over the recombinant LXCAR. Homology modeling and docking analysis revealed the molecular basis for the high catalytic activity of mutant LXCAR-S154Y toward BTAP. The results present here provide a promising alternative for economical and efficient production of chiral alcohols by engineered LXCAR.     

Enhanced biotransformation of 1,3-dichloro-2-propanol to epichlorohydrin via resin-based in situ product removal process

Biotransformation of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) by the whole cells of recombinant Escherichia coli expressing halohydrin dehalogenase was limited by product inhibition. To solve this problem and improve the ECH yield, a biotransformation strategy using resin-based in situ product removal (ISPR) was investigated. Seven macroporous resins were examined to adsorb ECH: resin HZD-9 was the best. When 10 % (w/v) HZD-9 was added to batch biotransformation, 53.3 mM ECH was obtained with a molar yield of 88.3 %. The supplement of the HZD-9 increased the ECH volumetric productivity from 0.5 to 2.8 mmol/l min compared to without addition of resin. In fed-batch biotransformation, this approach increased ECH from 31 to 87 mM. These results provide a promising basis for the biosynthesis of ECH.     

Chemical Constituents and Antimicrobial Activity of Indian Green Leafy Vegetable Cardiospermum halicacabum

The present study was carried out to analyze chemical constituents and antibacterial activity of ethanolic leaf extract of Cardiospermum halicacabum (ECH). The FT-IR spectrum confirmed the presence of alcohols, phenols, alkanes, alkynes, aliphatic ester and flavonoids in ECH. The GC–MS analysis revealed that ECH contained about twenty four compounds. The major chemical compounds identified were cyclohexane-1, 4, 5-triol-3-one-1-carboxylic acid, benzene acetic acid, caryophyllene, phytol and neophytadiene. The ECH was screened for its antibacterial activity against different bacterial strains and anti fungal activity against Candida albicans by agar well diffusion and minimum inhibitory concentration (MIC) assay. ECH exhibited antibacterial and antifungal activity. All the tested bacterial strains showed MIC values ranging from 80 to 125 μg of extract/ml and C. albicans showed 190 μg of extract/ml as a MIC. The maximum activity ECH was observed against human pathogen Staphylococcus aureus followed by Escherichia coli and the fish pathogen Aeromonas hydrophila. ECH exhibited moderate activity against some of the tested multidrug resistant strains.     

Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.     

Purification and Characterization of Two Epoxide Hydrolases from Corynebacterium sp. Strain N-1074

Enzymes II_a and II_b, which catalyze the conversion of epichlorohydrin (ECH) to 3-chloro-1,2-propanediol (MCP), were purified from Corynebacterium sp. strain N-1074, which catalyzes the formation of (R)-MCP from prochiral 1,3-dichloro-2-propanol via ECH. The specific activity of enzyme II_a for the formation of MCP from ECH was about 6.4-fold higher than that of enzyme II_b. Both enzymes catalyzed the conversion of 1,2-epoxides to the corresponding diol, although they differed in several enzymatic properties.     

Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic,Synergistic Lepidopteran-larvicidal and Nematicidal Activities

Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi9602_35-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi9602_35-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.     

Heterologous expression and structural characterization of two low pH laccases from a biopulping white-rot fungus Physisporinus rivulosus

The lignin-degrading, biopulping white-rot fungus Physisporinus rivulosus secretes several laccases of distinct features such as thermostability, extremely low pH optima and thermal activation for oxidation of phenolic substrates. Here we describe the cloning, heterologous expression and structural and enzymatic characterisation of two previously undescribed P. rivulosus laccases. The laccase cDNAs were expressed in the methylotrophic yeast Pichia pastoris either with the native or with Saccharomyces cerevisiae α-factor signal peptide. The specific activity of rLac1 and rLac2 was 5 and 0.3 μkat/μg, respectively. However, mutation of the last amino acid in the rLac2 increased the specific laccase activity by over 50-fold. The recombinant rLac1 and rLac2 enzymes demonstrated low pH optima with both 2,6-dimethoxyphenol (2,6-DMP) and 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonate). Both recombinant laccases showed moderate thermotolerance and thermal activation at +60 °C was detected with rLac1. By homology modelling, it was deduced that Lac1 and Lac2 enzymes demonstrate structural similarity with the Trametes versicolor and Trametes trogii laccase crystal structures. Comparison of the protein architecture at the reducing substrate-binding pocket near the T1-Cu site indicated the presence of five amino acid substitutions in the structural models of Lac1 and Lac2. These data add up to our previous reports on laccase production by P. rivulosus during biopulping and growth on Norway spruce. Heterologous expression of the novel Lac1 and Lac2 isoenzymes in P. pastoris enables the detailed study of their properties and the evaluation of their potential as oxidative biocatalysts for conversion of wood lignin, lignin-like compounds and soil-polluting xenobiotics.     

A short-chain carbonyl reductase mutant is an efficient catalyst in the production of (R)-1,3-butanediol

R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation–reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation–reduction cascade at larger scales.     

Thermal behaviour and tolerance to ionic liquid [emim]OAc in GH10 xylanase from Thermoascus aurantiacus SL16W

GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70–75 °C. The enzyme’s half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 °C, respectively. The melting point (T _m), as measured by DSC, was 78.5 °C, which is in line with a strong activity decrease at 75–80 °C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; T _m decreased by only 5 °C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The K _m of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of V _max was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10.     

Improving the Catalytic Activity and Thermostability of MAS1 Lipase by Alanine Substitution

MAS1 is a lipase isolated from Streptomyces sp. strain W007 with potential application in biotechnology. Structural analysis of MAS1 lipase showed that eight amino acids with bulkier side located in the substrate-binding pocket may be involved in affecting catalytic performance. Alanine substitutions of those residues were conducted to reduce steric clash of catalyzed pocket and probe their functional roles. The k_cat/K_m of mutants H108A, F153A, and V233A increased to 2.3-, 2.1-, and 1.4-fold, respectively. Interestingly, the half-life (60 °C) of F153A had shifted to 523 min after mutagenesis, which was fivefold enhancement toward that of MAS1 wide-type. Furthermore, higher hydrolysis ability of mutants H108A and F153A toward palm stearin of high melting temperature made them potentially applicable in oil/fat modification. Our work provided an example to obtain biocatalysts with desired catalytic behaviors by protein engineering.     

Crystal structure of the novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 reveals a special halide-stabilizing pair and enantioselectivity mechanism

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. Because DatA possesses a unique Asn-Tyr pair instead of the Asn-Trp pair conserved among the subfamily members, which was proposed to keep the released halide ion stable, the structural basis for its reaction mechanism should be elucidated. Here, we determined the crystal structures of DatA and its Y109W mutant at 1.70 and 1.95 Å, respectively, and confirmed the location of the active site by using its novel competitive inhibitor. The structural information from these two crystal structures and the docking simulation suggested that (i) the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for some halogenated compounds, such as 1,3-dibromopropane, mainly due to the electrostatic interaction between Trp109 and halogenated compounds and the change of substrate-binding mode caused by the interaction and (ii) the primary halide-stabilizing residue is only Asn43 in the wild-type DatA, while Tyr109 is a secondary halide-stabilizing residue. Furthermore, docking simulation using the crystal structures of DatA indicated that its enantioselectivity is determined by the large and small spaces around the halogen-binding site.     

Biochemical characterization of a thermostable β-1,3-xylanase from the hyperthermophilic eubacterium, Thermotoga neapolitana strain DSM 4359

The biochemical properties of a putative β-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against β-1,3-xylan and released β-1,3-xylobiose and β-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3–10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble β-1,3-xylan K _m value was 10.35 mg/ml and the k _cat value was 588.24 s^?1. The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

Structural insight into the substrate specificity of acyl-CoA oxidase1 from Yarrowia lipolytica for short-chain dicarboxylyl-CoAs

Acyl-CoA oxidase (ACOX) plays an important role in fatty acid degradation. The enzyme catalyzes the first reaction in peroxisomal fatty acid β-oxidation by reducing acyl-CoA to 2-trans-enoyl-CoA. The yeast Yarrowia lipolytica is able to utilize fatty acids, fats, and oil as carbon sources to produce valuable bioproducts. We determined the crystal structure of ACOX1 from Y. lipolytica (YlACOX1) at a resolution of 2.5 Å. YlACOX1 forms a homodimer, and the monomeric structure is composed of four domains, the Nα, Nβ, Cα1, and Cα2. The FAD cofactor is bound at the dimerization interface between the Nβ- and Cα1-domains. The substrate-binding tunnel formed by the interface between the Nα-, Nβ-, and Cα1-domains is located proximal to FAD. Amino acid and structural comparisons of YlACOX1 with other ACOXs show that the substrate-binding pocket of YlACOX1 is much smaller than that of the medium- or long-chain ACOXs but is rather similar to that of the short-chain ACOXs. Moreover, the hydrophilicity of residues constituting the end region of the substrate-binding pocket in YlACOX1 is quite similar to those in the short-chain ACOXs but different from those of the medium- or long-chain ACOXs. These observations provide structural insights how YlACOX1 prefers short-chain dicarboxylyl-CoAs as a substrate.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.     

Design,synthesis, docking and biological evaluation of 4-phenyl-thiazole derivatives as autotaxin (ATX) inhibitors

The autotaxin-lysophophatidic acid (ATX-LPA) signaling pathway is involved in several human diseases such as cancer, autoimmune diseases, inflammatory diseases neurodegenerative diseases and fibrotic diseases. Herein, a series of 4-phenyl-thiazole based compounds was designed and synthesized. Compounds were evaluated for their ATX inhibitory activity using FS-3 and human plasma assays. In the FS-3 assay, compounds 20 and 21 significantly inhibited the ATX at low nanomolar level (IC₅₀ = 2.99 and 2.19 nM, respectively). Inhibitory activity of 21 was found to be slightly better than PF-8380 (IC₅₀ = 2.80 nM), which is one of the most potent ATX inhibitors reported till date. Furthermore, 21 displayed higher potency (IC₅₀ = 14.99 nM) than the first clinical ATX inhibitor, GLPG1690 (IC₅₀ = 242.00 nM) in the human plasma assay. Molecular docking studies were carried out to explore the binding pattern of newly synthesized compounds within active site of ATX. Docking studies suggested the putative binding mode of the novel compounds. Good ATX inhibitory activity of 21 was attributed to the hydrogen bonding interactions with Asn230, Trp275 and active site water molecules; electrostatic interaction with catalytic zinc ion and hydrophobic interactions with amino acids of the hydrophobic pocket.     

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure–function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.