Enzymatic ketone reduction: mapping the substrate profile of a short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae

A short-chain alcohol dehydrogenase (YMR226c) from Saccharomyces cerevisiae was cloned and expressed in Escherichia coli, and the encoded protein was purified. The activity and enantioselectivity of this recombinant enzyme were evaluated with a series of ketones. The alcohol dehydrogenase (YMR226c) was found to effectively catalyze the enantioselective reductions of aryl-substituted acetophenones, α-chloroacetophenones, aliphatic ketones, and α- and β-ketoesters. While the enantioselectivity for the reduction of β-ketoesters was moderate, the acetophenone derivatives, aromatic α-ketoesters, some substituted α-chloroacetophenones, and aliphatic ketones were reduced to the corresponding chiral alcohols with excellent enantioselectivity. The enantiopreference of this enzyme generally followed Prelog’s rule for the simple ketones. The ester functionality played some role in determining the enzyme’s enantiopreference for the reduction of α- and β-ketoesters. The present study serves as a valuable guidance for the future applications of this versatile biocatalyst.     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

The kinetics and mechanism of homogeneous hydrogen transfer from alcohols to benzylideneacetone catalyzed by dichlorotris(triphenylphosphine)ruthenium(II)

Dichlorotris(triphenylphosphine)ruthenium(II) catalyzes the hydrogen transfer from alcohols to olefins. Kinetic studies were carried out at 170–190°C using the ruthenium(II) complex as homogeneous catalyst, benzyl alcohol, diphenylcarbinol, methylphenylcarbinol and benzoin as the hydrogen donors, benzylideneacetone as the hydrogen acceptor, and dibenzyl ether as a solvent. The IR spectra and GLC were used to monitor the reaction and the isotope effects were determined in order to elucidate the role of the catalyst and the mechanism of hydrogen transfer. In the reaction mixture RuCl₂(PPh₃)₃ is converted by the alcohols into RuH₂(CO) (PPh₃)₃, which then hydrogenates benzylideneacetone. The kinetic data are compatible with the expression. reaction rate = k_obs[Ru][olefin][alcohol] The rate-determining step of this reaction is considered to be the transfer of hydrogen from the alcohol to a ruthenium species.     

Cloning, Expression, and Characterization of β-mannanase from Bacillus subtilis MAFIC-S11 in Pichia pastoris

The β-mannanase gene (1,029 nucleotide) from Bacillus subtilis MAFIC-S11, encoding a polypeptide of 342 amino acids, was cloned and expressed in Pichia pastoris. To increase its expression, the β-mannanase gene was optimized for codon usage (mannS) and fused downstream to a sequence-encoding modified α-factor signal peptide. The expression level was improved by 2-fold. This recombinant enzyme (mannS) showed its highest activity of 24,600 U/mL after 144-h fermentation. The optimal temperature and pH of mannS were 50 °C and 6.0, respectively, and its specific activity was 3,706 U/mg. The kinetic parameters V _max and K _m were determined as 20,000 U/mg and 8 mg/mL, respectively, representing the highest ever expression level of β-mannanase reported in P. pastoris. In addition, the enzyme exhibited much higher binding activity to chitin, chitosan, Avicel, and mannan. The superior catalytic properties of mannS suggested great potential as an effective additive in animal feed industry.     

Evidence for a Molten Globule State in Cicer α-Galactosidase Induced by pH, Temperature, and Guanidine Hydrochloride

Physiologically as well as industrially, α-galactosidases are very important enzymes, but very little is known about the stability and folding aspect of enzyme. In the present study, we have investigated the temperature, pH, and guanidine hydrochloride (GuHCl) induced unfolding of Cicer α-galactosidase using circular dichroism and fluorescence spectroscopy. Strong negative ellipticities at 208, 215, and 222 nm indicate the presence of both α and β structures in Cicer α-galactosidase and showed that its secondary structure belongs to α?+?β class of proteins with 31 % α-helicity. For Cicer α-galactosidase the emission maximum was found to be 345 nm which suggests that tryptophan residues are less exposed to solvent. However, at pH?2.0, protein showed blue-shift. This state of protein lacked activity but it retained significant secondary structure. Enhanced binding of ANS at pH?2.0 indicated significant unfolding and exposure of hydrophobic regions. The unfolded state of Cicer α-galactosidase showed a red-shift of 15 nm with a concomitant decrease in the fluorescence intensity. The enzyme maintained its native structure and full activity up to 40 °C; however, above this temperature, denaturation was observed.     

Processing of β-Glucosidase–Silk Fibroin Nanoparticle Bioconjugates and Their Characteristics

Silk fibroin derived from Bombyx mori is a biomacromolecular protein with excellent biocompatibility. The aim of this work was to develop silk fibroin nanoparticles (SFNs) derived from the fibrous protein, which is a novel vector for enzyme modification in food processing. Silk fibroin was dissolved in highly concentrated CaCl₂ and subjected to lengthy desalting in water. The resulting liquid silk, which contained water-soluble polypeptides with molecular mass ranging from 10 to 200 kDa, and β-glucosidase were added rapidly into acetone. The β-glucosidase molecules were embedded into silk fibroin nanoparticles, forming β-glucosidase–silk fibroin nanoparticles (βG–SFNs) with a diameter of 50–150 nm. The enzyme activity of the βG–SFN bioconjugates was determined with p-nitrophenyl-β-d-glucoside as the substrate, and the optimum conditions for the preparation of βG–SFNs were investigated. The enzyme activity recovery of βG–SFNs was 59.2 % compared to the free enzyme (specific activity was 1 U mg^-1). The kinetic parameters of the βG–SFNs and the free β-glucosidase were the same. The βG–SFNs had good operational stability and could be used repeatedly. These results confirmed that silk protein nanoparticles were good carriers as bioconjugates for the modification of enzymes with potential value for research and development. The method used in this study has potential applications in food processing and the production of flavour agents.     

A polymer-supported diacetatobis(2-quinolylbenzimidazole)copper(II) complex as an efficient catalyst for oxidation of alcohols with tert-butyl hydroperoxide

A polymer-supported diacetatobis(2-quinolylbenzimidazole)copper(II) complex [PS–(QBIM)₂Cu(II)] was synthesized by functionalization of chloromethylated polystyrene cross-linked with 6.5 % divinyl benzene with 2-(2′-quinolyl)benzimidazole and subsequent treatment with Cu(OAc)₂ in methanol. The complex was characterized by physical, analytical and spectroscopic techniques. Electronic and ESR spectra, together with magnetic susceptibility measurements, indicated that the complex was paramagnetic with distorted octahedral geometry around the copper. The complex was found to be active toward oxidation of various alcohols including phenol, benzyl alcohol and cyclohexanol using 70 % aqueous tert-butyl hydroperoxide under mild conditions. Under the optimized reaction conditions, cyclohexanol gave 100 % conversion to cyclohexanone, benzyl alcohol gave 98 % yield of benzaldehyde and phenol gave 89 % yield of catechol and 4 % of hydroquinone. The complex was recycled five times without much loss in catalytic activity.     

Effect of mutations to amino acid A301 and F361 in thermostability and catalytic activity of the β-galactosidase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> VTCC-DVN-12-01

Background

Beta-galactosidase (EC 3.2.1.23), a commercially important enzyme, catalyses the hydrolysis of β-1,3- and β-1,4-galactosyl bonds of polymer or oligosaccharidesas well as transglycosylation of β-galactopyranosides. Due to catalytic properties; β-galactosidase might be useful in the milk industry to hydrolyze lactose and produce prebiotic GOS. The purpose of this study is to characterize β-galactosidase mutants from B. subtilis.

Results

Using error prone rolling circle amplification (epRCA) to characterize some random mutants of the β-galactosidase (LacA) from B. subtilisVTCC-DVN-12-01, amino acid A301 and F361 has been demonstrated significantly effect on hydrolysis activity of LacA. Mutants A301V and F361Y had markedly reduced hydrolysis activity to 23.69 and 43.22 %, respectively. Mutants the site-saturation of A301 reduced catalysis efficiency of LacA to 20–50 %, while the substitution of F361 by difference amino acids (except tyrosine) lost all of enzymatic activity, indicating that A301 and F361 are important for the catalytic function. Interestingly, the mutant F361Y exhibited enhanced significantly thermostability of enzyme at 45–50 °C. At 45 °C, LacA-361Y retained over 93 % of its original activity for 48 h of incubation, whereas LacA-WT and LacA-301Vwere lost completely after 12 and 24 h of incubation, respectively. The half-life times of LacA-361Y and LacA-301 V were about 26.8 and 2.4 times higher, respectively, in comparison to the half-life time of LacA-WT. At temperature optimum 50 °C, LacA-361Y shows more stable than LacA-WT and LacA-301 V, retaining 79.88 % of its original activities after 2 h of incubation, while the LacA-WT and LacA-301 V lost all essential activities. The half-life time of LacA-361Y was higher 12.7 and 9.39 times than that of LacA-WT and LacA-301 V, respectively. LacA-WT and mutant enzymes were stability at pH 5–9, retained over 90 % activity for 72 h of incubation at 30 °C. However, LacA-WT showed a little bit more stability than LacA-301 V and LacA-361Y at pH 4.

Conclusions

Our findings demonstrated that the amino acids A301V and F361 play important role in hydrolysis activity of β -galactosidase from B. subtilis. Specially, amino acid F361 had noteworthy effect on both catalytic and thermostability of LacA enzyme, suggesting that F361 is responsible for functional requirement of the GH42 family.

     

A New α-Galactosidase from Thermoacidophilic Alicyclobacillus sp. A4 with Wide Acceptor Specificity for Transglycosylation

An α-galactosidase gene (gal36A4) of glycosyl hydrolase family 36 was identified in the genome of Alicyclobacillus sp. A4. It contains an ORF of 2,187 bp and encodes a polypeptide of 728 amino acids with a calculated molecular mass of 82.6 kDa. Deduced Gal36A4 shows the typical GH36 organization of three domains—the N-terminal β-sheets, the catalytic (β/α)₈-barrels, and the C-terminal antiparallel β-sheet. The gene product was produced in Escherichia coli and showed both hydrolysis and transglycosylation activities. The optimal pH for hydrolysis activity was 6.0, and a stable pH range of 5.0–11.0 was found. The enzyme had a temperature optimum of 60 °C. It is specific for α-1,6-glycosidic linkages and had a K _m value of 1.45 mM toward pNPGal. When using melibiose as both donor and acceptor of galactose, Gal36A4 showed the transfer ratio of 23.25 % at 96 h. With respect to acceptor specificity, all tested monosaccharides, disaccharides, and oligosaccharides except for D-xylose and L-arabinose were good acceptors for transglycosylation. Thus, Gal36A4 may find diverse applications in industrial fields, especially in the food industry.     

Oxidation of aliphatic alcohols by triethylammonium chlorochromate in non-aqueous medium – A kinetic and mechanistic study

The oxidation of some aliphatic alcohols by triethylammonium chlorochromate (TriEACC) in dimethyl sulfoxide leads to the formation of the corresponding carbonyl compounds. The reaction is first order with respect to TriEACC. The reaction exhibited Michaelis–Menten type kinetics with respect to alcohol. The reaction is catalyzed by hydrogen ions. The hydrogen-ion dependence has the form: k_obs = a + b[H⁺]. The oxidation of [1,1-²H₂] ethanol (MeCD₂OH) exhibits a substantial primary kinetic isotope effect. Oxidation of aliphatic alcohol was studied in 19 different organic solvents. The solvent effect has been analysed using Kamlet’s and Swain’s multi-parametric equation. A suitable mechanism has been proposed.     

Oxidation of alcohols by TBHP in the presence of sub-stoichiometric amounts of MnO2

Commercially available activated MnO₂ has been investigated as a catalyst for the oxidation of alcohols (phenylethanol, 4-methyl- and 4-methoxybenzyl alcohol, trans-cinnamyl alcohol, cyclohexanol, menthol, perillyl alcohol and myrtenol) by TBHP/decane or TBHP/water in MeCN. The activity is highest for benzylic and allylic alcohols. Secondary alcohols yield ketones with good selectivities, while the aldehydes generated from primary alcohols are further oxidized. The process competes with the TBHP catalyzed decomposition. It thus requires the use of excess TBHP and high catalyst loadings to achieve high conversions. However, the low cost of the reagents makes this new protocol convenient for the oxidation of reactive secondary alcohols. The study also suggests that MnO₂ should be proscribed as a reagent to quench excess TBHP in oxidative processes when the synthetic target contains easily oxidizable alcohol functions and when carrying our detailed kinetic monitoring of oxidation processes.     

Efficient and selective oxidation of alcohols with <Emphasis Type="Italic">tert</Emphasis>-BuOOH catalyzed by a dioxomolybdenum(VI) Schiff base complex under organic solvent-free conditions

The catalytic activity of dioxidobis{2-[(E)-p-tolyliminomethyl]phenolato}molybdenum(VI) complex was studied, for the first time, in the selective oxidation of various primary and secondary alcohols using tert-BuOOH as oxidant under organic solvent-free conditions at room temperature. The effect of different solvents was studied in the oxidation of benzyl alcohol in this catalytic system. It was found that, under organic solvent-free conditions, the catalyst oxidized various primary and secondary alcohols to their corresponding aldehyde or ketone derivatives with high yield. The effects of other parameters such as oxidant and amount of catalyst were also investigated. Among different oxidants such as H₂O₂, NaIO₄, tert-BuOOH, and H₂O₂/urea, tert-BuOOH was selected as oxygen donor in the oxidation of benzyl alcohol. Also, it was found that oxidation of benzyl alcohol required 0.02 mmol catalyst for completion. Dioxomolybdenum(VI) Schiff base complex exhibited good catalytic activity in the oxidation of alcohols with tert-BuOOH under mild conditions. In this catalytic system, different primary alcohols gave the corresponding aldehydes in good yields without further oxidation to carboxylic acids.     

Expression and Characterization of a Novel Antifungal Exo-β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium cupreum</Emphasis>

A novel β-1,3-glucanase gene, designated Ccglu17A, was cloned from the biological control fungus Chaetomium cupreum Ame. Its 1626-bp open reading frame encoded 541 amino acids. The corresponding amino acid sequence showed highest identity (67 %) with a glycoside hydrolase family 17 β-1,3-glucanase from Chaetomium globosum. The recombinant protein Ccglu17A was successfully expressed in Pichia pastoris, and the enzyme was purified to homogeneity with 10.1-fold purification and 47.8 % recovery yield. The protein’s molecular mass was approximately 65 kDa, and its maximum activity appeared at pH 5.0 and temperature 45 °C. Heavy metal ions Fe²⁺, Mn²⁺, Cu²⁺, Co²⁺, Ag⁺, and Hg²⁺ had inhibitory effects on Ccglu17A, but Ba²⁺ promoted the enzyme’s activity. Ccglu17A exhibited high substrate specificity, almost exclusively catalyzing β-1,3-glycosidic bond cleavage in various polysaccharoses to liberate glucose. The enzyme had a Km of 2.84 mg/mL and Vmax of 10.7 μmol glucose/min/mg protein for laminarin degradation under optimal conditions. Ccglu17A was an exoglucanase with transglycosylation activity based on its hydrolytic properties. It showed potential antifungal activity with a degradative effect on cell walls and inhibitory action against the germination of pathogenic fungus. In conclusion, Ccglu17A is the first functional exo-1,3-β-glucanase to be identified from C. cupreum and has potential applicability in industry and agriculture.     

Effects of electron-withdrawing substituents on DPPH radical scavenging reactions of protocatechuic acid and its analogues in alcoholic solvents

The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of protocatechuic acid (3,4-dihydroxybenzoic acid) and its related catechols was examined. Compounds possessing strong electron-withdrawing substituents showed high activity. NMR analysis of the reaction mixtures of catechols and DPPH radical in methanol showed the formation of methanol adducts. The results suggest that high radical scavenging activity of catechols in alcohol is due to a nucleophilic addition of an alcohol molecule on o-quinones, which leads to a regeneration of a catechol structure. Furthermore, the radical scavenging activity in alcohols would largely depend on the electron-withdrawing/donating substituents, since they affect the susceptibility toward nucleophilic attacks on o-quinone.     

Volumetric properties of the boldine + alcohol mixtures at atmospheric pressure from 283.15 to 333.15 K: A new method for the determination of the density of pure boldine

Densities of boldine + alcohol binary mixtures were measured over the whole accessible range of boldine compositions at temperatures from 283.15 to 333.15 K using an Anton-Paar digital vibrating glass tube densimeter. The binary systems studied include, as a solvent, seven normal alcohols from n-C₁ to n-C₆, n-C₈, and isopropanol. The density of these systems has been found an increasing function of the boldine composition. A new methodology based on density data of solutions of solid solutes with normal alcohols is described in order to determine solid molar volume of pure solutes. This methodology was validated with pure solid naphthalene molar volumes data at 298.15 K, with an average uncertainty of 6%.     

Enantiomeric Separation of β-Amino Alcohols by Capillary Electrophoresis Using DM-β-CD as Chiral Selector

Enantiomeric separations of several β-amino alcohol drugs, i.e., phenylephrine, epinephrine, norepinephrine, synephrine, and chlorprenaline were performed by capillary electrophoresis using DM-β-CD as a chiral selector. Five test solutes were baseline resolved in six minutes. The effects of DM-β-CD concentration, pH value, ionic strength of the buffer, and the type of β-CDs on resolution were investigated. The results indicated that DM-β-CD is suitable for enantiomeric separation of β-amino alcohols containing a phenyl group on the chiral atom. Enantiorecognition mechanisms for test solutes are also discussed.     

A new method to prepare macroporous copolymer of glycidyl methacrilate and ethylene dimethacrilate as enzyme carrier

Macroporous copolymer of glycidyl methacrylate and ethylene dimethacrylate containing surface epoxy groups was firstly synthesized with dodecyl alcohol and cyclohexanol as liquid pore-forming agents and nanosize calcium carbonate as solid porogenic agent. The scanning electron microscopy was used to characterize their surface structure. Under the optimum conditions, β-galactosidase from Aspergillus oryzae was immobilized on the support obtained above, and the basic property and the kinetic data of the reaction on immobilized enzyme were determined. These data were compared with those obtained for the enzyme immobilized on the support prepared only with the liquid solution as pore-forming agent. Satisfactory results were obtained in enzyme activity, immobilization yield, pH stability, thermal stability, operational stability, and Michaelis constants K _M. The results indicated that the copolymer of glycidyl methacrilate and ethylene dimethacrylate newly prepared was more suitable to immobilize enzyme than the carrier synthesized with traditional method.     

β-d-Xylosidase from Geobacillus thermoleovorans IT-08: Biochemical Characterization and Bioinformatics of the Enzyme

The gene encoding a thermostable β-d-xylosidase (GbtXyl43B) from Geobacillus thermoleovorans IT-08 was cloned in pET30a and expressed in Escherichia coli; additionally, characterization and kinetic analysis of GbtXyl43B were carried out. The gene product was purified to apparent homogeneity showing M _r of 72 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme exhibited an optimum temperature and pH of 60 °C and 6.0, respectively. In terms of stability, GbtXyl43B was stable at 60 °C at pH 6.0 for 1 h as well as at pH 6–8 at 4 °C for 24 h. The enzyme had a catalytic efficiency (k _cat/K _M) of 0.0048?±?0.0010 s^?1 mM^?1 on p-nitrophenyl-β-d-xylopyranoside substrate. Thin layer chromatography product analysis indicated that GbtXyl43B was exoglycosidase cleaving single xylose units from the nonreducing end of xylan. The activity of GbtXyl43B on insoluble xylan was eightfold higher than on soluble xylan. Bioinformatics analysis showed that GbtXyl43B belonging to glycoside hydrolase family 43 contained carbohydrate-binding module (CBM; residues 15 to 149 forming eight antiparallel β-strands) and catalytic module (residues 157 to 604 forming five-bladed β-propeller fold with predicted catalytic residues to be Asp287 and Glu476). CBM of GbtXyl43B dominated by the Phe residues which grip the carbohydrate is proposed as a novel CBM36 subfamily.     

An efficient reduction protocol for the synthesis of β-hydroxycarbamates from β-nitro alcohols in one pot: a facile synthesis of (−)-β-conhydrine

An efficient and practical one-pot protocol for the reduction of β-nitro alcohols to their corresponding N-(tert-butoxycarbonyl) amino alcohols using Zn-NH₄Cl in aqueous methanol is described. Other reducible groups such as ketones and isolated double bonds remained intact. This methodology allows a short synthesis of (−)-β-conhydrine to be achieved.