Diastereoselective synthesis of (R)-(alkyl)-beta-D-galactopyranoside by using beta-galactosidase (Aspergillus oryzae) in low-water media

A beta-galactosidase (from Aspergillus oryzae) preparation viz. EPRP (enzyme precipitated and rinsed with propanol), obtained by the removal of bulk water by precipitation with n-propanol, showed higher biological activity than the lyophilized powder. FT-IR study confirmed that EPRP had retained the alpha-helical content of the native structure better than the lyophilized form. Use of this formulation of beta-galactosidase under low water conditions (temperature 55 degrees C, reaction time of 4 h) gave enantioselectivity, E > 1000 for the stereoselective synthesis of (R)-(1-phenylethyl)-beta-D-galactopyranoside, starting from racemic 1-phenylethanol and D-galactose. For racemic 2-octanol also, EPRP worked better. Under similar conditions, (R)-(2-octyl)-beta-D-galactopyranoside was formed with an enantioselectivity, E = 38.     

Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw

Wheat straw used in this study contained 44.24 +/- 0.28% cellulose and 25.23 +/- 0.11% hemicellulose. Alkaline H(2)O(2) pretreatment and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from wheat straw (8.6%, w/v) by alkaline peroxide pretreatment (2.15% H(2)O(2), v/v; pH 11.5; 35 degrees C; 24 h) and enzymatic saccharification (45 degrees C, pH 5.0, 120 h) by three commercial enzyme preparations (cellulase, beta-glucosidase, and xylanase) using 0.16 mL of each enzyme preparation per g of straw was 672 +/- 4 mg/g (96.7% yield). During the pretreatment, no measurable quantities of furfural and hydroxymethyl furfural were produced. The concentration of ethanol (per L) from alkaline peroxide pretreated enzyme saccharified wheat straw (66.0 g) hydrolyzate by recombinant Escherichia coli strain FBR5 at pH 6.5 and 37 degrees C in 48 h was 18.9 +/- 0.9 g with a yield of 0.46 g per g of available sugars (0.29 g/g straw). The ethanol concentration (per L) was 15.1 +/- 0.1 g with a yield of 0.23 g/g of straw in the case of simultaneous saccharification and fermentation by the E. coli strain at pH 6.0 and 37 degrees C in 48 h.     

Immobilization of lipases and assay in continuous fixed bed reactor

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type II). Two immobilization methods were also used, namely: 1). adsorption, using as support the following silica derivatives (150-300 microm e 450micro): phenyl, epoxy, amino and without derivation, and 2). covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/g(support) for CRL and 0.97 U/g(support) for PPL, and of transesterification, 2,8 U/g(support) for CRL and 1,9 U/g(support) for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40 degrees C and 50 degrees C and for PPL at 32 degrees C and 40 degrees C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continuous reactor. Lipases immobilized by covalent binding were used in the assays of operational stability in continuous reactors. For PPL in aqueous medium, at 32 degrees C, and CRL in organic medium at 40 degrees C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40 degrees C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40 degrees C the loss was 33% after 20 days. Comparing both sources with each other, very different results were obtained. Higher activity was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.     

Cross-linked protein-coated microcrystals as biocatalysts in non-aqueous solvents

Cross-linked protein-coated microcrystals (CLPCMCs) of subtilisin Carlsberg (SC), and lipases from Candida rugosa (CRL) and Burkholderia cepacia (BCL) were prepared by cross-linking protein-coated microcrystals (PCMCs). These showed enhanced reaction rates for transesterification in organic solvents of different polarity compared with PCMCs. CLPCMCs of SC were catalytically active up to 80°C in octane and gave 98% conversion in 6 h, while PCMCs gave only 58% conversion in 8 h and conversions did not further increase with time. The Fourier transform infrared spectra showed that the alpha-helical content of CLPCMCs (unlike that of PCMCs) of SC were similar to what is reported for free SC in water.     

Different properties of the lipases contained in porcine pancreatic lipase extracts as enantioselective biocatalysts

The porcine pancreatic lipase (PPL) extracts contain a mixture of several lipases. Their fractioning was performed by sequential adsorption via interfacial activation on supports with different hydrophobicity. A protein of 25 KDa was preferentially adsorbed on octyl-Sepharose, another protein of 33 kDa was mainly adsorbed on octadecyl-Sepabeads support, and the PPL was mainly adsorbed on the support bearing phenyl groups. The different immobilized preparations showed different properties and different response due to change in the experimental conditions. Thus, in the hydrolysis of (+/-)-2-hydroxy-4-phenylbutyric acid ethyl ester [(+/-)-1] to produce the corresponding acid [2], the octyl-25KDa preparation showed the best enantioselectivity (E) value (E = 7) at pH 5 and 25 degrees C, whereas the phenyl-PPL was the most enantioselective (E = 10) at pH 5, 4 degrees C, and 10% dioxane. Using different preparations at different pHs it was possible to resolve (+/-)-2-O-butyryl-2-phenylacetic acid [(+/-)-3] with a high E value (E > 100); for example, with octadecyl-33 KDa enzyme at pH 8.     

Optimized enzymatic synthesis of geranyl butyrate with lipase AY from Candida rugosa

Response surface methodology (RSM) and five-level, five-variable central composite rotatable design (CCRD) were used to evaluate the effects of synthetic variables, such as reaction time (1-9 h), temperature (25-65 degrees C), enzyme amount (10-50%), substrate molar ratio of geraniol to tributyrin (1:0.33-1:1), and added water amount (0-20%) on molar percent yield of geranyl butyrate, using lipase AY from Candida rugosa. Reaction time and temperature were the most important variables and substrate molar ratio had no effect on percent molar conversion. Based on contour plots, optimum conditions were: reaction time 9 h, temperature 35 degrees C, enzyme amount 50%, substrate molar ratio 1:0.33, and added water 10%. The predicted value was 100% and actual experimental value was 96.8% molar conversion. (c) 1996 John Wiley & Sons, Inc.     

大肠杆菌EP8—10转化苯丙酮酸生成L—苯丙氨酸的研究

E. coli EP8-10 was selected from the soil. It was able to produce the transaminase with high activity when it was cultivated on the medium containing peptone and beef extract. Optimum conditions of enzyme reaction was: phenylpyruvic acid's concentration of 0.3-0.5 mol/L, L-Asptaric acid used as amino donor, pH 8.5 37 degrees C. When phenylpyruvic acid was 0.3 mol/L, 48 g/L L-phenylalanine was produced after 6 h with 97% conversion rate.     

Enzymatic semisynthesis of [LeuB30] insulin

Experimental conditions for the preparation of [LeuB30] insulin by coupling of des-AlaB30 insulin with Leu-OBu(t) were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35-50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37 degrees C was almost the same or a little higher than that at 25 degrees C. With trypsin, the coupling yield at 37 degrees C after 24 h was considerably lower than at 25 degrees C. This was partly ascribed to the difference in concentration of organic cosolvent at 37 degrees C and 25 degrees C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [LeuB30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [LeuB30] insulin and human insulin.     

Protein-coated Microcrystals for use in Organic Solvents: Application to Oxidoreductases

Protein-coated microcrystals (PCMC), a biocatalyst preparation previously demonstrated to display substantially increased transesterification activity of proteases and lipases in organic solvents when compared to their as received counterparts [Kreiner M, Moore BD, Parker MC (2001) Chem. Commun. 12:1096--1097], was applied to oxidoreductases. Horse liver alcohol dehydrogenase (HLADH), catalase (CAT), soybean peroxidase and horseradish peroxidase were immobilised onto K₂SO₄ crystals and dehydrated in a 1-step process, resulting in PCMC. These PCMC preparations showed enhanced activity in different organic solvents in most types of reactions tested. The highest activation was observed with HLADH (50-fold as active as enzyme as received) and CAT (25-fold).     

Chemical modification with functionalized ionic liquids: a novel method to improve the enzymatic properties of Candida rugosa lipase

Chemical modification of lysine residues in Candida rugosa lipase (CRL) was carried out using five different functional ionic liquids, and about 15.4–25.0 % of the primary amino groups of lysine were modified. Enzymatic properties of the native and modified CRLs were investigated in olive oil hydrolysis reaction. Improved thermal stability, catalytic activity in organic solvents, and adaptability to temperature and pH changes were achieved compared with the native enzyme. CRL modified by [choline][H₂PO₄] showed the best results, bearing a maximum improvement of 16.7 % in terms of relative activity, 5.2-fold increase in thermostability (after incubation at 45 °C for 5 h), and 2.3-fold increase in activity in strong polar organic solvent (80 % dimethyl sulfoxide) compared with the native enzyme. The results of ultraviolet, circular dichroism and fluorescence spectroscopy suggested that the change of the secondary and tertiary structures of CRL caused by the chemical modification resulted in the enhancement of enzymatic performance. The modification of CRL with functional ionic liquids was proved to be a novel and efficient method for improving the enzymatic properties of CRL.     

l-Ribulose production from l-arabinose by an l-arabinose isomerase mutant from Geobacillus thermodenitrificans

Geobacillus thermodenitrificans, with a double-site mutation in L: -arabinose isomerase, produced 95 g L-: ribulose l(-1 ) from 500 g L: -arabinose l(-1) under optimum conditions of pH 8, 70 degrees C, and 10 units enzyme ml(-1) with a conversion yield of 19% over 2 h. The half-lives of the mutated enzyme at 70 and 75 degrees C were 35 and 4.5 h, respectively.     

Purification, immobilization, and stabilization of a lipase from Bacillus thermocatenulatus by interfacial adsorption on hydrophobic supports

A lipase from Bacillus thermocatenulatus (BTL2) cloned in E. coli has been purified using a very simple method: interfacial activation on a hydrophobic support followed by desorption with Triton. Only one band was detected by SDS-PAGE. The pure enzyme was immobilized using different methodologies. BTL2 adsorbed on a hydrophobic support (octadecyl-Sepabeads) exhibited a hyperactivation with respect to the soluble enzyme, whereas the other immobilized preparations suffered a slight decrease in the expressed activity. The soluble enzyme was very stable, but all immobilized preparations were much more stable than the soluble enzyme, the octadecyl-Sepabeads-BTL2 preparation being the most stable one in all conditions (high temperature or in the presence of organic cosolvents), maintaining 100% of the activity at 65 degrees C or 30% of dioxane and 45 degrees C after several days of incubation. The glyoxyl preparation, the second more stable, retained 80% of the initial activity after 2 days, respectively. The adsorption of this thermophilic lipase on octadecyl-Sepabeads permitted an increase in the optimal temperature of the enzyme of 10 degrees C.     

Crosslinked enzyme crystals of glucoamylase as a potent catalyst for biotransformations

Glucoamylase (E.C: 3.2.1.3, alpha-(1-->4)-glucan glucohydrolase) mainly hydrolyzes starch and has been extensively used in the starch, glucose (dextrose), and fermentation industries. Immobilized glucoamylase has an inherent disadvantage of lower conversion rates and low thermostability of less than 55 degrees C when used in continuous operations. We have developed crosslinked enzyme crystals (CLEC) of glucoamylase that overcome the above disadvantages, possess good thermal stability and retain 98.6% of their original activity at 70 degrees C for 1h, 77% activity at 80 degrees C for 1h, and 51.4% activity at 90 degrees C for 0.5h. CLEC glucoamylase has a specific activity of 0.0687 IU/mg and a yield of 50.7% of the original activity of the enzyme under optimum conditions with starch as the substrate. The crystals obtained are rhombohedral in shape having a size approximately 10-100 microm, a density of 1.8926 g/cm(3) and a surface area of 0.7867 m(2)/g. The pH optimum of the glucoamylase crystals was sharp at pH 4.5, unlike the soluble enzyme. The kinetic constants V(max) and K(m) exhibited a 10-fold increase as a consequence of crystallization and crosslinking. The continuous production of glucose from 10% soluble starch and 10% maltodextrin (12.5 DE) by a packed-bed reactor at 60 degrees C had a productivity of 110.58 g/L/h at a residence time of 7.6 min and 714.1g/L/h at a residence time of 3.4 min, respectively. The CLEC glucoamylase had a half-life of 10h with 4% starch substrate at 60 degrees C.     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.     

Enzymatic resolution of (S)-(+)-naproxen in a trapped aqueous-organic solvent biphase continuous reactor

A trapped aqueous-organic biphase system for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen methyl ester by Candida rugosa lipase in a trapped aqueous-organic biphase system. The reaction has been carried out in a laboratory-scale continuous-flow stirred tank reactor (CSTR). The staring material has been supplied in and remaining substrate recovered by organic phase. YWG-C(6)H(5), a poorly polar synthetic support, has been employed to immobilize the lipase and to restrict the aqueous phase. Lipase immobilized on YWG-C(6)H(5) containing aqueous phase has been added into the CSTR to catalyze the hydrolysis. A dialysis membrane tube containing a continuous flow closed-loop buffer has been applied in the CSTR for the extraction of product and recruiting of the aqueous part consumed. Various reaction conditions have been studied. The activity of immobilized enzyme was effected by the polarity of support, the substrate concentration, logP value of organic phase and the product inhibition. At steady-state operating conditions, an initial conversion of 35% has been obtained. The CSTR was allowed to operate continuously for 60 days at 30 degrees C with a 30% loss of activity. The hydrolysis reaction yielded (S)-(+)-Naproxen with >90% enantiomeric excess and overall conversion of 30%.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Enantioselective synthesis of phenylacetamides in the presence of high organic cosolvent concentrations catalyzed by stabilized penicillin G acylase. Effect of the acyl donor

Immobilization of penicillin G acylase on glyoxyl agarose and its further hydrophilization by physicochemical modification with ionic polymers has made it possible to perform the direct condensation between (+/-)-2-hydroxy-2-phenylethylamine [(+/-)-1] and different acyl donors in the presence of high concentrations of organic cosolvent (up to 90%) in the reaction medium. Using 50 mM phenyl acetic acid and these drastic reaction conditions, too harsh for any other PGA preparation, we have achieved an almost quantitative transformation (more than 99%) of 10 mM (+/-)-1 into the corresponding amide. Remarkably, the enantioselectivity of the enzyme immobilized on the amine was strongly dependent on the acyl donor employed. Thus, using phenylacetic acid (2), the enantioselectivity was almost negligible (1.3 favoring the S isomer), whereas using S-mandelic acid [(S)-4], the E factor reached a value of 21 (also favoring the S isomer). By using R-mandelic acid [(R)-4], we observed a different enantioselectivity (E was 3.6 favoring the R). At 4 degrees C, the E value reached a value higher than 100 when (S)-4 was used as the acyl donor. The reaction performed under these conditions allowed us to produce (2S,2'S)-N-2'-hydroxy-2'-phenyl)-2-hydroxyphenylacetamide [(2S,2'S)-7] with a diasteromeric excess higher than 98%.     

Enzymatic resolution for the preparation of enantiomerically enriched D-beta-heterocyclic alanine derivatives using Escherichia coli aromatic L-amino acid transaminase

An enzymatic resolution was carried out for the preparation of enriched beta-heterocyclic D-alanine derivatives using Escherichia coli aromatic L-amino acid transaminase. The excess of pyrazole, imidazole, or 1,2,4-triazole reacted with methyl-2-acetamidoacrylate in acetonitrile in the presence of potassium carbonate at 60 degrees C, directly leading to make the potassium salt of the corresponding N-acetyl-beta-heterocyclic alanine derivatives. After the acidic deprotection of the N-acetyl group, 10 mM of racemic pyrazolylalanine, triazolylalanine, and imidazolylalanine were resolved to D-pyrazolylalanine, D-triazolylalanine, and D-imidazolylalanine with 46% (85% ee), 42% (72% ee), and 48% (95% ee) conversion yield in 18 h, respectively, using E. coli aromatic L-amino acid transaminase (EC 2.6.1.5). Although the three beta-heterocyclic L-alanine derivatives have similar molecular structures, they showed different reaction rates and enantioselectivities. The relative reactivities of the transaminase toward the beta-heterocyclic L-alanine derivatives could be explained by the relationship between the substrate binding energy (E, kcal/mol) to the enzyme active site and the distance (delta, A) from the nitrogen of alpha-amino group of the substrates to the C4' carbon of PLP-Lys258 Schiff base. As the ratio of the substrate binding energy (E) to the distance (delta) becomes indicative value of k(cat)/K(M) of the enzyme to the substrate, the relative reactivities of the beta-heterocyclic L-alanine derivatives were successfully correlated with E/delta, and the relationship was confirmed by our experiments.     

Encapsulation of Trigonopsis variabilis D-amino acid oxidase and fast comparison of the operational stabilities of free and immobilized preparations of the enzyme

A one-step procedure of immobilizing soluble and aggregated preparations of D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is reported where carrier-free enzyme was entrapped in semipermeable microcapsules produced from the polycation poly(methylene-co-guanidine) in combination with CaCl2 and the polyanions alginate and cellulose sulfate. The yield of immobilization, expressed as the fraction of original activity present in microcapsules, was approximately 52 +/- 5%. The effectiveness of the entrapped oxidase for O2-dependent conversion of D-methionine at 25 degrees C was 85 +/- 10% of the free enzyme preparation. Because continuous spectrophotometric assays are generally not well compatible with insoluble enzymes, we employed a dynamic method for the rapid in situ estimation of activity and relatedly, stability of free and encapsulated oxidases using on-line measurements of the concentration of dissolved O2. Integral and differential modes of data acquisition were utilized to examine cases of fast and slow inactivation of the enzyme, respectively. With a half-life of 60 h, encapsulated TvDAO was approximately 720-fold more stable than the free enzyme under conditions of bubble aeration at 25 degrees C. The soluble oxidase was stabilized by added FAD only at temperatures of 35 degrees C or greater.     

Cloning, overexpression, and characterization of a thermoactive nitrilase from the hyperthermophilic archaeon Pyrococcus abyssi

Four open reading frames encoding putative nitrilases were identified in the genomes of the hyperthermophilic archaea Pyrococcus abyssi, Pyrococcus horikoshii, Pyrococcus furiosus, and Aeropyrum pernix (growth temperature 90-100 degrees C). The nitrilase encoding genes were cloned and overexpressed in Escherichia coli. Enzymatic activity could only be detected in the case of Py. abyssi. This recombinant nitrilase was purified by heat treatment of E. coli crude extract followed by anion-exchange chromatography with a yield of 88% and a specific activity of 0.14 U/mg. The recombinant enzyme, which represents the first archaeal nitrilase, is a dimer (29.8 kDa/subunit) with an isoelectric point of pI 5.3. The nitrilase is active at a broad temperature (60-90 degrees C) and neutral pH range (pH 6.0-8.0). The recombinant enzyme is highly thermostable with a half-life of 25 h at 70 degrees C, 9 h at 80 degrees C, and 6 h at 90 degrees C. Thermostability measurements by employing circular dichroism spectroscopy and differential scanning microcalorimetry, at neutral pH, have shown that the enzyme unfolds up to 90 degrees C reversibly and has a T(m) of 112.7 degrees C. An inhibition of the enzymatic activity was observed in the presence of acetone and metal ions such as Ag(2+) and Hg(2+). The nitrilase hydrolyzes preferentially aliphatic substrates and the best substrate is malononitrile with a K(m) value of 3.47 mM.