Enzymatic synthesis of γ-l-glutamyl-S-allyl-l-cysteine,a naturally occurring organosulfur compound from garlic,by Bacillus licheniformis γ-glutamyltranspeptidase

In the practical application of Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT), we describe a straightforward enzymatic synthesis of γ-L-glutamyl-S-allyl-L-cysteine (GSAC), a naturally occurring organosulfur compound found in garlic, based on a transpeptidation reaction involving glutamine as the γ-glutamyl donor and S-allyl-L-cysteine as the acceptor. With the help of thin layer chromatography technique and computer-assisted image analysis, we performed the quantitative determination of GSAC. The optimum conditions for a biocatalyzed synthesis of GSAC were 200 mM glutamine, 200 mM S-allyl-L-cysteine, 50 mM Tris–HCl buffer (pH 9.0), and BlGGT at a final concentration of 1.0 U/mL. After a 15-h incubation of the reaction mixture at 60 °C, the GSAC yield for the free and immobilized enzymes was 19.3% and 18.3%, respectively. The enzymatic synthesis of GSAC was repeated under optimal conditions at 1-mmol preparative level. The reaction products together with the commercially available GSAC were further subjected to an ESI-MS/MS analysis. A significant signal with m/z of 291.1 and the protonated fragments at m/z of 73.0, 130.1, 145.0, and 162.1 were observed in the positive ESI-MS/MS spectrum, which is consistent with those of the standard compound. These results confirm the successful synthesis of GSAC from glutamine and S-allyl-L-cysteine by BlGGT.     

Enzymatic synthesis of phenolic lipids in solvent-free medium using flaxseed oil and 3,4-dihydroxyphenyl acetic acid

The enzymatic synthesis of phenolic lipids (PLs) by transesterification of flaxseed oil with 3,4-dihydroxyphenyl acetic acid (DHPA) was investigated in solvent-free medium (SFM), using Novozym 435 from Candida antarctica as the biocatalyst. The effects of selected reaction parameters, water activity (a_w), enzyme concentration and agitation speed, were studied and optimized. Increasing the a_w of the reaction mixture from 0.18 to 0.38 resulted in a significant increase in the bioconversion yield from 62 to 77%. APCI–MS analysis confirmed the formation of six 3,4-dihydroxyphenyl acetoylated lipids, which were monolinolenyl, dioleyl, dilinolenyl, linoleyl linolenyl, oleyl linolenyl and oleyl linoleyl dihydroxyphenyl acetates. The highest enzymatic activity (178 nmol of PLs/g solid enzyme/min) was obtained using 40 mg of solid enzyme (400 PLU)/mL at agitation speed 150 rpm. Using the optimized conditions, the phenolic lipids showed a high relative proportion of linolenic acid (C_18:3 n?3) that increased from 57% in the flaxseed oil to 75 and 64% in the produced phenolic mono- and diacylglycerols, respectively. In addition, the synthesized phenolic lipids demonstrated a 7.2-fold lower radical scavenging activity than that of DHPA but half that of α-tocopherol.     

Subcritical water and dilute acid pretreatments for bioethanol production from Melaleuca leucadendron shedding bark

The feasibility of bioethanol production using the lignocellulose of the shedding bark of Melaleuca leucadendron (Paper bark tree) was investigated. The effects of pretreatment parameters (temperature, time and acid concentration) on the yields of sugars and inhibitors, and optimal pretreatment conditions were determined. At very low severity conditions (combined severity factor, CSF ≤ 0.335), 28% of xylan was recovered and this recovery increased with increasing CSF till it peaked to 64.4% (11.2 g xylose L⁻¹) at a CSF of 1.475. However, at CSF > 2.0, xylose yield declined due to degradation. Mild and progressive glucose yield was detected in prehydrolysate at CSF ≥ 1.514, and subsequent enzymatic hydrolysis allowed complete glucan solubilization. Implementing environmentally friendly subcritical water pretreatment at CSF ≤ 0.335 on the shedding bark, about 85% of glucan solubilization was achieved after enzymatic hydrolysis. An industrial Saccharomyces cerevisiae strain readily fermented crude hydrolysate within 12 h, yielding 24.7 g L⁻¹ ethanol at an inoculum size of 2% (v/v), representing a glucose to ethanol conversion rate of 0.475 g g⁻¹ (91% ethanol yield). Based on our findings, the shedding bark is a potential feedstock for bio-ethanol production.     

Development of an enzymatic synthesis approach to produce phloridzin using Malus x domestica glycosyltransferase in engineered Pichia pastoris GS115

Phloridzin has been an attractive target for the development of preparation methodologies due to its various applications as pharmaceutical ingredient and food additive. In this study, an enzymatic synthesis approach using engineered P. pastoris GS115 was developed to produce phloridzin. Four factors, identifying as key factors affecting the phloridzin yield, were optimized by response surface methodology as follows: induction time 121.8 h, pH 7.0, methanol 0.75%, and a rotation speed 194 rpm. The maximal yield of phloridzin reached 41.59 mg/L (95.31 μM) and the specific bioconversion rate of substrate was up to 98.50% with 1L working volume in a 5L bioreactor under the optimal conditions. Phloridzin with purity of 93.98% and recovery of 78.14% was achieved just using affinity chromatography and preparative high performance liquid chromatography (Pre-HPLC). These results confirm the enzymatic synthesis developed provides an efficient alternative to traditional approach for the preparation of phloridzin in vitro.     

Preparation of octyl β-d-xylobioside and xyloside by xylanase-catalyzed direct transglycosylation reaction of xylan and octanol

1-Octyl -d-xylobioside and xyloside were prepared by direct transglycosylation reaction of xylan and 1-octanol using purified xylanase from Aureobasidium pullulans. 2-Ethylhexyl -d- xylobioside and xyloside were also prepared in the same way. The maximum yields of 2-ethylhexyl -d-xylobioside and 2-ethylhexyl -d-xyloside were 110 and 54 mg/g xylan, respectively. The proposed mechanism for production of octyl xylobioside and xyloside involved the reaction of xylan and octanol by xylanase to produce octyl xylobioside and xylotrioside, the latter of which was simultaneously hydrolyzed by xylanase into octyl xyloside and xylobiose.     

Solvent free biocatalytic synthesis of isoniazid from isonicotinamide using whole cell of Bacillus smithii strain IITR6b2

A biocatalytic route for the synthesis of isoniazid, an important first-line antitubercular drug, in aqueous system is presented. The reported bioprocess is a greener method, does not involve any hazardous reagent and takes place under mild reaction conditions. Whole cell amidase of Bacillus smithii strain IITR6b2 having acyltransferase activity was utilized for its ability to transfer acyl group of isonicotinamide to hydrazine–2HCl in aqueous medium. B. smithii strain IITR6b2 possessed 3 folds higher acyltransferase activity as compared to amide hydrolase activity and this ratio was further improved to 4.5 by optimizing concentration of co-substrate hydrazine–2HCl. Various key parameters were optimized and under the optimum reaction conditions of pH (7, phosphate buffer 100 mM), temperature (30 °C), substrate/co-substrate concentration (100/1000 mM) and resting cells concentration (2.0 mg_dcw/ml), 90.4% conversion of isonicotinamide to isoniazid was achieved in 60 min. Under these conditions, a fed batch process for production of isoniazid was developed and resulted in the accumulation of 439 mM of isoniazid with 87.8% molar conversion yield and productivity of 6.0 g/h/g_dcw. These results demonstrated that enzymatic synthesis of isoniazid using whole cells of B. smithii strain IITR6b2 might present an efficient alternative route to the chemical synthesis procedures without the involvement of organic solvent.     

Production and characterization of a thermostable endo-type β-xylanase produced by a newly-isolated Streptomyces thermocarboxydus subspecies MW8 strain from Jeju Island

A xylanase-producing, Gram-positive, aerobic, and spore-forming bacterium was isolated from a soil sample collected from Jeju Island and was classified as a novel subspecies of Streptomyces thermocarboxydus on the basis of 16S rRNA gene sequence similarity, the results of DNA–DNA hybridization analysis, and phenotypic characteristics. The novel strain was named as S. thermocarboxydus subsp. MW8 (=KCTC29013 = DSM52054). This strain produced extracellular xylanase. Xylanase from the strain was purified to homogeneity and had an apparent molecular weight of 52 kDa. The NH₂-terminal sequence (Ala-Glu-Ile-Arg-Leu) was distinct from those of previously reported xylanases. The purified xylanase produced xylobiose as the end-product of birchwood xylan hydrolysis. The K_m and V_max values of the purified xylanase on birchwood xylan were 1.71 mg/ml and 357.14 U/mg, respectively. The optimum pH and temperature for the enzyme were found to be 7.0 and 50 °C, respectively, and the enzyme exhibited significant heat stability. In addition, the enzyme was active over broad pH ranges: 84% of the maximum activity at pH 5.0, 84–88% at pH 6.0, 88% at pH 8.0, and 75–81% (pH 9.0). These enzymatic properties may be very useful for use in bio-industrial applications.     

Production of xylo-oligosaccharides by immobilized-stabilized derivatives of endo-xylanase from Streptomyces halstedii

An endoxylanase from Streptomyces halstedii was stabilized by multipoint covalent immobilization on glyoxyl-agarose supports. The immobilized enzyme derivatives preserved 65% of the catalytic activity corresponding to the one of soluble enzyme that had been immobilized. These immobilized derivatives were 200 times more stable 200 times more stable than the one-point covalently immobilized derivative in experiments involving thermal inactivation at 60 °C. The activity and stability of the immobilized enzyme was higher at pH 5.0 than at pH 7.0. The optimal temperature for xylan hydrolysis was 10 °C higher for the stabilized derivative than for the non-stabilized derivative. On the other hand, the highest loading capacity of activated 10% agarose gels was 75 mg of enzyme per mL of support. To prevent diffusional limitations, low loaded derivatives (containing 0.2 mg of enzyme per mL of support) were used to study the hydrolysis of xylan at high concentration (close to 1% (w/v)). 80% of the reducing sugars were released after 3 h at 55 °C. After 80% of enzymatic hydrolysis, a mixture of small xylo-oligosaccharides was obtained (from xylobiose to xylohexose) with a high percentage of xylobiose and minimal amounts of xylose. The immobilized-stabilized derivatives were used for 10 reaction cycles with no loss of catalytic activity.     

Synthesis of deoxygenated α(1 → 5)-linked arabinofuranose disaccharides as substrates and inhibitors of arabinosyltransferases of Mycobacterium tuberculosis

Arabinosyltransferases (AraTs) play a critical role in mycobacterial cell wall biosynthesis and are potential drug targets for the treatment of tuberculosis, especially multi-drug resistant forms of M. tuberculosis (MTB). Herein, we report the synthesis and acceptor/inhibitory activity of Araf α(1 → 5) Araf disaccharides possessing deoxygenation at the reducing sugar of the disaccharide. Deoxygenation at either the C-2 or C-3 position of Araf was achieved via a free radical procedure using xanthate derivatives of the hydroxyl group. The α(1 → 5)-linked disaccharides were produced by coupling n-octyl α-Araf 2-/3-deoxy, 2-fluoro glycosyl acceptors with an Araf thioglycosyl donor. The target disaccharides were tested in a cell free mycobacterial AraTs assay as well as an in vitro assay against MTB H₃₇Ra and M. avium complex strains.     

Valorization of olive stones for xylitol and ethanol production from dilute acid pretreatment via enzymatic hydrolysis and fermentation by Pachysolen tannophilus

Olive stones are an agro-industrial by-product abundant in the Mediterranean area that is regarded as a potential lignocellulosic feedstock for sugar production. Statistical modeling of dilute-sulphuric acid hydrolysis of olive stones has been performed using a response surface methodology, with treatment temperature and process time as factors, to optimize the hydrolysis conditions aiming to attain maximum d-xylose extraction from hemicelluloses. Thus, solid yield and composition of solid and liquid phases were assessed by empirical modeling. The highest yield of d-xylose was found at a temperature of 195 °C for 5 min. Under these conditions, 89.7% of the total d-xylose was recovered from raw material. The resulting solids from optimal conditions were assayed as substrate for enzymatic hydrolysis, while fermentability of hemicellulosic hydrolysates was tested using the d-xylose-fermenting yeast Pachysolen tannophilus. Both bioprocesses were considerably influenced by enzyme loading and inoculum size. In the enzymatic hydrolysis step, about 56% of cellulose was converted into d-glucose by using an enzyme/solid ratio of 40 FPU g⁻¹, while in the fermentation carried out with a cell concentration of 2 g L⁻¹ a yield of 0.44 g xylitol/g d-xylose and a global volumetric productivity of 0.11 g L⁻¹ h⁻¹ were achieved.     

Metabolic engineering of Corynebacterium glutamicum for the de novo production of ethylene glycol from glucose

Development of sustainable biological process for the production of bulk chemicals from renewable feedstock is an important goal of white biotechnology. Ethylene glycol (EG) is a large-volume commodity chemical with an annual production of over 20 million tons, and it is currently produced exclusively by petrochemical route. Herein, we report a novel biosynthetic route to produce EG from glucose by the extension of serine synthesis pathway of Corynebacterium glutamicum. The EG synthesis is achieved by the reduction of glycoaldehyde derived from serine. The transformation of serine to glycoaldehyde is catalyzed either by the sequential enzymatic deamination and decarboxylation or by the enzymatic decarboxylation and oxidation. We screened the corresponding enzymes and optimized the production strain by combinatorial optimization and metabolic engineering. The best engineered C. glutamicum strain is able to accumulate 3.5 g/L of EG with the yield of 0.25 mol/mol glucose in batch cultivation. This study lays the basis for developing an efficient biological process for EG production.     

Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue

The generation of a fermentable hydrolysate from arabinoxylan is an important prerequisite for utilization of wheat hemicellulose in production of ethanol or other value added products. This study examined the individual and combined efficiencies of four selected, commercial, multicomponent enzyme preparations Celluclast 1.5 L (from Trichoderma reesei), Finizym (from Aspergillus niger), Ultraflo L (from Humicola insolens), and Viscozyme L (from Aspergillus aculeatus) in catalyzing arabinose and xylose release from water-soluble wheat arabinoxylan in an industrial fermentation residue (still bottoms) in lab scale experiments. Different reaction conditions, i.e. enzyme dosage, reaction time, pH, and temperature, were evaluated in response surface and ternary mixture designs. Ultraflo L treatment gave optimal arabinose release: treatment (6 h, 60 °C, pH 6) with this enzyme preparation liberated up to 46% by weight (wt.%) of the theoretically maximal arabinose yield from the substrate. Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose and catalyzed release of up to 25 wt.% of the theoretical maximum xylose yield (6 h, 60 °C, pH 4). Prolonged treatment for 24 h with a 50:50 mixture of Celluclast 1.5 L and Ultraflo L at 50 °C, pH 5 exhibited a synergistic effect in xylose release and 62 wt.% of the theoretically maximal xylose yield was achieved. Addition of pure β-xylosidase from T. reesei to the Ultraflo L preparation released the same amounts of xylose from the substrate as the 50:50 mixture of Celluclast 1.5 L and Ultraflo L. The data thus signified that the synergistic effect in xylose release between Celluclast 1.5 L and Ultraflo L is the result of a three-step interaction mechanism involving α-l-arabinofuranosidase and different xylan degrading enzyme activities in the two enzyme preparations.     

Two step ethanolysis: A simple and efficient way to improve the enzymatic biodiesel synthesis catalyzed by an immobilized–stabilized lipase from Thermomyces lanuginosus

In this study the immobilization and stabilization of a lipase from Thermomyces lanuginosus (TLL) on aldehyde-Lewatit (Lew-TLL) are described. TLL immobilization was rapid and over 90% of lipase activity was recovered after the immobilization. Lew-TLL was 10-fold more thermo stable than the commercial TLL preparation, Lipozyme TL-IM. The stabilized Lew-TLL was used for the enzymatic transesterification of ethanol and soybean oil. The transesterification was carried out following different strategies. First, with 7.5:1 molar ratio of ethanol:soybean oil, 15% immobilized enzyme and 4% water at 30 °C. In the presence of n-hexane, the transesterification reached 100% conversion, while in solvent-free system the yield was 75%. Second, at stoichiometric molar ratio, the yield was 70% conversion after 10 h of reaction in both systems. After this, transesterification was carried out by three stepwise additions of ethanol with a yield of 80% conversion, while a two step ethanolysis produced 100% conversion after 10 h of reaction in both solvent and solvent-free systems.     

Synthesis of methyl (R)-3-(4-fluorophenyl)glutarate via enzymatic desymmetrization of a prochiral diester

An efficient procedure for enzymatic desymmetrization of the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG) in an aqueous–organic phase was successfully developed to prepare methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG). Novozym 435 was selected as a highly efficient biocatalyst through lipase screening. The effects of various parameters in terms of co-solvent and its concentration, buffer pH, ionic strength and reaction temperature, on the reaction were investigated. It was found that 0.2 M phosphate buffer (pH 8.0) containing 20% MTBE (v/v) was the optimum reaction medium, and the optimum reaction temperature was 30 °C. Under the optimized reaction conditions, (R)-3-MFG was obtained in 95.6% ee value and 92.6% yield after 64 h when the concentration of 3-DFG and Novozym 435 were 200 mmol/l and 20 g/l respectively. Furthermore, Novozym 435 showed an excellent operational stability, retaining above 95% of the initial activity and enantioselectivity after 10 cycles of reaction. The developed method has a potential to be used for efficient enzymatic production of (R)-3-MFG.     

Novel minor lipase from Rhizopus chinensis during solid-state fermentation: Biochemical characterization and its esterification potential for ester synthesis

Rhizopus chinensis produces two lipases that catalyze ester synthesis when cultured under solid-state fermentation. The Lip2 was purified to homogeneity by ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. It has an apparent molecular weight of 33 kDa estimated from SDS–PAGE and 32 kDa calculated from analytical gel permeation, with synthetic activity and purification fold of 96.8 U/mg and 138.3, respectively. Maximum hydrolytic activity was obtained at pH 8.0–8.5 and 40 °C using pNPP as substrate. Slight activation of the enzyme was observed when Mn²⁺ is present. The enzyme was most active on p-nitrophenyl laurate (C12). The purified lipase exhibited maximum synthetic activity at pH memory of 6.0 and 30 ^oC. Most of ethyl esters synthesized by lyophilized enzyme achieved good yields (>90%), and caprylic acid served as the best acyl donor. The enzyme presented a particular affinity for ethanol, n-propanol and n-hexanol, with conversion of 92%, 93% and 92%, respectively, after 20 h incubation.     

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2 h vs 12 h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications.     

Expression of the lasB gene encoding an organic solvent-stable elastase in Pichia pastoris and potential applications of the recombinant enzymes in peptide synthesis

The lasB gene encoding a solvent-stable elastase from Pseudomonas aeruginosa (PAE) was isolated and heterologously expressed in Pichia pastoris, resulting in production of three heterogeneously glycosylated recombinant elastases (rPAEs). rPAEs showed higher solvent-stability and thermostability than native PAE, but these recombinant and native enzymes achieved similar values of specific activity (2393 U/mg and 2427 U/mg for rPAEs and the native one, respectively), apparent K_m (2.55 and 2.48 g/l for rPAEs and the native one, respectively) and k_cat (0.0489 and 0.0496/s for rPAEs and the native one, respectively) for casein hydrolysis. While rPAEs and their native counterpart displayed similar substrate specificity in bipeptide synthesis reactions in water-miscible organic solvents, the former gave higher synthesis rates and yields than the latter. The yields and rates of rPAEs-catalyzed bipeptide synthesis reactions substantially varied with the type of solvent, and dimethylsulfoxide (DMSO) was found to be more suitable for these reactions than methanol, ethanol, isopropanol, and n-butanol. The optimal reaction conditions for rPAEs-catalyzed Cbz-Ala-Phe-NH₂ synthesis were the presence of 50% (v/v) DMSO, and at pH 8.0 and temperature 20–30 °C.     

A novel thermostable cellulase free xylanase stable in broad range of pH from Streptomyces sp. CS428

A cellulase free thermostable xylanase from Streptomyces sp. CS428 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular xylanase was purified 26 fold with a 55% yield by CM Trisacryl cation exchange chromatography. The molecular mass of the enzyme (Xyn428) was approximately 37 kDa. Xyn428 was found to be stable over a broad pH range (4 to ～13.6) and to 50 °C and have an optimum temperature of 80 °C. Xyn428 had K_m and V_max values of 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, when beechwood xylan was used as substrate. N-terminal sequence of Xyn428 was INRTDHNENSYLEIHNNEAR. CS428 was grown on different agro waste xylan and produced 4197.1 U/mL of xylanase activity in 36 h of cultivation in wheat bran without supplements. Xyn428 activity was inhibited by Tris salt at concentrations above 20 mM, and produced xylose and xylobiose as major products. It was found to degrade agro waste materials by small unit of enzyme (20 U/g) as shown by electron microscopy. As being simple in purification, thermo tolerant, pH stability in broad range and ability to produce xylooligosaccharides show that Xyn428 has potential applications in industries as a biobleaching agent and for xylooligosaccharides production.     

Photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase from Saccharomyces cerevisiae and water-soluble zinc porphyrin

We evaluated the photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae and NAD⁺ photoreduction by the visible-light sensitization of zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) in the presence of methylviologen (MV²⁺), diaphorase, and triethanolamine (TEOA). When the sample solution containing ZnTPPS, MV²⁺, NAD⁺, diaphorase, and TEOA in potassium phosphate buffer solution was irradiated, the NADH produced increased with the irradiation time. After irradiation for 180 min, the conversion yield of NAD⁺ to NADH was about 60% under 0.1 mM NAD⁺ condition. The methanol production also depended on the conversion yield of NAD⁺ to NADH. After irradiation for 180 min, 0.38 μM of methanol was produced from formaldehyde (16 μM). The conversion ratio of formaldehyde to methanol was about 2.3%. This result indicates that a system for the photochemical synthesis of methanol from formaldehyde was developed with ADH and the NADH produced by the photosensitization of ZnTPPS in water media.     

Penicillin G acylase catalyzed acylation of 7-ACA in aqueous two-phase systems using kinetically and thermodynamically controlled strategies: improved enzymatic synthesis of 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ3-cephem-4-carboxylic acid

Penicillin G acylase (PGA) catalyzed acylation of 7-aminocephalosporanic acid (7-ACA) with R-mandelic acid and its derivatives gives 7-[(1-hydroxy-1-phenyl)-acetamido]-3-acetoxymethyl-Δ³-cephem-4-carboxylic acid. This compound is a useful intermediate for the synthesis of some 3′-functionalized cephalosporins. However, acylations catalyzed by PGA isolated from Escherichia coli give poor results both considering a kinetical or a thermodynamical approach. In order to improve this enzymatic acylation, polyethylene glycol (PEG 600)-ammonium sulphate aqueous two-phase systems have been studied with the aim to have, during the reaction, a continuous extraction of the acylation product outside of the enzyme environment (the ammonium sulphate phase). This strategy shifts the equilibrium in the thermodynamically controlled synthesis and prevents the hydrolysis of the synthesized antibiotic in the kinetically controlled synthesis. The best results were achieved using PEG 600 (80% in water) equilibrated with 4 M ammonium sulphate. In these conditions, the acylation product was completely partitioned in the PEG phase (K > 200), whereas the substrates maintained a suitable concentration in the enzyme environment. Both in the kinetic (88% yield) and the thermodynamic (75% yield) processes, the results obtained were sensitively improved in comparison with those achieved working in homogeneous solution (phosphate buffer). Using R-mandelic acid methyl ester, the yield increased from 65% (monophasic system) to 88%. The PEG solution, without isolation of the acylation product, was successfully used for the synthesis of Cefamandole and Cefonicid.