Enzymatic synthesis of cephalothin by penicillin G acylase*

Enzymatic synthesis of cephalothin from 7-aminocephalosporanic acid (7-ACA) and amide derivatives of 2-thienylacetic acid (2-TA) using penicillin G acylase (pen G acylase) was studied. Two amide derivatives of 2-TA namely 2-thienylacetamide (2-TAA) and 2-thienylacetohydroxamic acid (2-TAH) were used in this study. The main reason for choosing amide but not the methyl ester derivative of 2-TA for the enzymatic synthesis was to increase their solubilities in water. The solubility of 2-TA methyl ester (2-TAM), 2-TAA, and 2-TAH in aqueous solution is 8 +/- 0.05 mM, 87 +/- 0.75 mM and 120 +/- 1.65 mM, respectively. Enzymatic conversion of 2-TAH to cephalothin yielded side products but they were not found in the conversion of 2-TAA to cephalothin. The side products were derived from reactions between hydroxyamine and 7-ACA. The effects of pH, temperature, initial substrate concentrations and reaction time on the conversion of 2-TAA and 7-ACA to cephalothin were examined. The optimum reaction condition was determined at pH 6.5 and 10 approximately 15 degrees C. The best conversion yield of 72% was obtained when the initial concentration of 2-TAA and 7-ACA was at 0.4 M and 0.1 M, respectively. Furthermore, a one-step method was developed to purify cephalothin from the enzymatic reaction mixture with the purity of 91% and the recovery yield of 96%.     

Enzymatic synthesis of beta-lactam antibiotics using penicillin-G acylase in frozen media.

Penicillin-G acylase (EC 3.5.1.11) from Escherichia coli catalyzed the synthesis of various beta-lactam antibiotics in ice at -20 degrees C with higher yields than obtained in solution at 20 degrees C. The initial ratio between aminolysis and hydrolysis of the acyl-enzyme complex in the synthesis of cephalexin increased from 1.3 at 20 degrees C to 25 at -20 degrees C. The effect on the other antibiotics studied was less, leading us to conclude that freezing of the reaction medium influences the hydrolysis of each nucleophile-acyl-enzyme complex to a different extent. Only free penicillin-G acylase could perform transformations in frozen media: immobilized preparations showed a low, predominantly hydrolytic activity under these conditions.     

Enzymatic hydrolysis of whey proteins: I. Kinetic models

We have studied the enzymatic hydrolysis of whey proteins at pH 8 and50 degrees C with two proteases of bacterial origin, MKC Protease 660 L, and one of animal origin, PEM 2500 S. Our results show that a greater degree of hydrolysis is achieved under the same experimental conditions with the bacterial proteases than with the animal one. In our interpretation of the results we propose a mechanism in which the hydrolytic reaction is a zero-order one for the substrate, and the enzyme denaturalizes simultaneously via a second-order kinetic process due to free enzyme attacking enzyme bound to the substrate. Our results also indicate that there is an irreversible serine-protease inhibitor in whey proteins. (c) 1994 John Wiley & Sons, Inc.     

Enzymatic synthesis of beta-lactam antibiotics. I. Cefazolin]

Production of cefazolin by acyl transfer enzymatic synthesis with immobilised cefazolin synthetase from Escherichia coli as a biocatalyst acting in accordance with the mechanism including formation of the acyl-enzyme complex was shown possible. The process kinetic parameters and the ratio of the maximum conversion of the key amino acid and the initial concentrations of the substrate and nucleophile were determined. Correlation of the calculated and experimental data on the cefazolin yield in the enzymatic synthesis was good. The main physico-chemical properties of the substrates and the reaction products i.e. dissociation constants and solubility were investigated. The complex of the physico-chemical studies makes it possible to design a highly efficient technological process for production of cefazolin including not only the stage of the enzymatic synthesis but also the stage of separation of the reaction mixture components.     

Enzymatic synthesis of alkyl beta-D-xylosides by transxylosylation and reverse hydrolysis

The Trichoderma reesei beta-xylosidase (EC 3.2.1.37) is used to catalyze the production of alkyl beta-D-xyloside. Two general methods of production are tested and compared using the same enzyme: transglycosylation and reverse hydrolysis. Using both methods, primary, secondary, and tertiary alcohols are studied as acceptors. In kinetically controlled process (transglycosylation), the chosen donor is methyl beta-D-xyloside and primary, secondary, and tertiary alkyl alcohols are accepted. In the equilibrium-controlled synthesis, the donor is xylose whereas acceptors are only primary and secondary alcohols. The influence of the donor concentration is investigated in both processes. The yields of the kinetically controlled reactions are higher compared with those of the equilibrium-controlled synthesis. The specificity of the beta linkage is confirmed by proton nuclear magnetic resonance ((1)H NMR) analysis. (c) 1994 John Wiley & Sons, Inc.     

Enzymatic hydrolysis and biological activity of oligonucleotides containing 5-substituted pyrimidine bases.

Two series of alternating ODNs containing 5-n.alkyl-, alkenyl- and alkynyl-dU and -dC units have been prepared in order to study the kinetics of their hydrolysis by SV PDE and human serum, respectively. Both in (r5dUpdA)10 and (r5dCpdG)6 series the rate of hydrolysis decreased with increasing length of side-chain. Replacement of thymidines by 5-hexynyl-dU in different antisense oligomers resulted in considerably higher biological activity relative to that of the thymidine-containing counterparts.     

Bacillus sphaericus V-penicillin acylase I. Fermentation

Summary In an aerobic fermentor with a fermentation medium based on 4% corn steep liquor and salts, Bacillus sphaericus, strain NCTC 10338, produces an intracellular V-penicillin acylase, when pH is in the narrow interval 7.5–8.0. The strain grows well outside this pH range, but the growth is not associated with acylase activity in the cells. The enzyme was not induced by phenoxy acetic acid and was not repressed by glucose.     

Enzymatic preparation of cefaclor with immobilized penicillin acylase

Enzymatic syntheses of cefaclor by immobilized penicillin acylase under kinetic control were carried out. According to the initial reaction rate ratio of synthesis to hydrolysis (Vs/Vh), penicillin acylase from Alcaligenes faecalis was chosen as the suitable catalyst for the synthesis of cefaclor. The reaction conditions, such as temperature, pH, and substrate concentration were investigated based on their Vs/Vh values. In the process of preparing cefaclor, in situ product removal (ISPR) and acyl donor feeding were used to achieve high yield. At the optimal conditions, the yield of cefaclor was 90%. In addition, the product were separated and purified, the total yield of cefaclor was 61%.     

Enzymatic hydrolysis of molasses

Kinetic studies of the enzymatic hydrolysis of molasses were conducted using glucoamylase. Central Sugar Refinery SDN BHD contains 13-20% glucose. The molasses was diluted and the kinetic experiments were conducted at 67 degrees C with 100-1000 mg/l of glucoamylase. The glucose contents of the molasses were enhanced after hydrolysis of molasses solution with 1000 mg/l glucoamylase. A Lineweaver-Burk plot was obtained based on enzyme kinetic data. The rate constant, Km and maximum reaction rate, Vmax for 500 mg/l of glucoamylase were 100 mmol/l (18 g/l) and 5 mmol/l min (0.9 g/l min), respectively. The maximum reaction rate, Vmax for 1000 mg/l of glucoamylase was doubled, to 100 mmol/l (18 g/l) and the rate constant, Km was the same for 500 mg/l of glucoamylase. The substrate inhibition model was noncompetitive based on the resulting Lineweaver-Burk plot for enzyme concentration of 500 and 1000 mg/l.