Penicillinamidohydrolase in Escherichia coli. II. Synthesis of the enzyme, kinetics and specificity of its induction and the effect of O2.

The differential rate of synthesis of penicillinamidohydrolase (penicillin acylase -- EC 3.5.1.11) was studied in Escherichia coli growing in some chemically defined media and in a complex medium. The enzyme is synthesized at a constant rate only during the exponential phase of growth of cells. Its synthesis is induced most effectively (with respect to quantity) by phenylacetic acid. The induction lag of the enzyme synthesis in a medium with acetate corresponds to two generation times. The highest rate of the enzyme synthesis is reached in a medium containing phenylacetic acid as the only source of carbon and energy. The enzyme synthesis is fully repressed by an increased concentration of dissolved oxygen in the medium, even when Escherichia coli is cultivated in the medium with phenylacetic acid as the only carbon and energy source.

Penicillinamidohydrolase in Escherichia coli. I. Substrate specificity.

Substrate specificity of the bacterial penicillinamidohydrolase (penicillinacylase, EC 3.5.1.11) from Escherichia coli was determined by measuring initial rates of enzyme hydrolysis of different substrates within zero order kinetics. Some N-phenylacetyl derivatives of amino acids and amides of phenylacetic acid and phenoxyacetic acid of different substituted amides of these acids or amides, structurally and chemically similar to these compounds, served as substrates. Significant differences in ratios of initial rates of the enzyme hydrolysis of different substrates were found using a toluenized suspension of bacterial cells or a crude enzyme preparation, in spite of the fact that the enzyme is localized between the cell wall and cytoplasmic membrane, in the so-called periplasmic space. N-phenylacetyl derivatives are the most rapidly hydrolyzed substrates. Beta-phenylpropionamide and 4-phenylbutyramide were not utilized as substrates. The substrate specificity of the enzyme is discussed with respect to a possible use of certain colourless compounds as substrates, hydrolysis of which yields chromophor products suitable for a simple and rapid assay of the enzyme activity.

Penicillinamidohydrolase in Escherichia coli. III. Catabolite repression, diauxie, effect of cAMP and nature of the enzyme induction.

Synthesis of penicillinamidohydrolase (penicillin acylase, EC 3.5.1.11) in Escherichia coli is subjected to the absolute catabolite repression by glucose and partial repression by acetate. Both types of catabolite repression of synthesis of the enzyme in Escherichia coli are substantially influenced by cyclic 3',5'-adenosinemonophosphate (cAMP). Growth diauxie in a mixed medium containing glucose and phenylacetic acid serving as carbon and energy sources is overcome by cAMP. cAMP does not influence the basal rate of the enzyme synthesis (without the inducer). Derepression of synthesis of penicillinamidohydrolase by cAMP in a medium with glucose and inducer (phenylacetic acid) is associated with utilization of the inducer, due probably to derepression of other enzymes responsible for degradation of phenylacetic acid. Lactate can serve as a "catabolically neutral" source of carbon suitable for the maximum production of penicillinamidohydrolase. The gratuitous induction of the enzyme synthesis in a medium with lactate as the carbon and energy source and with phenylacetic acid is not influenced by cAMP; however, cAMP overcomes completely the absolute catabolite repression of the enzyme synthesis by glucose.

Cell aggregates of Escherichia coli with benzylpenicillin amidase activity.

Intact cells Escherichia coli CCM 2843, exhibiting substantial benzylpenicillin amidase activity, were bound mutually with supporting waste microbial cells, native or treated, to obtain an inexpensive biocatalyst for the production of 6-aminopenicillanic acid (6-APA). The bond was effected by glutaraldehyde (GA) and Sedipur CL-930 (PEI), without any carrier. The optimal concentration of GA was 2%, that of PEI 1%. The optimal biocatalyst was obtained by immobilization of productive cells with their fragments at a mass ratio of 4:1. The cell aggregates were used for hydrolysis of potassium benzyl-penicillin at a concentration of 5% to 6-APA. After 25 repeated batch conversions the degree of conversion did not decrease; its average value was 96.4%.

Effect of pyruvate decarboxylase activity and of pyruvate concentration on the production of 1-hydroxy-1-phenylpropanone in Saccharomyces carlsbergensis.

The assumption that the pyruvate decarboxylase activity of Saccharomyces carlsbergensis is the main limiting factor determining the formation rate and the total amount of d(--)-l-hydroxy-l-phenyl-propanone (phenylacetylcarbinol, PAC) produced was not confirmed. An increase of about 30% of the total amount of the PAC produced was obtained when 8.5% sodium pyruvate was gradually added. The total PAC production is probably influenced both by the pyruvate decarboxylase activity and the pyruvate concentration in the cells, the latter being actually the determining rate-limiting factor.

Immobilized cells.

Three basic types of immobilization (i.e. without carrier, entrapment and immobilization on the carrier surface) of microbial cells, nonmicrobial cell populations and subcellular organelles are reviewed. These are further developed into a number of combined and less frequently used techniques of immobilization and application of cell biocatalysts for industrial biotransformation in pharmacy, food industry and agriculture, including novel approached and some unpublished authors' results.

Enzymic synthesis of L-lysine from DL-alpha-amino-epsilon-caprolactam by new microbial strains.

The production of L-lysine from DL-alpha-amino-epsilon-caprolactam (DL-ACL) by new strains producing L-alpha-amino-epsilon-caprolactamase and aminocaprolactam racemase is described. Optimal conditions for hydrolysis of L-ACL by Cryptococcus sp. and for racemization of ACL by cells of a strain isolated in nature and identified as Pseudomonas sp. were determined. Synthesis of L-alpha-amino-epsilon-caprolactamase is induced by DL-ACL or L-lysine with the same effectivity. A positive effect of phosphates (potassium salts) on reduction of the induction lag was detected, the synthesis of this enzyme was found to be repressed by glucose and some possibilities of the reversion of this repressive effect were demonstrated. Under conditions optimal for the production of both enzymes a quantitative theoretical conversion of 10% aqueous DL-ACL to L-lysine by a mixture of native cells in a mass ratio of 1 : 2 (producer of ACL-hydrolase to producer of ACL-racemase) occurred in 8 h at 40 degrees C and pH 8.0.