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%.

Immobilization of Escherichia coli cells with penicillin-amidohydrolase activity on solid polymeric carriers.

Whole cells of Escherichia coli containing the enzyme penicillinamidohydrolase EC 3.5.1.11 were immobilized on the surface of modified macroporous copolymers of glycidylmethacrylate with ethylenedimethacrylate and of copolymers of methacrylaldehyde (MA) with divinylbenzene (DVB) by means of glutaraldehyde. These polymeric carriers were modified before cell binding by using ammonia or polyamines, especially ethylenediamine and hexamethylenediamine (HMDA). The highest specific activity and the largest yield in cell immobilization were achieved with the macroporous copolymer of MA and DVB modified with HMDA. The material thus obtained was used in repeated conversions of benzylpenicillin to 6-aminopenicillanic acid in a stirred batch reactor.

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for industrial biotransformation in pharmacy, food industry and agriculture, including novel approached and some unpublished authors' results.

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.

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.

Enzyme immobilization techniques on poly(glycidyl methacrylate-co-ethylene dimethacrylate) carrier with penicillin amidase as model.

Two types of bead-form macroporous carriers based on glycidyl methacrylate with ethylene dimethacrylate copolymers were used for the immobilization of penicillin amidase either directly or after chemical modification. Direct binding through oxirane groups, which is equally efficient at pH 4.2 and 7, is relatively slow and brings about an activity loss at low enzyme concentrations. The most efficient immobilization was achieved on glutaraldehyde-activated amino carrier, irrespective of whether the amino groups were formed by ammonia or 1,6-diaminohexane treatment of the original oxirane carrier. Hydrazine treatment gave lower immobilization yields. The same is true of the azide method independent of the length of the spacer. Most enzyme activity was preserved by coupling the carbodiimide-activated enzyme to the carrier with alkyl or arylamino groups at the end of a longer substituent. Immobilization on diazo-modified carrier gave average results. Rapid immobilization by a lysine-modified phosgene-treated carrier resulted in an activity loss. It is suggested that multipoint and very tight attachment of the enzyme molecule to the matrix decreased the activity. The immobilized activity is quite stable in solution and very stable upon lyophilization with sucrose.