Effect of amino acids on the production and activities of cephalosporin C acylase and penicillin V acylase from Aeromonas species ACY 95.

Amino acids altered the production and activities of cephalosporin C acylase and penicillin V acylase from Aeromonas species ACY 95 to a varying degree. DL-Tryptophan enhanced the cephalosporin C acylase formation by 222% while suppressed the penicillin V acylase formation by 68%.

Evaluation of product analogues for purification of penicillinase by affinity chromatography.

Among different matrices prepared, ampicilloic acid-polymer matrix offered 86.7% adsorption, 95% elution and 82.4% overall recovery of penicillinase. The structure of both the side chain and penicilloic or cephalosporoic acid moieties contribute to the affinity interactions.

Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95.

Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.

Expanded bed chromatography and radial flow column chromatography: the approaches for adsorption chromatography of biomolecules on industrial scale.

Adsorption of proteins directly from unclarified feed-stocks, has gained importance in the recovery of biomolecules on Industrial scale. Adsorption in expanded beds and radial flow gives significantly better results compared to conventional column chromatography methods for downstream processing of variety of proteins from particulate-containing feed-stocks, such as fermentation broths and cells extracts. The simple operation of these techniques reduces the complexity of downstream processing by eliminating steps such as filtration, centrifugation and concentration. One step purification, with simple equipment, these techniques enable to recover pure protein with 95% yield in biotechnological industry. The principles, operating procedures, adsorbents and applications of these techniques are discussed.

Molecular biology of ß-lactam acylases.

ß-Lactam acylases such as penicillin G acylases, penicillin V acylases and glutaryl 7-aminocephalosporanic acid acylases are used in the manufacture of 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid (7-ACA). Genetically-engineered strains producing 1050 U/g, 3200 U/g and 7000 to 10,000 U/I of penicillin G acylase, penicillin V acylase and glutaryl-7-ACA acylase, respectively, have been developed. The penicillin G acylase studied to date and the glutaryl-7-ACA acylase from Pseudomonas sp. share some common features: the active enzyme molecules are composed of two dissimilar subunits that are generated from respective precursor polypeptide; the proteolytic processing is a post-translational modification which is regulated by temperature; and the Ser residue at the N-terminus of the ß-sub-unit (Ser(290); penicillin G acylase numbering) is implicated as the active site residue. Protein engineering, to generate penicillin G acylase molecules and their precursors with altered sequences, and the structure-function correlation of the engineered molecules are discussed.

Comparative evaluation of determination of beta-lactam intermediates.

The Schiff's base formation between 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid and p-dimethylaminobenzaldehyde (PDAB) was investigated. The factors that affect the reaction such as concentration of PDAB, time and pH were studied and optimised for estimation of these intermediates.

Cephalosporin acylases: enzyme production, structure and application in the production of 7-ACA.

Cephalosporin acylases have application in the production of 7-aminocephalosporanic acid which forms a key raw material for the preparation of semisynthetic injectable cephalosporins. The enzymes are of industrial importance and hyperproducing genetically engineered strains have been constructed. Different aspects of these enzymes such as subunit structure, post translational modification, primary structure, substrate specificity and their importance in pharmaceutical industry are discussed.

Production, partial purification and characterisation of amyloglucosidase from a new variety of Aspergillus candidus Link var. aureus.

The production of amyloglucosidase by a new variety of Aspergillus candidus was studied on various natural carbon and nitrogen nutrient sources. Jowar starch and peanut meal were found to be the best nutrient sources of carbon and nitrogen respectively. The maximum enzyme productivity was observed at pH 6.5 and temperature 32 degrees C. Sodium fluoride was found to inhibit enzyme amyloglucosidase to an extent of 85%. Addition of trace metals viz. Zinc, copper and potassium in the medium impaired the production of the enzyme. A four fold purification of the enzyme was achieved with ammonium sulphate precipitation. The partially purified enzyme gave 84% conversion efficiency of starch to glucose in 10% starch solution without the addition of liquefying enzyme.

Production of extracellular acidic lipase by Rhizopus arrhizus as a function of culture conditions.

Thermophilic strain of Rhizopus arrhizus accumulates an acidic lipase in culture fluid when grown in a medium containing ground nut oil, milk powder and inorganic salts. Addition of 2.0% ground nut oil yielded the highest productivity of enzyme. Soyabean meal and arabinose were found to be the best nitrogen and carbon sources for enzyme production respectively. Addition of metal ions such as MnCl2, SnCl2 and CaCl2 increased the enzyme productivity by 4 fold. The enzyme productivity in the fermenter was much higher (310 U/ml) than in shake-flask (180 U/ml). Crude lipase preparation showed pH and temperature activity optima at 3.5 and 45 degrees C respectively. The enzyme is thermostable and highly active in hydrolysing triglycerides and failed to hydrolyse-methyl esters of caprylate and palmitate.

Production of carbohydrases by Sclerotium rolfsii.

Coproduction of alpha-amylase, beta-amylase, amyloglucosidase, cellulase, xylanase, pectinase and beta-galactosidase by Sclerotium rolfsii was studied on various polysaccharides. Starch induced alpha-amylase, beta-amylase, amyloglucosidase and beta galactosidase; cellulose induced cellulase, xylanase, pectinase and beta-galactosidase; and pectin induced pectinase and beta-galactosidase. None of the enzymes studied except beta-galactosidase were induced on xylan. Group controlled mechanism for production of carbohydrases by Sclerotium rolfsii is suggested.

Beijerinckia indica var. penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity.

Beijerinckia indica var. penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, gamma-irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.

Role of side chain moiety in the hydrolysis of penicillins by beta-lactamase.

Various beta-lactam compounds and structurally related moieties were examined as substrates of beta-lactamase from Bacillus cereus 5/B NCTC 9946. The enzyme was specific for penicillins and none of the cephalosporins were hydrolysed. Electronic environment of allylic carboxy group in dihydrothiazine ring restricts the acceptance of cephalosporins as substrates. The efficiency of hydrolysis of penicillins is dependent on dense resonating electronic environment of phenyl ring present in the side chain, flexibility of the side chain and the distance between the phenyl ring and carbonyl group in the side chain.

Persistence of Candida sp. 115 during hydrolysis of penicillin G and metabolism of phenylacetic acid.

The growth of Candida sp. 115 was investigated on the constituents of penicillin G hydrolysis reaction mixture. Neither penicillin G nor 6-aminopenicillanic acid was degraded or utilised for growth. The yeast accepted phenylacetic acid, sodium acetate and glucose as growth substrates. Phenylacetic acid was metabolised via p-hydroxy phenylacetic acid, which was the only accumulated metabolite. The enzymes responsible for hydroxylation of phenylacetic acid were induced by phenylacetic acid and sodium acetate.

Enzymatic hydrolysis of 7-phenylacetamidodesacetoxycephalosporanic acid by immobilized penicillin G acylase.

Enzymatic parameters such as pH, temperature and substrate concentration were studied for the hydrolysis of 7-PADCA by penicillin G acylase. Optimum pH and temperature were 8.0 and 50 degrees C, respectively. Km value of soluble and immobilized enzyme for 7-PADCA was 2.3 x 10(-5) M and 7.5 x 10(-5) M, respectively. At 7-PADCA concentration of 5% and an IME: 7-PADCA ratio of 1:2.5, the hydrolysis was complete in 110 min.

A comparative evaluation of ELISA and latex agglutination tests for detection of human chorionic gonadotropin (hCG) in urine for pregnancy.

PIP: To resolve questions about the relative sensitivity and specificity of the latex agglutination and enzyme-linked immunosorbent assay (ELISA) for the detection of human chorionic gonadotropin (hCG) in pregnancy, urine samples from 265 cases of suspected pregnancy were tested simultaneously by both methods. Included in the sample were women whose menstrual period ranged from 5-60 days late. In 243 cases (92%), identical results were obtained with both tests (142 positive and 101 negative). In an additional 12 cases, urine samples containing low levels of hCG when tested by the latex agglutination test showed negative results, while the same samples showed positive pregnancy (subsequently confirmed clinically) when tested by ELISA. Finally, 10 urine samples with red blood cells or pus cells gave false positive results on the latex agglutination test, but these cells did not interfere in the ELISA test. The lag period after the last menstrual period was 5-7 days for ELISA compared with 20 days for the latex agglutination test. The sensitivity of the former test was 0.5 IU hCG/ml of urine and 3.5 IU hCG/ml of urine for the latter test. Other advantages of the ELISA test are its stability (reagents are stable at room temperature, while those of the latex agglutination test are not), and its very clear and easy to read end point. The only advantage of the latex agglutination test was the time required for performing the test (5 minutes, compared with 40-50 minutes for ELISA).^ieng