Bernard J. Abbott

Preparation of Pharmaceutical Compounds by Immobilized Enzymes and Cells

Publisher Summary This chapter discusses the preparation of pharmaceutical compounds by immobilized enzymes and cells. Immobilized enzymes and cells can be used to prepare many pharmaceutical compounds. Industrial exploitation of enzymes and microorganisms traditionally has been accomplished by using intact microorganisms or soluble cell free enzyme preparations. These processes are not very efficient because the catalysts (enzymes or microorganisms) are used for just one batch reaction or fermentation. Additional uses are not feasible because: enzymes and cells are relatively unstable and may lose activity during a fermentation or reaction; and conventional recovery methods are either expensive or cause denaturation and loss of catalytic activity. If enzymes and microorganisms are to be reused effectively, their stability must be improved, and inexpensive non-destructive recovery methods must be developed. Immobilization offers a means of achieving both objectives.

Microbiological transformation of cannabinoids.

Microorganisms were screened for their ability to modify 2 synthetic cannabinoid substrates (I andII). Structure analyses revealed that microorganisms transformed the substrates by (a) primary oxidation of the side chain, β-oxidation of the side chain, ketone formation on the side chain or cyclohexene ring, (b) secondary hydroxylation on the side chain, (c) aromatization of the cyclohexene ring, and (d) tertiary hydroxylation at the b/c ring juncture.

Cephalosporin acetylesterase (Bacillus subtilis).

Publisher Summary This chapter discusses the assay procedure and properties of cephalosporin acetylesterase (Bacillus subtilis). Cephalosporin esterase activity is readily assayed by titration with an automatic pH star. The reaction generates acetic acid that causes a drop in pH of the reaction mixture. A standardized KOH solution is automatically added to maintain the pH at a preset value (usually pH = 7.0). The amount of KOH added per unit of time is directly proportional to the reaction rate. The rate of KOH addition is automatically recorded on a moving chart, and the initial reaction rate can be determined from the slope of the line. The enzyme is extremely stable. It may be stored in an unbuffered solution at 25° and pH - 7.0 for 3 weeks with little or no loss of activity. In addition to cephalosporins, the enzyme will hydrolyze mono- and triacetin, α-naphthyl acetate, and glucose pentaacetate. The enzyme does not hydrolyze casein, acetanilide, p-nitrophenylacetate, p-nitrophenylsulfate, and it is not inhibited by arsenilic acid or bis (p-nitrophenyl) phosphate.

Enzymatic removal of a cephalosporin methyl ester blocking group

Abstract Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C 4 methyl ester blocking group on 7-aminodesacetoxycephalosporanic acid (7-ADCA). Only one culture, Streptomyces capillispira Mertz and Higgens nov. sp., produced an enzyme that catalysed the reaction. Enzyme synthesis in a defined mineral salts medium was repressed by NH 3 and amino acids. Under optimum fermentation conditions, the maximum rate of substrate hydrolysis was 6 × 10 −10 mol min −1 mg −1 cell. The enzyme was recovered from the mycelia and partially purified by gel filtration. Kinetic studies by pH-stat titration indicated that the pH optimum was 7.5–8.5, the temperature optimum was 25–30°C, and the substrate K m value was 2.3 mg ml −1 . The reaction products, 7-ADCA and methanol, were weak competitive inhibitors of the enzyme with K 1 values of 6.63 and 0.188 mg ml −1 , respectively. The enzyme also hydrolysed cefaclor and cephalexin methyl esters but did not hydrolyse cephalosporin ethyl esters. With further improvements in enzyme yields and stability, enzymatic deblocking of cephalosporins could provide an alternative to chemical deblocking processes.