The metabolism of cyclohexanol by Nocardia globerula CL1

Abstract

1. Nocardia globerula CL1, isolated by enrichment on cyclohexanol and grown with it as carbon source, oxidized it with a Q(o2) of 39mul/h per mg dry wt. and the overall consumption of 2.2mumol of oxygen/mol of substrate. Cyclohexanone, 2-hydroxycyclohexan-1-one dimer and cyclohexane-1,2-dione were oxidized with Q(o2) values similar to that for cyclohexanol whereas in-caprolactone and 6-hydroxycaproate were oxidized very slowly and adipate not all. 2. Disrupted cell suspensions could not be shown to catalyse the conversion of cyclohexanol into cyclohexanone. 3. A cyclohexanol-induced cyclohexanone oxygenase (specific activity 0.55mumol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mol of NADPH and 1mol of O(2) in the presence of 1mol of cyclohexanone. NADPH oxidation did not occur under anaerobic conditions. The only detected reaction product with 25000g supernatant was 6-hydroxycaproate. 4. Extracts of cyclohexanol-grown cells contained a lactone hydrolase (specific activity 15.6mumol hydrolysed/min per mg of protein), which converted in-caprolactone into 6-hydroxycaproate. 5. Incubation of 6-hydroxycaproate with 25000g supernatant in the presence of NAD(+) resulted in NAD(+) reduction under anaerobic conditions, oxygen consumption under aerobic conditions and the conversion of 6-hydroxycaproate into adipate. 6. Cyclohexanone oxygenase fractions devoid of in-caprolactone hydrolase catalysed the stoicheiometric formation of in-caprolactone from cyclohexanone in the presence of excess of NADPH. 7. The reaction sequence for the oxidation of cyclohexanone by N. globerula CL1 is: cyclohexanol --> cyclohexanone --> in-caprolactone --> 6-hydroxycaproate --> adipate. 8. It is suggested that the adipate may be further dissimilated by beta-oxidation.

The Metabolism of trans‐Cyclohexan‐1,2‐diol by an Acinetobacter Species

“…As a check on the efficient functioning of the pH-stat system 5 pmol of I-oxa-2-0x0-cycloheptane were added to the vessel (C) followed by 0.12 mg of 25000 x g supernatant from cyclohexanol-grown N . gloherulu CL1 (D) that contains the lactone hydrolase [8] dicated that an alternative reaction to the hydrolytic cleavage of cyclohexan-1 ,2-dione was being catalysed by this protein. The probable identity of this reaction is indicated by the work of Schwarzenbach and Wittwer [25] and Long and Bakule [19,26].…”

“…For growth of N. globerula CL1 and Acinetobacter NCIB 9871 on cyclohexanol the carbon source was supplied and cells were grown as previously described [8]. For growth of the trans-cyclohexanol-1,2-diol isolates trans-cyclohexan-I ,2-diol was supplied at 1.5 g/l or sodium acetate at 3 g/l; both compounds being added before sterilization of the medium.…”

“…After 15 h growth this was either harvested for experimental purposes or used to inoculate 10 1 of medium in a Microferm Laboratory Fermenter (New Brunswick Scientific Co., U.S.A.) supplied with sterile air at 5 l/min and stirred at 250 rev./ min. Cells were harvested after 15 h growth, washed and resuspended for disruption as previously described [8]. When larger crops of cells were required and 10-1 culture was used to inoculate 30 1 of medium in a 45-1 capacity polythene vessel adapted to fit the Microferm M F 114 fermenter.…”

“…Standard elective culture procedures were used with trans-cyclohexan-1,2-diol as carbon source at 1 g/1 in basal salts medium, pH 7.1 [8].…”

Multiple Forms of Cyclohexanone Oxygenase from Nocardia globerula CL1

“…Cyclohexanone 1,2-monooxygenase of Nocardiu globerula CL1 grown with cyclohexanol as sole source of carbon has been purified by conventional techniques [1,2]. It has a molecular weight of 53000, consists of a single polypeptide chain is a flavoprotein with FAD as prosthetic group and has an absolute specificity for NADPH as electron donor.…”