Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Combination of the tod and the tol pathways in redesigning a metabolic route of Pseudomonas putida for the mineralization of a benzene, toluene, and p-xylene mixture.

Construction of a hybrid strain which is capable of mineralizing components of a benzene, toluene, and p-xylene mixture simultaneously was attempted by redesigning the metabolic pathway of Pseudomonas putida. Genetic and biochemical analyses of the tod and the tol pathways revealed that dihydrodiols formed from benzene, toluene, and p-xylene by toluene dioxygenase in the tod pathway could be channeled into the tol pathway by the action of cis-p-toluate-dihydrodiol dehydrogenase, leading to complete mineralization of a benzene, toluene, and p-xylene mixture. Consequently, a hybrid strain was constructed by cloning todC1C2BA genes encoding toluene dioxygenase on RSF1010 and introducing the resulting plasmid into P. putida mt-2. The hybrid strain of P. putida TB105 was found to mineralize a benzene, toluene, and p-xylene mixture without accumulation of any metabolic intermediate.     

Ginkgolide B production in cultured cells derived from Ginkgo biloba L. leaves

Callus cultures and cell suspension cultures derived from Ginkgo biloba L. leaves produced ginkgolidc B. In cell suspension cultures, the production reached a maximum by the 13th day of subculture and followed by a sharp decrease. The medium of Murashige and Skoog induced the highest ginkgolide B content in cultures while the medium of Schenk and Hildebrandt promoted cell growth. For the maximal production of ginkgolide B, cells were cultured in Murashige and Skoog medium modified to contain 1.0 mg/l of -naphthaleneacetic acid, 0.1 mg/1 of kinetin, 30 g/1 sucrose and 1.25 mM potassium phosphate with a molar ratio of ammonium to nitrate ions of 1 3.Abbreviations B5 Gamborg et al (1968) medium - GKB Ginkgolide B - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic aicd - SH Schenk and Hildebrandt (1972) medium     

Effects of prostaglandins, dibutyryl cAMP, LHRH, estrogens, progesterone, and potassium on output of prostaglandin F2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha, hCG, estradiol, and progesterone by placental minces

In order to compare the endocrine response of placental minces to luteinizing hormone releasing hormone (LHRH) and dibutyryl cAMP (dbcAMP) and to screen for effects of potential stimulatory and inhibitory substances, the simultaneous outputs of PGF2 alpha, 13, 14-dihydro-15-keto-prostaglandin F2 alpha (PGFM), progesterone, 17 beta-estradiol, and hCG were evaluated during a 4 hour incubation in 5 placentas. The output of hCG was highest for 12-week placentas, intermediate for a 16 week placenta, and lowest for term placentas. The output of 17 beta-estradiol by 12 and 16 week placentas in the presence of 30 microM dehydroepian-drosterone sulfate (DHEAS) was greater than that by term placentas. Progesterone output was apparently independent of gestational age although some variation between 12-week placentas was demonstrated. Output of PGF2 alpha was lower in 12 and 16-week placentas than in term placentas and that of PGFM was lower in 12-week placentas than in term placentas. LHRH (100 nM) produced stimulation of PGF2 alpha output (P less than .005) and a trend toward inhibition of progesterone output (which failed to achieve statistical significance) but no stimulation of hCG under these conditions. Stimulation of the outputs of hCG (P less than .005) and PGF2 alpha (P less than .001) and inhibition of that of progesterone (P less than .005) was produced by 20 mM dbcAMP. DHEAS inhibited output of progesterone (P less than .01) and PGF2 alpha (P less than .01). There were no effects of potassium, estrogens, progesterone, or prostaglandins on output of any measured substance.     

Ligninolytic activity of Phanerochaete chrysosporium: Physiology of suppression by NH 4 + and l-glutamate

Previous research showed that addition of nutrient nitrogen to ligninolytic (stationary, nitrogen-starved) cultures of the wood-decomposing basidiomycete Phanerochaete chrysosporium causes a suppression of lignin degradation. The present study examined early effects on nitrogen metabolism that followed addition of NH ₄ ⁺ and l-glutamate at concentrations that yield similar patterns of suppression. Both nitrogenous compounds were rapidly assimilated (>80% in 6 h). Both caused an initial 80% or greater increase in the intracellular glutamate pool and had similar effects in increasing the specific activities of NADP- and NAD-glutamate dehydrogenases and glutamine synthetase. Differences between the effects of added NH ₄ ⁺ and glutamate showed that suppression was not correlated with intracellular pools of arginine or glutamine, nor was the maintenance of an elevated glutamate pool required to maintain the suppressed state. While a portion of the initial glutamate suppression could be attributed to an effect on central carbon metabolism through glutamate catabolism by NAD-glutamate dehydrogenase, the long term suppression by glutamate and the suppression by NH ₄ ⁺ were more specific. Suppression by NH ₄ ⁺ or glutamate in the presence or absence of protein synthesis (cycloheximide) followed essentially identical kinetics during 12 h. These results indicate that nitrogen additions cause a biochemical repression of enzymes associated with lignin degradation. Results are consistent with the hypothesis that nitrogen metabolism via glutamate plays a role in initiation of repression.Non-Standard Abbreviations DMS 2,2-dimethylsuccinate - TCA trichloroacetic acid     

Mutants of Cellulomonas which produce increased levels of β-glucosidase

Summary Mutants have been isolated from a strain of Cellulomonas which are capable of producing up to 26-fold higher levels of -glucosidase than the parent under certain growth conditions.     

The production of 6-aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.