Studies on cell-free metabolism: Ethanol production by a yeast glycolytic system reconstituted from purified enzymes

A reconstituted glycolytic system has been established from individually purified enzymes to simulate the conversion of glucose to ethanol plus CO₂ by yeast. Sustained and extensive conversion occurred provided that input of glucose matched the rate of ATP degradation appropriately.ATPase activity could be replaced by arsenate, which uncoupled ATP synthesis from glycolysis. The mode of uncoupling was investigated, and it was concluded that the artificial intermediate, 1-arseno-3-phosphoglycerate, has a half-life of no more than a few milliseconds. Arsenate at 4 mM concentration could simulate the equivalent of 10 μmol ml^?1 min^?1 of ATPase activity.The reconstituted enzyme system was capable of totally degrading 1 M (18% w/v) glucose in 8 h giving 9% (w/v) ethanol. The levels of metabolites during metabolism were measured to detect rate-limiting steps.The successful operation of the reconstituted enzyme system demonstrates that it is possible to carry out complex chemical transformations with multiple enzyme systems in vitro.     

Molecular identification of a novel inulinolytic fungus isolated from and grown on tubers of Helianthus tuberosus and statistical screening of medium components

Of 24 fungi belonging to more than five genera isolated from tubers of rotten Helianthus tuberosus, 11-inulinolytic active isolates were able to develop halo zones around their fungal colonies, indicating inulinase activity. Alternaria, Aspergillus, Fusarium, Pencillium and Trichoderma were the most common inulinolytic genera, representing more than 90?% of the total positive inulinolytic fungi. Aspergillus tamarii and Pencillium citrinum quantitatively recorded better growth (5.5 and 4.7?mg?ml^?1) and inulinase production (21.53 and 20.15?U?ml^?1) in submerged culture. The enzyme preparation showed also invertase activity. Aspergillus tamarii, as the most potent producer of inulinase, was identified using the Inter Transcribed Spacer marker. The sequence comparisons showed that our molecularly identified strain (GU295949) is related more closely to A. tamarii strains of the gene bank. Statistical screening using the fractional factorial Plackett-Burman design with 12 run was applied for screening ten variables, the low levels of pH (4.8), inoculum size (10³ spore?g^?1), NH₄NO₃ (1.0?mg?g^?1) and MgSO₄ (0.12?mg?g^?1), were the most significant variables on A. tamarii inulinase production. The high inulinase/invertase ratio (1.841?C4.293) classified the enzyme preparation as inulinases, which can be used efficiently in production of fructose syrup from tubers of H. tuberosus.     

Effect of adrenalectomy on cellular calcium metabolism and on the response to adrenergic stimulation of hepatocytes isolated from male and female rats

The effects of adrenalectomy on cell calcium metabolism and on the effects of epinephrine on cAMP, phosphorylase a activity, and calcium efflux were studied in hepatocytes isolated from adult male and female rats. Adrenalectomy increased the total calcium of hepatocytes, all exchangeable calcium pools, and all calcium fluxes between the cellular pools in both sexes. After adrenalectomy, basal cAMP was elevated, phosphorylase a + b was decreased, but basal phosphorylase a activity was not changed. In adrenalectomized males and at all concentrations of epinephrine studied (1·10^?8?1·10^?5M) stimulation of calcium efflux was decreased and cAMP accumulation was enhanced, while the resulting phosphorylase a activation was depressed. In hepatocytes from adrenalectomized females there was a similar increase in cAMP accumulation induced by epinephrine, and a decrease in the stimulation of calcium efflux; however, the depression in phosphorylase a activation was much less and was significant only at 1·10^?8 and 1·10^?5M epinephrine. In the male, while activation of phosphorylase a shifted from a pure α-adrenergic response mediated by calcium to one also involving a cAMP-mediated β-adrenergic response, the contribution of the attenuated calcium signal was still significant. Hepatocytes from female rats did not show a comparable α- to β-shift, since the relative contribution of calcium and cAMP to phosphorylase activation was similar in sham-operated and adrenalectomized animals.     

Antimycotic Activity Potentiation of Allium sativum Extract and Silver Nanoparticles against Trichophyton rubrum

A natural and biocompatible extract of garlic as a support, decorated with silver nanoparticles, is a proposal to generate an effective antifungal agent against dermatophytes at low concentrations. Silver nanoparticles (AgNPs) with a diameter of 26±7 nm were synthesized and their antimycotic activity was examined against Trichophyton rubrum (T. rubrum), inhibiting 94 % of growth at a concentration of 0.08 mg ml^?1. Allium sativum (garlic) extract was also obtained (AsExt), and its MIC was 0.04 mg ml^?1. To increase the antifungal capacity of those systems, AsExt was decorated with AgNPs, obtaining AsExt‐AgNPs. Using an AsExt concentration of 0.04 mg ml^?1 in independent experiments with concentrations from 0.01 to 0.08 mg ml^?1 of AgNPs, it was possible to inhibit T. rubrum at all AgNPs concentrations; it proves a synergistic effect between AgNPs and AsExt. Even if 1 % of the minimum inhibitory concentration of AsExt (0.0004 mg ml^?1) is used, it was possible to inhibit T. rubrum at all concentrations of AgNPs, demonstrating the successful antimycotic activity potentiation when combining AsExt and AgNPs.     

The effect of calmodulin and troponin-C on activities of homogeneous liver phosphoprotein phosphatases

The effect of calmodulin was determined on activities of two homogeneous liver phosphoprotein phosphatases with phosphorylase a and phosphorylated histones as substrates. Calmodulin in the absence or presence of calcium had no effect on the dephosphorylation of phosphorylase a by either phosphatases. However, calmodulin inhibited the dephosphorylation of histones catalyzed by both phosphatases. No difference was found whether the reactions were carried out in the absence or presence of calcium. The effect of calmodulin on histone dephosphorylation was variable depending on (i) the absence or presence of KCl and Mg²⁺, and (ii) the concentration of histone in the reaction mixture. In the presence of KCl and Mg²⁺ at a histone concentration of 0.1 mg/ml, calmodulin inhibited the enzyme activity. At 1 mg/ml histone, lower concentrations of calmodulin activated whereas higher concentrations of calmodulin inhibited the enzyme activity. Similar, but relatively less, effect was observed with troponin-C. In the absence of KCl and Mg²⁺, calmodulin as well as troponin-C activated the enzyme activity. The optimal concentration of calmodulin (or troponin-C) was approximately 30–50% of histone concentration in the reaction mixture. Calcium alone or with calmodulin (or troponin-C) had no additional effect on enzyme activities. DNA and RNA, two negatively charged nucleic acids, also showed similar effects on histone dephosphorylation. Depending on whether KCl and Mg²⁺ were absent or present in the reaction mixtures, both nucleic acids either activated or inhibited the dephosphorylation of histones.     

Disruption of the allosteric phosphorylase a regulation of the hepatic glycogen-targeted protein phosphatase 1 improves glucose tolerance in vivo

Type 2 diabetes is characterised by elevated blood glucose concentrations, which potentially could be normalised by stimulation of hepatic glycogen synthesis. Under glycogenolytic conditions, the interaction of hepatic glycogen-associated protein phosphatase-1 (PP1–G_L) with glycogen phosphorylase a is believed to inhibit the dephosphorylation and activation of glycogen synthase (GS) by the PP1–G_L complex, suppressing glycogen synthesis. Consequently, the interaction of G_L with phosphorylase a has emerged as an attractive anti-diabetic target, pharmacological disruption of which could provide a novel mechanism to lower blood glucose levels by increasing hepatic glycogen synthesis. Here we report for the first time the in vivo consequences of disrupting the G_L–phosphorylase a interaction, using a mouse model containing a Tyr284Phe substitution in the phosphorylase a-binding region of the G_L protein. The resulting G_L^Y284F/Y284F mice display hepatic PP1–G_L activity that is no longer sensitive to allosteric inhibition by phosphorylase a, resulting in increased GS activity under glycogenolytic conditions, demonstrating that regulation of G_L by phosphorylase a operates in vivo. G_L^Y284F/Y284F and G_L^Y284F/+ mice display improved glucose tolerance compared with G_L^+/+ littermates, without significant accumulation of hepatic glycogen. The data provide the first in vivo evidence in support of targeting the G_L–phosphorylase a interaction for treatment of hyperglycaemia. During prolonged fasting the G_L^Y284F/Y284F mice lose more body weight and display decreased blood glucose levels in comparison with their G_L^+/+ littermates. These results suggest that, during periods of food deprivation, the phosphorylase a regulation of G_L may prevent futile glucose–glycogen cycling, preserving energy and thus providing a selective biological advantage that may explain the observed conservation of the allosteric regulation of PP1–G_L by phosphorylase a in mammals.     

Persistence and spread of mycoplasma in axenic callus tissue cultures of tobacco (Nicotiana glauca Grah.) in the presence of kinetin and IAA in nutrient medium

A technique is described for the establishment of infectious axenic callus cultures originated from stems of tobacco plants suffering from a mycoplasma disease, potato witches' broom. When stem pieces from a diseased tobacco plant (Nicotiana glauca Grah.) are culture on a modified nutrient medium according toMurashige andSkoog (1962) with added kinetin (0.64 mg or 2.56 mg 1 000 ml^?1) and IAA (2 mg or 4 mg 1 000 ml^?1), the pathogen persists and spreads in the newly formed callus tissue. The presence of the pathogen was proved by implantation of callus slices into stems of tomato plants which indicate the disease very reliably. Reconstituted tobacco plants too recovered the infectious agent of potato witches' broom; 27 plants from the 35 reconstituted plants were diseased. Similar results were obtained with Crimean yellows. Calli were cultured and subcultured from 1 to 18 monthsin vitro and some infectious cultures are maintained and their investigation goes on. We suppose that kinetin plays a very important role for infectivity preservation in callus cultures.     

Effect of Explant Source on Axillary Shoot Multiplication During Micropropagation of a Rare Medicinal Plant — Wattakaka volubilis (L f) Stapf

Nodal explants of in vivo plants and in vitro seedlings of Wattakaka volubilis were cultured on Murashige and Skoog medium fortified with various concentrations of cytokinins — BA (0.5–5 mg l^?1), KN (0.5–10 mg l^?1),TDZ (0.05–1 mg l^?1) either singly or in combination with NAA (0.1 mg l^?1). KN proved best for inducing healthy shoots in both in vitro and in vivo derived explants. Maximum number of shoots (14.1±0.84) with 80% regeneration frequency was obtained from nodal explants of seedlings cultured on 5 mg 1^?1 KN + 0.1 mg l^?1 NAA. In vivo nodal explants produced a maximum of 4.2 shoots on MS medium fortified with 2 mg l^?1 BA+0.1 mg l^?1 NAA. The differentiated shoots from both could be rooted with 85% frequency on 1/2 strength MS medium (1% sucrose) with 0.6% agar + 1 mg l^?1 IBA + 0.2 mg l^?1 KN. Rooted shoots were transplanted to vermiculite-soil (3:1) mixture in polyethylene covered pots with 45% transplantation success. Peroxidase isozymes (native PAGE) analysis helped to verify the variation in regenerated plants.     

Glycosis in the eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas. Rate-limiting steps and activation at fertilization.

The content of glycolytic intermediates and of adenine nucleotides was measured in eggs of the echiuroid, Urechis unicinctus and the oyster, Crassostrea gigas, before and after fertilization. On the whole, the profile of the change in each glycolytic intermediate in Urechis eggs upon fertilization was found to be essentially similar to that in oyster eggs. Calculation of the mass action ratio for each glycolytic step from the amounts of glycolytic intermediates determined suggests that there are at least three limiting enzymes in the glycolysis system in unfertilized and fertilized eggs of each species examined. Phosphorylase (EC 2.4.1.1), phosphofructokinase (EC 2.7.1.11), and pyruvate kinase (EC 2.7.1.40) may be rate-limiting enzymes for the glycolysis system in Urechis eggs as well as in oyster eggs. These enzymes are thought to be activated upon fertilization, though even the reactions of the enzymes in fertilized eggs do not reach a state of equilibrium. In eggs of Urechis and oyster, phosphorylase is the first enzyme to be activated following fertilization. In Urechis eggs, pyruvate kinase is activated after the instant increase in the phosphorylase activity upon fertilization, followed by phosphofructokinase activation. In oyster eggs, however, pyruvate kinase and phosphofructokinase seem to be stimulated simultaneously, subsequent to phosphorylase activation upon fertilization. The mechanism controlling phosphorylase and pyruvate kinase activity is unknown, but the phosphofructokinase activity in both species may be regulated by the intracellular concentration of adenine nucleotides, since the enzyme activity is enhanced along with a decline in the phosphate potential in the eggs of both Urechis and of oyster.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Renierol from marine sponge Haliclona.SP.: A natural inhibitor of xanthine oxidase with hypouricemic effects

The purpose of this study was to evaluate the inhibitory effect of renierol, extracted from marine sponge Halicdona.SP., on xanthine oxidase (XO) and its hypouricemic effect in vivo. Renierol and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid and superoxide radical from xanthine. Renierol inhibited XO in a concentration-dependent and competitive manner. IC₅₀ value was 1.85 μg·ml^{? 1} through the measuring of uric acid and was 1.36 μg.ml^{? 1} through the measuring of superoxide radical. Renierol was found to have an in vivo hypouricemic activity against potassium oxonate-induced hyperuricaemia in mice. After oral administration of renierol at doses of 10, 20 and 30 mg.kg^{? 1}, there was a significant decrease in the serum urate level (4.08 ± 0.09 mg.dl^{? 1}, P < 0.01), (3.47 ± 0.11 mg.dl^{? 1}, P < 0.01) and (3.12 ± 0.08 mg.dl^{? 1}, P < 0.01), when compared to the hyperuricaemic control (6.74 ± 0.23 mg.dl^{? 1}). Renierol was a potent XO inhibitor with hypouricemic activity in mice.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Production of α-Amylase by the Ruminal Anaerobic Fungus Neocallimastix frontalis

α-Amylase production was examined in the ruminal anaerobic fungus Neocallimastix frontalis. The enzyme was released mainly into the culture fluid and had temperature and pH optima of 55°C and 5.5, respectively, and the apparent K_m for starch was 0.8 mg ml⁻¹. The products of α-amylase action were mainly maltotriose, maltotetraose, and longer-chain oligosaccharides. No activity of the enzyme was observed towards these compounds or pullulan, but activity on amylose was similar to starch. Evidence for the endo action of α-amylase was also obtained from experiments which showed that the reduction in iodine-staining capacity and release in reducing power by action on amylose was similar to that for commercial α-amylase. Activities of α-amylase up to 4.4 U ml⁻¹ (1 U represents 1 μmol of glucose equivalents released per min) were obtained for cultures grown on 2.5 mg of starch ml⁻¹ in shaken cultures. No growth occurred in unshaken cultures. With elevated concentrations of starch (>2.5 mg ml⁻¹), α-amylase production declined and glucose accumulated in the cultures. Addition of glucose to cultures grown on low levels of starch, in which little glucose accumulated, suppressed α-amylase production, and in bisubstrate growth studies, active production of the enzyme only occurred during growth on starch after glucose had been preferentially utilized. When cellulose, cellobiose, glucose, xylan, and xylose were tested as growth substrates for the production of α-amylase (initial concentration, 2.5 mg ml⁻¹), they were found to be less effective than starch, but maltose was almost as effective. The fungal α-amylase was found to be stable at 60°C in the presence of low concentrations of starch (≤5%), suggesting that it may be suitable for industrial application.     

Optimization of the composition of the nutrient medium for cellulase and protein biosynthesis by thermophilic Aspergillus fumigatus NRC 272

An active strain of Aspergillus spp. has been selected for the production of cellulolytic enzymes and proteins when grown on peracetic acid-treated wheat straw. This strain produced a considerable amount of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] in the extracellular supernatant and exhibited good overall cellulolytic activity, as measured using filter paper and Avicel as substrates. Also, under the same conditions the strain showed high activities of β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37). The maximum enzyme yields (carboxymethylcellulose activity 26.4 units ml^?1, filter paper activity 2.26 units ml^?1 and Avicel activity 16.82 units ml^?1; β-d-glucosidase 9.09 units ml^?1 and β-d-xylosidase 1.92 units ml^?1) were obtained after 96 h incubation at 45°C.     

Development of Bed Reactor Using Brick Dust Immobilized CIVI-Cellulase from Seeds of Cowpea (Vigna sinensis L)

Cellulase extracted from seeds of Cowpea (Vigna sinensis L var VITA-4) was partially purified and immobilized on brick dust as solid support via glutaraldehyde. The percentage retention of the enzyme activity on brick dust was nearly 85%. After immobilization specific activity of the enzyme increased from 0.275 to 0.557 U mg^?1 protein with about 2 fold enrichment. The optimum pH and temperature of soluble enzyme were determined as pH 4.6 and WC, respectively whereas immobilized enzyme showed at pH 5.0 and 37°C, respectively. The V_max values for soluble and immobilized enzyme were determined as 6.67 and 1.25 mg min^?1, respectively whereas K_m values were 4.35 and 4.76 mg ml^?1, respectively. The immobilized enzyme displayed higher thermal stability than soluble enzyme and retained about 50% of its initial activity after 12 reuses. Immobilized enzyme was packed in an indigenously designed double walled glass bed reactor for continuous production of reducing sugars.     

Interaction of muscle glycogen phosphorylase with pyridoxal 5'-methylenephosphonate

Rabbit muscle glycogen phosphorylase (EC 2.4.1.1) was reconstituted with pyridoxal 5′-methylenephosphonate with ca. 25% restoration of enzymatic activity. The modified enzyme has very similar chemical and physical properties to native phosphorylase including UV and fluorescence spectra, quaternary structure, high energy of activation in the reconstitution reaction, optimum pH and susceptibility to phosphorylase kinase in the b to a conversion. While V_max is reduced to ca. one-fifth, affinities for the substrate glucose 1-P and the effector AMP are increased. This is the first analog of pyridoxal 5′-P modified in the 5′-position found to restore catalytic activity to apophosphorylase.     

Control of platelet glycogenolysis activation of phosporylase kinase by calcium

An apparent enigma during platelet aggregation is that increased glycogenolysis occurs despite a fall in cyclic AMP levels. Activation by a classical cascade is therefore unlikely, and an alternative stimulus for phosphorylase a formation was sought. It was found that low levels of Ca²⁺ markedly activate phosphorylase b kinase from human platelets, with a K_a of 0.89 μM Ca²⁺, which is similar to that for the skeletal muscle enzyme. The kinase activity is unstable, and on enzyme ageing there is a 50% loss in activity with the K_a decreasing to 0.33 μM Ca²⁺.In unstimulated platelets, phosphorylase a was 13.3% of total measured activity, and glycogen synthetase I was 32.3%. Aggregation induced by ADP did not change the percentage of I synthetase, while increasing that for phosphorylase a. Dibutyryl cyclic AMP did, as expected, increase the percentage of both phosphorylated enzymes.These findings suggest that the natural activator of platelet glycogenolysis during aggregation is Ca²⁺, which directly stimulates phosphorylase b kinase without altering glycogen synthetase activity. The cyclic AMP-dependent protein kinase does not appear to be involved.     

Enzymatic removal of a cephalosporin methyl ester blocking group

Over 7000 microorganisms were screened to find an enzyme source for the hydrolysis of a C₄ 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 NH₃ 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 K₁ 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.     

Carbohydrate metabolism in Manduca sexta during late larval development

The carbohydrate metabolism in Manduca sexta underwent significant changes during late larval development. Approximately 10% of fat body glycogen phosphorylase was active during the feeding period of the 5th instar, pharate-pupal development and after the pupal moult; it is concluded that glycogen synthesis prevailed. During the last larval and the pupal moult, as well as the wandering stage the percentage of active phosphorylase was significantly increased indicating that fat body glycogen stores were broken down to supply substrates to meet the demands of carbohydrate metabolism. In the course of the last larval moult and the wandering stage the fat body glycogen content decreased significantly from about 300 to about 200 μg mg⁻¹ dry mass substantiating that carbohydrates were released from the fat body. Prior to phosphorylase activation, the concentrations of total haemolymph sugars decreased significantly from about 12 to about 6 mg trehalose equivalents ml⁻¹ (last larval moult) and from about 18 to about 12 mg ml⁻¹ (wandering stage), and increased again slightly when phosphorylase was activated. The haemolymph glucose concentration decreased significantly from about 1.1 to 0.3 mg ml⁻¹ (last larval moult) and in the course of the 5th-instar feeding period from about 1.1 to 0.2 mg ml⁻¹, and remained at this level until the beginning of adult development. The amount of chitosan present in the cuticle increased steadily during the feeding period of the 5th instar from about 10 to 110 mg. It appears that fat body glycogen might be broken down during the last larval moult and the wandering period to provide substrates for chitin synthesis. A dramatic decrease in the amount of chitosan was observed prior to the pupal moult.