Kinetic Studies of β-Galactosidase Induction

The kinetics of β-galactosidase induction in E. coli ML 3 have been studied. Following addition of inducer, the rate of enzyme synthesis accelerates from the uninduced to a steady-state rate. At saturating concentration of inducer the time constant (T_c) for this process is 2.5 to 3 minutes. With decreasing inducer concentration (I), increasing time constants are observed. I/I + K′ approximates I/T_c. The steady-state rate of β-galactosidase synthesis is approximated by I²/I² + K². K′ and K have been estimated for IPTG and TMG. The kinetics of β-galactosidase production after the removal of inducer by dilution or after the addition of glucose have been investigated. A transition time of 2.5 to 3 minutes is observed before enzyme synthesis slows or stops. These results are consistent with the hypothesis that the enzyme-forming unit is unstable.     

Induction and glucose repression of endo-β-xylanase in the yeast Trichosporon cutaneum SL409

Extracellular endo-1,4-β-xylanase (EC 3.2.1.8) synthesis in the yeast Trichosporon cutaneum SL409 is inducible. The enzyme can be induced in washed glucose-grown cells by xylan or xylose. Methyl β-d-xylopyranoside, a synthetic analogue of xylobiose, however, was not an inducer of xylanase in Trichosporon cutaneum SL409. The induction of xylanase by xylan lasted longer and the final total activities were significantly higher compared to the induction by xylose. Xylanase induction was subject to glucose repression.     

Inactivation of prophage lambda repressor in vivo.

Jacob &; Monod (1961) postulated that prophage A induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in vitro (Roberts et al., 1978), we wanted to determine the action of the “cellular inducer” in vivo. Our results have led to a new model, which defines the relationship between the “cellular inducer” and the recA gene product.In order to quantitate the action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet light irradiation, thymine deprivation and temperature shift-up of tif-1 mutants.The kinetics of repressor decay in wild-type monolysogens indicate that repressor inactivation is a relatively slow cellular process that takes a generation time to reach completion. Incomplete inactivation of the repressor without subsequent prophage development may occur in a cell. We call this phenomenon detected at the biochemical level “subinduction”. In hyperimmune lysogens. subinduction is always the case.A high cellular level of A repressor that prevents prophage λ induction does not prevent induction of a heteroimmune prophage such as 434 or 80. Although the cellular inducer does not seem specific for any inducible prophage, it does not inactivate two prophage repressors present in a cell in a random manner. We have called this finding “preferential repressor inactivation”. Preferential repressor inactivation may be accounted for by considering that the intracellular concentration of a repressor determines its susceptibility to the action of the inducer.In bacteria with varying repressor levels, a fixed amount of repressor molecules is inactivated per unit of time irrespective of the initial repressor concentration. The rate of repressor inactivation depends on the catalytic capacity of the cellular inducer that behaves as a saturated enzyme. In wild-type bacteria the cellular inducer seems to be produced in a limited amount, to have a weak catalytic capacity and a relatively short half-life. The amount of the inducer formed after tif-1 expression is increased in STS bacteria overproducing a tif-1-modified RecA protein. This result is an indication that a modified form of the RecA protein causes repressor inactivation in vivo.From the results obtained we propose a model concerning the formation of the cellular inducer. We postulate that the cellular inducer is formed in a two-step reaction. The is model visualises how the RecA protein can be induced to high cellular concentrations, even though the RecAp protease molecules remain at a low concentration. The latter accounts for the limited proteolytic activity found in vivo.     

Regulation of isopropylmalate isomerase synthesis in Neurospora crassa.

The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate.     

Effect of aromatic compounds on the production of laccase and manganese peroxidase by white-rot basidiomycetes

Three white-rot fungi displayed a wide diversity in their response to supplemented aromatic compounds. Pyrogallol stimulated Cerrena unicolor laccase and manganese peroxidase (MnP) synthesis in synthetic medium 2.5- and 2-fold, respectively, whereas 2,4,6-trinitrotoluene (TNT) brought about a 2.8-fold increase in laccase yield by Trametes versicolor in submerged fermentation of ethanol production residue. No effect of the tested aromatic compounds on enzyme secretion by Ganoderma lucidum in mannitol-containing medium was detected. Nevertheless, G. lucidum is a potent producer of laccase in submerged fermentation of wheat bran and enzyme synthesis can be further increased by supplementation of medium with an appropriate inducer. The structure and the concentration of aromatic compounds play an important role in the regulation of enzyme synthesis. The supplementation of synthetic medium with 0.03–0.3 mM TNT or hydroquinone increased the differential rate of laccase synthesis by C. unicolor from 1,267 to 3,125–8,630 U mg biomass^?1 day^?1. Moreover, the same aromatic compound may function as either an inducer or a repressor, depending on the fungus and enzyme studied. Thus, hydroquinone increased 3-fold T. versicolor laccase activity decreasing 2- and 8-fold the yields of MnP and endoglucanase, respectively.     

Induction of Lipomyces starkeyi Dextranase

Lipomyces starkeyi ATCC 20825 is a derepressed mutant derived from L. starkeyi ATCC 12659. It requires the presence of an inducer before it produces dextranase. This study was undertaken to determine the most efficient, commercially feasible method for inducing this enzyme. The following compounds induced dextranase synthesis: 1-O-β-methyl-glucopyranoside, 1-O-α-methyl-glucopyranoside, dextran, isomaltopentose, isomaltotetraose, isomaltotriose, and isomaltose. 1-O-β-Methyl-glucopyranoside was found to be a gratuitous inducer. Early in the growth phase, cells produced higher specific levels of enzyme than they did in late log phase. The length of exposure of the yeast cells to the inducer also affected the amount of dextranase produced. The maximum amount of enzyme was produced after 12 h of exposure to the inducer. The saturation concentration was the same for all inducers tested, i.e., approximately 1 mg of inducer for every 2 × 10⁸ cells.     

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels

Chromatography of extracts of maize on diethylaminoethyl-cellulose resolves starch synthase activity into two fractions (Ozbun, Hawker, Preiss 1971 Plant Physiol 48: 785-769). Only starch synthase I is capable of synthesis in the absence of added primer and the presence of 0.5 molar citrate. This enzyme fraction has been purified about 1,000-fold from maize kernels homozygous for the endosperm mutant amylose-extender (ae). Because ae endosperm lacks the starch-branching enzyme which normally purifies with starch synthase I, the final enzyme fraction was free of detectable branching enzyme activity. This allowed a detailed characterization of the citrate-stimulated reaction. The citrate-stimulated reaction was dependent upon citrate concentrations of greater than 0.1 molar. However, the reaction is not specific for citrate and malate also stimulated the reaction. Branching enzyme increased the velocity of the reaction about 4-fold but did not replace the requirement for citrate. Citrate reduced the K_m for the primers amylopectin and glycogen from 122 and 595 micrograms per milliliter, respectively, to 6 and 50 micrograms per milliliter, respectively. The enzyme was found to contain 1.7 milligrams of anhydroglucose units per enzyme unit. Thus reaction mixtures contained 1 to 5 micrograms (5 to 25 micrograms per milliliter) of endogenous primer. The citrate-stimulated reaction could be explained by an increased affinity for this endogenous primer. The starch synthase reaction in the absence of primer is dependent upon several factors including endogenous primer concentration, citrate concentration as well as branching enzyme concentration.     

Chitinase is an inducible enzyme in Beauveria bassiana

Sterilization of chitin by autoclaving or boiling causes release of d-glucosamine and N-acetylglucosamine from the macromolecule and these solubilized components actually function as the inducers for synthesis of chitinase. The insoluble macromolecule is not an inducer of chitinase since sterilization by dry heat or chloroform will not bring about release of the amino sugars or induction of the enzyme. Free glucosamine, N-acetylglucosamine, and chitobiose are all good inducers of chitinase. Most sustained synthesis of the enzyme occurs in an autoclaved chitin-salts medium.     

Half-of-the sites reactivity in the catalytic mechanism of yeast glyceraldehyde 3-phosphate dehydrogenase

Novick &; Weiner (1957) proposed a model in which induction of the lac operon with suboptimal concentrations of inducer generates a population containing both uninduced and fully induced cells. The latter arise as cells acquire the galactoside transport system, thus initiating an autocatalytic cycle of induction since this permease can transport an inducer for its own synthesis. Evidence in favor of this model has been obtained from direct measurements of the enzyme content of individual cells, using a fluorogenic assay sensitive to one molecule of β-d-galactosidase. Fully induced cells, at the predicted frequency, were found in suboptimally induced populations of wild type strains, and of a strain lacking thiogalactoside transacetylase, but not of a strain lacking galactoside permease. In the wild type, the frequency of cells with an enzyme content intermediate between uninduced and fully induced levels was greater than the frequency predicted for cells within the autocatalytic cycle of induction. According to the model, then, in some of these cells, induction of β-d-galactosidase has occurred without formation of the permease necessary to initiate accumulation of inducer.     

The specificity of induction of α-galactosidase from Saccharomyces carlsbergenensis

A number of sugars and derivatives have been tested for their ability to induce the synthesis of α-galactosidase from Saccharomyces carlbergensis. Besides galactose and the substrates of the enzyme melibiose, raffinose and stachyose, D-galacturonic acid, L-arabinose, D-tagatose, methyl-α-D-galactoside, lactose and isopropyl-β-D-thiogalactoside were able to act as inducers. Of these, metyl-α-D-galactoside, lactose, isopropyl-β-D-thiogalactoside and L-arabinose have been shown to be gratuitous inducers with which kinetic studies of induction have been carried out. Lactose was the most efficient inducer, giving a maximal differential rate of synthesis of the enzyme of 110 mU/10⁷ cells at a concentration of 190 mM, followed by L-arabinose (60 mU/10⁷ cells at a concentration of 180 mM, followed by L-arabinose (60 mU/10⁷ cells at 40 mM), isopropyl-β-D-thiogalactoside (43 mU/10⁷ cells at 60 mM) and metyl-α-D-galactoside (25 mU/10⁷ cells at 150 mM). The concentration of inducer required to obtain half-maximal induction was similar for lactose, L-arabinose and isopropyl-β-D-thiogalactoside and about 5-fold higher for methyl-α-D-galactoside. The property of the compounds to act as inducers was compared to their ability to interact with the enzyme and the results discussed in terms of the molecular structures which are recognized by the enzyme and by the induction machinery.     

Evolution of a regulated operon in the laboratory

The evolution of new metabolic functions is being studied in the laboratory using the EBG system of E. coli as a model system. It is demonstrated that the evolution of lactose utilization by lacZ deletion strains requires a series of structural and regulatory gene mutations. Two structural gene mutations act to increase the activity of ebg enzyme toward lactose, and to permit ebg enzyme to convert lactose into allolactose, an inducer of the lac operon. A regulatory mutation increases the sensitivity of the ebg repressor to lactose, and permits sufficient ebg enzyme activity for growth. The resulting fully evolved ebg operon regulates its own expression, and also regulates the synthesis of the lactose permease.     

FACTORS AFFECTING THE INTRACELLUALR SYNTHESIS OF KYNURENINE

Near the time of pupation, autofluorescent kynurenine globules appear in the cells in the anterior region of the fatbody of Drosophila melanogaster. It has been reported previously that kynurenine synthesis may be induced in an additional group of fat cells by feeding the precursor tryptophan to Drosophila larvae, and that this induction of kynurenine production viewed within the fat cells is correlated with an increase in tryptophan pyrrolase activity. In the present report, conditions are outlined which result in the appearance of kynurenine in all of the fat cells. The number of cells in the fatbody which contain kynurenine is influenced by the quantity of tryptophan included in the diet, as well as by the developmental stage at the time of treatment and the duration of the feeding period on the inducer. Physical barriers modifying permeability, such as the membranous layer noted surrounding the fatbody, may be a factor in the regulation of the time and nature of the cellular induction of kynurenine synthesis. Another factor to be considered is the possibility of interference with the availability of tryptophan as a substrate or inducer for this synthesis within the cell. It is suggested that the occurrence of pteridines in some of the fat cells may modify the response of these cells to produce kynurenine, since pteridines as electron acceptors can complex with tryptophan as an electron donor. Kynurenine may be produced in the fat cells under in vitro conditions when they are incubated with L-tryptophan, but kynurenine is not formed when fat cells are incubated with D-tryptophan. The in vitro studies further demonstrate that induction of kynurenine synthesis may occur in fat cells isolated from young larvae in contrast, to in vivo conditions in which inducer does not effect an earlier appearance of kynurenine in the larval fatbody.     

Optimization of some variables that affect the synthesis of laccase by Pleurotus ostreatus

The influence of initial pH, concentration of yeast extract, inducer, type of enzyme releaser and buffer system on the composition of a medium for laccase production by Pleurotus ostreatus DM-1513 was investigated. A 2⁵ full factorial experimental design was initially employed to evaluate the effects of these variables on the enzyme synthesis. Data analysis showed that low pH and high yeast extract concentration values, as well as the absence of both an inducer and a buffer system, had positive effects on the secreted enzyme levels, whereas the type of enzyme releaser did not have a significant effect. The highest levels of laccase activity (489–540?U/l) were obtained in optimization experiments using media with initial pH between 6.0 and 6.5 and yeast extract concentrations of 0–0.25%