Besides lignin, plant cell walls are made of polysaccharides: pectin, cellulose and hemicellulose. Most members of the Erwinia chrysanthemi group are secreting hydrolytic enzymes such as pectinases, proteases, cellulases and phospholipase (Starr and Chatterjee). Soft-rot disease and plant tissue maceration probably result from concerted activity of some or all of these enzymes.

The structural gene encoding the major endoglucanase (EGZ) of Erwinia chrysanthemi strain 3665 has been isolated by hybridization using a DNA fragment of E. chrysanthemi strain 3937 previously cloned in the λL47-1 vector and expressing an endoglucanase activity. Furthermore, genes homologous to celZ and celY of strain 3665 were also isolated and subcloned from strain 3937 into Escherichia coli. They were shown to encode enzymes similar to EGZ and EGY of strain 3665.

The structural gene coding for a new endo-beta-1,4-glucanase of Erwinia chrysanthemi strain 3665, previously identified in a cosmid library, was subcloned into pUC18. The gene is expressed from a 1.9 X 10(3)-base-pair insert and its direction of transcription was determined. The properties of the gene product purified from cell-free extracts of Escherichia coli have been studied. The purified protein has an endoglucanase activity but is significantly different from the major endoglucanase Z secreted by E. chrysanthemi strain 3665. The new enzyme was designated as endoglucanase Y and the related gene celY. In E. coli, most of the endoglucanase activity was found in the periplasmic space.

Erwinia chrysanthemi produced several pectate lyases (EC 4.2.2.2) and endocellulases (EC 3.2.1.4) which were largely secreted into the culture medium. Mutants deficient in the secretion mechanism for these enzymes were obtained by chemical and insertion mutagenesis. Further study of one such mutant revealed that both enzyme activities were retained simultaneously within the periplasmic space.

The extracellular carboxymethyl-cellulase of Erwinia chrysanthemi, strain 3665, had a marked tendency to form aggregates when concentration and/or storage time of culture supernatant were increased. In submitting an unconcentrated glycerol culture supernatant to ion exchange chromatography, one major endo-β-1,4-glucanase could be isolated with a high degree of purity and partially characterized. The molecular size was 45 kd. The pI was 4.3. The enzyme rapidly decreased the viscosity of carboxymethyl-cellulose with a slow increase in the reducing sugars produced. It displayed its highest activity towards carboxymethyl-cellulose at a pH between 6.2 and 7.5. It had a significant capacity to hydrolyze amorphous cellulose such as phosphoric acid-swollen cellulose. The major products of this degradation were cellobiose and cellotriose. It exhibited a very low activity on microcrystalline cellulose. Glucose and cellobiose did not affect significantly its activity against carboxymethyl-cellulose.

Erwinia chrysanthemi strain 3665 growing aerobically in a mineral salts medium containing various carbon sources constitutively secreted low levels of carboxymethyl-cellulase activity. Increased production of this activity was triggered by conditions which reduced the growth rate. The results obtained with continuous culture suggested that this production was controlled by a mechanism similar to catabolite repression. However, other factors might be implicated in the regulation of cellulase production.

A cell-bound β-glucosidase (β-d-glucoside glucohydrolase; EC 3.2.1.21) from Clostridium thermocellum was purified to apparent homogeneity. A molecular weight of about 43000 gel fluration of the native enzyme on Ultrogel AcA34. A constant ratio of aryl-β-glucosidase and cellcouse throughout purification, similar heat stabilities, pH profiles and sensitivity to different hiibitor and competitive inhibition of the aryl-β-gluosidase and the β suggest that the same enzyme accounts for the aryl-β-glucosidase and the cellobiase activities. However, the affinity for cellobiose was very much lower than for p-β-d-glucoside. The β-glucosidase had maximum rates at pH 6·0 to 6·5 for both activities. The enzyme was specific for substrates with the both activities. β-configuration. particulary and β-1,3 and β-1,2 linkages. The enzyme did not hydrolyse carboxymethylcellulose or cellulose, but hydrolysed cello-oligosaccharides. It was strongly inhibited by d-glucono-δ-lactone was sensitive to thiol reagents. When preparations of C. thermocellm cellulase were supplemented with purified β-glucosidase, glucose was the predominant product of cellulose and the rate of saccharification was increased.

A mechanism of initiation of glycogen biosynthesis in Escherichia coli has been previously postulated: In a first step, the glucosyl groups would be transferred into an acceptor protein from UDPglucose or ADPglucose by two glucosyl transferases, distinct from the glycogen synthase. In this work, the activity of transfer from UDPglucose into a methanol-insoluble fraction could not be found in the crude extracts of six independently isolated glycogen synthase-deficient mutants of E. coli K-12. Purified E. coli K-12 glycogen synthase was able to catalyze the unprimed reaction from ADPglucose and UDPglucose but at a very low rate; the rate with UDPglucose is 6–7% the rate observed with ADPglucose. With these two substrates, the unprimed reaction was strongly stimulated by the simultaneous presence of salts and branching enzyme. However the activity with UDPglucose increased rapidly at low concentrations of branching enzyme and was inhibited at physiological concentrations whereas the activity with ADPglucose reached a maximum only at these concentrations. Consequently, the relative activities found with ADPglucose and UDPglucose varied with the branching enzyme concentration. Transfer from UDPglucose was inhibited by low concentrations of ADPglucose and high concentrations of glycogen. These results suggest that the same enzyme, namely the glycogen synthase, catalyzes the unprimed transfer from ADPglucose and UDPglucose and that ADPglucose is probably the most important physiological donor in glycogen biosynthesis in E. coli.

A β-glucosidase was isolated from Clostridium thermocellum; the enzyme was localized in the periplasmic space.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.

The debranching enzyme (EC 3.2.1.-) from Escherichia coli K12 was purified 312-fold with a 21% yield, DEAE-cellulose and DEAE-Sephadex chromatography were used for purification. The preparation was homogeneous and showed only a single band of protein and activity upon polyacrylamide gel electrophoresis. The enzyme hydrolyzed 1,6-alpha-glucosidic linkages in phosphorylase and beta-amylase limit dextrins prepared from glycogen and amylopectin. Small branched oligosaccharides were also hydrolyzed. Amylopectin was also completely hydrolyzed but the enzyme showed only a very low activity with glycogen as the substrate. The enzyme cannot be classified as a pullulanase because it has practically no activity with pullulan. But it also differs from the bacterial isoamylases described in other studies because of its inability to hydrolyze glycogen. The optimal pH is about 5.6. The optimal growth conditions for the synthesis of the enzyme by E. coli were also examined in the present studies.

Conference Article| April 01 1975 A Debranching Enzyme in Escherichia coli* R. JEANNINGROS; R. JEANNINGROS 1Laboratoire de Chimie Bactérienne, C.N.R.S., 13274 Marseille Cedex 2, France Search for other works by this author on: This Site PubMed Google Scholar N. CREUZET; N. CREUZET 1Laboratoire de Chimie Bactérienne, C.N.R.S., 13274 Marseille Cedex 2, France Search for other works by this author on: This Site PubMed Google Scholar C. FRIXON; C. FRIXON 1Laboratoire de Chimie Bactérienne, C.N.R.S., 13274 Marseille Cedex 2, France Search for other works by this author on: This Site PubMed Google Scholar J. CATTANEO J. CATTANEO 1Laboratoire de Chimie Bactérienne, C.N.R.S., 13274 Marseille Cedex 2, France Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1975) 3 (2): 336–337. https://doi.org/10.1042/bst0030336 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation R. JEANNINGROS, N. CREUZET, C. FRIXON, J. CATTANEO; A Debranching Enzyme in Escherichia coli. Biochem Soc Trans 1 April 1975; 3 (2): 336–337. doi: https://doi.org/10.1042/bst0030336 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search © 1975 Biochemical Society1975 Article PDF first page preview Close Modal You do not currently have access to this content.

A particulate fraction of an E. coli mutant catalyses the transfert of glucose from ADP-glucose to glycogen but also to a methanol-insoluble product in the absence of primer. The last reaction requires the presence of albumin and either high concentrations of salts or a protein factor. This factor is present in the 158,000 x g supernatant of DF 2000 mutant and in the extracts of mutants lacking glycogen synthase.

A mutant of E. coli lacking both D -glucose-phosphate isomerase and D -glucose-6-phosphate dehydrogenase synthesizes glycogen when supplemented with D -glucose. The cells grown on D -gluconate, without D -glucose, contain all the enzymes for glycogen synthesis but are devoid of glucoside primer. However, this strain is able to synthesize glycogen rapidly and without lag when it is supplemented with D -glucose. This de novo synthesis does not seem to be limited by the energy charge of the cells.

The chapter presents the current views and concepts of regulation of enzyme activity and expression on bacterial glycogen synthesis. The structural genes of the glycogen biosynthetic enzymes of Escherichia coli and of Salmonella typhimurium are isolated and more information bearing on the genetic regulation of glycogen synthesis in E. coli is accumulated. Many bacterial species accumulate glycogen in either stationary phase or under limited growth conditions with excess carbon in the media. For several bacterial species (E. coli, Agrohactcrium tumcfacicws, Arthrohacter, Enterohacter aerogenes), it has been shown that the rate of glycogen accumulation in stationary phase increases when growth ceases due to depletion of a nutrient required for growth. The site of regulation of glycogen synthesis in bacteria is at the ADPglucose pyrophosphorylase step. Many of the physical and chemical properties of the bacterial ADPglucose pyrophosphorylases are summarized. Effects of cAMP and cAMP receptor protein and effects of ppGpp on the expression of glycogen biosynthetic genes are discussed.

Waste-based polyhydroxyalkanoate (PHA) production by bacterial enrichments generally follows a three step strategy in which first the wastewater is converted into a volatile fatty acid rich stream that is subsequently used as substrate in a selector and biopolymer production units. In this work, a bacterial community with high biopolymer production capacity was enriched using glycerol, a non-fermented substrate. The substrate versatility and PHA production capacity of this community was studied using glucose, lactate, acetate and xylitol as substrate. Except for xylitol, very high PHA producing capacities were obtained. The PHA accumulation was comparable or even higher than with glycerol as substrate. This is the first study that established a high PHA content (≈70 wt%) with glucose as substrate in a microbial enrichment culture. The results presented in this study support the development of replacing pure culture based PHA production by bacterial enrichment cultures. A process where mixtures of substrates can be easily handled and the acidification step can potentially be avoided is described.