Depolymerase Enzyme Research Articles

Protein organization on the PHA inclusion cytoplasmic boundary

Polyhydroxyalkanoate (PHA) cellular inclusions consist of polyesters, phospholipids, and proteins. Both the polymerase and the depolymerase enzymes are active components of the structure. Recently, proteins associated with these inclusions have been described in a number of bacterial species. In order to further clarify the structure and function of these proteins in relation to polymer inclusions, ultrastructural studies of isolated polymer inclusions were initiated. The surface boundary characteristics of polymer inclusions, produced by several genera of bacteria, two different Pseudomonas putida deletion mutants and by Escherichia coli recombinants, were examined. The recombinant E. coli carried either the PHB biosynthesis operon ( phaCAB) from Ralstonia eutropha alone, or both this operon and a gene encoding an inclusion surface protein of R. eutropha ( phaP). The results support two suggestions: (i) specific genes in the PHA gene cluster code for the proteins forming the surface boundary arrays which characterize the polymer inclusion; and (ii) transfer of such a gene would result in subcellular compartmentalization of accumulating polymer. Although the proteins appear to serve a similar function among different genera, nevertheless, the different surface proteins are encoded by a variety of non-homologous genetic sequences.

The bgl1 gene encoding extracellular beta-glucosidase from Trichoderma reesei is required for rapid induction of the cellulase complex.

We have used a targeted gene deletion event to remove the coding region for the bgl1 gene encoding an extracellular beta-glucosidase from the genome of the cellulolytic fungus Trichoderma reesei. The bgl1 null mutants were used to investigate the role of beta-glucosidase in the hydrolysis of cellulose and induction of the other cellulolytic enzyme components. In the absence of extracellular beta-glucosidase, growth of bgl1 null strains on several carbon sources was the same as that of the parent (as measured by mycelial dry weight). However, levels of extracellular protein and total endoglucanase production were seen to lag relative to those levels observed in the control strain. The mRNA levels of the CBHI, CBHII, EGI, and EGII cellulase genes (cbh1, cbh2, egl1 and egl3) showed a corresponding lag in induction, suggesting that the absence of extracellular beta-glucosidase has an effect on the co-ordinate regulation of the other cellulase genes at the level of transcription. The addition of a potent inducer of the cellulase complex (sophorose) resulted in normal rates of cellulase gene mRNA production and extracellular protein release. This indicates that the absence of beta-glucosidase is not affecting some intrinsic cellular ability to produce mRNA or secrete protein. These data suggest that a functional beta-glucosidase is at least partially responsible for the efficient induction of the depolymerase enzymes of the cellulase complex. The observation that the cellulase complex is induced, albeit after a lag, suggests that other enzymes are present that can substitute for the function of beta-glucosidase during induction.

Poly(β-hydroxybutyrate) stereoisomers: a model study of the effects of stereochemical and morphological variables on polymer biological degradability

Abstract : In this study, we have prepared poly(Beta-hydroxybutyrate), PHB, random stereocopolymers from beta-butyrolactone, BL, using a diethylzinc/water (1.0/0.6) catalyst system. (R)-and (S)-BL were synthesized in high enantiomeric purity (>98% ee(enantiomeric excess), and approximately 5% racemization occurred at the methine stereocenters upon polymerization. The PHB stereoisomers produced had R repeat unit compositions of 95, 90, 85, 81, 77, 67, and 50%. In addition, a 50% (R)-PHB stereoisomer with a predominantly syndiotactic repeat unit placement was prepared in our laboratory. The relative degradability of these PHB stereoisomers was studied with a PHB depolymerase enzyme isolated from Penicillium funiculosum. This enzyme has been shown to catalyze the hydrolysis of (R)-PHB, but does not show activity for the enantiomeric substrate (S)-PHB.

Use of a bacteriophage-encoded glycanase enzyme in the generation of lipopolysaccharide O side chain deficient mutants of Escherichia coli O9:K30 and Klebsiella O1:K20: role of O and K antigens in resistance to complement-mediated serum killing

Coliphage K30 lysates contain free and phage-associated forms of a bacteriophage-encoded capsule depolymerase (glycanase) enzyme, active against the serotype K30 capsular polysaccharide of Escherichia coli. The free glycanase has been purified to apparent homogeneity. The molecular weight of the enzyme was estimated at 450,000, and when heated in SDS at 100 degrees C, the enzyme dissociated into two subunits of 90,000 and 52,000. The glycanase enzyme was used as a reagent to reversibly degrade the capsular layers on cells of Escherichia coli O9:K30 and Klebsiella O1:K20. This treatment rendered these bacteria sensitive to their respective lipopolysaccharide-specific bacteriophages, coliphage O9-1 and Klebsiella phage O1-3. This novel approach facilitated isolation of lipopolysaccharide O antigen side chain deficient mutants which retained the ability to synthesize the capsule. The response of defined mutants, O+:K-, O-:K+, and O-:K-, to exposure to nonimmune rabbit serum was measured. Results showed that the primary barrier against complement-mediated serum killing in both Escherichia coli O9:K30 and Klebsiella O1:K20 was the O antigen side chains of the lipopolysaccharide molecules. In both strains, the capsule played no role in the determination of serum resistance.

Factors affecting the formation of polysaccharide depolymerase and glycoside hydrolyase enzymes by Butyrivibrio fibrisolvens NCDO 2249

Specific activities of hemicellulose‐degrading polysaccharide depolymerase and glycoside hydrolase enzymes were measured in batch and continuous cultures of Butyrivibrio fibrisolvens NCDO 2249 grown on cellobiose or a hemicellulosic carbohydrate. Enzyme activities were influenced by the growth substrate and by the rate and stage of growth of the micro‐organism. In cellobiose batch cultures specific activities were maximal as the growth rate declined and in the initial stages of the stationary phase. The growth substrate did not affect the range of glycoside hydrolases formed, although specific activities were substrate‐dependent, with activity increases (up to 200‐fold) occurring in enzymes essential for effective substrate utilization. Appreciable xylanase activity was present only in xylan‐grown cultures. The substrate effects were also evident in chemostat cultures. The activity response of the nine enzymes monitored to growth rate changes differed in that while the activity of some enzymes, including xylanase, declined at high dilution rates the activities of others were not growth rate‐dependent and were maintained over the range of dilution rates examined. Exocellular activities were detected only in spent media from cultures grown with a polymeric (hemicellulosic) carbohydrate.

Characterisation of coliphage K30, a bacteriophage specific for Escherichia coli capsular serotype K30

Coliphage K30, a bacteriophage specific for strains bearing the Escherichia coli serotype K30 capsular polysaccharide, produced plaques surrounded by extensive haloes, a characteristic of phage which produce capsule depolymerase (glycanase) enzymes. Klebsiella K20, a strain producing a capsular polysaccharide chemically identical to that of E. coli K30, was not lysed by coliphage K30, although the bacteriophage encoded glycanase enzyme did degrade the K20 polysaccharide. Morphologically, coliphage K30 belonged to Bradley group C. The coliphage K30 particle comprised 20 structural polypeptides which varied from 9.5–136 kDa and genomic DNA of 38.7 ± 1.0 kb.

Characterisation of coliphage K30, a bacteriophage specific forEscherichia colicapsular serotype K30

Coliphage K30, a bacteriophage specific for strains bearing the Escherichia coli serotype K30 capsular polysaccharide, produced plaques surrounded by extensive haloes, a characteristic of phage which produce capsule depolymerase (glycanase) enzymes. Klebsiella K20, a strain producing a capsular polysaccharide chemically identical to that of E. coli K30, was not lysed by coliphage K30, although the bacteriophage encoded glycanase enzyme did degrade the K20 polysaccharide. Morphologically, coliphage K30 belonged to Bradley group C. The coliphage K30 particle comprised 20 structural polypeptides which varied from 9.5–136 kDa and genomic DNA of 38.7 ± 1.0 kb.

Subcellular distribution of polysaccharide depolymerase and glycoside hydrolase enzymes in rumen ciliate protozoa

The distribution of polysaccharide depolymerase and glycoside (acid) hydrolase activity in nine genera of rumen entodiniomorphid and holotrich ciliate protozoa was examined by differential centrifugation. Sedimentable activity was detected in all of the protozoa examined and occurred principally in fractions that were prepared by centrifugation at 1000g for 10 min, 10,000g for 10 min, and 20,000g for 20 min (fractions F1, F2, and F3). Acid phosphatase was present in these subcellular fractions which contained membrane-bound vesicles 0.1–0.8 μm in size. The enzyme location profile of the subcellular fractions differed within the genera examined. The distribution of the enzyme activity in the subcellular fractions indicated the presence of distinct populations of hydrolase-containing organelles and other functional vesicles in the rumen ciliates.

Subcellular distribution of glycoside hydrolase and polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum

The distribution of 12 acid hydrolase and two polysaccharide depolymerase enzymes in the rumen entodiniomorphid ciliatePolyplastron multivesiculatum, isolated from the ovine rumen 2 h after feeding, was examined by differential and density-gradient centrifugation. Approximately 60%–70% of the recovered activity was sedimentable in fractions prepared by centrifugation at 103g for 10 min (F1) and 104g for 10 min (F2) with 25%–35% of the acid hydrolases and 15%–20% of acid phosphatase and the polysaccharidases remaining nonsedimentable (in fraction F5) after centrifugation at 105g for 60 min. Approximately 60% of the sedimentable activity was located in fraction F1. Latency of the hydrolase activity was demonstrated. After isopycnic centrifugation in sucrose density gradients, the hydrolytic enzymes cosedimented in acid phosphatase-containing, membrane-bound, pleomorphic lysosomelike vesicles 0.1–1.0 μm in size, with a mean equilibrium density of 1.17 (1.15–1.19) g/ml.

Partial Purification and Characterization of a Polysaccharide Depolymerase Associated with Phage-Infected Erwinia amylovora

Erwinia amylovora infected with bacteriophage ERA103 produced an enzyme which degraded the extracellular polysaccharide of noninfected cells. The depolymerase enzyme was purified 15-fold by a procedure which included ammonium sulfate precipitation, ultracentrifugation, CM-Sephadex batchwise separation, Sephadex G-50 column chromatography, and Sephacryl S-200 column chromatography. The enzyme had a molecular weight of approximately 21,000 and a pH optimum of 6.0. Activity was enhanced by supplements of 2-mercaptoethanol or dithiothreitol.

Formation of polysaccharide depolymerase and glycoside hydrolase enzymes byBacteroides ruminicola subsp.ruminicola grown in batch and continuous culture

The activity of ten polysaccharide depolymerase and glycoside hydrolase enzymes was monitored inBacteroides ruminicola subsp.ruminicola throughout the growth cycle and over a range of dilution rates in carbon-limited continuous (chemostat) culture. The enzymes were principally cell associated, and the specific activities increased throughout the growth cycle to reach maximum levels in the late exponential and stationary growth phases. In chemostat-grown cells the activities were growth rate dependent and were highest at the lowest dilution rates examined (0.025/h), but remained constant over a wide range of growth rates (D=0.05–0.15/h). The specific activities were lower in cells with a generation time of 3 h (D=0.225/h). The major metabolites formed from xylose, in batch and continuous culture, were lactic, acetic, and succinic acids, with traces (∼1%–2% of total acid production) of branched and straight-chain C3−C5 volatile fatty acids. The proportions of the metabolites produced varied with the stage and rate of growth.

POLYSACCHARIDE DEGRADING ENZYMES IN MICROBIAL POPULATIONS FROM THE LIQUID AND SOLID FRACTIONS OF BOVINE RUMEN DIGESTA

Polysaccharide depolymerase and glycoside hydrolase enzymes involved in the degradation of plant structural polysaccharides were most active in the adherent particle-associated microorganisms, whereas soluble saccharides were metabolized by the liquid phase and nonadherent populations. The activities were constant confirming the stability of the populations. Key words: Polysaccharidase, glycosidase, particle-associated microorganisms

Mobilization of recently formed forest soil organic sulfur

Soils from a mixed mature hardwood forest were assayed for their capacity to mobilize sulfur which had been previously immobilized into a nonsalt-extractable (insoluble) form. These soils rapidly released soluble organic sulfur and inorganic sulfate from this fraction. It is suggested that the former component is a depolymerization product of a more complex organic sulfur matrix. The activity of preformed extracellular depolymerase and sulfohydrolase enzymes in the soil may be responsible for the depolymerization and subsequent desulfation of the organic sulfur matrix. This is supported by observations that treatment of soil samples with sodium sulfate, sodium azide, erythromycin, or candicidin failed to inhibit the capacity of A1-horizon soils to mobilize the organic sulfur fraction. The rates and final levels of sulfur mobilization increased with an increase in temperature and decreased with sample depth.

Interaction between Rhizobium japonicum phage M-1 and its receptor.

The receptor for phage M-1 was present in the exopolysaccharide (EPS) of Rhizobium japonicum D211. The EPS was a heteropolysaccharide consisting of glucose, galactose, glucuronic acid, and glucosamine units. These monosaccharides prevented phage-cell attachment indicating that they may mimick the receptor. Phage-cell attachment was either stimulated or inhibited by Mg2+ and Ca2+ depending upon their concentration. An enzyme which depolymerized the exopolysaccharide releasing oligosaccharides was detected in the phage-infected cell lysates. A comparison of the properties of adsorption and those of the depolymerase enzyme indicated that the latter was a component of the phage and appeared to be involved in the phage-receptor interaction.

Role of pectic and cellulolytic enzymes in the invasion of the soybean by Rhizobium japonicum.

Past workers have suggested pectic enzyme involvement in the invasion of legumes by Rhizobium. However, no role for pectic acid, pectin, or methyl cellulose depolymerase enzymes in the invasion of R. japonicum was suggested by the current study. Seedling inoculation with infective bacteria did not result in increased enzyme activity. Rhizobium japonicum cell-free culture extracts and 3-indoleacetic acid did not affect the activation, induction, or binding of these enzymes.

Desulfation of sulfated polyanions by rat gastric mucosa.

SummaryRat stomach mucosal scrapings contain enzymes which attack chondroitin 4-and 6-sulfates, heparain, sulfated polyanions from gastric and duodenal mucosa and aryl sulfates. Inorganic and organic sulfates were liberated from the sulfated polyanions. Fractionation with (NH4)2SO4 afforded enrichment of the sulfatase and depolymerase enzymes in the precipitate obtained at 60–72% saturation. Enzyme activities were stable for at least two months when stored in pH 4.0 acetate buffer under toluene at 5°.

Exopolysaccharide colanic acid and its occurrence in the Enterobacteriaceae.

A study of strains from the genera Salmonella, Escherichia, and Aerobacter has shown that under appropriate conditions many strains produce an exopolysaccharide slime of identical composition, which has been identified as colanic acid on the basis of its chemical composition and its sensitivity to certain bacteriophage-induced depolymerase enzymes. Chemical analysis shows that the polysaccharide contains O-acetyl groups in addition to the sugars glucose, galactose, fucose, and glucuronic acid. Mild acid hydrolysis has led to the isolation of a beta-glucosylfucose in addition to glucuronic acid containing oligosaccharides. Many strains were found to synthesize colanic acid under normal conditions of growth or under conditions favoring polysaccharide synthesis, whereas others only synthesized colanic acid when the control mechanism was derepressed by p-fluorophenylalanine.