Glucan 4-glucanohydrolase Research Articles

Application of Synergistic Phenomena for Enhanced Production of Alpha 1,4 D- Glucan Glucanohydrolase Using Submerged Fermentation.

The study revealed enhanced production of α 1, 4 D glucan glucanohydrolase utilizing the synergistic phenomena of bacterial hetero-culture. For this purpose, 101 hetero-cultures were screened qualitatively and quantitatively. The bacterial hetero-culture showing the highest amylolytic potential was identified as Bacillus subtilis and Bacillus amyloliquefeciens by 16S rDNA sequencing technique. Different fermentation media were evaluated and M 5 gave maximum GGH production. Different physicochemical parameters like incubation time, temperature, initial pH and inoculum size was optimized. The optimal enzyme production was obtained at 24 h, 37oC, pH 7.0 and 3% inoculum size. Glucose (3%), ammonium sulphate (1.5%) and yeast extract (2.0%) was selected as best carbon and nitrogen source, respectively. The novelty of the present piece of research was the application of the hetero-culture technique for enhanced GGH production using submerged fermentation which was not experienced before with these strains.

Polymeric amylase nanoparticles as a new semi-synthetic enzyme system for hydrolysis of starch

α-Amylase (EC 3.2.1.1; α-d-1,4,glucan glucanohydrolase) catalyzes the hydrolysis of α-d-(1,4)-glucosidic linkages in starch, glycogen, and various malto-oligosaccharides, by releasing α-anomeric products. In this study, a novel method has been developed to prepare nanoprotein particles that carry α-amylase as a monomer by using a photosensitive microemulsion polymerization process. The nanostructured α-amylase with photosensitive features have been characterized by fluorescence spectroscopy, transmission electron microscopy (TEM) and Zeta Sizer. The fluorescence intensity of amylase nanoparticles was determined to be 658a.u. at 610nm and the average particle size of nanoamylase was found to be about 71.8nm. Both free α-amylase and nanoparticles were used in the hydrolysis of starch under varying reaction conditions such as pH and temperature that affect enzyme activity and the results were compared to each other. Km values were 0.26 and 0.87mM and Vmax values were 0.36IUmg−1 and 22.32IUmg−1 for nanoenzyme and free enzyme, respectively. Then, thermal stability, storage stability and reusability were investigated and according to the results, activity was preserved 60% at 60°C; 20% at 70–80°C temperature values and 80% after 105days storage. Finally after 10cycles, the activity was preserved 90% and this novel enzymatic polymeric amylase nanoparticle has showed considerable potential as reusable catalyst.

ChemInform Abstract: Synthesis of Aryl 3-O-β-Cellobiosyl-β-D-glucopyranosides for Reactivity Studies of 1,3-1,4-β-Glucanases.

Abstract A series of substituted aryl β -glycosides derived from 3- O - β -cellobiosyl- d -glucopyranose with different phenol-leaving group abilities as measured by the pK a of the free phenol group upon enzymatic hydrolysis has been synthesised. Aryl β -glycosides with a pK a of the free phenol leaving group>5 were prepared by phase-transfer glycosidation of the per- O -acetylated α -glycosyl bromide with the corresponding phenol, whereas the 2,4-dinitrophenyl β -glycoside was obtained by condensation of 1-fluoro-2,4-dinitrobenzene with the partially acetylated trisaccharide followed by acid de- O -acetylation. The aryl β -glycosides have been used for reactivity studies of the wild-type Bacillus licheniformis 1,3-1,4- β - d -glucan 4-glucanohydrolase. The Hammett plot log k cat versus pK a is biphasic with an upward curvature at low pK a values suggesting a change in transition-state structure depending on the aglycon.

Fluorescence study on Interactions of α–Crystallin with the Molten Globule State of 1, 4–β–D–Glucan Glucanohydrolase from Thermomonospora sp. induced by guanidine hydrochloride

In this paper, the interaction between alpha- crystallin and molten globule structure of 1,4-beta-D-Glucan Glucohydrolase (TSC) from an alkalothermophilic Thermomonospora sp. was investigated mainly by fluorescence quenching spectra, circular dichroism and three dimensional fluorescence spectra under simulative physiological conditions. Denaturation studies using GdnCl indicated that TSC folds through a partially folded state that resembles molten globule at 1.8 M GdnCl. The chaperone activity of alpha- crystallin was employed to study refolding of TSC. Here we studied the refolding of GdnCl denatured TSC from its molten globule state (TSC-m complex) in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and ANS binding studies, suggest that alpha-crystallin formed a complex with a putative intermediate molten globule-like intermediate in the refolding pathway of TSC. Reconstitution of the active TSC was observed on cooling the alpha-crystallin * TSC-m complex to 4 degrees C. Addition of alpha-crystallin to the molten globule-like intermediate of TSC (TSC-m complex) complex initiated the refolding of TSC with 69% recovery of the biological activity of the enzyme.

Coexpression of rumen microbial β-glucanase and xylanase genes in Lactobacillus reuteri

The aim of this study was to clone and coexpress two rumen fibrolytic enzyme genes in Lactobacillus reuteri. The ability of the genetically modified strain to degrade beta-glucan and xylan was evaluated. The Fibrobacter succinogenes beta-glucanase (1,3-1,4-beta-D: -glucan 4-glucanohydrolase [EC 3.2.1.73]) gene and the Neocallimastix patriciarum xylanase gene, xynCDBFV, were constructed to coexpress and secrete under control of the Lactococcus lactis lacA promoter and its secretion signal and then transformed into L. reuteri Pg4, a strain isolated from the gastrointestinal tract of broiler chickens. The transformed L. reuteri strain acquired the capacity to break down soluble beta-glucan and xylan. The introduction of the recombinant plasmids and production of beta-glucanase and xylanase did not affect cell growth. To the best of our knowledge, this is the first report of coexpression of rumen microbial fibrolytic enzyme genes in L. reuteri.

Biochemical characterization of a novel β-1–3, 1–4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan

A bifunctional high molecular weight (Mr, 64,500 Da) β-1–3, 1–4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.

Purification and characterization of extracellular alpha-amylase from Aspergillus fumigatus

Extracellular alpha-amylase [ (1 → 4)-α- d- glucan glucanohydrolase, EC 3.2. 1.1] from Aspergillus fumigatus (Aspergillus sp. K-27) was purified to homogeneity by anion-exchange (DEAE-cellulose) and affinity ( α-cyclodextrin-Sepharose) chromatography. The purified enzyme, a glycoprotein with 15% carbohydrate content, showed an isoelectric point of 3.7, a molecular weight of 65,000 (as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and an amino acid composition with a high number of neutral hydrophobic residues. Alpha-amylase activity on α- d- glucans in solution was optimal at pH 5.5, and the enzyme was stable at 40°C. It hydrolyzed amylose and amylopectin, with respective K m of 0.42 and 7.7 mg mL −1 and k cat/ K m of 3.4 and 2.5 mL, mg −1 min −1. The major end-products of maltohexaose, degradation were glucose and maltose. Maltotriose, maltotetraose, and maltopentaose were formed as intermediate products with an α-anomeric configuration. Despite its ability to slowly degrade some α-(1 → 6) linkages, this purified enzyme should be classified as an alpha-amylase.

Stereochemical course and structure of the products of the enzymic action of endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis.

The stereochemical course of the reaction catalysed by endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) has been determined by 1H n.m.r. The enzyme-catalysed hydrolysis of barley beta-glucan proceeds with overall retention of the anomeric configuration, indicating that the enzyme operates through a double-displacement mechanism. The structures of the final oligosaccharide products, 3-beta-O-cellobiosyl D-glucopyranoside and 3-beta-O-cellotriosyl D-glucopyranoside, have been completely assigned by 1H- and 13C-n.m.r. spectroscopy.

Optimization studies for the production of α-amylase using cheese whey medium

Cheese whey has been utilized as a carbon source for α-amylase (EC 3.2.1.1; 1,4-α- d- glucan glucanohydrolase) production by Bacillus sp. The medium and culture conditions have been optimized for maximum enzyme yield. The optimum concentrations of the medium constituents were found to be 2% corn starch, 3% corn gluten, 0.1% MgSO 4 · 7H 2O, 0.1% KH 2PO 4, 0.2% NaCl, and 0.02% CaCl 2 in cheese whey. About 2,690 DUN ml −1 of α-amylase enzyme was obtained in a shake flask in 72 h from optimized cheese whey medium at pH 7.0. In a stirred-tank bioreactor, up to 4,360 DUN ml −1 of α-amylase was produced in 30 h from the optimized medium at a high aeration rate of 2.2 vvm.

Chromosomal mapping of the Glb 1 locus encoding (1 → 3),(1 → 4)-β- D-glucan 4-glucanohydrolase EI in barley

A study of (1 → 3),(1 → 4)-β- D -glucan 4-glucanohydrolase (EBG) variation in samples from natural populations of Hordeum spontaneum (the wild progenitor of barley), using isoelectric focusing and immunoblotting, had identified some additional variants for these enzymes. Using a variant for EBG enzyme El and appropriate backcross material the linkage relationships between the gene locus encoding this variant (Glb 1) and established isozyme loci have been established. Results show that the Glb 1 locus is located on barley chromosome 5 and is tightly linked to the malate dehydrogenase locus (Mdh 1) which occurs on the long arm of this chromosome.

Production of cellulase and detection of Avicel-adsorbing carboxymethylcellulase from a mesophilic fungus Humicola grisea Fb

Two mesophilic fungal strains, identified as Humicola grisea were isolated from forest soil. Strain Humicola grisea Fb digested a variety of lignocellulosic growth substrates. The amount of cellulase enzymes induced on processed cellulose was higher than on lignocellulose. This strain exhibited high carboxymethylcellulase [ endo-1,4-β- d- glucanase, ( 1,4-( 1,3:1,4)-β- d- glucan 4-glucano hydrolase, EC 3.2.1.4)], filter paper degrading activity, and Avicelase activity in cultures on cellulose powder, Avicel and filter paper. The culture filtrate of cellulose powder-grown H. grisea Fb hydrolysed filter paper to the extent of 8% and carboxymethylcellulose and Avicel to 33% and 6.5% in 3 h respectively. The carboxymethylcellulase activity in the culture filtrate ( 7.41 U ml −1 ) was adsorbed onto Avicel to the extent of 22%. The carboxymethylcellulase activity in the culture filtrate was optimally active at 50°C and pH 5.0.

Kinetic studies of amylase and biomass production by calvatia gigantea

Production of alpha-amylase (alpha-4, glucan 4-glucanohydrolase, EC 3.2.1.1) by microorganisms has been practiced for many years in small and large scale operations and the literature on this enzyme is voluminous. Aspergillus niger and Aspergillus oryzae have been reported as the main fungal species used for commercial production of the enzyme. On the other hand, large volumes of low-cost agricultural products such as acorn (the perisperm-free dry seed contains approximately 60% starch) are wasted in many countries and provide a challenge to biotechnology to efficiently utilize these rich sources of starch for the production of high added value products like enzymes. C. gigantea is an edible puffball excreting high levels of alpha-amylase when cultivated on different sources of starch containing elevated quantities of toxic tannic compounds. This fungus has been employed for the production of microbial protein from wastes and acorns containing high levels of toxic tannic compounds. The same fungus was also reported to grow on both hydrolyzable and condensed tannins as sole carbon sources. The present work was undertaken to investigate certain kinetic characteristics of alpha-amylase and biomass production by C. gigantea grown on soluble and acorn starch in a lab fermenter. (Refs. 18).

Enhancement of α-amylase production by immobilized Bacillus subtilis in an airlift fermenter

Bacillus subtillis ATCC 21770 was entrapped in a carrageenan gel, especially formulated for immobilization. Bacterial growth and α-amylase (1,4-α- d- glucan glucanohydrolase EC 3.2.1.1) production were tested. The bead suspensions were submitted to two aeration modes, one consisting of bubbling air into a round flask, the other involving sparging of air into an airlift fermenter. The latter system, which produces microbubbles, gave 40–70% increase in enzyme production over the former and a doubling of bacterial density within the beads was measured. The use of CaCl 2 instead of KCl as polymerization agent led to a better yield of α-amylase.

Production of β- d-glucosidase, endo-1,4- β- d-glucanase and d-xylanase from straw by Aspergillus fumigatus IMI 255091

Aspergillus fumigatus IMI 255091 has been found to grow well on ground straw as the principal substrate, Production of β - d -glucosidase ( β - d -glucoside glucohydrolase, EC 3.2.1.21), endo-1,4- β - d -glucanase [1,4-(1,3;1,4)- β - d -glucan 4-glucanohydrolase, EC 3.2.1.4] and d -xylanase (1,4- β - d -xylan xylanohydrolase, EC 3.2.1.8) was maximal within 7 days with 4% (w/v) straw. Comparisons were made with both a conventional agitated fermenter and an air-lift fermenter. The latter gave the highest yields. The β - d -glucosidase was partially purified by fractionation and its K m determined. This was found to differ from that of other β - d -glucosidases reported in the literature.

A strain of Saccharomyces cerevisiae which grows efficiently on starch

Saccharomyces cerevisiae BRG 530 is a meiotic segregant of S. cerevisiae NCYC 361 (syn. Saccharomyces diastaticus ). While NCYC 361 grew poorly on soluble starch, BRG 530 utilized 62% of starch for growth in batch culture, yielding 0.86 g biomass (g starch used) −1 . In comparison, Lipomyces kononenkoae CBS 2514, which produces α-amylase (1,4-α- d -glucan glucanohydrolase, EC 3.2.1.1) in addition to glucoamylase (1,4-α- d -glucan glucohydrolase, EC3.2.1.3) and possesses debranching activities, utilized starch more completely (92%). However, with CBS 2514, the specific growth yield was lower than with BRG 530 at 0.65 g biomass (g starch used) −1 , and only 15% of L. kononenkoae biomass was protein compared with 41% for the S. cerevisiae strain. Therefore, even with incomplete utilization of substrate, BRG 530 was more efficient in producing yeast protein from starch. More complete (96%) utilization of starch by BRG 530 was achieved by adding α-amylase; this has little effect on the efficiency of substrate conversion to biomass. The properties of BRG 530 suggest that a strain of S. cerevisiae could be developed for the production of yeast single-cell protein or other growth-associated products from starchy substrates.

A strain of Saccharomyces cerevisiae which grows efficiently on starch

Saccharomyces cerevisiae BRG 530 is a meiotic segregant of S. cerevisiae NCYC 361 (syn. Saccharomyces diastaticus ). While NCYC 361 grew poorly on soluble starch, BRG 530 utilized 62% of starch for growth in batch culture, yielding 0.86 g biomass (g starch used) −1 . In comparison, Lipomyces kononenkoae CBS 2514, which produces α-amylase (1,4-α- d -glucan glucanohydrolase, EC 3.2.1.1) in addition to glucoamylase (1,4-α- d -glucan glucohydrolase, EC3.2.1.3) and possesses debranching activities, utilized starch more completely (92%). However, with CBS 2514, the specific growth yield was lower than with BRG 530 at 0.65 g biomass (g starch used) −1 , and only 15% of L. kononenkoae biomass was protein compared with 41% for the S. cerevisiae strain. Therefore, even with incomplete utilization of substrate, BRG 530 was more efficient in producing yeast protein from starch. More complete (96%) utilization of starch by BRG 530 was achieved by adding α-amylase; this has little effect on the efficiency of substrate conversion to biomass. The properties of BRG 530 suggest that a strain of S. cerevisiae could be developed for the production of yeast single-cell protein or other growth-associated products from starchy substrates.

Use of cellulase antibodies to study leaf abscission

The primary leaves of the bean (Phaseolus vulgaris c.v. red kidney) are shed as the result of cell wall breakdown in the abscission zones found at the base of the lamina and at the stem–petiole junction. Horton and Osborne1 suggested that cellulase (B l : 4 glucan 4-glucanohydrolase) was involved in this wall hydrolysis because its activity increased considerably during abscission. Although this result has been repeated on a wide range of plant material2, critical scrutiny of the data has led several authors3,4 to question the direct involvement of the enzyme in wall weakening. The following objections have been raised: (1) cellulase may be present at relatively high levels before abscission is induced5, (2) although wall breakdown is confined to two or three layers of cells, cellulase is usually found over a wider area of the petiole, (3) the increase in cellulase which accompanies abscission may not take place until cell separation is essentially complete3, (4) cellulase may increase without concomitant breakdown of the abscission layer4. One possible explanation for these anomalies is that these assays failed to distinguish between the various isozymes of cellulase, of which only one is postulated to be involved in abscission6. Recently, the specific isozyme thought to participate in bean leaf abscission (9.5 cellulase) has been purified and monospecific rabbit antibodies raised against it7. These antibodies do not cross-react with the other form (s) of cellulase and thus can be used both to quantify changes in 9.5 cellulase during abscission and to determine cytochemically the distribution of the enzyme. Using these antibodies, we have now made observations consistent with the participation of cellulase in the wall hydrolysis which leads to fracture.

Reduction of the formation of dental plaque and gingivitis in humans by crude mutanase

The effect of a Trichoderma harzianum enzyme preparation containing mutanase (alpha-1,3 glucan glucanohydrolase) on plaque accumulation and composition and on occurrence of gingivitis was assessed in 20 persons in a double-blind cross-over investigation. The enzyme preparation was administered in chewing gum. Two test periods of 1 week were preceded by scaling and cleansing of the teeth, oral hygiene instruction, and controlled hygiene for at least 3 weeks. Oral hygiene measures were discontinued during the test periods, while the persons chewed six pieces of chewing gum per day, one half using enzyme-containing gum, and the other half using placebo gum. The test periods were identical, only enzyme gum was used instead of placebo, or vice versa. Evaluations of plaque and gingivitis showed that less plaque had accumulated and less gingivitis developed during the enzyme than during the placebo period, but bacteriologic studies of interproximal plaque did not reveal differences that could explain the clinical findings. Treatment with the enzyme preparation caused some local side effects, but no primary skin irritation, delayed hypersensitivity, nor anti-enzyme IgE was detected in any of the persons.

Determination of the carbohydrate content of porcine pancreatic amylase

Porcine pancreatic α-amylase (α-1,4 glucan 4-glucanohydrolase, EC 3.2.1.1) is shown to be a glycoprotein. Both molecular forms I and II of amylase contain 0.75 mole of fucose, 0.45 mole of galactose and 0.52 mole of mannose per mole of enzyme. Analyses of amino sugars indicate 1.2 and 0.8 residues of glucosamine bound per mole of amylase I and amylase II, respectively. A method for identification and quantitation of neutral sugars by gas chromatography of the trimethylsilyl derivatives of their corresponding alditols is proposed.

Some aspects of the specificity and action pattern of β-1,3-glucan glucanohydrolase from Spisula sachalinensis

1. 1.|The study of some aspects of the specificity and action pattern of laminarinases (β-1,3glucan glucanohydrolase, EC 3.2.1.6) from Spisula sachalinensis, which we had previously referred to as laminarinases III and IV (V. V. Sova et al., Biochim. Biophys. Acta, 212 (1970) 111) has been carried out. Both enzymes can probably be ascribed to endo- β-(1→3)-glucan glucanohydrolases. 2. 2.|Some data on homogeneous laminarinase IV have been reported.