Regulation of Cellobiase Secretion in Termitomyces clypeatus by Co-Aggregation with Sucrase

Regulated secretory proteins are sorted via selective co-aggregation in eukaryotes. Cellobiase (C) of the filamentous fungus Termitomyces clypeatus remained co-aggregated with sucrase (S), and only one isoform of each of the enzymes was present in intra- and extracellular extracts. Kinetics of secretion of sucrase increased in vivo and in vitro in secreting (Sc) medium and decreased under non-secreting (NSc) conditions similar to those observed for cellobiase. In the Sc condition, total enzyme production and activity ratios of cellobiase and sucrase (C/S) in cell-bound, extra- and intracellular preparations increased with time and were significantly higher from those obtained in non-secretory media. It was concluded that secretion of sucrase in culture medium is under same cellular regulation as that of cellobiase, and sucrase is involved in regulating extracellular release of cellobiase through co-aggregation in the fungus

In vitro probiotic characterization of Lactobacillus casei isolated from marine samples

The present study evaluated probiotic potentials of marine bacteriocinogenic lactic acid bacteria (LAB).Three marine LAB isolates, designated SB71, SB73 and SB93, were characterized as Lactobacillus casei.They possessed features like high level of gastrointestinal survival, inability to form biogenic amines,adherence to Caco-2 cells and marked cholesterol assimilation. To the best of our knowledge, this is the first report about marine probiotic bacteria from the Indian subcontinent. Tolerance of the three isolates to NaCl, bile and low pH was noteworthy. EPS from SB93 markedly disrupted bacterial adherence and promoted remediation of cadmium and lead. Broad-spectrum antimicrobial and antiadhesive activities were exhibited by the SB93 bacteriocin and the active principle survived autoclaving. All three bacteriocins exhibited antimicrobial activity against Vibrio cholerae as opposed to previous results. Earlier reports mentioned that the bacteriocinogenic isolates were cultivated for 24 h following which antimicrobial action of bacteriocins (in cell-free culture supernatants) was noted. However, in the present study, the test LAB isolates were cultivated for a period of up to 5 days and bacteriocins (in cell-free supernatants collected daily from the cultures) continued to exhibit antimicrobial activity, thus advocating for their potential lasting effect in diseased individuals

Mustard Stalk and Straw: A New Source for Production of Lignocellulolytic Enzymes by the Fungus Termitomyces Clypeatus and as a Substrate for Saccharification

Agro residue of mustard obtained as mustard stalk and straw (MSS) was investigated for the first time for production of lignocellulolytic enzymes by Termitomyces clypeatus and also for use as substrate for saccharification. MSS with high cellulose and hemicellulose content was utilized as sole source of carbon by the fungus for productions of enzymes such as (CMcase, �-glucosidase, xylanase and �-xylosidase) in submerged fermentation. Production of enzymes were further increased by 2–10 folds on supplementation with common agro-residues such as wheat bran and rice straw (MWR) in 1:1:1 ratio and by using alkali treated MSS (TMSS). The enzymes obtained from MWR and TMSS media could saccharify 10% (w/v) wheat bran up to 53% and 58% in 24 h, and xylan up to 52% and 81% in 12 h, respectively. MSS was used for saccharification by enzymes of T. clypeatus grown in cellulose media after pretreatment with hot water and NaCl respectively, where extent of saccharification was doubled to 80% by salt treatment as compared to that with hot water. The results indicated that MSS can be used as a potential and cheap renewable raw material from India for production of bio-ethano

Increased Enzyme Secretion by 2-Deoxy-D-Glucose in Presence of Succinate by Suppression of Metabolic Enzymes in Termitomyces Clypeatus.

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and nonsecreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-D-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-D-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-D-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-D-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value

Interference of Sugars in the Coomassie Blue G dye Binding Assay of Proteins

The presence of sugars causes significant deviation from the actual absorbance of proteins in the Bradford protein assay. In these studies, polysaccharides and disaccharides at milligram levels mimicked proteins in microgram equivalents. Monosaccharides, which individually did not show any absorbance, interfered significantly by sequestering the dye species. The studies demonstrated that in a mixture of sugars and proteins, sugar interference was much higher than expected from sugar molecules’ individual contribution. Estimated protein values were increased 2 to 4 times after precipitation from fungal culture broths. Thus, in carbohydrate-rich samples, protein concentrations should be ascertained by precipitation from crude extracts and resolubilization in a noninterfering buffe

Enhanced Activity and Stability of Cellobiase (b-Glucosidase: EC 3.2.1.21) Produced in the Presence of 2-deoxy-D-glucose from the Fungus Termitomyces Clypeatus

Generally less glycosylation or deglycosylation has a detrimental effect on enzyme activity and stability. Increased production and secretion of cellobiase was earlier obtained in the presence of the glycosylation inhibitor 2-deoxy-D-glucose in filamentous fungus Termitomyces clypeatus [Mukherjee, S.; Chowdhury, S.; Ghorai, S.; Pal, S.; Khowala, S. Biotechnol. Lett. 2006, 28, 1773–1778]. In this study the enzyme was purified from the culture medium by ultrafiltration and gel-permeation, ion-exchange and high-performance liquid chromatography, and its catalytic activity was six times higher compared to the control enzyme. Km and Vmax of the purified enzyme were measured as 0.187 mM and 0.018 U mg�1, respectively, using pNPG as the substrate. The enzyme had temperature and pH optima at 45 �C and pH 5.4, respectively, and retained full activity in a pH range of 5–8 and temperatures of 30–60 �C. Interestingly less glycosylated cellobiase was resistant towards proteolytic as well as endoglycosidase-H digestion and showed higher stability than native enzyme due to increased aggregation of the protein. The enzyme also showed higher specific activity in the presence of cellobiose and pNPG and less susceptibility towards salts and different chemical agents. The b-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries

In situ Reversible Aggregation of Extracellular Cellobiase in the Filamentous Fungus Termitomyces clypeatus

Cellobiase (E.C. 3.2.1.21), is a widely exploited industrial glycosidase with a major role in biofuel industry. Its stability and shelf life are major bottlenecks in achieving a superior formulation for industry. In the filamentous fungus Termitomyces clypeatus, the enzyme is secreted in a co-aggregated form with sucrase; the separation of this co-aggregation results in substantial loss of the enzyme’s activity. The aim of the present study was to examine the mode of aggregation of the secreted cellobiase-sucrase coaggregate and its role in the stabilization of cellobiase. Transmission electron microscopy and dynamic light scattering of purified co-aggregates revealed reversible, concentration driven self-aggregation of the extracellular enzymes to form larger entities. However, the intracellular enzyme aggregates were rigid, non-interacting, and possessed a higher percentage of disulphide bonds. Circular dichroic spectra of the two coaggregates indicated no significant difference in secondary structures. Self-association increased the stability of extracellular aggregates towards heat by 1.5 fold, SDS by 4 ~ 7 fold, and chaotropic agents, by 1.5 ~ 2 fold, than the intracellular counterpart. The Km of extracellular aggregate varied between 0.29 and 0.45 mM as a result of spontaneous aggregation and disaggregation, whereas that of intracellular aggregate was 0.22 mM irrespective of its concentration status. In situ detection of cellobiase in native PAGE revealed two activity bands of the extracellular enzyme, which indicated a minimum of two active dissociated aggregate species, as compared to a single band for the intracellular enzyme. These studies are believed to improve the understanding of aggregation of the fungal glycosidases, which remains to be a blackbox, to increase the efficacy of these enzyme

Characterization of a Novel Low Molecular Weight Sucrase From Filamentous Fungus Termitomyces Clypeatus

An extracellular sucrase from the culture filtrate of filamentous basidiomycota Termitomyces clypeatus grown on high sucrose (5%, w/v) was purified by gel filtration chromatography, ion exchange chromatography and HPGPLC. The biochemical properties, molecular weight and conformation of sucrase produced were significantly different from the sucrase earlier purified from sucrose (1%, w/v) mediumin the fungus. Purified sucrase was characterized as a low molecular weight protein of 13.5 kDa as approximated by SDS-PAGE and HPGPLC and exhibited predominantly random coil conformation in far-UV CD spectra. The enzyme was optimally active at 47 8C and pH 5.0. Km and catalytic activity of the enzyme for sucrose were found to be 3.5 mM and 1.06 U/mg/mM, respectively. The enzyme was maximally active towards sucrose than to raffinose and sucrase activity was significantly inhibited by bivalent metal ions and reducing group agents. The results indicated that due to changes in aggregation pattern, molecular organization of purified sucrase, produced in high sucrose medium, was altered and was different from the previously reported enzyme. This is the first report of a sucrase of such low size showing activity

Comparative elucidation of properties of sucrase-cellobiase co-aggregate produced in media containing sucrose by <i style="">Termitomyces clypeatus</i>

468-479Production profiles and characterization of the sucrase-cellobiase (S-C) co-aggregates from the filamentous fungus Termitomyces clypeatus were compared in media containing 1% and 5% sucrose to understand the effect of cellular regulation over secretion and aggregation of these two industrially important glycosidases. The enzymes were secreted constitutively but in a high sucrose medium (5%), cellobiase secretion was reduced to a basal level of 0.03 U/mL. In intracellular, cell-bound and extracellular milieus, S/C ratios gradually declined in a predictable trend indicating participation of more cellobiase subunits for secretion. Sucrase was secreted via vacuoles in the fungus, following the same route as that of cellobiase and thus co-aggregates of S-C were present in the vacuolar fraction. The extracellular co-aggregates showed similar molecular sizes (>550 kDa) on zymography; however, SDS-PAGE revealed substantial difference in their subunit assemblies. Sucrase from the 5% medium showed a 2.6 times lower Km than 1% medium. These observations demonstrated the formation of a unique S-C co-assembly, optimally suited to its needs and accommodation of the constituent subunits, to be used for biotechnological applications

Cellobiase from Termitomyces clypeatus: activity and secretion in presence of glycosylation inhibitors

In presence of the glycosylation inhibitors, 2-deoxy-D-glucose (1 mg/ml), tunicamycin (30 lg/ml), 1-deoxynojirimycin (30 lg/ml) and D-glucono-d-lactone (1 mg/ml), total cellobiase activity, in the extracellular, intracellular and cell bound fractions, of the fungus Termitomyces clypeatus grown in 20 ml cellobiose medium (1%, w/v) increased by 50-, 1.8-, 2.4-, 1.3-fold, respectively, with respect to control medium (16.3 U). The inhibitors also stimulated secretion of 95% of the total protein in culture medium, except D-glucono-d-lactone which released 60% of the total protein. 2-Deoxy-D-glucose (1 mg/ml) led to production of extracellular cellobiase up to 40 U/ml, whereas in absence of the inhibitors only 0.59 U/ml enzyme was detected