Suman Khowala

Purification and characterization of a thermostable intra-cellular β-glucosidase with transglycosylation properties from filamentous fungus Termitomyces clypeatus

An intra-cellular beta-glucosidase was purified to homogeneity by gel filtration, ion exchange chromatography and HPGPLC from mycelial extract of Termitomyces clypeatus in the presence of the glycosylation inhibitor 2-deoxy-d-glucose. CD spectroscopy demonstrated that the purified enzyme exhibited alpha-helical conformation. MALDI-TOF identified the enzymes molecular weight as 6688Daltons, but SDS-PAGE and immunoblotting indicated that the enzyme formed aggregates. The enzyme also showed unique properties of co-aggregation with sucrase in the fungus. The enzyme showed around 80% stability up to 60 degrees C and residual activity was 80-100% between pH ranges 5-8. The enzyme had higher specific activity against p-nitrophenyl-d-glucopyranoside than cellobiose and HPLC showed that the enzyme possesses transglycosylation activity and synthesizes cello-oligosaccharides by addition of glucose. The enzyme will be useful in synthetic biology to produce complex bioactive glycosides and to avoid chemical hazards. This is the first report of a beta-glucosidase enzyme with such a low monomeric unit size.

Secretion of β-glucoside by Termitomyces clypeatus: Regulation by carbon catabolite products

Abstract Casamino acids, irrespective of carbon source used, highly stimulated extracellular production of β-glucosidase by T. clypeatus , which produced very low amounts of enzyme in the presence of 1% (w/v) sugar including xylose in minimal growth medium. Casamino acids did not affect the rate of sugar uptake or improve glucose transport by mushroom, but they significantly reduced the intral extracellular enzyme ratio by increasing the secretion of the enzyme from the cell pool into the culture filtrate. No phosphoenol pyruvate-mediated transport of sugars was detectable in the fungi. Few amino acid utilized as carbon source by the mushroom and Krebs cycle acids (poorly supporting growth) showed activities on enzyme production similar to that of casamino acids. Nonmetabolized glucose analogue glucosamine also increased extracellular enzyme production. Liberation of enzyme from washed mycelia was stimulated by the presence of glutamate in the incubation mixture and was also found to be insensitive to cycloheximide (30 μg ml −1 ). Regulation of the excretion of β-glucosidase in T. clypeatus by glucose catabolic product(s) was indicated.

Purification and characterisation of κ-casein specific milk-clotting metalloprotease from Termitomyces clypeatus MTCC 5091

Milk-clotting enzymes are valued as chymosin-like protease substitutes for cheese making industries. An extracellular metalloprotease (AcPs) with high milk-clotting activity was purified from edible mushroom Termitomyces clypeatus and characterised. AcPs was preferentially active towards κ-casein, analysed by Urea-PAGE and LC-ESI-MS, whereas the degradation of α and β-casein components by AcPs proceeded slowly justifying its suitability for cheese making. RP-HPLC peptide profiling revealed that the AcPs activity on milk casein was similar to that of a commercial milk coagulant. The enzyme exhibited pH and temperature optima at 5.0 and 45 °C, respectively and showed a pI value of 4.6. One- and two dimensional zymographies revealed a single polypeptide band with proteolytic signal. The MALDI-TOF/MS followed by peptide mass fingerprinting revealed homology with a predicted protein of Populus trichocarpa. To our knowledge, this is the first report on a metalloprotease from T. clypeatus, and the results indicate that this enzyme can be considered as a potential substitute for chymosin in cheese manufacturing.

Enhanced activity and stability of cellobiase (β-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. K(m) and V(max) 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 degrees C and pH 5.4, respectively, and retained full activity in a pH range of 5-8 and temperatures of 30-60 degrees 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 beta-glucosidase can be considered as a potentially useful enzyme in various food-processing, pharmaceutical and fermentation industries.

Saccharification of xylan by an amyloglucosidase of Termitomyces clypeatus and synergism in the presence of xylanase

SummaryAn amyloglucosidase from a mycelial culture of the mushroom Termitomyces clypeatus hydrolysed larch wood xylan independently and synergistically with an endo-β(1→4) xylanase of the same fungus. The glucoamylase saccharified xylan predigested with xylanase at a faster rate compared to that of xylanase acting on amylase-digested xylan. However, overall saccharification of xylan in both cases was the same. Only glucose was liberated from xylan by amylase digestion whereas xylose, xylobiose and other oligosaccharides were liberated during xylanase digestion. The synergistic response of enzyme combinations was reflected in the liberation of glucose from xylan, rather than xylose. Glucoamylase and xylanase activities on soluble and insoluble fractions of larch wood xylan with different xylose and glucose contents suggested that synergism in xylanolysis by the presence of glucoamylase was dependent on the activity of the participating xylanase on the xylan preparation. It is suggested that possibly α-glucosidic linkages are present in xylan and that amyloglucosidase might be involved in xylanolysis.

Secretion of Cellobiase Is Mediated via Vacuoles in Termitomyces clypeatus

The majority of cellobiase activity in Termitomyces clypeatus was localized in vacuolar fractions of the fungus under secretory and nonsecretory conditions of growth. Activities of marker proteins for subcellular organelles, e.g., vacuoles, cytosol, ER, and mitochondria, in mycelial extracts from the secreting conditions increased by approximately 20, 12, 5, and 2.5 times, respectively, as compared to those obtained from mycelium grown in nonsecreting conditions. The average size and concentration of vacuoles visualized by electron microscopy were also increased in secreting conditions in the fungus. The specific activity of cellobiase in vacuoles isolated in Ficoll‐sucrose gradient, as obtained from mycelial growth in secretory medium, was more than 40 times higher in comparison to that found from nonsecretory medium. The results indicated that subcellular localization of cellobiase in vacuoles is regulated by the cellular signaling prevailing in the fungus. Mycelial extraction of intracellular proteins by hand grinding and by bead‐beater from cells frozen in the presence or absence of liquid nitrogen was also compared. Maximum recovery of intracellular protein was obtained with the bead‐beater under aerobic conditions in the absence of nitrogen. Highest recovery of vacuoles up to 85% was obtained by single‐step ultracentrifugation of the mycelial extract of the fungus in Ficoll‐sucrose gradient. The method appeared to be useful for separation of other subcellular organelles in filamentous fungi.

Hetero-aggregation with sucrase affects the activity, stability and conformation of extra- and intra-cellular cellobiase in the filamentous fungus T. clypeatus

Cellobiase (C) from T. clypeatus was found to be aggregated with sucrase (S) in extra- and intra-cellular fractions, when co-aggregates of the enzyme with sucrase with different activity ratios (C/S) were obtained during purification. The co-aggregates were compared for their activity, stability, and kinetic parameters with a purified sucrase-free cellobiase preparation. The specific activity and stability of both the extra- and intra-cellular enzyme decreased significantly in the absence of sucrase. The catalytic activity (Vmax/Km) of sucrase-free cellobiase were decreased by 4236 and 652 fold compared to the crude enzyme in culture filtrate and mycelial extracts respectively. The stability of the enzyme also decreased versus pH, temperature and in the presence of chaotropic agents such as SDS, Gdn.HCl and urea after disaggregation from sucrase. Optimum temperatures of the free intra- and extra-cellular cellobiase were shifted to 47°C from 45°C after the removal of sucrase from the co-aggregates, whereas optimum pH of the free enzyme and co-aggregates remained the same. Intra-cellular cellobiase had very high affinity for sucrase and it was difficult to separate them. Cellobiase preparations from extra- and intra-cellular fractions were analysed by circular dichroism and light scattering spectroscopy and it was concluded that co-aggregation with sucrase was responsible for a change in conformation of cellobiase in the aggregates. The conformation of intra-cellular enzyme preparations were also different from those in the extra-cellular fractions. Instant regain of cellobiase activity in intra- and extra-cellular preparations were obtained on the addition in vitro of free sucrase from the respective fractions to the incubation mixture. The experiments suggested that hetero-aggregation with sucrase regulates the activity and stability of cellobiase in the fungus.

Regulation of endo-1,4-β-glucanase secretion fromTermitomyces clypeatus by carbon catabolic product(s)

Secretion of CMCase byTermitomyces clypeatus was only observed in the presence of a gluconeogenic amino acid, a citrate-cycle acid, maleate, subinhibitory concentrations of glucosamine, or fluoride in the medium. The enzyme was not secreted in the presence of caffeine or IBMX or theophylline, and these phosphodiesterase inhibitors lowered the secretion of CMCase by glutamate. The presence of both glucosamine and glutamate in a cellulose medium were, however, antagonistic to CMCase secretion. In a growth medium, xylose and glucose were equivalent carbon source for the fungus while succinate was a poor source and strongly repressed growth at higher concentrations. Growth ofT. clypeatus was highly favored in media containing xylose/glucose with succinate/glutamate. During growth ofT. clypeatus in a glucose medium, the intracellular glucose level was stabilized by the presence of succinate, glutamate or glucosamine in the medium. All these observation suggested that a negative cellular regulation, mediated by carbon catabolic product(s), existed inT. clypeatus which regulated the secretion of CMCase. A transient but significant increase of intracellular cAMP and cGMP levels was observed at the onset of mycelial growth in glucose and glucose/maleate media, respectively.

Regulation (Alteration) of Activity and Conformation of Sucrase by Coaggregation with Cellobiase in Culture Medium of Termitomyces clypeatus

Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS–PAGE and HPGPLC. Km and Vmax of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 °C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.

Purification and Characterization of an Extracellular β-Xylosidase of Termitomyces clypeatus†

Termitomyces clypeatus liberated β‐xylosidase (EC 3.2.1.37) optimally in xylan medium but poorly in cellulose medium. The enzyme activity reached 5–6% of that of xylanase liberated in xylan medium. The culture filtrate enzyme, purified 5‐fold by ammonium sulfate precipitation, BioGel P‐200, and DEAE‐Sephadex anion exchange chromatographies at pH 5.0, was homogeneous (190 kDa) in polyacrylamide gel electrophoresis (PAGE) and in high‐performance gel permeation liquid chromatography (HPGPLC) but contained high amounts of cellobiase and sucrase and gave multiple protein bands in SDS‐PAGE (SDS = sodium dodecyl sulfate) . The aggregate was subsequently resolved by DEAE−anion exchange chromatography at pH 6.0 into a number of β‐xylosidase fractions with decreasing sucrase contents. The sucrase free β‐xylosidase fraction was subsequently purified to 55.6‐fold by hydrophobic interaction chromatography on a phenyl−sepharose column. The enzyme was a homogeneous 94 kDa protein, both in SDS−PAGE and HPGPLC. The physicochemical properties of the enzyme were similar to those of other fungal β‐xylosidases, and the enzyme had no unrelated glycosidase activities. The purified (94 kDa) and aggregated forms (190 kDa) of β‐xylosidase had the same pH optima (5.0), temperature optima (60 °C), substrate specificities, and sensitivities toward end product inhibition by xylose or to the actions of SDS, urea, and guanidine hydrochloride. But aggregated enzyme was reasonably stable in the pH and temperature ranges where purified enzyme was completely inactive. The protein−protein aggregation appeared to confer additional stability to the β‐xylosidase toward extracellular denaturing conditions.