Rajib Majumder

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.

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 enzymes.

Evaluation of in vitro antioxidant, anticancer and in vivo antitumour activity of Termitomyces clypeatus MTCC 5091

Abstract Context: Termitomyces clypeatus (Lyophyllaceae) is a filamentous edible mushroom, having ethnomedicinal uses. However, information about the antioxidant, anticancer and antitumour properties of this mushroom remains to be elucidated. Objective: The study examines the in vitro antioxidant, anticancer and in vivo antitumour activity of T. clypeatus. Materials and methods: Antioxidant activity was evaluated with seven in vitro assays. Cytotoxicity of T. clypeatus was tested against a panel of cancer cells lines including U373MG, MDA-MB-468, HepG2, HL-60, A549, U937, OAW-42 and Y-79 using MTT assay. The antitumour activity of aqueous extract was evaluated against Ehrlich ascites carcinoma (EAC) tumour model in Swiss albino mice. Results: HPLC analysis of aqueous extract revealed the presence of sugar entities. Termitomyces clypeatus showed excellent in vitro antioxidant activity. Termitomyces clypeatus was found cytotoxic against all cancer cells, among which it showed higher activity against U937 (IC50 25 ± 1.02 μg/mL). Treatment of EAC-bearing mice with varied doses of aqueous extract significantly (p < 0.01) reduced tumour volume, viable tumour cell count and improved haemoglobin content, RBC count, mean survival time, tumour inhibition and % increase life span. The enhanced antioxidant status in treated animals was evident from the decline in the levels of lipid peroxidation, increased levels of glutathione, catalase and superoxide dismutase. Discussion: The analyzed data indicate that the aqueous extract of T. clypeatus exhibits significant antitumour activity, which might be due to the antioxidant effects on EAC bearing hosts. Conclusion: Termitomyces clypeatus possesses anticancer activity, valuable for application in food and drug products.

Trehalose induced structural modulation of Bovine Serum Albumin at ambient temperature

Trehalose is a well-known protein stabilizing osmolyte. The present study has been designed to understand the interaction of trehalose with BSA at ambient temperature. Steady state fluorescence and life-time analysis along with CD, DLS and ITC have been employed to show that trehalose causes surface-associated structural perturbation of BSA to promote its compaction. Trehalose at 0.1M concentration resulted in increased solvent exposure of one of the two tryptophans of BSA with a 5nm redshift in emission and enhanced susceptibility to acrylamide quenching with an increase in KSV from 2.61M-1to 5.16M-1. 0.5M trehalose resulted in reduced accessibility of tryptophan and destabilization of ANS binding (Forster radius increased from 24Å to 27.36Å for tryptophan-ANS FRET) indicating shielding of BSA in trehalose matrix. Simultaneously, there was compaction of BSA as shown by increased alpha-helicity from 45.85% to 48.81%, decreased thioflavin-T binding and reduction in hydrodynamic radius from 9.69nm to 6.59nm. Trehalose induced solution viscosity resulted in significant decrease in binding affinity of BSA towards curcumin and resveratrol. The results are in unison with the preferential exclusion and vitrification models to explain protein stabilization by trehalose and also points at the structure-function trade-off of proteins in presence of trehalose.

AkP from mushroom Termitomyces clypeatus is a proteoglycan specific protease with apoptotic effect on HepG2

Termitomyces clypeatus is an edible mushroom, prized for its therapeutic values and as producer of industrially important enzymes. However, the biomedical efficacies of anticancer proteases have not been reported yet. The present study aimed to purify and characterize a serine protease (AkP) from T. clypeatus for investigating cytotoxic potency on HepG2, Hep3B, and compared the effect on normal hepatic L-02 cells. Purification and biochemical characterization of AkP were evaluated by three stage chromatography, 1D/2D-SDS-PAGE, 1D zymography, far-UV CD spectral analysis, N-terminal sequencing, MALDI-TOF/MS-MS analysis and enzyme kinetics studies. AkP could cleave the growth promoting cell surface proteoglycans of HepG2, corroborated by RP-HPLC analysis. AkP (IC50: 75±1.18nM) mediated anti-proliferative activity solely on HepG2 cells through the induction of apoptosis. Augmentation of apoptosis was attributed to up-regulation of p53 and Bax protein expression succeeded by caspase-3 activation. Serine protease inhibitor phenyl methane sulfonyl fluoride (PMSF) inhibited both its proteolytic activity and cytotoxicity on HepG2. These findings demonstrate that AkP could be an effective biomolecule for killing of cancer cells by p53 restoration and surface proteoglycans cleavage.

Prevention of protein aggregation by extracellular fungal sucrase of Termitomyces clypeatus

Abstract Objective: Extracellular sucrase from Termitomyces clypeatus is known to impart stability and enhance activity of cellobiase, another secreted glycosidase of the fungus through co-aggregation with cellobiase. To explore whether sucrase can bind with some proteins like Insulin, BSA, alcohol dehydrogenase (ADH), carbonic anhydrase and whey proteins and prevent their dithiothreitol (DTT) induced/thermal aggregation and/or loss of activity measuring by spectrophotometry, gel filtration assay and activity assays. Methods: MALDI-TOF and dynamic light scattering were used to assess the monomeric and aggregated molecular size of sucrase. Thermostatted spectrophotometric assays, gel filtration assays were used to study protein aggregation. Fluorescence of bound ANS was used to monitor temperature induced structural changes in sucrase together with determination of melting temperature. Results: The mass of the monomeric unit of sucrase as 6649 Da. Enzyme inhibited DTT induced aggregation of insulin and suppressed the thermal aggregation of carbonic anhydrase, ADH and whey proteins, respectively by 83%, 68% and 70% at 70°C. Sucrase also protected about 84% activity of ADH. Conclusion: An extracellular fungal sucrase with a low monomeric size can efficiently prevent protein aggregation. The studies can impart knowledge about potential therapeutic applications of this industrially important enzyme in protein misfolding disorders.

Depletion of Cr(VI) from aqueous solution by heat dried biomass of a newly isolated fungus Arthrinium malaysianum: A mechanistic approach

For the first time, the heat dried biomass of a newly isolated fungus Arthrinium malaysianum was studied for the toxic Cr(VI) adsorption, involving more than one mechanism like physisorption, chemisorption, oxidation-reduction and chelation. The process was best explained by the pseudo-second order kinetic model and Redlich-Peterson isotherm with maximum predicted biosorption capacity (Qm) of 100.69 mg g−1. Film-diffusion was the rate-controlling step and the adsorption was spontaneous, endothermic and entropy-driven. The mode of interactions between Cr(VI) ions and fungal biomass were investigated by several methods [Fourier Transform-Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD) and Energy-Dispersive X-ray spectroscopy (EDX)]. X-ray Photoelectron Spectroscopy (XPS) studies confirmed significant reduction of Cr(VI) into non-toxic Cr(III) species. Further, a modified methodology of Atomic Force Microscopy was successfully attempted to visualize the mycelial ultra-structure change after chromium adsorption. The influence of pH, biomass dose and contact time on Cr(VI) depletion were evaluated by Response Surface Model (RSM). FESEM-EDX analysis also exhibited arsenic (As) and lead (Pb) peaks on fungus surface upon treating with synthetic solutions of NaAsO2 and Pb(NO3)2 respectively. Additionally, the biomass could also remove chromium from industrial effluents, suggesting the fungal biomass as a promising adsorbent for toxic metals removal.

Gastric Pathology and Metalloproteinases

The spectrum of gastric pathologies involves heterogeneity with respect to biochemical mechanisms and clinical outcome and is globally common. Each year, 5–6 million people worldwide are affected by gastric ulcer, gastric cancer and inflammatory bowel diseases, and mortality rate being >50% shows steep increase in incidence. Hence, understanding the underlying pathogenesis and better therapeutic strategies remain the major challenges in gastroenterology field. Current knowledge of gastric pathology reveals that extracellular proteases vastly influence functional irregularities of cells along with their responses to microenvironment. Based on studies on metalloproteinases and their inhibitors, it is well accepted about their important roles in physiological developmental processes as well as pathological conditions. From past several years of extensive research on matrix, metalloproteinases (MMPs) establish their critical role in several cellular functions including proliferation, apoptosis and angiogenesis. MMPs are a family of “molecular scissors” with ambivalent actions and ability to cleave extracellular matrix (ECM) proteins that in turn facilitate tissue remodelling. Approximately, 27 subtypes of MMPs are there having mutual interaction among each of them in gastrointestinal disorders. Functional overlap between the MMPs leads to non-specificity, which makes designing MMP inhibitors more difficult. Thus, specific MMP inhibitors would be promising therapeutic tool against inflammatory diseases including gastric diseases. This chapter illustrates the new insights into mechanism of MMP regulation in gastrointestinal inflammatory disorders encompassing clinical trials for MMP inhibitors and new therapeutic strategies by targeting specific MMP(s) to control gastrointestinal pathologies.