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.

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

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.

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 ß-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.

Enhancement of extracellular cellobiase activity by reducing agents in the filamentous fungus Termitomyces clypeatus.

Extracellular cellobiase activity of Termitomyces clypeatus increased from 2.9 U ml(-1) to 4.4 and 4.1 in presence of dithiothreitol (DTT) and ß-mercaptoethanol (ME), respectively, with a decrease in Km from 0.4 to 0.3 mM (DTT) and 0.35 mM (ME). Catalysis was further enhanced if the reduced enzyme was alkylated and activity increased from 11.4 U ml(-1) (control) to 15.2 (DTT+N-ethylmaleimide) and 15.3 (DTT+iodoacetamide) using p-nitrophenyl-ß-D-glucopyranoside and from 14.6 U ml(-1)(control) to 21.9 (DTT+N-ethylmaleimide) and 18.7 (DTT+iodoacetamide) using cellobiose. The reduced enzyme showed 17 % lesser glucose inhibition. CD and tryptophan fluorescence showed no change in secondary structure was caused by DTT up to 50 mM. Cysteine content of the enzyme was 24 %. It is postulated that reduction of disulphide bonds allows better substrate affinity for cellobiase. The studies describe a novel and simple method to increase cellobiase activity for industrial applications.

Effect of Alocasia indica tuber extract on reducing hepatotoxicity and liver apoptosis in alcohol intoxicated rats.

The possible protective role of ethanolic extract of A. indica tuber (EEAIT) in hepatotoxicity and apoptosis of liver caused by alcohol in rats was investigated. Treatment of rats with alcohol (3 g ethanol per kg body weight per day for 15 days intraperitoneally) produced marked elevation of liver biomarkers such as serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (γ-GT), and total bilirubin levels which were reduced by EEAIT in a dose-dependent manner. Furthermore, EEAIT improved antioxidant status (MDA, NO, and GSH) and preserved hepatic cell architecture. Simultaneous supplementation with EEAIT significantly restored hepatic catalase (CAT) and superoxide dismutase (SOD) activity levels towards normal. The studies with biochemical markers were strongly supported by the histopathological evaluation of the liver tissue. EEAIT also attenuated apoptosis and necrosis features of liver cell found in immunohistochemical evaluation. HPLC analysis of the extract showed the presence of three major peaks of which peak 2 (RT: 33.33 min) contains the highest area (%) and UV spectrum analysis identified it as flavonoids. It is therefore suggested that EEAIT can provide a definite protective effect against chronic hepatic injury caused by alcohol in rats, which may mainly be associated with its antioxidative effect.

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 non-secreting 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.