Improved Production and Properties of β-glucosidase Influenced by 2-deoxy-D-glucose in the Culture Medium of Termitomyces clypeatus

Shakuntala Ghorai,Sumana Mukherjee,Soumya Mukherjee,Suman Khowala 한국생물공학회 2011 Biotechnology and Bioprocess Engineering Vol.16 No.2

Increased production, secretion, and activity of β-glucosidase in the filamentous fungus Termitomyces clypeatus was achieved in presence of the glycosylation inhibitor 2-deoxy-D-glucose (0.05%, w/v) during submerged fermentation. Enzyme activity increased to 163 U/mL by adding mannose (2 mg/mL) to the medium. Such a high enzyme activity has not been achieved without mutation or genetic manipulation. The K_m and V_max of the enzyme in culture medium were determined to be 0.092 mM and 35.54 U/mg, respectively, with p-nitrophenyl β-D-glucopyranoside as substrate, confirming its high catalytic activity. The enzyme displayed optimum activity at pH 5.4 and 45°C. The enzyme was fairly stable between acidic to alkaline pH and retained about 75 ~ 65% residual activities between pH 4 and 10.6 and demonstrated full activity at 45ºC for 3 days. The enzyme was also stable in the presence of Zn^2+ and Mg^2+ and 80% of the residual activity was observed in the presence of Mn^2+, Ca^2+, K^+, Cu^2+,EDTA, and sodium azide. Around 70% of the activity was retained in the presence of 2 M guanidium HCl and 3 M urea, whereas the activity was 5 and 2 times higher in the presence of 4 mM beta-mercaptoethanol and 50 mM DTT,respectively. The enzyme obtained from the culture filtrate showed potential cellulose saccharifying ability which increased further when supplemented with commercial cellulase. Thus, this enzyme could be used without any additional downstream processing for commercial cellulase preparation and production of bioethanol or for other biotechnological applications.

Nursing Status in Nepal

( Shakuntala Thanju ) 부산대학교 간호과학연구소 2017 글로벌 건강과 간호 Vol.7 No.1

HOW CAN AQUACULTURE CONTRIBUTE TO HEALTHY DIETS OF THE POOR?

Shakuntala Haraksingh Thilsted 한국수산과학회 양식분과 2015 한국수산과학회 양식분과 학술대회 Vol.2015 No.5

Toward unrestricted use of public genomic data

Amann, Rudolf I.,Baichoo, Shakuntala,Blencowe, Benjamin J.,Bork, Peer,Borodovsky, Mark,Brooksbank, Cath,Chain, Patrick S. G.,Colwell, Rita R.,Daffonchio, Daniele G.,Danchin, Antoine,de Lorenzo, Victor American Association for the Advancement of Scienc 2019 Science Vol.363 No.6425

<P>Despite some notable progress in data sharing policies and practices, restrictions are still often placed on the open and unconditional use of various genomic data after they have received official approval for release to the public domain or to public databases. These restrictions, which often conflict with the terms and conditions of the funding bodies who supported the release of those data for the benefit of the scientific community and society, are perpetuated by the lack of clear guiding rules for data usage. Existing guidelines for data released to the public domain recognize but fail to resolve tensions between the importance of free and unconditional use of these data and the “right” of the data producers to the first publication. This self-contradiction has resulted in a loophole that allows different interpretations and a continuous debate between data producers and data users on the use of public data. We argue that the publicly available data should be treated as open data, a shared resource with unrestricted use for analysis, interpretation, and publication.</P>

Evidence of an Alternative Route of Cellobiase Secretion in the Presence of Brefeldin A in the Filamentous Fungus Termitomyces clypeatus

( Banik Samudra Prosad ),( Swagata Pal ),( Sudeshna Chowdhury ),( Shakuntala Ghorai ),( Suman Khowala ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.4

Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 μg/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at 50oC, 93.3% activity at pH 9, 63.64% activity in the presence of 1M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe.

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

Samudra Prosad Banik,Swagata Pal,Shakuntala Ghorai,Sudeshna Chowdhury,Rajib Majumder,Soumya Mukherjee,Suman Khowala 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.5

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. 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.

Predictive modeling as a tool to assess polymer–polymer and polymer–drug interactions for tissue engineering applications

Lakshmi Yaneesha Sujeeun,Nowsheen Goonoo,Kaylina Marie Moutou,Shakuntala Baichoo,Archana Bhaw‑Luximon 한국고분자학회 2023 Macromolecular Research Vol.31 No.4

The success of tissue engineering scaffolds for wound healing relies on balanced physicochemical properties and the addition of small molecules. These two require an in-depth understanding of the interactions between the different components of the scaffolds for favorable and synergistic actions. Thus, the choice of polymeric blends is crucial to tune the properties of the resulting scaffolds. Miscibility of the polymer blends can be used to assess polymer–polymer interactions for effective scaffold engineering and cell–material interactions. The focus of this study was to apply machine learning (ML) methods to classify and predict the miscibility of polymer blends, namely poly(hydroxybutyrate-co-valerate)/fucoidan (PHBV/FUC), polyhydroxybutyrate/kappa-carrageenan (PHB/KCG), and cellulose acetate/polyamide (CA/PA). Physicochemical parameters assessed through Fourier transform infrared spectroscopy (FTIR), thermal analysis, and mechanical properties were used as input data. Depending on blend film compositions, the polymers were either partially miscible or completely immiscible. Six supervised classification algorithms were trained on the data with Scikit-learn. The random forest classifier outperformed the other algorithms with the optimal performance metrics. A simple multiple linear regression model was applied to polymer–drug interaction data. Preliminary results indicated that regression models could be correlated with ultraviolet (UV) absorbance of polymer–drug solutions.