Open Access Original Research Article

In Silico Evaluation of the Structural Dynamics of Beta-Amylase from Sweet Potato (Ipomoea batatas)

David Akintayo Obe, Toluwase Hezekiah Fatoki

Asian Journal of Biotechnology and Bioresource Technology, Page 1-10
DOI: 10.9734/ajb2t/2021/v7i330100

Background: Sweet potato tubers are invaluable crop that could serve both dietary and industrial purposes owing to its high β-amylase content. β-amylases play essential role in plant carbohydrate metabolism as well as in many industrial applications such as the malting process in the brewing and distilling industries.

Aim: This study aims at better understanding of the evolutionary and molecular properties, and structural dynamics of β-amylase of sweet potato using in silico approach.

Methodology: 16 of the 250 sequences that are at least 69% identity to the query sequence (P10537) were manually selected from UniProt database for further analysis.

Result: It has theoretical isoelectric point of 4.97 and molecular weight of 56 kDa. The root-mean-square fluctuation (RMSF) of sweet potato β-amylase showed possible conservation of the amino acid residues 105-130 and 260-345, with highest fluctuation in C-terminal loop (residues 443-498). The catalytic role of Glu187 and Thr344 in β-amylase of sweet potato has been elucidated, and it provided the missing link in the previously available mechanisms, while Cys96 is essential for the inactivation of enzyme activity.

Conclusion: Elucidation of molecular mechanisms of expression and catalytic activity, together with the understanding of physicochemical properties of β-amylase from sweet potato will help in development of useful applications that are of industrial importance.

Open Access Original Research Article

Molecular Cloning of Selected Regions of Catalytic Domain of CtsK Gene in Escherichia coli – A Feasibility Study

Hasanka Madubashetha, Ruwini Cooray, P. D. S. U. Wickramasinghe, Lakshan Warnakula, Nimali De Silva

Asian Journal of Biotechnology and Bioresource Technology, Page 11-35
DOI: 10.9734/ajb2t/2021/v7i330101

Cathepsin K (CatK), encoded by CtsK gene in human, is involved in bone remodeling through ossification. The objective of the work conducted here was to express catalytic domains of CtsK gene in bacterial expression system as an initial step, facilitating recombinant production of human CatK for downstream applications in pharmacology. Four healthy human blood samples were collected. Genomic DNA was extracted using FlexiGene® whole blood DNA extraction kit. Upon quantification of DNA through NanodropTM spectrophotometer, sufficient quantity and quality was observed. CtsK gene was amplified by Polymerase Chain Reaction (PCR) using two pairs of primers tagged with restriction endonuclease sites of Sal1 and HindIII facilitating molecular cloning and visualized by Agarose Gel Electrophoresis (AGE). Two different bands of size 545bp and 265bp were observed. The bands were dissected and gel purified using GenaxxonTM gel purification kit and sequentially double digested by restriction enzymes; SalI and HindIII.  Vector PBS was also subjected to sequential double digestion using same enzymes and visualized via AGE. Double digested insert of size 265bp and vector were ligated using T4 DNA Ligase (all enzymes from PromegaTM). On another trail, ligation of the PCR product with band size 265bp to pGEM-TTM easy vector system (from PromegaTM) was also done and transformed to Top10 Escherichia coli competent cells for expression separately. Cells were grown in LB media in presence of XGAL, IPTG and Ampicillin and transformed cells were screened. In the restriction enzyme digestion and ligation setup, since the insert and vector were both double digested, it is confirmed that white colonies obtained were Escherichia coli cells were transformed with the desired recombinant vector and is therefore confirmatory. In the case of pGEM-TTM ligation, a colony PCR was done using the white colonies obtained and product size was confirmed via AGE. In conclusion, the objective of study was successfully achieved, by expressing a catalytic domain of CtsK. Developments and improvements could be made for expression of entire CatK gene and downstream production of the Cathepsin K protein for effective therapeutic purpose.

Open Access Original Research Article

Preservation of Indigenous Fungal Cultures by Freeze Drying Technique using Skim Milk and Honey as the Protectants

G. B. Olukotun, B. B. Adamu, O. J. Asake, S. S. Leh-Togi Zobeashia

Asian Journal of Biotechnology and Bioresource Technology, Page 36-42
DOI: 10.9734/ajb2t/2021/v7i330102

COVID-19 pandemic movement restrictions as part of the control measures put in place by countries in Sub-Saharan Africa (SSA) has implications on many sectors which include very high costs of importing materials owing to high exchange rates as well as non- availability of microbial cultures for food/ industrial productions, medical/academic research and applications because of fear of new microbial infections and bio-terrorism. Overcoming these challenges in SSA requires developing better policies and packages to confront the challenges by way of rebooting their strategies and policies for sustainable economic growth through reawakening the potentials they have. The maintenance and production of reliable pure microbial cultures with desirable quality is a key operation and is the first significant stage in the success of fungal applications in any scientific venture where they are used. For many years the process of freeze-drying (lyophilisation) has been employed primarily with the purpose of preserving living materials, including microorganisms, for extended periods of time; this is possible because, upon dehydration, substances no longer change as a consequence of the usual turnover of metabolic reactions characteristic of the living condition. This study assessed lyophilization (freeze drying) of some indigenous filamentous fungal cultures (Spores) using skim milk and honey (supports being maize and cassava flours) as the protectants and then critically compared the two protective media for the preservation of active filamentous fungi spores for the first time, and from the data obtained, their applications were optimised. Some fungal isolates(Aspergillus sp., Mucor sp., Penicillum sp., Fusarium sp., Rhizopus sp., Cladosporium sp. Alternaria sp. and Geotricum sp.) previously purified and identified were obtained from the Products Development Unit, National Biotechnology Development Agency, Abuja, Nigeria and were re-validated where their viability and purity were confirmed. The samples were Freeze Dried in the respective medium and monitored immediately after. After the freeze‐drying, the residual water contents were between 0.20-0.32%, 0.39-0.49% and 0.33-0.41%, respectively, with skim milk, Honey with Cassava Starch and honey with Maize Starch. The driedspore viabilities were between 96% (Aspergillus spores in Reconstituted Skim Milk) and 21% (Geotricum spores in Honey with Cassava Starch. Among the protective agents used, skim milk was found to be the best protectant.Each fungal isolate was considered viable if the rate of growth present was the same as that of the original culture and if the morphology of the colony matched the fungal identification documented for each species. All lyophilized fungal species were found viable from both type of lyophilized materials, skim milk and honey and showed the initial colony characteristics and growth rates.

Open Access Original Research Article

Diagnostic of the Cooking and Storage Systems of a Cassava-Derived Food Locally Called “fufu” in the City of Yaoundé, Cameroon

Jorelle Jeanne Bimem Adjele, Annick Manuela Bengue Baomog, Raissa Hermine Hell, Hippolyte Tene Mouafo, Maxwell Wandji Nguedjo, Alex Dimitri Kamgain Tchuenchieu, Gabriel Nama Medoua

Asian Journal of Biotechnology and Bioresource Technology, Page 43-55
DOI: 10.9734/ajb2t/2021/v7i330103

Fufu is among the most consumed traditional dishes derived from cassava in Cameroon. However, the processing of cassava into flour used in the preparation of fufu, the home storage of that flour and the cooking practices might lead to microbial contamination and hence constitute a potential health risk for consumers. The aim of this study was to diagnose the cooking systems of fufu in the city of Yaoundé taking into consideration the processing of granules, its storage systems and the cooking practices. A cross-sectional study was conducted in the city of Yaoundé. A total of 74 participants were submitted to a survey by the means of a structured questionnaire. Information on the socio-demographic characteristics of the population, the nature and quality of raw material used to cook fufu, the cooking processes, the storage of cooked fufu and complaints associated with the consumption of fufu were collected and statistically analyzed. Results showed that the majority of participants were women (82.4%) with university education level (73%). Most of the participants preferred granules of fermented cassava as raw material. The odor was the main purchasing criteria of raw materials which were generally stored at room temperature (25±2°C) in the household for 1 to 2 weeks. 76.05% of participants were aware of the impact of microorganisms on the color and odor of the raw material. Two cooking processes of fufu were identified, the heating duration being the main difference. The majority of participants used a heating duration of less than 15 min as it leads to slightly rigid, less elastic and not sticky fufu highly appreciated in the Centre and Sud regions of Cameroon. Leftovers fufu eaten by participants without any heating process and leftovers fufu reheated for a short duration before being consumed might be the causes of digestive troubles associated with its consumption. Prospective studies should be conducted to optimize the cooking process of fufu taking into consideration its microbiological (sporulated microflora) and sensorial quality.

Open Access Original Research Article

Evaluation of Natural Oil Adducts in Alkyd-Based Varnish Emulsion and Effect on Rowan (Sorbus torminalis) Wood

Halil Turgut Sahin

Asian Journal of Biotechnology and Bioresource Technology, Page 56-63
DOI: 10.9734/ajb2t/2021/v7i330104

In these days, preparation of eco-friendly wood surface coating agents has become important topic for environmental concerns. In this study, an alternative surface coating formulations was prepared with adducts of oils of apricot kernel (A), sesame (S) and grape seed (G) were mixed with oil modified alkyd-based synthetic varnish system. These emulsions were applied on Rowan (Sorbus  torminalis)  wood and exposed artificial UV irradiation. However, all emulsion coated samples show lower water sorptions regardless of conditions or level of treatments. Increasing adduct charges from 5.0% to 10% have not considerably effects for water sorptions. The reducing water sorption properties of 35.5%, 35.2% and 39.4% were obtained with 10% sesame oil-varnish coated samples of 10Sı and 10 Sıı in one- and two-time coatings and three-times coated samples of 5Sııı in 5.0% sesame oil-varnish emulsion coatings, respectively. Moreover, selected oil adducts into varnish had one and two point lowering effects on coated surface hardness (in 4H-5H levels).  The UV exposure seems to not much influence on surface hardness properties. The similar tendency were also observed for cross-cut resistance that marginally similar adhesion (scratch resistance)  for both control and UV exposured samples.  Increasing coatings and charge of apricot kernel oil in varnish emulsion looks like more effective than other varnish formulations, in terms of gloss stability. However, increasing adducts charges and UV radiation time, have more less similar effects on all coated and UV irradiated samples.