Journal of Applied Biotechnology Reports
Volume:8 Issue: 2, Spring 2021

Polyphenol oxidase (PPO) is a copper-containing enzyme that can be used for different applications including wastewater treatment and biosensing. Given these wide arrays of application, the reaction and biochemical characteristics of the enzyme must be known to further determine its other applications and to control the essential factors during processing. The purpose of this research was to review the different factors that influence the effective extraction and characterization of PPO from plant materials. The pH of the extraction mixture, extraction temperature, type of buffer, mass to solvent ratio, extraction time, and additives are the factors that influence the effective extraction of PPO from plant materials. Since PPOs taken from different plant sources have varied protein structures, these factors have different effects during extraction. The isolated PPO from the extraction process can be characterized based on its activity as a function of pH, temperature, and type of substrate, and on the values of its kinetic parameters (Km and Vmax). PPO isolated from different plant sources shows varied optimum pH, optimum temperature, substrate affinity, and kinetic parameter values.

The ocean is considered to be an immense reservoir of biological and microbial diversity on the planet. In marine biospheres, microbial communities are ecologically significant as intermediaries of energy. By decomposing the dead as well as decaying organic matter with the assistance of microbial communities, it plays an indispensable role in the nutrient regeneration cycles of marine ecosystems. Marine environments associated with microorganisms such as bacteria, fungi, and bacterial virus have renowned potential to produce novel bioactive natural products and chemically diverse secondary metabolites like antibiotics, antifungal, antiviral, antitumor, anticancer, and also different hydrolyzing enzymes, namely, protease, lipase, amylase, chitinase, etc. Hence, the bioprospecting for these compounds is of greater importance. Numerous effective and efficient applications of marine microbial metabolites contribute to the fields of pharmaceuticals, biotechnological, agricultural, cosmetics industries, and so on. This review attempts to summarize the present status of bioprospecting marine microorganisms and their role in natural product discovery.

Microorganisms are ubiquitous in nature. They are found across diverse biosphere and greatly vary among them. Several beneficial microbes engaging in degradation and their services to the ecosystem have not been fully explored. This tangled module could be resolved by Meta 'omics' approach and analysing their molecular interaction with the environment. In our day-to-day life, human beings are exposed to various xenobiotics in the form of drugs/pharmaceuticals, pesticides, artificially flavoured food and beverages. Newer diseases are also emerging due to environmental pollutants. Bioremediation offers an effective way to resilient our fragile planet. Next-generation sequencing became an ultimate technique to unravel the significance of the microbiome in remediating polluted lands and sludges. Integrating the meta omics data would open new perspectives in the clean-up of toxic contaminants from our environment. Through this review, we attempted to explore the potential of meta-omics approaches in deciphering the eavesdropping of complex microbes. This review provides novel insights into the Meta-omics techniques that aid in the field of bioremediation.

Analysis of genome sequences to search for encoded proteins and motifs is the most widely used technique for the prediction of new drug and vaccine targets. It can effectively leverage computational techniques to deliver effective and pragmatic advantages in the search for new drugs and vaccines. We examine the diversity and evolution of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) isolates from different geographical parts of India using phylogenetic tree analysis. A dataset of 172 Indian SARS-CoV-2 genome sequences was collected from a database and a phylogenetic tree was constructed. From the phylogenetic analysis, we identified 6 different clusters and from each cluster, we have chosen 10 genome sequences to find open reading frames (ORFs) and common encoded proteins. We found 4 encoded proteins that are common among the 60 genome sequences and they correspond to ORF7a protein, membrane glycoprotein, surface glycoprotein, and nucleocapsid phosphoproteins. Our results highlight that there are 6 conserved motifs with a high frequency of occurrence suggesting that potentially use in further study. The encoded proteins and their detected sequential motifs might be useful for screening potential drugs and vaccine candidates of SARS-CoV-2 Indian isolates in the current epidemic situation.

Wide applications in research, clinical and cosmetic industry of human epidermal growth factor (hEGF) made it a research interest target. Its production in different expression systems has shown several limitations. Recombinant expression of hEGF in E. coli is always accompanied by inclusion body formation. The object of this study is to the evaluation of a chromatography-independent approach for the production of EGF in E. coli as soluble form. In order to evaluate a chromatogram independent purification approach for recombinant hEGF production in a soluble form, the hEGF gene was fused to an elastin-like protein (ELP) and expressed in E. coli BL21 (DE3) using pET26b expression vector for secretion the product into periplasmic space. Periplasmic protein content analysis confirmed that the recombinant protein is secreted into the periplasm. The purification process was done by using 0.4 M ammonium sulfate in two cycles of inverse phase transition (ITC). After two cycles of purification, purity reached more than 95%. Western blotting analysis with the monoclonal anti-EGF antibody has confirmed the accuracy of EGF. Biological activity of the purified protein was investigated on NIH-3T3 cell line and results indicated EGF-induced proliferation in treated cells. Our results showed periplasmic expression is the proper approach to the production of soluble recombinant hEGF. By using ELP fused to EGF, the purification process was established without applying chromatography which will result in decreasing in final costs. This study introduced a new economic and efficient approach to the production and purification of recombinant hEGF.

Endo-β-1,4-glucanase is the first enzyme in the conversion of cellulose to fermentable sugars. The objectives of this study were to clone and characterize a thermostable Endo-β-1,4-glucanase enzyme of Bacillus subtilis DR-8806 obtained from water samples from Dig Rostam, a hot mineral spring in Kerman, Iran. Endo-β-1,4-glucanase gene from a thermostable Bacillus subtilis bacterium was cloned and expressed in Escherichia coli. The recombinant proteins of the expression cell were tested by western blotting analysis. The enzymatic activity of the recombinant endoglucanase was measured using dinitrosalicylic acid method and carboxymethyl cellulose as substrate. Bioinformatics analysis was done to characterize domain organization and protein family through Pfam search server and PROSITE. Based on 16S ribosomal RNA sequence analysis, Bacillus is characterized and named as Bacillus subtilis DR-8806. Western blot analysis verified the recombinant endoglucanase by detecting a specific band of ~55kDa. Amino acid homology analysis of the protein showed 99% homology with that of endoglucanase from Bacillus subtilis. The optimum temperature for enzyme reaction was attained at a temperature of 55°C. The cellulolytic activity of Endo-β-1,4-glucanase protein determined 8.5 IU ml-1. It showed that endoglucanase amino acid sequence contains a glycosyl hydrolase family 5, linker domain, and a cellulose-binding type 3 domain. The GH5 domain also contained a glycosyl hydrolase catalytic core. It is possible to consider the purified Endo-β-1,4-glucanase of B. Subtilis DR-8806 as an efficient cellulose producer. Further research is required to examine the industrial applications of this study.

 In historical cases, mass disasters, missing person’s identification, and Archaeogenetic investigations, the bone, teeth, and hair samples are often the best and the only biological material available for DNA typing. In this study, we demonstrated an extremely effective protocol for DNA extraction of hair.  Obtaining genetic information from hair samples for DNA typing involves the following steps: sterilization, DNA extraction, quality control, PCR, and profiling. After extracting aDNA (Ancient DNA) from the hair, autosomal STR analyses were performed for each sample using the AmpFℓSTR® MiniFiler™ and Identifiler™ kits.  Qualitative analysis of DNA by electrophoresis showed the excellent quality of the extracted samples so that the electrophoresed bands were clearly visible. The results demonstrated that the FDEH method is cheaper and faster than commercial kits; moreover, this new method enhances DNA recovery from hair. Because of simple protocol and high quality, this method can be utilized in Medical.

The ongoing global pandemic of coronavirus disease (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus- 2 (SARS CoV-2) has jeopardized our health system and leaving everyone in disarray.  Despite the diligent cumulative effort of academia, there is hardly any light in the end tunnel so far in developing efficient and sustainable treatment options to tackle this public health threat. Therefore, designing a suitable vaccine to overcome this hurdle calls for immediate attention. The current study aimed to design a multi-epitope based vaccine using immunoinformatics tools. We approached the structural proteins: S, E, and M proteins of SARS CoV-2 since they facilitate the infection of the virus into a host cell. By using different bioinformatics tools and servers, the multiple B-cell and T-cell epitopes were predicted potential for the required vaccine design. The phylogenetic analysis provides in-depth knowledge on ancestral molecular changes and the molecular evolutionary relationship of S, E, and M proteins. Based on the antigenicity and surface accessibility of the spike (S), envelope (E), and membrane (M) proteins, eight epitopes were selected by various B cell and T cell epitope prediction tools.  Molecular docking was executed to interpret the binding interactions of these epitopes from where three potential epitopes WTAGAAAYY, YVYSRVKNL, and GTITVEELK were finalized with their noticeable higher binding affinity scores -9.1,-7.4, and -7.0  kcal/mol, respectively. It is noteworthy to mention that the targeted epitopes are believed to cover 91.09% of the population coverage worldwide. In sum, we identified the three most potential epitopes at length, which might be turned to our purpose of designing the peptide-based vaccine against SARS CoV-2.

Although radiation is recognized as the most effective nonsurgical treatment, the outcomes and control rates are generally poor. However, a combination of radiation therapy with hyperthermia and chemotherapy can improve the efficacy of treatment. The aim was to explore the potential of morphological and gradient-based features on microscopic images in improving the identification accuracy of subtle differences in cell structure during different treatments. Fifty single-cell images were used for each group and treatment regimen. The groups were individually subjected to: 1) hyperthermia at 43°C; 2) temozolomide (TMZ) chemotherapy at 10% inhibitory concentration; 3) radiotherapy at 2Gy; 4) combination of TMZ chemotherapy and hyperthermia; 5) combination of radiotherapy and hyperthermia; 6) combination of TMZ chemotherapy and radiotherapy; and 7) combination of TMZ chemotherapy, radiotherapy, and hyperthermia. Morphological and gradient-based features were extracted from each cell. The area under the receiver operating characteristic curve (AUC) was calculated for each significant feature to evaluate the performance of cell change detection. According to AUCs, gradient-based features showed superior performance to morphological features in identifying cell changes during all treatment regimens in all groups. In this regard, the AUC of the gradient-mean feature exceeded 0.599 for all groups. The ratio of maximum to minimum cell diameter was the best morphological feature, with an AUC above 0.588 for all groups. Quantitative analysis of features is a reliable indicator of damage, with the potential to characterize cell changes during treatment regimens.

Inulinases are β-fructohydrolase enzymes that catalyze the hydrolysis of inulin. Recently, this enzyme has gained much importance mainly due to its ability to produce high-density fructose syrup using inulin as a raw material. In the current study, screening of inulinase-producing microorganisms was carried out from the rhizosphere soil of the Dahlia plant and rotten garlic samples.   The inulinase activity was detected with the help of 3,5-dinitrosalicylic acid (DNSA) and Seliwanoff’s method, and the organism showing the highest potential was selected for further optimization studies. The optimum culture conditions for inulinase production, by the test fungal culture, were observed when 5% inoculum was added to the minimal medium (pH 5.5) containing 1% inulin/ costus root powder as a carbon source and 0.15% NaNO3/ NH4Cl as a nitrogen source, and incubated at 30°C for 48h under shaker conditions (200 rpm). Maximum enzyme activity was observed at pH level of 5 and temperature level of 45°C, with thermal stability noted between 35°C-55°C. The I/S value of the crude enzyme was calculated to be 0.45 indicating true inulinase activity. It showed no significant inhibition in the presence of metal ions such as Zn+2, Mg+2, and Fe+3. The Ca+2 ions showed partial inhibition whereas Cu+2 ions showed an enhancement in the enzyme activity. These factors may present the test fungal culture isolated in the present study to be a potential candidate for the production of thermo-tolerant and metal resistant inulinase enzyme in order to be used for various biotechnological processes.

Coenzyme Q10 is one of the antioxidants with a worldwide market. Nowadays the coenzymeQ10 production has been considered by fermentation using microorganisms. In this study, the Response Surface Methodology was used to optimize culture composition for coenzyme Q10 production by a previously isolated bacterium, Gluconobacter japonicus FM10. A central composite design was employed to optimize the culture composition including sorbitol, yeast extract, peptone, KH2PO4, and MgSO4 for coenzyme Q10 production. The dry cell weight and coenzymeQ10 concentration were monitored as response variables and the desirability function approach was applied to obtain the optimum level for each factor. Results showed that an average, 3 mg/L of coenzyme Q10 was obtained when the optimized culture composition was employed (110 g/L of sorbitol, 25 g/L of yeast extract, 35 g/L of peptone, 0.5 g/L of KH2PO4, and 0.55 g/L of MgSO4). In addition, the expected dry cell weight reached 6 g/L in the presence of 90 g/L of sorbitol, 17.5 g/L of yeast extract, 35 g/L of peptone, 0 g/L of KH2PO4, and 1.7 g/L of MgSO4. The results of regression analysis revealed that the concentrations of peptone and sorbitol were the most effective factors in producing coenzyme Q10 and dry cell weight, respectively.

Understanding the association among various disorders has remarkably improved their diagnosis and therapies. It has been observed that autism and inflammatory bowel diseases cause a sort of inflammation. Exploring the relationship between them will lead to discovering important involved genes and in turn will eventually help discover any possible common therapeutic protocols. The aim of the present study was to determine the correlation between autism spectrum disorders and inflammatory bowel diseases. The common genes associated with autism spectrum disorders and inflammatory bowel diseases were retrieved from DisGeNET. SFARI databases and were subjected to an in silico data analysis framework to explore predictive genes and the related pathways. Eleven genes including HLA-DRB1, MTHFR, PON1, IL6, MTOR, SETD2, GSTM1, APC, IFNG, SERPINE1, and MAPK1 regulated by YY1 and IRF1 transcription factors were characterized as discriminating molecules which by further screening were enriched in pathways mostly involved in neutrophil apoptosis, neutrophil homeostasis, chemokine biosynthesis and the regulation of immune system response. According to findings it can be stated that the identified common genes were associated with a wide range of pathogenic mechanisms.