Purification, characterization and application of collagenolytic protease from Bacillus subtilis strain MPK.

A new extracellular protease from Bacillus subtilis strain MPK with collagenolytic activity was isolated and purified. Fish skin which otherwise would be treated as waste is used as substrate for the production of protease. Using various techniques such as ammonium sulphate precipitation and ion exchange chromatography, protease was purified and characterized subsequently. Protease of approximately 61 kDa molecular weight was purified by 135.7-fold with 18.42% enzyme recovery. The protease showed effective properties like pH and temperature stability over a broad range with optimum pH 7.5 and temperature 60 °C. Km and Vmax were found to be 1.92 mg ml-1 and 1.02 × 10-4 mol L-1 min-1, respectively. The protease exhibited stability in various ions, surfactants, inhibitors and organic solvents. Subsequently, the protease was successfully utilized for collagen hydrolysis to generate collagen peptides; thus, the produced protease would be a potential candidate for multifaceted applications in food and pharmaceutical industries due to its significant characteristics and collagenolytic properties.

Biochemical and in silico structural properties of a thermo-acid stable ß-glucosidase from Beauveria bassiana.

ß-glucosidase hydrolyses the glycosidic bonds in cellobiose and cello-oligosaccharides, a critical step in the saccharification for biofuel production. Hence, the aim of this study was to gain insights into the biochemical and structural properties of a ß-glucosidase from Beauveria bassiana, an entomopathogenic fungus. The ß-glucosidase was purified to homogeneity using salt precipitation, ultrafiltration, and chromatographic techniques, attaining a specific activity of 496 U/mg. The molecular mass of the enzyme was then estimated via SDS-PAGE to be 116 kDa, while its activity pattern was confirmed by zymography using 4-methylumbelliferyl-ß-d-glucopyranoside. Furthermore, the pH optima and temperature of the enzyme were found to be pH 5.0 and 60 °C respectively; its activity was significantly enhanced by Mg2+ and Na+ and was found to be relatively moderate in the presence of ethanol and dichloromethane. Molecular docking of the modelled B. bassiana ß-glucosidase structure with the substrates, viz., 4-nitrophenyl ß-d-glucopyranoside and cellobiose, revealed the binding affinity energies of -7.2 and -6.2 (kcal mol-1), respectively. Furthermore, the computational study predicted Lys-657, Asp-658, and Arg-1000 as the core amino acid residues in the catalytic site of the enzyme. This is the first investigation into a purified ß-glucosidase from B. bassiana, providing valuable insights into the functional properties of carbohydrases from entomopathogenic fungal endophytes.

Diverse origins of fibrinolytic enzymes: A comprehensive review.

Fibrinolytic enzymes cleave fibrin which plays a crucial role in thrombus formation which otherwise leads to cardiovascular diseases. While different fibrinolytic enzymes have been purified, only a few have been utilized as clinical and therapeutic agents; hence, the search continues for a fibrinolytic enzyme with high specificity, fewer side effects, and one that can be mass-produced at a lower cost with a higher yield. In this context, this review discusses the physiological mechanism of thrombus formation and fibrinolysis, and current thrombolytic drugs in use. Additionally, an overview of the optimization, production, and purification of fibrinolytic enzymes and the role of Artificial Intelligence (AI) in optimization and the patents granted is provided. This review classifies microbial as well as non-microbial fibrinolytic enzymes isolated from food sources, including fermented foods and non-food sources, highlighting their advantages and disadvantages. Despite holding immense potential for the discovery of novel fibrinolytic enzymes, only a few fermented food sources limited to Asian countries have been studied, necessitating the research on fibrinolytic enzymes from fermented foods of other regions. This review will aid researchers in selecting optimal sources for screening fibrinolytic enzymes and is the first one to provide insights and draw a link between the implication of source selection and in vivo application.

Biochemical engineering for elemental sulfur from flue gases through multi-enzymatic based approaches - A review.

Flue gases are the gases which are produced from industries related to chemical manufacturing, petrol refineries, power plants and ore processing plants. Along with other pollutants, sulfur present in the flue gas is detrimental to the environment. Therefore, environmentalists are concerned about its removal and recovery of resources from flue gases due to its activation ability in the atmosphere to transform into toxic substances. This review is aimed at a critical assessment of the techniques developed for resource recovery from flue gases. The manuscript discusses various bioreactors used in resource recovery such as hollow fibre membrane reactor, rotating biological contractor, sequential batch reactor, fluidized bed reactor, entrapped cell bioreactor and hybrid reactors. In conclusion, this manuscript provides a comprehensive analysis of the potential of thermotolerant and thermophilic microbes in sulfur removal. Additionally, it evaluates the efficacy of a multi-enzyme engineered bioreactor in this process. Furthermore, the study introduces a groundbreaking sustainable model for elemental sulfur recovery, offering promising prospects for environmentally-friendly and economically viable sulfur removal techniques in various industrial applications.

Multicystic Solitary Fibrous Tumor of the Kidney: A Case Report With Review of Literature.

Solitary fibrous tumor (SFT) is a rare mesenchymal neoplasm known to occur at various soft tissue and visceral locations. Kidney is one of the rare locations for these tumors with around 64 cases being available in the literature. Most of the renal SFTs are tan-white, solid, firm, unencapsulated, and lobulated masses. A predominantly cystic renal SFT has never been reported in the literature. Herein we describe a case of multicystic renal SFT in a 44-year-old male with the characteristic CD34 + /STAT6 + immunophenotype. A careful gross and microscopic examination is warranted while dealing with cystic spindle cell neoplasms of the kidney and SFT should always be considered in the differential diagnosis. STAT6 immunohistochemistry is quite specific for the diagnosis. Moreso, a detailed immunopanel is necessary to exclude other spindle cell neoplasms of the kidney because of significant therapeutic and prognostic implications.

Cellulosic Ethanol Production Using Waste Wheat Stillage after Microwave-Assisted Hydrotropic Pretreatment.

One of the key elements influencing the efficiency of cellulosic ethanol production is the effective pretreatment of lignocellulosic biomass. The aim of the study was to evaluate the effect of microwave-assisted pretreatment of wheat stillage in the presence of sodium cumene sulphonate (NaCS) hydrotrope used for the production of second-generation bioethanol. As a result of microwave pretreatment, the composition of the wheat stillage biomass changed significantly when compared with the raw material used, before treatment. Microwave-assisted pretreatment with NaCS effectively reduced the lignin content and hemicellulose, making cellulose the dominant component of biomass, which accounted for 42.91 ± 0.10%. In post pretreatment, changes in biomass composition were also visible on FTIR spectra. The peaks of functional groups and bonds characteristic of lignins (C-O vibration in the syringyl ring, asymmetric bending in CH3, and aromatic skeleton C-C stretching) decreased. The pretreatment of the analyzed lignocellulosic raw material with NaCS resulted in the complete conversion of glucose to ethanol after 48 h of the process, with yield (in relation to the theoretical one) of above 91%. The highest observed concentration of ethanol, 23.57 ± 0.10 g/L, indicated the high effectiveness of the method used for the pretreatment of wheat stillage that did not require additional nutrient supplementation.

Isolation, biochemical characterization, and development of a biodegradable antimicrobial film from Cirrhinus mrigala scale collagen.

Collagen is a promising candidate for food and pharmaceutical applications due to its excellent biocompatibility, low antigenicity, and controlled biodegradability; however, its heavy price restricts its utilization. Fish scales generated during the processing are generally regarded as waste material and an environmental pollutant, though they are a promising source of collagen. In the present study, Cirrhinus mrigala scales were demineralized and extracted for acid-soluble collagen (ASC) using acetic acid, with a collagen yield of 2.7%. UV-Vis spectra, SDS-PAGE, FTIR analyses, and amino acid composition confirmed the type I nature of the collagen extracted. The denaturation temperature of the collagen was found to be 30.09 °C using differential scanning calorimetry (DSC). The collagen was highly soluble at acidic pH and lower NaCl concentrations while its solubility was lowered in alkaline conditions and NaCl concentrations above 0.5 M. The collagen exhibited good emulsifying potential with an emulsion activity index (EAI) and emulsion stability index (ESI) of 21.49 ± 0.22 m2 g-1 and 15.67 ± 0.13 min, respectively. Owing to the good physicochemical characteristics of the extracted collagen, collagen-chitosan-neem extract (CCN) films were prepared subsequently which showed good antimicrobial activity against Bacillus subtilis NCIM 2635, Staphylococcus aureus NCIM 2654, Escherichia coli NCIM 2832, and Pseudomonas aeruginosa NCIM 5032, suggesting the potential of collagen in the development of antimicrobial films. These results demonstrate that the collagen from fish waste could be valorized and used effectively along with chitosan and neem extract for the synthesis of novel biodegradable films with antimicrobial efficacy.

Beauveria bassiana Xylanase: Characterization and Wastepaper Deinking Potential of a Novel Glycosyl Hydrolase from an Endophytic Fungal Entomopathogen.

Beauveria bassiana is an entomopathogenic fungus widely used as a biopesticide for insect control; it has also been shown to exist as an endophyte, promoting plant growth in many instances. This study highlights an alternative potential of the fungus; in the production of an industrially important biocatalyst, xylanase. In this regard, Beauveria bassiana SAN01 xylanase was purified to homogeneity and subsequently characterized. The purified xylanase was found to have a specific activity of 324.2 U·mg-1 and an estimated molecular mass of ~37 kDa. In addition, it demonstrated optimal activity at pH 6.0 and 45 °C while obeying Michaelis-Menton kinetics towards beechwood xylan with apparent Km, Vmax and kcat of 1.98 mg·mL-1, 6.65 µM·min-1 and 0.62 s-1 respectively. The enzyme activity was strongly inhibited by Ag2+ and Fe3+ while it was significantly enhanced by Co2+ and Mg2+. Furthermore, the xylanase was shown to effectively deink wastepaper at an optimal rate of 106.72% through its enzymatic disassociation of the fiber-ink bonds as demonstrated by scanning electron microscopy and infrared spectroscopy. This is the first study to demonstrate the biotechnological application of a homogeneously purified glycosyl hydrolase from B. bassiana.

Cold plasma for the preservation of aquatic food products: An overview.

Cold plasma (CP) is an upcoming technology implemented for the preservation of highly perishable foods, especially aquatic food products (AFPs). The high moisture content, high-quality protein with all essential amino acids and unsaturated fatty acids makes AFP more susceptible to microbial spoilage and oxidation of lipids and proteins. Spoilage lowers the nutritive value and could generate toxic components, making it unsafe for consumption. In recent times, the rising demand for food products of aquatic origin with preserved quality and extended shelf-life has been recorded. In addition, minimally or nonthermally processed and preserved foods are gaining great attention. CP technology has demonstrated an excellent ability to inactivate microorganisms without promoting their resistance and triggering some deteriorative enzymes, which are typical factors responsible for the spoilage of AFP. Consequently, CP could be recommended as a minimal processing intervention for preserving the quality of AFP. This review focuses on different mechanisms of fish spoilage, that is, by microorganisms and oxidation, their inhibition via the application of CP, and the retention of quality and shelf-life extension of AFP.

Characterisation, pathogenicity and hydrolytic enzyme profiling of selected Fusarium species and their inhibition by novel coumarins.

Three Fusarium species isolated locally were characterised by the amplification of their rDNA ITS region, host specificity, and hydrolytic enzyme production. The strains were identified as Fusarium pseudoanthophilum, which is being reported for the first time in South Africa, as well as F. foetens and F. fujikuroi. All the three strains were capable of infecting vegetables such as tomatoes, bell and cayenne peppers, belonging to the Solanaceae family. The Fusarium strains also showed significant production of cell wall degrading enzymes in vitro, such as amylase, cellulase, xylanase, and polygalacturonase, thus highlighting the possibilities of these enzymes as pathogenic factors. Subsequently, the strains were discovered to be susceptible to three halogenated coumarins. The most effective of the tested coumarins, 6-bromo3-2,2-dibromoacetyl-2H-chromen-2-one, showed MIC values of 0.125, 0.0625 and 0.125 mg/ml against F. foetens, F. pseudoanthophilum and F. fujikuroi, respectively. The antifungal potentials of the halogenated coumarins were confirmed in silico through PASS analysis, toxicity prediction and docking studies. Findings from this study demonstrate the use of these coumarins as potential control agents against the Fusarium species and other pathogenic fungi in general.

Environmental Impacts of Microplastics and Nanoplastics: A Current Overview.

The increasing distribution of miniaturized plastic particles, viz. microplastics (100 nm-5 mm) and nanoplastics (less than 100 nm), across the various ecosystems is currently a subject of major environmental concern. Exacerbating these concerns is the fact that microplastics and nanoplastics (MNPs) display different properties from their corresponding bulk materials; thus, not much is understood about their full biological and ecological implications. Currently, there is evidence to prove that these miniaturized plastic particles release toxic plastic additives and can adsorb various chemicals, thereby serving as sinks for various poisonous compounds, enhancing their bioavailability, toxicity, and transportation. Furthermore, there is a potential danger for the trophic transfer of MNPs to humans and other higher animals, after being ingested by lower organisms. Thus, this paper critically analyzes our current knowledge with regard to the environmental impacts of MNPs. In this regard, the properties, sources, and damaging effects of MNPs on different habitats, particularly on the biotic components, were elucidated. Similarly, the consequent detrimental effects of these particles on humans as well as the current and future efforts at mitigating these detrimental effects were discussed. Finally, the self-cleaning efforts of the planet via a range of saprophytic organisms on these synthetic particles were also highlighted.

Plastic biodegradation: Frontline microbes and their enzymes.

Plastic polymers with different properties have been developed in the last 150 years to replace materials such as wood, glass and metals across various applications. Nevertheless, the distinct properties which make plastic desirable for our daily use also threaten our planet's sustainability. Plastics are resilient, non-reactive and most importantly, non-biodegradable. Hence, there has been an exponential increase in plastic waste generation, which has since been recognised as a global environmental threat. Plastic wastes have adversely affected life on earth, primarily through their undesirable accumulation in landfills, leaching into the soil, increased greenhouse gas emission, etc. Even more damaging is their impact on the aquatic ecosystems as they cause entanglement, ingestion and intestinal blockage in aquatic animals. Furthermore, plastics, especially in the microplastic form, have also been found to interfere with chemical interaction between marine organisms, to cause intrinsic toxicity by leaching, and by absorbing persistent organic contaminants as well as pathogens. The current methods for eliminating these wastes (incineration, landfilling, and recycling) come at massive costs, are unsustainable, and put more burden on our environment. Thus, recent focus has been placed more on the potential of biological systems to degrade synthetic plastics. In this regard, some insects, bacteria and fungi have been shown to ingest these polymers and convert them into environmentally friendly carbon compounds. Hence, in the light of recent literature, this review emphasises the multifaceted roles played by microorganisms in this process. The current understanding of the roles played by actinomycetes, algae, bacteria, fungi and their enzymes in enhancing the degradation of synthetic plastics are reviewed, with special focus on their modes of action and probable enzymatic mechanisms. Besides, key areas for further exploration, such as the manipulation of microorganisms through molecular cloning, modification of enzymatic characteristics and metabolic pathway design, are also highlighted.

Enhanced xylanase and endoglucanase production from Beauveria bassiana SAN01, an entomopathogenic fungal endophyte.

This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.

Production, Purification and Characterization of a Novel Bacteriocin Produced by Bacillus subtilis VS Isolated from Mango (Mangifera indica L)

Abstract Bacteriocin has been identified as an excellent alternative to chemical preservatives due to its astonishing antimicrobial activity against food spoiling and food-borne pathogens. So there is a need to identify the newer and potent sources of bacteriocin producers. This study aims the isolation of potent bacteriocin producing microorganism from fresh fruits and vegetables, its production, purification, and characterization. Firstly, 43 isolates were analysed for its antimicrobial potential, out of which7 were found to inhibit the growth of various pathogens. Considering the results of antimicrobial activity; the microorganism isolated from mango was regarded as the most potent one; which was identified as Bacillus subtilis VS.70% ammonium sulphate precipitated and dialysed bacteriocin was purified using DEAE cellulose and sephadex G75 chromatography. Bacteriocin was purified by 24.64 fold with 8.65% recovery and its molecular weight was found to be 31.2kDa. The Purified bacteriocin was found to be stable at broad pH and temperature. It was found to be degraded by various proteases studied confirming its proteinaceous nature. Considering all these attributes; the purified bacteriocin isolated from Bacillus subtilis VS can be exploited by various food industries.

Biotechnological potential of Beauveria bassiana as a source of novel biocatalysts and metabolites.

Beauveria bassiana though widely perceived as an entomopathogenic fungus has also been found in nature to be endophytic. As entomopathogens, the life cycle of different B. bassiana strains are organized and adapted as pathogens to their invertebrate hosts while as endophytes they maintain a symbiotic relationship with their plant hosts. To fulfill these aforementioned ecological roles, this fungus secretes an array of enzymes as well as secondary metabolites, which all have significant biological roles. Basically, chitinases, lipases and proteases are considered to be the most important of all the enzymes produced by B. bassiana. However, studies have also shown their ability to produce other vital enzymes which include amylase, asparaginase, cellulase, galactosidase etc. Previous reports on this filamentous fungus have laid more emphasis on its entomopathogenicity, its endophytism and its highly acclaimed application in the biological control of pests. This review, however, is the first to fully assess the enzyme-secreting potential of this entomopathogenic fungus and its use as a novel source of several industrial biocatalysts and other important biochemicals. This article highlights the inherent properties of the fungus to degrade various biopolymers as well as its relative safety for human use. Some of the important factors have raised the possibilities of exploitation for industrial production and as safe hosts for gene expression.

Statistical media optimization for the production of clinical uricase from Bacillus subtilis strain SP6.

In this study, a potent uricase producing organism was isolated by a thorough screening and identified as Bacillus subtilis strain SP6 by using 16s rDNA sequencing. Response surface methodological optimization was employed for the enhanced production of uricase from newly isolated Bacillus subtilis strain SP6. In media optimization studies, Plackett Burman (PB) design was used for the selection of the critical media components; which were further optimized using central composite design (CCD). Lactose, soya peptone, uric acid and FeSO4.7H2O were found to be the critical factors influencing the enzyme production. Optimum uricase production with these factors was deduced using central composite design. Significant level of the factors were 12.2 g/L of lactose, 12.79 g/L of soya peptone, 2.55 g/L of uric acid and 0.00325 g/L FeSO4.7H2O. Use of statistical optimization upsurges uricase yield from 1.2 U/ml to 15.87 U/ml enhancing the overall production by 13.23 fold; which confirms that the model is effective for process optimization.

Use of statistical experimental methods for optimization of collagenolytic protease production by Bacillus cereus strain SUK grown on fish scales.

In this study, novel and cheap sources like fish scales and molasses were used for the production of collagenolytic protease. Statistical optimization of different parameters for the production of collagenolytic protease by Bacillus cereus strain SUK has been carried out using response surface methodology (RSM). Three most significant medium components identified by Plackett-Burman (PB) were fish scales, molasses, and incubation time, which were further optimized using central composite design (CCD). The medium having fish scales 9.38 g l-1, molasses 2.42 g l-1, and incubation time of 67.34 h was found to be optimum for maximum collagenolytic protease production. B. cereus strain SUK has shown multiple plant growth-promoting traits, whereas degraded fish scale hydrolysates (FSHs) were having antimicrobial as well as plant growth-promoting abilities. The collagenolytic efficiency of this isolate can be exploited in an eco-friendly process of bioconversion of fish waste, representing an alternative way of waste management that could be used to produce various value-added products, such as collagenolytic protease, microbial biomass, amino acids, protein hydrolysates, and collagen peptides.

Synergistic effect of Chryseobacterium gleum sp. SUK with ACC deaminase activity in alleviation of salt stress and plant growth promotion in Triticum aestivum L.

Soil salinity is major abiotic stresses affecting morphological, biochemical and physiological processes of plant growth. Chryseobacterium gleum sp. SUK isolated from salt-stressed soil exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity with IAA (indole acetic acid), siderophore, ammonia, hydrogen cyanide production, 2% salt tolerance and fungal cell wall degrading enzyme production (cellulase, protease). The isolate also showed a poultry feather degrading activity which is the main waste material of poultry industry and opulent source of proteins, amino acids, nitrogen, phosphorous, calcium, potassium, manganese, zinc and copper. Application of feather-degraded lysate with the degrading isolate, C. gleum sp. SUK denotes triple role of bioformulation to surmount salinity stress, management of poultry waste disposal and utilization of feathers degraded products as a biostimulant for better growth of plants as well as strain SUK having multifarious plant growth promoting traits. Wheat crops exposed to salt stressor were inoculated with studied bioformulation. Results of plant analysis showed improvement in root and shoot length, fresh and dry weight, chlorophyll, proteins, amino acids, phenolics, flavonoids content and decreased level of proline. In addition, Na+ uptake was decreased and K+ uptake was increased. Therefore, application of novel bioformulation could increase the yield of crops by ameliorating growth of plants and alleviating the salinity stress.

Response surface methodology-based optimization of production media and purification of α-galactosidase in solid-state fermentation by Fusarium moniliforme NCIM 1099.

Response surface methodology was used to enhance the production of α-galactosidase from Fusarium moniliforme NCIM 1099 in solid-state fermentation. Plackett-Burman design was employed for selection of critical media constituents which were optimized by central composite rotatable design. Wheat bran, peptone and FeSO4·7H2O were identified as significant medium components using PB design. Further CCRD optimized medium components as wheat bran; 4.62 µg, peptone; 315.42 µg, FeSO4·7H2O; 9.04 µg. RSM methodological optimization increased the enzyme production from 13.17 to 207.33 U/g showing 15.74-fold enhancement. The α-galactosidase was purified by 70% fractionation followed by DEAE anion exchange column chromatography which yields 23.33% with 28.68-fold purification. The molecular weight of α-galactosidase was 57 kDa which was determined by SDS-PAGE analysis. Purified enzyme has optimum pH of 4.0 and was found to be stable in wide pH range of 3.0-9.0. Its optimum temperature was 50 °C, whereas its activity remains above 50% up to 2 h at 75 °C. Hg2+ was found to be a potent inhibitor and Mg2+ acted as an activator of enzyme. No significant change was observed in enzyme activity for galactose concentration, ranging from 1 to 100 mM. The K m values of enzyme for substrates p-nitrophenyl-α-D-galactopyranoside, melibiose and raffinose were 0.20, 1.36, and 3.66 mM, respectively. Low K m and stability to various physiological conditions of enzyme represents its potential which can be exploited in various industrial applications.