Since last century or so anthropogenic activities have intensely metamorphosed the earth’s ecosystem and resulted into major environmental changes. Widespread interference of human related activities have resulted in major problems including environmental pollution, land degradation, global warming/climate change, paucity of potable water supply and biodiversity loss. These issues have directly affected the quality and sustainability of the ecosystems. In addition, these activities have resulted in loss of habitats resulting in mass extinction of species which in itself is a matter of great concern. Studies and data clearly show that if present trends continue the conditions are expected to worsen in the coming time and human civilization itself will be in trouble. To minimize this crisis, possible green solutions like use of microbes and biotechnological tools are gaining importance and need further attention in order to lessen or remediate the harmful effects of anthropogenic activities thus ensuring environmental sustainability.

Environmental sustainability: challenges and viable solutions

Marine bacterial species contribute to a significant part of the oceanic population, which substantially produces biologically effectual moieties having various medical and industrial applications. The use of marine-derived bacterial pigments displays a snowballing effect in recent times, being natural, environmentally safe, and health beneficial compounds. Although isolating marine bacteria is a strenuous task, these are still a compelling subject for researchers, due to their promising avenues for numerous applications. Marine-derived bacterial pigments serve as valuable products in the food, pharmaceutical, textile, and cosmetic industries due to their beneficial attributes, including anticancer, antimicrobial, antioxidant, and cytotoxic activities. Biodegradability and higher environmental compatibility further strengthen the use of marine bio-pigments over artificially acquired colored molecules. Besides that, hazardous effects associated with the consumption of synthetic colors further substantiated the use of marine dyes as color additives in industries as well. This review sheds light on marine bacterial sources of pigmented compounds along with their industrial applicability and therapeutic insights based on the data available in the literature. It also encompasses the need for introducing bacterial bio-pigments in global pigment industry, highlighting their future potential, aiming to contribute to the worldwide economy.

An Overview on Industrial and Medical Applications of Bio-Pigments Synthesized by Marine Bacteria.

The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi. The presence of aprN, a gene-encoding nattokinase, was verified with PCR method. The predicted amino acid sequence was aligned with homologous sequences, and a phylogenetic tree was constructed. Nattokinase was sublimated with ammonium sulfate, using a DEAE-Sepharose Fast Flow column, a CM-Sepharose Fast Flow column and a Sephadex G-75 gel filtration column. SDS-PAGE analysis indicated that the molecular weight of the purified nattokinase from Bacillus subtilis MX-6 was about 28 kDa. Fermentation of Bacillus subtilis MX-6 nattokinase showed that nattokinase production was maximized after 72 h; the diameter of clear zone reached 21.60 mm on the plasminogen-free fibrin plate. Nattokinase production by Bacillus subtilis MX-6 increased significantly after supplementation with supernatant I, supernatant II and soy peptone but decreased substantially after the addition of amino acids. This result indicated that the nattokinase production by B. subtilis MX-6 might be induced by soybean polypeptides. The addition of MgSO4 and CaCl2 increased B. subtilis MX-6 nattokinase production.

Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6.

Synthetic coloring agents have been broadly utilized in several industries such as food, pharmaceuticals, cosmetic and textile. Recent surveys on the potential of teratogenicity and carcinogenicity of synthetic dyes have expressed concerns regarding their use in foods. Worldwide, food industries have need for safe, natural and new colorings to add variety to foods and make them appealing to consumers. Natural colorings not only expand the marketability of the food product, but also add further healthful features such as antibacterial, antioxidant, anticancer and antiviral properties. Novel microbial strains should be explored to meet the increasing global search of natural pigments and suitable techniques must be developed for the marketable production of new pigments, using microbial cultures, viz., fungi, and bacteria. To address the issue of the natural coloring agents, this review presents the recent trends in several studies of microbial pigments, their biological properties and industrial applications.

Microbial pigments as an alternative to synthetic dyes and food additives: a brief review of recent studies.

Several microbial proteases were reported for dehairing; however, they become unstable and lose the activity at alkaline conditions during the process. Hence, there is a need for protease that withstands its activity at high pH. This work attempts to screen and isolate alkaline protease from marine Streptomyces. An alkaline protease producing strain Streptomyces sp. GS-1 was isolated from the backwaters of Munanbam and Valapad, Kerala, India. Growth characteristics and protease production were investigated in 10 different basal media (BM) supplemented with four different substrates viz. skim milk, casein, wheat bran and rice bran. Maximum protease production was detected in BM2 medium with wheat bran after 9.5 days of incubation. The activity of extracted protease was found to be 341 U/ml and a correlation coefficient of 0.9532 between growth and enzyme production was observed. The crude enzyme exhibited maximum activity at 45 °C and pH 8.5. The Km and Vmax values of the enzyme were determined as 5.29% and 32.25 μmol l−1 min−1 mg−1, respectively, using 1% casein as substrate. The enzyme sustained its activity at high temperature (50 °C for 8 h), alkaline pH (8–10) and in the presence of metal ions and organic solvents. The purified enzyme was inhibited by phenylmethylsulphonyl fluoride (PMSF) suggesting it belongs to serine protease. The enzyme displayed dehairing property and successfully removed the hair follicles as tested in goat hide. Thus, the enzyme extracted from Streptomyces sp. GS-1 was found to be effective in dehairing during leather processing.

Extraction and characterization of alkaline protease from Streptomyces sp. GS-1 and its application as dehairing agent

The existing study is purposeful on the intracellular pigment extraction from actinomycetes isolated from Kovalam Beach regions of Chennai, Tamil Nadu, India. Only one actinobacterial isolate showed pigmented growth out of total 4 isolates. Ethyl acetate as the solvent was used in cell disruption technique for the extraction of intracellular pigments. UV-Visible spectrophotometry, FT-IR spectroscopy, HPLC and GC-MS were used for the partial characterization of the pigment. The extracted pigment was applied for the preparation of lip balm and assessing its textile dyeing property. In addition, the extracted pigment was analysed for antioxidant, antibacterial activity, MTT assay and haemolytic activity. On optimization, dextrose and maltose were the best carbon sources. The finest nitrogen sources were found to be casein and peptone. The optimum temperature range was 35°C -40°C and optimal pH was found to be between 6.0 and 8.0. The obtained results showed potent antioxidant activity and found to be non-toxic to human erythrocytes.

https://iopscience.iop.org/article/10.1088/1757-899X/263/2/022049/pdf

Pigment from Streptomyces bellus MSA1 isolated from marine sediments

Nattokinase (NK) is a serine protease enzyme of the subtilisin family. It exhibits a strong fibrinolytic activity. The fibrinolytic enzymes from Bacillus sp. have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process including plasmin activation. In the present study, VIT garden soil was collected and subjected to isolation process in order to screen for the NK production. Screening for NK enzyme was performed by radial caseinolytic assay. The production of NK enzyme was done in two different production medium for comparative studies. The NK enzyme was purified by gel permeation chromatography. The activity of the purified NK was checked by clot lysis and casein digestion assay. To investigate the structural basis of NK and fibrinogen interaction and also to identify the best binding mode, molecular dynamics and docking studies were performed. Based on the morphological and biochemical characterization, the isolate was identified as Bacillus sp. The overall purification fold of NK was about 3 with the specific activity of 664U/mg and 9.9% yield. Homogeneity of the purified enzyme was analyzed and confirmed by the single band obtained in SDS-PAGE. Molecular weight of the purified protease was estimated as 25 kDa. Purified NK enzyme exhibited 97% of effective clot lysis activity. The NK was docked in to the knob region of the fibrinogen at its binding site using Dock server. A total of 26 residues of fibrinogen and 29 residues of NK constitute the interface region. However, 9 residues of fibrinogen (THR238, MET264, LYS266, ARG275, THR277, ALA279, ASN308, MET310, and LYS321) and 8 residues of NK (GLY61, SER63, THR99, PHE189, LEU209, TYR217, ASN218, and MET222) are involved in intact binding. A significant amount of NK enzyme was obtained from Bacillus sp. The docking analysis revealed that the NK and fibrinogen adopt an extended binding pattern and interacts with the crucial residues to exhibit their activity.

In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.

The current research work aims at optimization, production, purification and evaluation of fibrinolytic extracellular protease from Bacillus subtilis VITMS2 isolated from fermented milk of Vigna unguiculata. The optimal production was achieved at 4.0% inoculum, pH7.0, 30 °C with (1% w/v) sucrose, (2% w/v) soya bean meal and (2% w/v) malt extract and 10 mM of CaCl2, MgSO4, Na2HPO4 and K2HPO4. The clear cell-free supernatant was purified using conventional ammonium sulphate salt fractionation (75%), ultrafiltration, ion-exchange (DEAE Sepharose FF) and gel filtration (Sephadex G-50). The molecular mass was determined to be 29 kDa using SDS-PAGE analysis. The purified enzyme showed strong fibrinolytic activity with a specific activity of 2418.85 U/mg and has a yield of 12.01%. The enzyme was highly stable up to 60 °C and a pH range of 10.0 until 72 h of incubation. The purified enzyme showed 97.4% in vitro thrombolytic activity. The Km and Vmax values of the enzyme was determined to be 0.0114 mM and 147.8 µmol min−1 using the chromogenic substrate S-7388. IC50 of ace inhibition was assessed to be 0.06 mg/mL suggesting anti-hypertensive property of the fibrinolytic enzyme. The above-obtained ace-inhibition results was supported by in silico molecular docking studies which revealed better binding affinity of nattokinase with a HADDOCK score of − 22.0 ± 8.5 confirms affinity towards angiotensin converting enzyme.

Fibrinolytic and ACE Inhibitory Activity of Nattokinase Extracted from Bacillus subtilis VITMS 2: A Strain Isolated from Fermented Milk of Vigna unguiculata

The main aim of the review is to recapitulate the effects of thrombotic diseases, clotting disorders, and the methodology of prospecting a range of thrombolytics from various sources and thrombolytic therapies. Thrombolytic medications are administered to infringe dangerous clots inside blood vessels when there is deprivation in this haemostatic balance. Certain bioactive compounds present in herbs, plant leaves, roots and fruits exhibit thrombolytic activities. Serine proteases, metalloproteases and some cysteine proteases possess thrombolytic properties. These proteases are found in plants, humans, animals and microbes as well. Thrombolytics which are of clinical importance are discussed in this article along with its variants and biological sources exhibiting thrombolytic activity. Alternative therapies followed in recent days are also discussed, including future prospects of research on thrombolytics. Knowledge of thrombolytic biodiversity plays a decisive role in choosing safe, economic and effective thrombolytics for scale up and commercialization.

Focalization of thrombosis and therapeutic perspectives: a memoir

Equilibrium of biodiversity is essential for the maintenance of the ecosystem as they are interdependent on each other. The decline in biodiversity is a global problem and an inevitable threat to the mankind. Major threats include unsustainable exploitation, habitat destruction, fragmentation, transformation, genetic pollution, invasive exotic species and degradation. This review covers the management strategies of biotechnology which include sin situ, ex situ conservation, computerized taxonomic analysis through construction of phylogenetic trees, calculating genetic distance, prioritizing the group for conservation, digital preservation of biodiversities within the coding and decoding keys, molecular approaches to asses biodiversity like polymerase chain reaction, real time, randomly amplified polymorphic DNA, restriction fragment length polymorphism, amplified fragment length polymorphism, single sequence repeats, DNA finger printing, single nucleotide polymorphism, cryopreservation and vitrification.

https://iopscience.iop.org/article/10.1088/1757-899X/263/2/022047/pdf

S. Merlyn Keziah

Papers

Pigment from Streptomyces bellus MSA1 isolated from marine sediments

In vitro and in silico studies on fibrinolytic activity of nattokinase: A clot buster from Bacillus sp.

Fibrinolytic and ACE Inhibitory Activity of Nattokinase Extracted from Bacillus subtilis VITMS 2: A Strain Isolated from Fermented Milk of Vigna unguiculata

Focalization of thrombosis and therapeutic perspectives: a memoir

Essentials of Conservation Biotechnology: A mini review