Review on Microbial Sources, Production, Purification and Potential Industrial Applications of Laccases

Laccase (EC 1.10.3.2) is a multicopper blue oxidase which are involved in the oxidation of a broad range of organic substrates, including phenols, polyphenols, anilines, and even certain inorganic compounds by a one-electron transfer mechanism. Laccases are widely distributed in bacteria, fungai, insects and higher plants. There are mainly two production techniques for cultivation of laccase such as submersed fermentation and solid- state fermentation. This paper briefly discuss the effect of carbon source, effect of nitrogen source, effect of inducers, effects of surfactants, effect of agitator, influence of metal ions and use of agro-industrial waste in production medium. The paper also discussed the purification techniques such as ammonium sulphate precipitation for extraction purpose followed by dialysis and ion-exchange chromatography as well characterization techniques. Laccases are known to show application ranging from pharmaceutical industries to textile sector as well as in biosensor development.

Study on Purification and Characterization of Polyphenol Oxidase from Acetes chinensis

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis, was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant Km was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn2+ and K+ could enhance the activity of PPO; that Ba2+ and Ca2+ could inhibit the activity of PPO; and that Cu2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

Isolation, purification and characterization of mannan degrading enzyme from a new isolate Acinetobacter baummanni

A mannan-degrading microbe was isolated from rotting porang tubers (Amorphophallus muelleri Blume). Molecular identification using 16S-rRNA sequence analysis revealed that the isolate showed 99.67% similarity with Acinetobacter baummanni. A crude enzyme from ammonium sulphate precipitation was used for preliminary characterization. The characterization results showed that the enzyme activity is optimum at 45 °C, and stable at 35-50 °C, while the optimum pH is 7, and stable at pH 5-7. The substrate with the highest relative activity was found in guar gum which was 137.512%. The enzyme activity was inhibited by Ca, Na, K ions, and increased by Mn2+ ions.

Bacterial Protease Inhibitors as Antibacterial agents to prevent Bacterial Infections Associated with Biofilms

Isolation of protease inhibitor producing bacteria from microbial mat and investigating its anti-biofilm potential against biofilm producing organism was selected as the main objective of the present study. Protease inhibitor (PI) was produced from bacterial isolates and purified using ammonium sulphate precipitation methods. Primary and secondary protease inhibitor assay was carried out separately to confirm the inhibition of protease enzyme activity both qualitatively and quantitatively. Antibacterial activity and anti-biofilm assay was performed to determine the biofilm prevention capabilities of PI. Three isolates (B1PI, B2PI and B3PI) were screened and B2PI bacterial culture was selected based on the results of primary and secondary protease inhibitor assay. Maximum trypsin inhibition of 77.5±0.25% was recorded for the isolate B2PI. Antibacterial activity of the B2PI protease inhibitor fractions exhibited inhibitory zones of 22.3±1.04mm and 20.2±0.25mm against Escherichia coli and Staphylococcus aureus respectively. Anti-biofilm assay of protease inhibitor fractions expressed 31.2μl/ml of MBIC against Escherichia coli and Staphylococcus aureus. The results conclude that, the protease inhibitor from the microbial mat isolate will be an effective alternative to the commercial antibiotics either alone or in combination with other drugs synergistically which shall be studied elaborately in future.

Carbohydrate-binding specificity of concanavalin lectin from Canavalia spp.

The carbohydrate-binding specificity of lectins from the four accessions of Canavalia seeds (C. ensiformis (ConA), C. cathartica (ConC), C. gladiata (ConG) and C. rosea (ConM) was studied by hemagglutination inhibition assay using monosaccharides, disaccharides and sugar derivatives. Canavalia seed extracts subjected to ammonium sulphate precipitation and the fraction with higher specific activity were analysed for carbohydrate-binding specificity and its mitogenic potential. All four lectins exhibited similar carbohydrate-binding specificity in agglutination inhibition which is in line with docking experiments. Dmannose and D-maltose were highly specific than other sugars. The results of computational method revealed differences in the affinity towards various carbohydrates. Mitogenic activity of all four lectins in human lymphocytes showed varied mitotic index. Among the four lectins studied, binding affinity to mannose and proliferation index was in the order ConG>ConA>ConM>ConC accounting to the efficacy of biological functions of highly similar analogues.

Purification, Characterization of an Entomopathogenic Fungal Lectin from Purpureocillium Lilacinum, and its Involvement in Pathogenesis Leading to Mycotic Keratitis

A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non reducing conditions. PCL is a blood group non specific lectin and has highest affinity towards Chitin, Mucin, asialo mucin, Fetuin with a MIC of 0.15µg/mL and also recognizes L-fucose, galactose, lactose, N-acetly galactosamine, Hyaluronic acid. PCL is stable up to 60 ºC and within the pH range 4–8. To understand its role in pathogenesis, effect of PCL was evaluated on Human Corneal Epithelial Cells (HCECs). PCL showed strong glycan mediated binding to HCECsand PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.

Thermal stability of purified superoxide dismutase from liver of commercially valuable fish, Labeo rohita Exposed to Pb+Cr mixture

In this experiment, effect of lead (Pb) + chromium (Cr) mixture on superoxide dismutase (SOD) in the liver of Labeo rohita at a concentration of 11.1 mgL-1 was observed. The ammonium sulphate precipitation and ion exchange chromatography techniques were successfully used to purify SOD. After purification, SOD activity of control and Pb+Cr treated fish was noted as 581.00 and 645.45 UmL-1, respectively while the specific activity was 1383.33 and 1613.62 Umg-1, respectively. The fold purification value of SOD was 2.75 and 2.45 for control and stressed fish, respectively. The recovery was calculated as 77.06 and 57.43% for control and stressed fish, respectively. The results of kinetic characterization showed that SOD form control and exposed fish had maximum activity at pH 6.5 and 7.0. Temperature also had a significant effect on activity of SOD. The SOD activity was measured maximum at 30°C for both control and Pb+Cr exposed fish. The Km value of liver SOD for control and Pb+Cr treated L. rohita was calculated as 1.48 and 0.62 mM, respectively. The value of Vmax for SOD from liver of control and Pb+Cr exposed fish was 1000 and 570 U mL-1, respectively. The enthalpy of denaturation (∆H*) for liver SOD from control and Pb+Cr exposed L. rohita was computed as 3.492 and 2.802 KJ mol-1 at 40°C, respectively and these values were dropped off with increasing the temperature until it remains 3.251 and 2.561 KJ mol-1 at 70°C, respectively. The free energy of thermal denaturation (ΔGº) of liver SOD was slightly increased with increasing temperature until 75°C which shows its resistance against heat. The values of ΔGº was observed as 58.03 and 57.95 KJ mol-1 for control and exposed fish at 40°C, respectively while the same was increased upto 62.37 and 62.00 KJ mol-1 at 70°C, respectively. It was concluded from negative value of ΔS* (entropy of inactivation) that the SOD is stable thermodynamically.

Anti-trypsin and antihypertensive activity of protein extracts from Nigella sativa seeds

Protease inhibitors (PIs) are a ubiquitous, diverse group of molecules present in multiple forms in all organisms. These inhibitors inactivate proteases from predators/pathogens in addition to regulating intracellular proteolysis. In addition to intracellular localization, storage organs of plants are also a potential site of protease inhibitors. Proteins with trypsin inhibitory activity were isolated from Nigella sativa seed extracts by ammonium sulphate precipitation. Extraction conditions were optimized by choosing an optimum solvent, temperature and incubation period. The highest inhibitory activity of protein extracts was achieved by using 50 mM Tris buffer as solvent and overnight incubation of the suspension at 4°C. The crude seed extract fractionated at 60% ammonium sulphate concentration exhibited highest trypsin inhibitory activity, i.e., 60.15 ± 2.95 %, which was comparable to soybean trypsin inhibitor used as positive control. Ammonium sulphate precipitation of crude extract yielded 39.83-fold purification. Partially purified trypsin inhibitor exhibited 2.39±0.23 TIU mg-1. Additionally, Nigella sativa protein extracts were also investigated for their health-promoting effects against two important proteases, α- Dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE). Highest inhibitory activity against ACE was shown by the crude extract of N. sativa. Among AS fractions, 30% ammonium sulphate concentration exhibited highest inhibition activity against ACE and DPP-IV. Our results suggest that the widely believed role of N. sativa in control of hypertension may at least be partially shared by inhibition of ACE. This is the first study conducted to evaluate the biological activity of N. sativa protein extracts suggesting a potential use of N. sativa proteins in management of hypertension as well as an important source of trypsin inhibitor. Further identification, purification and characterization of different bioactive compounds from N. sativa are being carried out.

Extraction, Purification of Tyrosinase and Tyrosinase Inhibitory Activity of Four Medicinal Plants from Nanded District (MS), India

Tyrosinase has an important role in melanin formation, is responsible for the production of colour pigments of skin, hair, and eye. In the presents study, tyrosinase was isolated from Mushrooms, isolation of enzyme was done by acetone precipitation procedure and precipitation of enzyme was done with ammonium sulphate precipitation method. Plants selected for extraction were Azadirachta indica (Neem), Manikara zapota (Chiku), Annona squamosa (Sitaphal), and Hibiscus Rosa-sinesis (China rose). For phytochemical screening Alkaloids-Mayer’s Test, Flavonoids (Shinoda Test, Alkaline Reagent Test), sugar (Benedict’s reagent Test), Glycosides (Borntrager's Test), Phenolic compounds Test (Ferric chloride Test, Gelatin Test, Lead Acetate Test). Mushroom tyrosinase inhibitory assay was determined by the spectroscopic method. The study shows the tyrosinase inhibitory activity of selected medicinal plants.

Isolation and Screening of Pectinase Producing Bacteria from Soil Sample

The vast majority of the industrial use of enzymes is covered from microorganisms. Microorganisms are favoured in industry because of their several advantages for example rapid growth, short life expectancy and simplicity in doing genetic alterations. Microbial enzymes are thus amply provided, very much standardized and promoted by many companies. Among various enzymes, Pectinases hold an exceptional place because of its different uses in various sectors like food, textile and biofuel industries.A total of 25% of total enzyme market is being shared by Pectinase alone.The current study was carried out to evaluate the pectinase activity of the pectinolytic bacteria. 40 Bacterial strains were isolated from different soil samples and screened for Pectinase production. Primary and Secondary screening showed 3 potential isolates I38 , I39 and I40 showing pectin degradation on Vincent’s media. Further, extracellular pectinase was partially purified by ammonium sulphate precipitation and dialysis. Sequential ammonium sulphate saturations from 20-80% i.e. (20, 40, 60 and 80%) showed 60% ammonium sulphate was optimum for precipitation of intracellular enzyme whereas 80% was optimum for extracellular enzyme.