Lignolytic and hemicellulolytic enzyme cocktail production from Bacillus tequilensis LXM 55 and its application in pulp biobleaching.

Bioprocessing of pulp requires lignolytic as well as hemicellulolytic enzymes. The present study is the first report of a cocktail of laccase (L), xylanase (X), and mannanase (M), from a single bacterium for pulp biobleaching. A novel strain Bacillus tequilensis LXM 55 produced thermo-alkali stable L + X + M. On optimization higher enzyme yield (IUml-1/fold increase) of laccase (396.35/24.16), xylanase (212.95/81.90) and mannanase (153.33/102.90) were achieved in the cocktail. Treatment of pulp with cocktail of enzymes led to 49.35% reduction in kappa number and considerable enhancement in the brightness (11.59%), whiteness (4.11%), and other pulp properties. Most importantly, no mediator system was required for the application of laccase. 40% less chlorine consumption was required to obtain the paper of the same quality as that of pulp treated without enzyme but with 100% chlorine. Therefore, this cocktail of enzymes is highly suitable for pulp biobleaching in the paper mill.

Lime and Sulphide-Free Dehairing of Animal Skin Using Collagenase-Free Alkaline Protease from Vibrio metschnikovii NG155.

The objective of this work was to isolate a microorganism producing alkaline protease that can be used as an ecofriendly alternative to chemicals in dehairing process of leather manufacture. Alkaline protease producing bacterium Vibrio metschnikovii NG155 was isolated from soil samples of leather industry. The protease was highly effective in dehairing of goat skin, completely eliminating the use of lime and sulfide. Histological studies of the skin after dehairing showed that the enzyme did not damage the collagen layer and brought good fiber opening. Absence of collagenase activity was confirmed by reacting pure collagen with the enzyme and analyzing it on SDS PAGE, which showed no degradation of collagen. The enzyme was stable in a wide range of pH (7-11) and temperature (10-50 °C), which makes it suitable for industrial application.

Commercialization potential of PET (polyethylene terephthalate) recycled nanomaterials: A review on validation parameters.

Polyethylene Terephthalate (PET) is a polymer which is considered as one of the major contaminants to the environment. The PET waste materials can be recycled to produce value-added products. PET can be converted to nanoparticles, nanofibers, nanocomposites, and nano coatings. To extend the applications of PET nanomaterials, understanding its commercialization potential is important. In addition, knowledge about the factors affecting recycling of PET based nanomaterials is essential. The presented review is focused on understanding the PET commercialization aspects, keeping in mind market analysis, growth drivers, regulatory affairs, safety considerations, issues associated with scale-up, manufacturing challenges, economic viability, and cost-effectiveness. In addition, the paper elaborates the challenges associated with the use of PET based nanomaterials. These challenges include PET contamination to water, soil, sediments, and human exposure to PET nanomaterials. Moreover, the paper discusses in detail about the factors affecting PET recycling, commercialization, and circular economy with specific emphasis on life cycle assessment (LCA) of PET recycled nanomaterials.

Cloning, expression and characterization of laccase from Bacillus licheniformis NS2324 in E. coli application in dye decolorization.

Laccase gene from Bacillus licheniformis NS2324 was cloned and expressed in E. coli by using pUC 18 as cloning vector and pet 15b as expression vector. The purified recombinant laccase (rLacNS2324) showed a molecular mass of 66 KDa. The optimum pH and temperature for rLacNS2324 was found to be pH 8 and 40 °C respectively. The half life of rLacNS2324 at pH 7, 8 and 9 is 24 h. The half life of laccase at 45 °C is 8 h. Laccase activity was increased in the presence of Cu2+ (135.3%), Mn2+ (283.76%), and Co2+ (199.96%) at 5 mM of concentration, but inhibited to 17.01% in the presence of 5 mM Zn2+ ions. rLacNS2324 was found tolerant to NaCl and NaI. Among the inhibitors, it was found to be tolerant to EDTA, however, its activity was inhibited in the presence of sodium azide, dithiothreitol and ß-mercapethanol. rLacNS2324 was able to decolorize a bromophenol blue by 85% and phenol red by 75% in 1 h without any mediator. Methylene blue was almost completely degraded (99.28% decolorization) by 10 IUml-1 of laccase at 40 °C, pH 8.0 and in time 4 h. Overall rLacNS2324 showed ability to be used industrially to decolorize dyes in an eco-friendly and cost effective way.

Purification and characterization of a novel white highly thermo stable laccase from a novel Bacillus sp. MSK-01 having potential to be used as anticancer agent.

Laccases are multicoopper oxidases catalyzing the oxidation of phenolic as well as non-phenolic compounds. Laccases show typical blue color due to the presence of covalent Type 1 Cu-Cys bond which absorbs at 600 nm. However, recently some white laccases have also been identified which lacks typical spectra of blue laccases and do not show peak at 600 nm. In the present study, a novel white laccase was isolated from Bacillus sp. MSK-01. MSK laccase was purified and characterized in detail and the purified laccase was referred to MSKLAC. It has a molecular weight of 32 KDa. UV-visible spectrum of purified MSKLAC do not show characteristic peak at 600 nm and bend at 330 nm. The enzyme was repressed by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and ß-mercaptoethanol. The laccase was highly thermo-stable enzyme having optimum temperature of 75 °C and could treasure more than 50% activity even at 100 °C. The optimum pH for ABTS and guaiacol was 4.5 and 8.0 respectively. MSKLAC was stable in the presence of most of the metal ions and surfactants. The effect of MSKLAC on lung cancer cell line was also assessed. It was observed that MSKLAC is inhibitory to lung cell cancer line. Thus, MSKLAC has potential to be used as an anti-proliferative agent to cancer cells.

Response surface based optimization of laccase production from Bacillus sp. MSK-01 using fruit juice waste as an effective substrate.

Laccases are multicopper oxidases containing four copper atoms per monomer distributed in three redox sites. Because of its tremendous applications in different areas, isolation of new laccases with wide range of industrial implementation. The present study focuses on the optimization of laccase production from Bacillus sp. MSK-01 under solid state fermentation conditions using fruit juice waste as the substrate. MSKLAC was produced extracellularly by the bacteria. This laccase was able to oxidize ABTS and syringaldazine. Various nutritional and environmental factors were utilized for increasing the enzyme yield. Plackett Burman was used to study the influence of input parameters on laccase yield. Tween-80, initial moisture ratio and magnesium sulphate were the major influencing factor affecting laccase yield. Central composite design of RSM was used for the modelling of experiment. Desirability approach was used to optimize laccase yield. Maximum laccase yield of 1645 IUg-1 was obtained when 0.55% of tween -80, 1:2.34 initial moisture ratio and 300µM magnesium sulphate was used. A 470 fold increase in the yield of laccase from unoptimized condition was obtained.

Immobilization of laccase-ABTS system for the development of a continuous flow packed bed bioreactor for decolorization of textile effluent.

Decolourization of textile effluent is always being a problem of major concern for the safe disposal of effluent water in river stream. Due to intense colour of the effluent water, even after the treatment, disposal of the water in river stream is under strict environmental regulation. Laccase, a natural oxidase, have the miraculous power to decolourize different dyes with or without mediators. However, stability and cost of implementation of the enzyme at industrial conditions make the entire process unfeasible at industrial scale. In the present study, a highly thermo-alkali-stable, metal, surfactant and organic solvent tolerant laccase from Bacillus sp. MSK-01 was immobilized in Cu-alginate bead with ABTS mediator under standardized conditions. A continuous flow packed bed bioreactor was formulated to develop a continuous method of the treatment of effluent water. Results showed 66% reduction in the colour of the effluent. UV-VIS spectrum analysis of the treated and untreated samples showed the formation of the degradation products of dyes due to the action of laccase. The developed process can be made useful for on site industrial application.

An alkaline bacterial laccase for polymerization of natural precursors for hair dye synthesis.

In the present study, an extracellular alkali stable laccase (Lac DS) from Bacillus subtilis DS which has pH optima at 8.5 using p-phenylenediamine (PPD) as substrate has been reported. Lac DS retained 70% activity for 4 h at pH 8.5 and 90% activity for 24 h at 55 °C. The enzyme yield was enhanced by optimization of fermentation conditions. A 746-fold increase in yield was observed under optimized conditions using 150 µM MgSO4, 1.2% yeast extract, 0.35% tryptone, and 150 µM vanillic acid. Lac DS was used to polymerize natural dye precursor catechol, pyrogallol, syringaldehyde, syringic acid, ferulic acid and gallic acid to develop a range of natural hair colors such as black, golden yellow, and reddish brown. The results indicate that alkaline Lac DS is a suitable candidate to develop a user-friendly and commercially applicable hair dyeing process in the area of cosmetic industry.

An extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4, with a potential to biobleach softwood pulp.

Degradation of residual lignin in kraft pulp by chemical bleaching is implicated in causing environmental pollution. The use of thermo- and alkali-tolerant bacterial laccases is considered to be important biological alternative to chemical processing. Laccases from Bacillus species have shown promise in this respect but their intracellular/spore bound presence make their industrial application economically unfeasible. We report here on a novel extracellular active thermo-alkali-stable laccase (SN4 laccase)  which is active at 90 °C and pH 8.0 using 2,6-dimethoxyphenol as substrate from Bacillus tequilensis SN4. SN4 laccase retained 27 % activity for 5 min at 100 °C and more than 80 % activity for 24 h at 70 °C. The enzyme is also stable at a higher pH (9.0-10.0). Enzyme production was optimized by submerged fermentation. Relatively high yields (18,356 nkats ml-1) of SN4 laccase was obtained in a medium containing 650 µM MnSO4, 350 µM FeSO4, and 3.5 % ethanol. A 764-fold increase in laccase activity was observed under optimal conditions. In addition, reduction in kappa number and increase in brightness of softwood pulp by 28 and 7.6 %, respectively, were observed after treatment with SN4 laccase without a mediator. When N-hydroxybenzotriazole was used as a mediator, the kappa number was decreased to 47 % and brightness was increased to 12 %.

Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4.

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (kcat/Km) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and ß-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2'-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu2+, Co2+, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.