Improvement of degummed ramie fiber properties upon treatment with cellulase secreting immobilized A. larrymoorei A1.

Degummed ramie fiber is widely used in the textile industry. Cellulase enzyme can be effectively used for bio-polishing of the ramie fiber. We immobilized Agrobacterium larrymoorei A1, a potent extra-cellular cellulase producing bacteria, in Ca-alginate. The production of enzyme significantly increased with increasing alginate concentration and reached a maximum activity of 0.28 IU/ml at 20 g/l, which was 32% higher as compared to free cells. These immobilized cells were used on ramie fibers. Scanning electron micrograph (SEM) and differential interference contrast (DIC) studies showed increased smoothness and orientation of surface structure of the fibers after 19.5 h. The single fiber tenacity was almost same as compared to non-treated fiber and the initial modulus increased by 24.01%. The remarkable reusability of these immobilized cells provides a cost effective method for treatment of natural fibers containing cellulose.

Arg²³5 is an essential catalytic residue of Bacillus pumilus DKS1 pectate lyase to degum ramie fibre.

After 24 h of incubation with only purified pectate lyase isolated from Bacillus pumilus DKS1 (EF467045), the weight loss of the ramie fibre was found to be 25%. To know the catalytic residue of pectate lyase the pel gene encoding a pectate lyase from the strain Bacillus pumilus DKS1 was cloned in E. coli XL1Blue and expressed in E. coli BL21 (DE3) pLysS. The pel gene was sequenced and showed 1032 bp length. After purification using CM-Sepharose the enzyme showed molecular weight of 35 kDa and maximal enzymatic activity was observed at 60°C and a pH range of 8.5-9.0. Both Ca²(+) and Mn²(+) ions were required for activity on Na-pectate salt substrates, while the enzyme was strongly inhibited by Zn²(+) and EDTA. The deduced nucleotide sequence of the DKS1 pectate lyase (EU652988) showed 90% homology to pectate lyases from Bacillus pumilus SAFR-032 (CP000813). The 3D structure as well as the catalytic residues was predicted using EasyPred software and Catalytic Site Atlas (CSA), respectively. Site directed mutagenesis confirmed that arginine is an essential catalytic residue of DKS1 pectate lyase.

Large-scale degumming of ramie fibre using a newly isolated Bacillus pumilus DKS1 with high pectate lyase activity.

A combined (enzymatic and chemical) process using a Bacillus pumilus strain (DKS1), isolated from the soil, was used to degum ramie bast fibres. After 24 h of incubation with the isolated pectinolytic strain using a low-cost medium, the weight loss of the ramie fibre was found to be 25% under small scale. High activity of pectate lyase was detected in the culture supernatants; 400 kg of ramie fibres was degummed with 24% weight loss in large-scale degumming under field conditions. No cellulase activity was found. Microbial intervention followed by mild (0.1%) alkali treatment showed high percentage of weight loss from the ramie fibre. Bacterial degumming followed by chemical treatment resulted in an increase of single fibre tenacity (cN/tex) by more than 20.81% as compared to non-degummed (decorticated) fibre samples. Scanning electron micrographs (SEM) and fluorescence microscope showed that after Bacillus pumilus DKS1 treatment the surface of the decorticated ramie fibre becomes very smooth. These results indicate the process provides an economical and eco-friendly method for the small scale as well as large-scale degumming of decorticated ramie fibre. This study has great relevance to the textile as well as paper industry.

Thermodynamic characterization of a highly thermoactive extracellular pectate lyase from a new isolate Bacillus pumilus DKS1.

An extracellular pectate lyase (EC 4.2.2.2) was purified from the culture filtrate of a newly isolated Bacillus pumilus DKS1 grown in pectin containing medium. Using ion-exchange and gel filtration chromatography, this enzyme was purified and found to have a molecular weight of around 35kDa. The purified enzyme exhibited maximal activity at a temperature of 75 degrees C and pH 8.5. The presence of 1mM calcium and manganese enhanced pectate lyase activity and was strongly inhibited by zinc, nickel and EDTA. The thermal inactivation studies revealed an entropy-enthalpy compensation pattern below a critical temperature. The alkaliphilicity and high thermostability of this pectate lyase may have potential implications in fibre degumming.

Enhanced functionality and stabilization of a cold active laccase using nanotechnology based activation-immobilization.

A simple nanotechnology based immobilization technique for imparting psychrostability and enhanced activity to a psychrophilic laccase has been described here. Laccase from a psychrophile was supplemented with Copper oxide nanoparticles (NP) corresponding to copper (NP-laccase), the cationic activator of this enzyme and entrapped in single walled nanotube (SWNT). The activity and stability of laccase was enhanced both at temperatures as low as 4°C and as high as 80°C in presence of NP and SWNT. The enzyme could be released and re-trapped (in SWNT) multiple times while retaining significant activity. Laccase, immobilized in SWNT, retained its activity after repeated freezing and thawing. This unique capability of SWNT to activate and stabilize cold active enzymes at temperatures much lower or higher than their optimal range may be utilized for processes that require bio-conversion at low temperatures while allowing for shifts to higher temperature if so required.

Improved production of reducing sugars from rice husk and rice straw using bacterial cellulase and xylanase activated with hydroxyapatite nanoparticles.

Purified bacterial cellulase and xylanase were activated in the presence of calcium hydroxyapatite nanoparticles (NP) with concomitant increase in thermostability about 35% increment in production of d-xylose and reducing sugars from rice husk and rice straw was obtained at 80°C by the sequential treatment of xylanase and cellulase enzymes in the presence of NP compared to the untreated enzyme sets. Our findings suggested that if the rice husk and the rice straw samples were pre-treated with xylanase prior to treatment with cellulase, the percentage increase of reducing sugar per 100g of substrate (starting material) was enhanced by about 29% and 41%, respectively. These findings can be utilized for the extraction of reducing sugars from cellulose and xylan containing waste material. The purely enzymatic extraction procedure can be substituted for the harsh and bio-adverse chemical methods.

Thermostability, pH stability and dye degrading activity of a bacterial laccase are enhanced in the presence of Cu2O nanoparticles.

The present study relates to a nanotechnology enabled method in which purified laccase from Escherichia coli AKL2 was supplemented with 100 µM copper oxide nanoparticles (Cu(2)O) (NP-laccase). The activity, half life and stability of NP-laccase were enhanced by 4, 42 and 36-fold respectively at high temperature (80 °C) and also over a wide range of pH (4-12) than laccase (in the presence of 0.18 mM CuSO(4)). Thermodynamic analysis of the nanoparticle-induced enzyme stability revealed an enhanced entropy-enthalpy compensation at 80 °C, which reflected the maintenance of its native structure. This was further supported by CD studies. The enhanced activity and thermostability of NP-laccase can be utilized for efficient decolorisation of dyes (both phenolic and azo).

Degumming of ramie fiber and the production of reducing sugars from waste peels using nanoparticle supplemented pectate lyase.

Banana, citrus and potato peels were subjected to treatment with hydroxyapatite nanoparticle (NP) supplemented purified pectate lyase (NP-PL), isolated from Bacillus megaterium AK2 to produce reducing sugar (RS). At both 50 and 90°C production of RS by NP-PL was almost twofold greater than that by untreated pectate lyase (PL) from each of the three peels. The optimal production of RS from banana and citrus peels were after 24 and 6h of incubation while it was 24 and 4h for potato peels at 50 and 90°C, respectively, on NP-PL treatment. NP-PL could degum raw, decorticated ramie fibers as well as enhance fiber tenacity and fineness. The weight loss of the fibers were 24% and 31% better (compared to PL treatment) after 24 and 48 h of processing. These findings have potential implications for the bio-ethanol, bio-fuel and textile industries.

Nanotechnology enabled enhancement of enzyme activity and thermostability: study on impaired pectate lyase from attenuated Macrophomina phaseolina in presence of hydroxyapatite nanoparticle.

In this paper we show that hydroxyapatite nanoparticles (NP) can not only act as a chaperon (by imparting thermostability) but can serve as a synthetic enhancer of activity of an isolated extracellular pectate lyase (APL) with low native state activity. The purified enzyme (an attenuated strain of Macrophomina phaseolina) showed feeble activity at 50°C and pH 5.6. However, on addition of 10.5 µg/ml of hydroxyapatite nanoparticles (NP), APL activity increased 27.7 fold with a 51 fold increase in half-life at a temperature of 90°C as compared to untreated APL. The chaperon like activity of NP was evident from entropy-enthalpy compensation profile of APL. The upper critical temperature for such compensation was elevated from 50°C to 90°C in presence of NP. This dual role of NP in enhancing activity and conferring thermostability to a functionally impaired enzyme is reported for the first time.

Improvement of thermostability and activity of pectate lyase in the presence of hydroxyapatite nanoparticles.

The activity and half-life of pectate lyase (PL) from Bacillus megaterium were nine- and 60-fold, respectively, higher at 90 °C in the presence of hydroxyapatite nanoparticles (NP-PLs) than in the presence of 1mM CaCl(2). Thermodynamic analysis of the nanoparticle-induced stability revealed an enhanced entropy-enthalpy compensation by the NP-PLs since a reciprocal linearity of the enthalpy-entropy change to 90 °C was observed. Without nanoparticles, the linearity range was 70 °C. Such compensation reflected the maintenance of the native structure of proteins. The remarkable enhancement of activity and stability of the NP-PL system at high temperatures may be utilized commercially e.g. in the food industry or the processing of natural fibers that may require a thermotolerant enzyme.

Cloning of feather-degrading minor extracellular protease from Bacillus cereus DCUW: dissection of the structural domains.

Bacterial extracellular proteases play an important role in cell survival and cell-cell communication. A high-molecular-mass minor extracellular protease (Vpr) from a feather-degrading bacterium, Bacillus cereus DCUW, has been reported by our laboratory. In the present study, we cloned and expressed Vpr in Escherichia coli. Complete nucleotide sequencing of this gene predicted that the protease is a member of the serine protease family, and smart domain analysis revealed that the protease consists of an N-terminal signal sequence for secretion, a subtilisin_N sequence that is a signature for N-terminal processing, a catalytic S_8 peptidase domain, and finally a long C-terminal protease-associated (PA) region containing nine intrinsically disordered subdomains. Four truncated constructs of the Vpr protease were cloned and expressed in E. coli. We found that the catalytic domain (amino acid residues 172-583) is sufficient for protease activity. Maturation of the Vpr protease needed both N-terminal and C-terminal processing. We have demonstrated that the oligomerization property is associated with the C-terminal protease-associated domain and also shown that the substrate-binding specificity to raw feather resides in this domain.

Degradation of raw feather by a novel high molecular weight extracellular protease from newly isolated Bacillus cereus DCUW.

Biotreatment of feather wastes and utilization of the degraded products in feed and foodstuffs has been a challenge. In the present study, we have demonstrated the degradation of feather waste by Bacillus cereus DCUW strain isolated during a functional screening based microbial diversity study on East Calcutta Wetland Area. A high molecular weight keratinolytic protease from feather degrading DCUW strain was purified and characterized. Moreover, utilization of degraded products during feather hydrolysis was developed and demonstrated. The purified keratinolytic protease was found to show pH and temperature optima of 8.5 and 50 degrees C, respectively. PMSF was found to inhibit the enzyme completely. The purified enzyme showed molecular weight of 80 kDa (from SDS-PAGE). The protease was found to have broad range substrate specificities that include keratin, casein, collagen, fibrin, BAPNA and gelatin. The protease was identified as minor extracellular protease (Vpr) by RT-PCR and northern blotting techniques. This is the first report describing the characterization of minor extracellular protease (Vpr) and its involvement in feather degradation in B. cereus group of organisms.

Bacterial diversity of East Calcutta Wet land area: possible identification of potential bacterial population for different biotechnological uses.

The extent of microbial diversity in nature is still largely unknown, suggesting that there might be many more useful products yet to be identified from soil microorganisms. This insight provides the scientific foundation for a renewed interest in examining soil microorganisms for novel commercially important products. This has led us to access the metabolic potential of soil microorganisms via cultivation strategy. Keeping this in mind, we have performed a culture-dependent survey of important soil bacterial community diversity in East Calcutta Wetland area (Dhapa Landfill Area). We describe isolation of 38 strains, their phenotypic and biochemical characterization, and finally molecular identification by direct sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA gene products. We have isolated and identified strains able to fix nitrogen, produce extracellular enzymes like protease, cellulase, xylanase, and amylase, and solubilize inorganic phosphates. Some isolates can synthesize extracellular insecticidal toxins. We find a good correlation between biochemical and phenotypic behavior and the molecular study using 16S rRNA gene of the isolates. Furthermore, our findings clearly indicate the composition of cultivable soil bacteria in East Calcutta Wetland Area.