SYNOPSIS

Biotechnology is gaining ground rapidly due to the various advantages that it offers over conventional chemical processes especially regarding environment and cost involvement. Industrial enzymes represent the heart of biotechnology processes. According to a recent report from Business Communications Company, (BCC 2008) the global market for industrial enzymes increased from $2.2 billion in 2006 to $2.3 billion by the end of 2007, $2.7 billion by 2012 and $4.9 billion by 2013 . New and emerging applications have helped drive demand for enzymes and the industry is responding with a continuous stream of innovative products. The industrial enzyme market is divided into three application segments: technical enzymes, food enzymes and animal feed enzymes. Technical enzymes for detergent, pulp and paper manufacturing, have a largest segment with approximately 52% market share and the largest share of the enzyme market has been held by alkaline proteases. Proteases cover the 60% of total enzyme market and amongst the most valuable commercial enzyme. Alkaline proteases hold a great potential for application in the detergent and leather industries and there is an ever increasing trend to develop environment friendly technologies ,. Plants, animals and microbes are the main sources for protease production. The preferred sources of proteases are microbes because of their rapid growth and the ease with which they can be genetically manipulated to generate new enzymes with altered properties and are currently being utilized by the detergent industry eg. Serine proteases produced by Bacillus strains ,. Proteases from several bacteria have been purified and characterized ,,,. Genus Pseudomonas a gram-negative bacterium that predominantly produces alkaline proteolytic enzymes and the proteases has been purified ,. Fungal alkaline proteases are advantageous because of the ease of downstream processing to prepare a microbe-free enzyme at low cost production., . The bulk of the plant proteases eg Papain, Bromelain and Ficin have major application in the food industry where they are added at different stages of production. Thermostable serine proteases named wrightin from the latex of the plant Wrightia tinctoria, Carnein from the latex of the weed Ipomoea carnea spp. fistulosa (Morning glory) and Milin from the latex of Euphorbia milii, too have found applications in food and other biotechnology industries ,,.

SYNOPSIS 1

During leaf senescence protein degradation is enhanced, endoproteases have been isolated from alfalfa, oat and barley senesced leaves which are involved in the process of protein degradation during foliar senescence ,,,. A 70-kDa serine protease was identified from artificially senescing parsley leaves this protease activity is low in young leaves, was found to increase considerably in parallel to the advance of senescence and the reduction in the protein content of the leaves [21].Senescing leaves show dominance of proteolytic enzymes belonging to four major classes, which are common in mammals, and microbes. Senescence induced protein breakdown has been well reported in many plant systems, it results in availability of transportable nitrogen ,, and degradation of protein ,,. However, till date there are no reports of utilization of protease in industry from this abundant senesced leaf waste. The innovative aspect of the present work was to identify and isolate alkaline proteases from various biological sources such as senesced leaves of regional plants that are currently not used in agriculture and from soil microbes with a purpose to have positive effect for solid waste management. The purified enzyme would be checked for their potential industrial application. The first objective of the present work was to identify new sources of alkaline proteases eg: i. ii. From senesceing leaves as it shows dominance of proteolytic enzymes. Soil sample of the poultry waste site which is rich in organic waste was selected

for Screening of microbial isolate that can produce alkaline protease. Secondly, check for application of isolated enzymes in industry processes. Since there are no reports available on the use of plant proteases in detergent industry we were interested in exploiting the use of plant proteases for its commercial application in detergent industry. We chose senesced leaves of Lantana camara after initial screening for protease from various plants. Lantana camara is most commonly occurring weed in the world and the proteases extracted from this weed would be cost effective.

SYNOPSIS 2

We describe an easy protocol of production and potential application of caseinolytic, thermostable alkaline protease by utilizing the senesced leaves of common weed L. camara. We also report the purification of a novel extracellular alkaline protease which was produced by bacterium, Pseudomonas thermaerum GW1 isolated from soil. Protease from senesced leaves of the weed Lantana camara was purified by a two-step procedure involving ammonium sulfate precipitation and Sephadex G-250 gel permeation chromatography. The Sephadex G-250 fraction of senesced leaves of Lantana camara showed 28.31 fold with a yield of 6.19%. The enzyme was shown to have a low molecular weight of 43 kda by SDS-PAGE. It was strongly activated by metal ions such as Cu 2+, Zn2+, Mg2, Co2+ and Mn2+. It remained active at 60C, pH 10.5 even after 1 hour of incubation when casein was used as substrate. The compatibility of the enzyme was studied with commercial and local detergents, 60% activity of the enzyme was retained even after 1 h of incubation at pH 10.0. The easy availability of the senesced leaves of this common weed makes it a cheaper enzyme source and potential additive in detergents , Secondly an extracellular protease was purified and characterized from Pseudomonas thermaerum GW1 a new strain identified isolated from soil of Poultry waste site. Based on biochemical characteristics, nucleotides homology and phylogenetic analysis the microbe was detected to Pseudomonas thermaerum and its nearest homolog was found to be Pseudomonas aeruginosa. The strain produces extracellular protease in the culture media that was maintained at 37C and at 140 rpm. The media was harvested for protease after 48 hrs of incubation at 37C in basal media supplemented with 1% casein. Enzyme was purified by ammonium sulphate precipitation and DEAE-cellulose chromatography. The molecular weight of the enzyme was estimated to be 43,000 daltons as shown by casein zymography studies. The optimum pH for the proteolytic activity was pH 8.0 and enzyme remained stable between pH 5 -11 at 60C. Interestingly Mn2+ (5mM) strongly activated enzyme activity by 5 fold, while Cu2+, Mg2+and Ca2+ moderately activated enzyme activity, where as Zn2+, Fe2+ and Hg2+ inhibited enzyme activity. The protease produced was stable in presence of 50 % (v/v) ethylacetate and acetone and however showed 50% reduction on enzyme activity in the presence of glycerol. Isopropanol, methanol and benzene increased protease activity by 2.7, 1.3 and 1.1 fold respectively suggesting its potential industrial application , .

SYNOPSIS 3

Future studies regarding upgrading the protease production technology from laboratory to a large-scale process, allowing for a new green industrial process to be developed especially where enzymatic treatment of protein fibers, like hair, wool and silk is involved so that there is significant reduction in the chemical use and cost. Development of new formulations for industrial and domestic wool carpet cleaning, garment washing, dyeing processes for protein fibers by pre-treatment with the proteases can be performed. The amino acid sequence determination of purified alkaline protease from senesced leaves of Lantana camara and Pseudomonas thermaerum would be performed and checked for innovative application in other biotechnology industries.

SYNOPSIS 4

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