PRODUCTION OF MICROBIAL PROTEASE AND ITS APPLICATION IN LEATHER SOLID WASTE BIOTREATMENT

Abstract

The demand of leather items is increasing day by day with the growi ng population. Although, leather industry provides the necessities, such as leather shoes and garments while using the byproducts of the meat industry, but the by-products generated during the process of leather making and the so lid waste coming out of leather industry while making va luab le leather items cannot be discarded. Only 25% of the wet salted hides are converted into commercial leather, while 70% becomes chromium containing leather waste, and the remainder of the hides (5%) is lost in waste water as fat, so luble protein, and so lid suspended pollutants. Chrome shavings contribute 50% of the chromium containing solid wastes produced by leather industry whi ch are di sposed mostly in landfills and some of them are used for making of leather boards, artificial leather. An increasing environmental concern and decreasing number of disposal sites has trigge red research to recover protein (collagen) and chromium from the chrome shav ings. Four strains of Bacillus sp. (SALt , SAL2, SAL3, and SAL4) were iso lated from so il and tannery wastes and screened for the protease producing ability. The SAL1 strain gave the maximum zone of hydrol ys is when inoculated on agar plates containing 1 % casein as substrate. The strain was identi fied as Bacillus subtilis on the basis of biochemical tests and molecular characterization. The BadZhls strain SAL 1 was cultured in shake flask and various parameters were studied for the optimum production of protease. Among the three different media, used for the production of protease, the medium containing casein, gelatin and glycerol gave the optimum production i.e. 285 proteolytic units/miligram (PU/mg). Among the various parameter tested for optimization of protease production, incubation period of 72hours,pH 8.5,temperature 37°C, 10% inoculum of 24 hours gave the maximum production of protease. Among the different carbon sources such as sucrose, fructose, manitol, dextrose, sorbitol , glycero l, glucose, maltose and lactose, maximum protease production was observed in case of glycerol and among all the nitrogen sources such as tryptone, peptone, beef extract, yeast extract and gelatin, maximum production was observed when yeast extract was used as nitrogen source. Addition of metal cations in the medium has a very profound effect on the production of protease. As a result of optimization studies, the yield of alkaline protease by the strain could be increased approximately up to 35 %.( 387 PU/mg). ix This protease from Bacillus strain SALI has been purified to homogeneity as judged by SDSPAGE. The protease was purified to homogeneity by a combination of ammonium sulfate precipitation, DEAE Sephacryl ion exchange and Phenyl Sepharose hydrophobic interaction chromatography. The protease was purified up to 11.18 fold and had a specific activity of 4250 PU/mg. The enzyme was a monomeric protease with a relative molecular mass of 27 kDa as determined by SDS-PAGE. Proteolytic activity of the enzyme was detected by gelatin zymography, which gave a very clear protease activity zone on gel. Molecular mass of purified protease was also determined by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) that corresponded to the mass determined by SDS-PAGE. The homogeneity of enzyme was also checked by capillary electrophoresis which gave one peak confirming the purity of the enzyme. The enzyme exhibited its optimal activity at 60°C and at pH 9. The enzyme was stable in the pH range of7.0-10.0 and was able to maintain its stability at 50°C for 1 hour. The proteases from Bacillus strain SAL1 were then tested for collagenolytic activity on leather dust. Maximum leather dust degradation was observed when leather dust was incubated with 5000 PU/mg of enzyme for 84 hours and the extent of degradation was measured by the release of hydroxyproline (9.0 flg/ml).This indicated that proteases produced from Bacillus strain SAL 1 can be used successfully in the biotreatment of leather dust. The protease from Bacillus strain SAL 1 was also used for the biotreatment of chrome shavings. In this work, hydrolysis of chrome shaving was done by two ways. One is one step process in which hydrolysate protein is extracted by treatment with alkaline proteolytic enzyme at pH 8.5, temperature 60°C, and agitation for 3 hours. Other is two-step process in which gelable proteins as well as hydrolysate proteins are extracted by treatment with alkaline proteolytic enzyme at pH 8.5, temperature 60 °c, and agitation for 11 hours. Hydrolysate proteins extracted from both processes contained high values of TKN (18.83%and 17.98% respectively) and showed a wide distribution of molecular weights (from16-97KDa). Because of the high nitrogen content, the isolated collagen hydrolysate has potential use as a fertilizer and as animal feed additives. The effect of protease on the finished leather was also studied by applying it on the scraps of shoe upper leather. Firstly they were treated at high alkaline condition at 60°C at pH 12, and x then the pH was decreased for the enzyme reaction. This process produced hydrolysate protein and chrome sludge. Because of the high nitrogen contents (15.87%), the isolated collagen hydrolysate has potential use as a fertilizer and as animal feed additives. The most ideal way to minimize the waste generated from the leather industry is to convert the by-products into reusable items. For example, the hair pulp and shavings from the tannery solid waste, which are uSLIally disposed, contain significant quantities of useful proteins like keratin and co llagen, respectively. These proteins have demonstrated uses in pharmaceuticals, adhesives, cosmetics, films, encapsulations, etc. By employing the protease for the biodegradation of s<?lid waste we can reduce them into gelatin and protein hydrolysate which can be used in organic nitrogenolls fertilizer and animal feed and in preparation of films and adhesive. In this way, we can convert the harmful environmental pollutants into useful commercial items and find a way to make leather industry more profitable and less harmful to the environment.