Bioprocess Development for Production of Proteases by Bacillus Licheniformis

Abstract

Bacterial proteases have enormous technical applications in food, pharmaceuticals and leather industries. Alkaline protease, however, has got important applications in eco-friendly leather processing. The bulk application of this hydrolyzing enzyme demands its cost effective production. Hence the present study was aimed at the development of a bioprocess for production of the alkaline protease by Bacillus licheniformis MZK05 strain which was previously isolated from tannery effluents and identified by 16S rRNA gene sequence analysis. Since the production of the enzyme by the wild BlMZK05 strain was not adequate in a liquid medium namely Alkaline Protease Producing Broth (APPB), strain improvement through random mutagenesis followed by optimization of the enzyme fermentation by statistical methods were investigated. The mutation protocol involved the treatment of the BlMZK05 cells of exponential phase with ultra violet (UV) irradiation and ethyl methanesulfonate (EMS) under varied combinations viz. combinatorial, single or sequential with different doses and exposure periods. The potential mutants were selected from colonies of distinct characteristics, their clear zone ratio of casein hydrolysis on Skim Milk Agar (SMA), production capacity of the enzyme in liquid APPB medium and test for prolonged reproducible enzyme production ability. The distinct colonies were selected after mutagenic treatments that resulted in varied level of death rates. The highest death rates were revealed with EMS treatments for overnight. Of 182 colonies with distinct phenotypic characteristics, 100 mutants which exhibited casein hydrolysis in varying ratio yielded the enzyme activity between 46 – 270 U/ml in liquid APPB medium in shake flasks at 37°C, pH 7.5 and 150 rpm. The mutants with rather larger, raised and irregular colony appearance with respect to the parental organism produced greater protease activity than the other mutants. Most of the mutants tested for the reproducibility of their enzyme production capability for a prolonged period of about twelve months, were found inconsistent as “rise and fall” except two mutants namely B. licheniformis MZK05M9 (BlM9) and B. licheniformis MZK05EO17 (BlEO17). These mutants exhibited persistent enzyme activities of 135±3 U/ml and 235± 4 U/ml, respectively. The enzymes of two mutants BlM9 and BlEO17 were thus evaluated for their possible applications in leather processing based on their hydrolyzing capability of nonstructural (albumin, globulin and elastin) and structural (collagen) leather proteins. Both the mutants were capable of hydrolyzing all but BlM9 did not act on collagen indicating the suitability of BlM9’s enzyme in leather manufacturing processes. In this regard, optimization of the fermentation of alkaline protease by BlM9 was performed using statistical methods which demonstrated effective medium components molasses as carbon source, soybean meal as nitrogen source and the salts NaCl, MgSO4.7H2O and K2HPO4 by Plackett-Burman design. The concentrations of these medium components were optimized by Response Surface Methodology (RSM) based on Central Composite Design (CCD) in shake culture at pH 7.5, agitation 150 rpm and temperature 37°C. The optimum values for the tested variables for the maximum alkaline protease production were found as molasses (0.92%), soybean Meal (0.79%), NaCl (0.125%), MgSO4.7H2O (0.125%) and K2HPO4 (0.59%) with the protease activity in the optimized medium 761 U/ml predicted by statistical software Minitab Version 17. The experimental value of the enzyme activity as obtained 765 U/ml was comparable and thus validated the predicted values of the software. The granular sizes of the soybean meal affected the enzyme production revealing 4.7 mm mesh size supported the enzyme production 5 % higher than that of the mixed sizes between 6 to 4 mm. Fermentation in 7.0 L bioreactor cultivation with optimized medium at pH 7.5 and 37°C under cascade control of dissolved oxygen concentration, a high level of enzyme activity 1020 ± 10 U/ml was obtained after 28 hrs at stationery phase. This enzyme activity was 1.7 fold higher than that found in Molasses Soybean meal medium optimized by “one variable-at-a time” method. The enzyme was stable up to 30 days at 4°C where as the enzyme lost its activity to 50% at 30°C after 40 days. However, it exhibited 100% stability up to 18 days at 30oC in the presence of 0.5% polyethylene glycol. The partially purified protease had a pH optimum of 8.5 and temperature optimum of 55°C and the molecular mass of the enzyme was 27.2 kDa as judged by SDS–PAGE. According to the inhibition profiles obtained with the various protease inhibitors, it was confirmed that the partially purified protease belongs to the serine protease type. The activity of partially purified enzyme was enhanced by calcium, magnesium, barium, potassium and manganese ions and strongly inhibited by mercury ion. In addition, the protease showed remarkable stability in the presence of 1% SDS; 1, 3 and 5% Triton X-100 and H2O2, which comprise the common bleach-based detergent formulation suggesting its possible usage in detergent as cleansing aids. The application of the enzyme in dehairing of animal skin in leather processing demonstrated that treatment with enzymes removed 85% of hair from goat skin after 24 hrs under mild shaking condition at room temperature where as the treatment with enzymes and 5% lime together resulted in 100% dehairing under similar conditions. Sodium sulfide along with lime also removed 100% hair faster (with 20 hrs) than other two treatments. The grain surface of the enzyme treated skin was smoother and silkier than that of the chemical treated skin as revealed by Scanning Electron Microscopy. The quality of the enzyme was found equally efficient to a commercial enzyme in bating of animal hide as proved by different physical tests such as tensile strength, percent of elongation, stitch tears strength, water vapor permeability, grain crack strength and tongue tear strength tests. In addition, the stability profile (pH, temperature and surfactants) also revealed its suitability for application in detergent industry. Thus these results will be a useful basis for commercial production of the alkaline protease by BlM9 in Bangladesh.