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Title: Isolation and Characterization of Thermostable Protease Producing Bacteria from Tannery Industry Effluent

Author: Fariha Natasha

Supervisor: Dr. Md. Nural Anwar

Co-Supervisor: Shoma Dutta

Research Field: Environmental Microbiology

Batch: 11th

Session: 2010-2011


Abstract

Confronted with increasing legal and social pressures, no tanner can afford the luxury of not being familiar with the main issues and principles of environmental protection pertaining to tannery operations. Tanneries are typically characterized as pollution intensive industrial complexes which widely varying, high-strength wastewaters. Industries therefore are looking for ways to reduce pollution at the source as a way of avoiding costly treatment and reducing environmental liability and compliance costs. The scope of industrial enzyme is growing. Among these enzymes, proteases are well known enzyme which widely used in leather industry. As enzymes have proved to solve unresolved technical issues relating to pollution control, the present study was undertaken to screen for organisms which could produce extracellular thermostable proteases.
Organisms were isolated from the tannery effluents which were collected from Madina tannery of Chittagong. The physicochemical parameters of the sample were studied. The pH and temperature of the collected samples was found to range between 6.92 to 6.98 and 27˚C to 28˚ C respectively. The BOD and COD of the samples were found to range from 286 mg/l to 450.5 mg/l and 289.33 mg/l to 388 mg/l respectively. For the different controlling points, the bacterial load was found to vary from 2.5108 × 106 cfu/ml to 2.5663 × 107 cfu/ml respectively. A total of 14 isolates were obtained and among which five isolates were found protease degraders ensuring by egg albumin degradation, gelatin hydrolysis and skim milk casein hydrolysis test and were designated as C1SD2, D1SD1, O2SD1, O4SD1 and O4SD2 finally for the study. The stability of the protease was also studied under high temperatures. The enzyme production was found to be between 40 ̊ C to 65 ̊ C. Among them, 3 isolates, O4SD1, C1SD2 and O2SD1 were stable up to 65 ̊ C and with optimum temperature 37 ̊ C, 40 ̊ C and 50 ̊ C respectively. Various cultural, morphological and biochemical tests revealed that the organisms to be Bacillus pasteuri, Bacillus brevis and Bacillus laterosporus respectively.
The isolates C1SD2, O2SD1 and O4SD1 showed highest activity on the liquid media described by Amar and Halvorson, 1975 (547.5 U/ml, 301.5 U/ml and 237 U/ml respectively). The optimum yield was achieved after 96 hours culture at pH 8.0 for all the isolates. The isolate C1SD2 (Bacillus pasteuri) showed highest protease activity after growth at 50˚ C and the others showed highest activity at 37˚C. The isolate C1SD2 showed maximum activity at 0.5% yeast extract and 1% tryptone containing medium. The isolate O2SD1 showed highest activity with 1.5% glucose and peptone containing medium. 1.5% glucose and 2% ammonium di hydrogen phosphate were found to induce protease production by the isolate O4SD1.
The isolate C1SD2 showed highest activity at pH 8 and temperature 45˚ C, for 60 minutes with 1.5% casein as substrate. The isolate O2SD1 exhibited maximum activity at pH 6 and temperature 35˚C, for 40 minutes with substrate concentration of 2.5% and BSA as substrate. On the other hand, the isolate O4SD1 showed maximum activity at pH 6 and temperature 35˚C for 60 minutes with substrate concentration of 1% and BSA as substrate. All the isolates exhibited a high degree of inhibition with PMSF and a little with EDTA and so classified as serine-type of protease with some mettalo nature.
The effect of various metal salts (such as CaCl2, MgCl2, ZnSO4, FeCl3 and MnSO4) and activators on enzyme activity was studied.
The heavy metal resistance pattern and the antibiotic sensitivity of the isolates were also assessed. The isolates, C1SD2, O2SD1 and O4SD1 were found highly resistant to lead, chromium and arsenic. The isolates exhibited less resistance to mercury. The isolates showed resistance against penicillin-G, cefixime, amoxicillin and ampicillin. Precipitation steps were carried out with 50 to 70% ammonium sulphate. The desired protein was precipitated from the enzyme extract by using 50% to 70% ammonium sulfate.
The amino acids serine (Rf 0.20-0.31), glutamic acid (Rf o.37), leucine (Rf 0.76), tyrosine (Rf 0.50-0.53) and valine (Rf 0.56) were detected by using TLC.
The present study shows the existence of Bacillus strains which can produce industrially important thermostable enzyme which would also make it possible to reduce the use of harmful chemicals and wastes in the environment because these can be replaced by such types of proteases.