Approach towards sustainable leather: Characterization and effective industrial application of proteases from Bacillus sps. for ecofriendly dehairing of leather hide.

Tanneries are one of the most polluted industries known for production of massive amount of solid and liquid wastes without proper management and disposal. In this project we demonstrated the ecofriendly single step dehairing of leather hides with minimum pollution load. In this study, Bacillus species (Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P) capable of producing proteases was successfully isolated by employing the new optimized selective media named M9-PEA as confirmed by 16sRNA genes sequencing. Sequence of 1493 bp long 16S rRNA genes of Bacillus paralicheniformis strain BL.HK and Bacillus cereus strain BS. P was submitted to GenBank under the accession number OP612692.1, OP612721.1 respectively The Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P produced extracellur proteases of 28 and 37 KDa as resolved by SDS-PAGE respectively. The enzymes showed temperature optima at 50 °C and 55 °C and pH optima at 8.5, 9.5 respectively. The Proteases of Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P were employed for dehairing of animal hides. The process resulted in significant removal of interfibriller substances without damage to collagen layer after one hour treatment, which was confirmed by histology, scanning electron microscopy. The quantification of various skin constituents (collagen, uronic acid, hexosamines, and GAGs) and pollution load parameters revealed that enzymatic treatment are more reliable. The results of skin application trials at industrial level with complete elimination of chemicals remark the biotechnological potential of these proteases for ecofriendly dehairing of animal hides without affecting the quality of the leathers produced.

Environmental impact and diversity of protease-producing bacteria in areas of leather tannery effluents of Sialkot, Pakistan.

Massive discharge of wastes produced by the processing of leather so far confers the most important environmental challenge facing the tanneries worldwide. Waste material from tanneries mostly consists of skin remnants and proteinaceous substances as by-products of leather processing. In these conditions, protease-producing bacteria play a vital role in degrading wastes in this sludge. Therefore, an investigation was made to study the effect of long-term tannery sludge contamination on the diversity of both protease-producing microbes and of bacterial extracellular proteases near tanneries of Sambrial and Sialkot. The high amount of carbon and nitrogen in the soil samples reflected their effect on the diversity of the microbial communities in these areas. Phylogenetic analysis based on 16S rRNA gene sequences suggest that the isolated proteolytic bacteria belonged to 9 different genera including Pseudomonas (26.19%), Proteus (19.04%), Serratia (16.66%), Klebsiella (14.28%), Providencia (9.52%), Achromobacter (7.14%), Enterobacter (2.38%), Myroides (2.38%), and Acinetobacter (2.38%). Enzyme activity showed that among all Pseudomonas and Proteus showed relatively high protease production, and inhibition studies revealed that proteases produced by all isolates were strongly inhibited by serine and/or metalloprotease inhibitors, and a smaller proportion was inhibited by inhibitors of cysteine and/or aspartic proteases. Furthermore, isolated bacteria revealed promising degradation activities against casein and/or gelatin with only a few that could hydrolyze elastin, suggesting proteases produced by these isolated bacteria belong to different classes of proteases, i.e., serine and metalloproteases. This study provided new insights on the community structure of cultivable protease-producing bacteria near tannery sludge of Sambrial and Sialkot. This study would be beneficial not only for establishing the way for effective degradation of tannery slugs but also for questing the novel properties of proteases for a future technological application.