Production of different proteases from fish gut microflora utilizing tannery fleshing

Abstract

Three alkaline protease-producing strains designated as ANFLR1, NPLR1, and PROLR15 were isolated from Labeo rohita fish gut. These strains are able to produce alkaline protease using tannery fleshing (TF) as the sole carbon and nitrogen source and were identified as Bacillus megaterium, Serratia marcescens, and novel Pontibacter sps. Proteases from these organisms were purified to electrophoretic homogeneity following ammonium sulphate precipitation, ion exchange, and column chromatography. SDS-PAGE revealed molecular weights of the proteases to be 46 kDa (ANFLR1), 52 kDa (NPLR1), and 58 kDa (PROLR15). The optimum pH and temperature for the protease activity of ANFLR1, NPLR1, and PROLR15 were found to be 10. 5, 11.5, 9, and 70 • C, 60 • C, and 50 • C, respectively. The maximum protease activities at the optimum conditions were 420 U/mL (ANFLR1), 550 U/mL (NPLR1), and 530 U/mL (PROLR15). Inhibition of the NPLR1 protease by pepstatin confirmed aspartate-type enzymatic activity. Fe 3+ enhanced the activity of PROLR15 protease. Unlike all other microbial proteases known so far, the PROLR15 enzyme did not require Ca 2+ for activity and thermal stability. SDS-PAGE and scanning electron microscopy analyses confirmed the conversion of high molecular weight substrate (TF) to low molecular weight peptides by these proteases. The alkaline metalloprotease production by novel Pontibacter sps. and aspartate protease production by S. marcescens remain unexplored. Hence, TF with its relatively abundant availability can be beneficially utilized for alkaline protease production through the fish gut microbial fermentation processes.C. Sumathi et al. Eng. Life Sci. 2012, 12, No. 2, 223-237 proteases [3,9,[12][13][14][15]. The strains identified are featherdegrading Bacillus licheniformis [16-18], Bacillus subtilis [19], Bacillus cereus [20], Bacillus pseudofirmus [21] keratinolytic proteases that are useful in dehairing of animal hides, B. subtilis [22-24], Bacillus velesensis [25], B. cereus [26], B. licheniformis [27], and Bacillus pumilus [28], which subsequently yield the edible protein hydrolysates from fish, lean meat, Zein, fresh whey [12], and shrimp shell wastes [29,30] through fermentation processes [31][32][33]. Isolation of three different alkaline proteaseproducing bacterial strains ANFLR1, NPLR1, and PROLR15 after adaptation with TF is a novel approach which has not been explored yet. Alkaline proteases generally belong to the group of proteases, which have either a serine center or a metallo-type, exhibiting a wide pH range (pH 6-13). Similarly, Gram-negative bacilli Serratia sp. cultivated in proteinaceous substrates generally produce two different kinds of extracellular proteases-a major metalloprotease and a minor serine protease [10,34]. This is the first approach to reveal aspartate alkaline protease production by a Serratia strain. Furthermore, the present work explores the unexploited application of alkaline protease production by a novel Pontibacter strain PROLR15 isolated from the novel source Labeo roh...

Factors affecting mass transfer of protease in pelt during enzymatic bating process

“…These results suggest that employment of proteases with lower molecular weight is beneficial to enhancement of protease transfer rate in bating process. This is accordant with the former researches that the trypsin from pancreas works better than other proteases from microorganisms owing to its lower molecular weight [36,37] and moderate proteolytic activity [14]. Therefore, to improve the bating performance, besides screening proteases with high specificity in hydrolyzing non-collagenous proteins, a possible direction would be to develop proteases which possess proper proteolytic activity and have a molecular weight as low as possible.…”