Introduction

A phenomenal increase has been observed in recent years in the use of alkaline protease as industrial catalysts for various biochemical processes. Microorganisms represent an attractive source for producing an abundant and uninterrupted supply of proteases through fermentation methods. Various organic substrates have been studied for the production of alkaline protease. This study deals with the hydrolysis and fermentation of animal fleshing (tannery solid waste) by the enzymatic activity of non pigmented strain of Serratia marcescens ANFLR2 isolated from the gut of Labeo rohita, the fresh water fish. The digestion of food mainly depends on the enzymatic action of the gut microbial flora, which can change its profile enzymes according to the type of food the host feeds on. The focal theme of the present investigation was to adapt the fish gut symbiotic bacteria to animal fleshing and isolation of Serratia marcescens for hydrolyzing the same.

Materials and methods

Isolation of protease producing bacteria

Labeo rohita was acclimatized with animal fleshing as a feed for 30 days before sampling the fish. The acclimatized fish were sacrificed and immersed in 1% iodine solution for surface sterilization.  The gastro-intestinal tracts were dissected under aseptic conditions using sterilized instruments. The gut contents were washed using sterile water. About one gram of the separated guts was immediately homogenized in a surface sterilized mortar and pestle with 10 ml of sterilized saline solution. The homogenized gut sample was serially diluted with saline solution and plated on skim milk agar medium and incubated for 24 to 48 hours at
37⁰C. The colonies which produced a zone of hydrolysis were picked up and streaked onto agar slants for further screening.

Screening of Animal fleshing hydrolyzing bacteria

The strains showing the highest proteolytic activity were inoculated in the liquid minimal media containing 1% ANFL. The protein content and protease activity were determined for every 24 hours for 4 days. The strain which reflected the maximum protein content and protease activity was selected and identified using standard biochemical assays.

Biochemical test and 16SrRNA analysis

The DNA was extracted from 24 hour old LB broth following the modified method of Hykin et al. 1994. The extracted DNA was amplified using two forward and two reverse primers in nested PCR. The purified DNA product, of approximately 1.5 kb, was sequenced using five forward and one reverse primer as described earlier. The deduced sequence was subjected to BLAST search tool, for the closest match in the database. Phylogenetic analysis was performed by subjecting the deduced sequence to the 16 SrRNA database to obtain the closely related sequences.

Fermentation of animal fleshing

The substrate ANFL was collected from a commercial tannery in Chennai and processed as described earlier (Ganesh kumar et al., 2008). The fermentation was carried out in a 2000 mL fermentation chamber. The production medium consisted of NaCl (1.4g/L), NH₄Cl (0.005g/L), K₂HPO₄ (1.25g/L), KH₂PO₄ (0.9g/L) and 10g of animal fleshing.  The medium was inoculated with Serratia marcescens ANFLR2 of 10% concentration. The fermented medium was concentrated using ammonium sulfate precipitation, dialyzed and lyophilized for further use.

Characterization of the Serratia marcescens ANFLR2 protease

Molecular weight determination of the purified protease

The purified protease was subjected to SDS PAGE analysis to determine the molecular weight of the purified protease using high molecular weight protein markers.

Effect of pH on Protease activity

The partially purified protease was dissolved in pure double distilled water. About  0.5 mL aliquots of enzyme were distributed in test tubes and incubated for 1 hour with various buffers of pH range 3 to 12. The protease activity of the aliquots were determined after one hour at different pH.

Effect of temperature on Protease activity

The partially purified proteases were dissolved in appropriate buffer and incubated at different temperatures (4, 20, 28, 37, 45, 55, 65 and 75⁰C) for 60 minutes to determine the stability of the enzyme at these temperatures.

Effect of Ca⁺ ions, metal ions, inhibitors and surfactants on protease activity

The enzyme was dissolved in appropriate buffers and incubated with different concentration (1mM, 5mM, and 10mM) of metal ions such as MgCl₂, MnSO₄, CaCl₂, CuSO₄, ZnSO₄ and CaCl₂, inhibitors (EDTA, Dithiothreitol and Polymethane Sulfonyl Fluoride (PMSF))  and surfactants (SDS, Tween 20, Tween 80 and Triton X 100) for 60 minutes to evaluate the effect of these components on the activity and stability of the proteases.

Results and Discussion

The isolates  belonged to  gram negative rod, non motile and  non spore forming bacteria, exhibiting positive response to citrate, catalase and oxidase tests. The isolate had 98% similarity to Serratia marcescens strains and was assigned a Genbank accession number HM584905 and given a strain name as Serratia marcescens ANFLR2.  The protease enzyme produced by the non pigmented Serratia marcescens ANFLR2  have higher activity and stability in alkaline pH range of about 8 to 10 with maximum activity  at 9 (Fig. 1).

Fig. 1.  The relative activity of Serratia protease as a function of pH

The enzyme was  highly stable at high temperatures  in the range 50⁰C to 65⁰C (Fig. 2).

The protease activity produced by the fermentation process was 220 U at 370C and pH 8. On the addition of 1% triton X 100 to the medium, the activity was increased to 300 U. Further addition of CaCl2 to the medium enhanced the protease activity to 370 U. The protease activity was inhibited by 10mM of PMSF having residual activity of 38% indicating that it could be classified under serine proteases. Ca2+ ions enhanced the stability of the protease to retain about 89% of relative activity after 60 minutes.  The  stability of the protease was also evaluated in the presence of 1% Triton X 100 which retained about 85% of the relative activity of the protease after 60 minutes. This may be interpreted that  the protease produced should be effective enough  to break all the disulfide and hydrogen bonds present in animal fleshing, because the bacteria  was  isolated  from fish  gut which were degrading  high molecular proteins to the assimilating level to the fishes. The symbiotic association of the bacteria with that of the fish might have induced this capacity to degrade such high molecular mass animal proteins by producing high molecular weight protease of 64 Kda (Fig. 3).

The molecular weight reported by us is higher than the values reported in literature for Serratia sp. (40-58kda).  Hence, the alkaline protease produced by the non  pigmented Serratia marcescens is a novel enzyme having high molecular mass which is capable of cleaving the complex animal proteins.

kDa

Fig. 3. Molecular Weight Profile of Serratia Protease

Conclusion

This study confirms the capacity of the non pigmented Serratia marcescens to produce a novel protease against animal fleshing to hydrolyze it. The protease produced by the Serratia marcescens ANFLR2 was characterized as a thermo tolerant alkaline serine protease having a molecular mass of around 64 kda which has not been cited in literature so far described.

References

Ganesh Kumar, A., Nagesh, N., Prabhakar, T. G. and Sekaran, G. (2008) Purification of extracellular acid protease and analysis of fermentation metabolites by Synergistes sp. utilizing proteinaceous solid waste from tanneries. Biores. Technol. 99, 2364 - 2372.

Hykin, P. G., Tobal, K. and McIntyre, G. (1994) The diagnosis of delayed post-operative endophthalmitis by polymerase   chain reaction of bacterial DNA in vitreous samples. J Med. Microbiol, 40, 408 - 15.