Microbial proteases from gelatin production system for the degradation of keratin-containing materials
This study aimed to isolate microorganisms from gelatin production systems, an extreme environment, which were prospected to isolate bacteria that secreted keratin degrading proteases. Keratin protein-containing materials are major by-products of the livestock and poultry processing industries. Keratin protein may have potential applications in food and nutrition; however, keratins are resistant to degradation by many common proteases, including those in the digestive system. Prospecting for proteases that possess keratin-degrading capability would enable utilization of keratin protein. Specialized environments such as that used for gelatin production, involves alkaline pre-treatment of animal hide in conditioning baths that leads to the release of animal skin, hair, and bone debris. The bath liquor and debris may harbour bacteria that may secrete keratinases in response to keratin containing materials in the environment. The isolation of protease-secreting microorganisms was achieved by the cultivation of the above materials at 37oC in either (1) liquid broth Tris TSB (tryptic soy broth (TSB) containing 50 mM Tris with final media pH 8.5 or 10.8), or (2) on milk TSA agar (tryptic soy agar (TSA) containing 50 mM Tris and 1% (w/v) low-fat milk with final media pH approximately 8.8). Microorganisms were not able to be isolated at pH 10.8, but some species grew on Tris TSB at pH 8.5 and on milk TSA. Microbial colonies on milk TSA were collected and identified by 16S rRNA gene sequencing. Sequence comparison with online databases showed that microorganisms isolated from conditioning bath liquors (CBLs) were primarily from Bacillus genus, while those isolated from animal hide, hair and bone debris were members of Aeromonas genus. The microbial communities associated with two types of CBLs which were used to pre-treat the animal hide for 5 and 45 days, i.e. CBL C5 and CBL D45 respectively were investigated via metagenomic 16S V3-V4 rRNA amplicon sequencing using an Illumina MiSeqTM platform. The 16S rRNA gene libraries were demultiplexed, denoised/clustered, and then taxonomically classified using reference sequences from the Silva 16S databases using Qiime2 software. The results showed that both CBLs contained numerous genera of microbes made up predominantly of Burkholderiaceae, Enterobacteriaceae, and Carnobacteriaceae. And, these may not be viable microorganisms. The alpha and beta diversity of CBLs were also not substantially different. Two strains of Bacillus licheniformis that were encoded CP7 and DP1 were chosen for the studies, due to the generally recognized as safe (GRAS) status of many members of this bacterial species. The strains were evaluated for optimum growth conditions and nutrient requirement for the production of extracellular proteases, assayed using either casein or extracted soluble wool keratin as substrates. The results indicated that the optimum cultivation conditions for protease production by CP7 was 50oC, pH 7.5, 200 rpm, 24 h in sucrose, wool, NaCl and K2HPO4 (SWNK) media containing (w/v) 1.5% sucrose, 0.434 % wool, 5% NaCl and 70 mM K2HPO4, and for DP1, 50oC, pH 9.0, 200 rpm and 24 h in sucrose-malt extract, NaCl and K2HPO4 (SMNK) media containing 0.5% sucrose, 0.5% malt extract, 5% NaCl, and 70 mM K2HPO4. Only CP7 was found to be capable of producing keratinase(s) with the capability to degrade native chicken feather and wool. CP7 cell pellet was also investigated but was found to have quite a low level of proteases. The secreted CP7 proteases were harvested from the growth media and concentrated by ammonium sulphate precipitation. Two major protease containing fractions were obtained by anion exchange chromatography (Q-FPLC), which exhibited the capability of degrading keratin-containing materials. The target protease fractions were further fractionated using gel permeation (S100-FPLC) and analysed by reversed-phase chromatography (RP-HPLC). The protein and hydrolytic profiles of the protease fractions were investigated using SDS-PAGE and gelatin zymography. Protein gel bands were excised and subjected to in-gel digestion and LC-MS/MS for protein identification. Two protease fractions (QP0 SP2 and QP1 SP3) analysed were found to contain protease related to B. licheniformis Subtilisin Carlsberg. The optimal activity of the proteases was found to be 60oC, pH 10.0 and pH 6.0-10.0, respectively. This research has provided further insight into culturable protease-producing microorganisms in such gelatin production alkaline environment. Although the proteases analysed in this study have similar sequence homology to subtilisin Carlsberg, they appear to require different conditions for optimal activity. Various metal ions, protease inhibitors, reducing agents, and a detergent were also used to characterize both QP0 SP2 and QP1 SP3.
Advisor: Bekhit, Alaa El-Din Ahmed; Carne, Alan; Agyei, Dominic
Degree Name: Doctor of Philosophy
Degree Discipline: Food Science
Publisher: University of Otago
Keywords: Protease; keratinase; keratin; Gelatin; Bacillus; licheniformis; extremophile
Research Type: Thesis