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1. Full name: Pham Thuy Linh
2. Sex: Female
3. Date of birth: 27 July, 1981
4. Place of birth: Hà Nội
5. Admission decision number: No 2259/SĐH, dated 07 December, 2006
6. Changes in academic process: Extension number: 2514/QĐ-CTSV.
7. Official thesis title: “Investigation on production of recombinant enterocin P as food preservation”.
8. Major: Microbiology
9. Code: 62 42 40 01
10. Supervisors: - Associate Professor. PhD.Trương Nam Hải
- Professor. PhD. Phạm Văn Ty
11. Summary of the new findings of the thesis
The investigation gave following results: 1) Gene entP encoding for peptide EntP of E. faecium P13 was artificially synthesized, cloned and transformed into 3 expression systems. 2) In E. coli ER2566, EntP in fusion with MxeIntein were formed mostly as inclusion bodies with no antibacterial activity. EntP released from the cleaving of intein inhibited the growth of L. monocytogenes and S. aureus. 3) Gene hisentP was expressed in P. pastoris strain GS115 whiles gene entP and hisentP was successfully expressed in strains X33. X33 showed higher expression than GS115 and none of difference on the peptides productivity was identified in two X33 recombinant strains producing EntP and HisEntP. Appropriate X33hisentP fermenting conditions for production of EntP were determined to be: peptone and yeast extract-contain medium, pH 6, gene expression inducing at OD600 of 50, maximum air flow. Antibacterial activity of flask cultivation supernatant was 100 AU/ml. 5) Strain X33hisentP was grown in 10 and 80-liter fermentative equipments. Antibacterial activities were 50 and 25 AU/ml, respectively. 6) HisentP was collected most effectively by ethanol precipitation at concentration of 90%. HisentP remained activities after the heating of 60 minutes at 100oC and 15 minutes at 121oC as well as after 8 hours of exposure to pH between 2 and 11. HisentP was cleaved quickly in the presence of pepsin, papain, proteinase K, bromelin, trypsin and a-chymotrypsin resulting in the disappearance of its antibacterial activity. HisentP can inhibit L. monocytogenes, S. aureus, L. fermentum and E. faecium. 7) HisentP did not cause acute and sub acute toxicity in tested rats.
12. Practical applicability
This is the first systematic investigation of producing recombination bacteriocin in Vietnam, which results in recombinant protein HisentP with antibacterial and biochemical characteristics similar to natural enterocin P. HisentP can firstly extend food preservation shelf, which can be developed to biological preservation additives. More over, data from the study is useful scientific information for continue applicative investigations in the future.
13. Futher research directions
Optimizing the fermentation procedures to increase recombinant HisentP biosynthesis efficiency and decrease the costs of input material to decline the price of products.
Enhancing HisentP recovery efficiency from fermentation supernatant as well as optimizing the HisentP cleavage and HisentP purification for food application.
Investigating on preservation pattern with different kinds of food such as fruit, meat, products from meat, etc using recombinant EntP in various methods: immersing, spraying, additives, wrapping film, etc.
14. Thesis-related publications
Tran Ngoc Tan, Đinh Thu Hang, Nguyen Thanh Nhan, Pham Thuy Linh, Đo Thi Huyen, Le Van Truong, Truong Nam Hai (2008), “Synthesis and expression of a gene encoding circular enterocin AS48in Escherichia coli”, Journal of biotechnology 6(3), pp. 367-373.
Nguyen Thanh Nhan, Le Thu Ngoc, Đo Thi Huyen, Tran Ngoc Tan, Pham Thuy Linh, Le Van Truong, Truong Nam Hai (2009), “Cloning and expression of enterocin P fused with CBD-SSP Intein in Escherichia coli ER2566”, Journal of biotechnology 7(2), pp. 27-33.
Pham Thuy Linh, Le Thu Ngoc, Tran Ngoc Tan, Nguyen Thanh Nhan, Do Thi Huyen, Le Van Truong, Truong Nam Hai (2009), “Expression of a gene encoding enterocin P of Enterococcus faecium in Pichia pastoris GS115”, National conference on genetically modified organisms and biosafety 2009, pp. 145-149.
Nguyen Thanh Nhan, Pham Thi Minh Đuc, Le Thu Ngoc, Le Van Truong, Đo Thi Huyen, Tran Ngoc Tan, Pham Thuy Linh, Truong Nam Hai (2010), “Construction of recombinant plasmid from expresstion vector pAC7 containing entP gene fused with fragments of baamylF1 và baamylF2 for production of enterocin P in Bacillus subtilis 168”, Journal of biotechnology 8(1), pp. 13-20.
Pham Thuy Linh, Nguyen Khanh Hoang Viet, Do Thi Huyen, Tran Ngoc Tan, Nguyen Thanh Nhan , Le Van Truong, Pham Van Ty, Truong Nam Hai (2010), “Synthesis and expression of a gene encoding enterocin P of Enterococcus faecium in Pichia pastoris X33”, Journal of biotechnology 8(2), pp.221-226.
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