ISSN 2736-1756
Advanced Journal of Microbiology Research ISSN 2241-9837 Vol. 12 (11), pp. 001-009, November, 2018. © International Scholars Journals
Full Length Research Paper
Expression and identification of a small recombinant beefy meaty peptide secreted by the methylotrophic yeast Pichia pastoris
Song-Rong Zeng1,2, Yan-Ping Wang1*, Yong-Qing Yang1,3 and Jian Zhang1
1College of Food Engineering and Biotechnology, Key Laboratory of Food Nutrition and Safety of the Ministry of Education, Tianjin Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Tianjin 300457, P. R. China.
2Yingdong College of Life Sciences, Shaoguan University, Shaoguan 512005, P. R. China.
3Handan Municipal Center for Disease Prevention and Control, Handan 056002, P. R. China.
Accepted 10 November, 2018
Abstract
A tasty peptide, beefy meaty peptide (BMP), which was initially isolated from beef digested by papain, has potential as a new flavor enhancer. BMP is a small peptide consisting of eight amino acids. In this study, DNA encoding BMP was synthesized and cloned into the expression vector pPICZ A to obtain the recombinant expression vector pPICZ A-BMP. The recombinant vector was linearized and then integrated into the genome of Pichia pastoris strain X-33 by electroporation. The recombinant strain P.p2 was then incubated in BMGY medium and then induced in BSM medium for expression of BMP. The fermentation broth was centrifuged and the supernatant was purified by gel-filtration chromatography. Purified recombinant BMP was then tested by high performance liquid chromatography- mass spectrometry (HPLC-MS). After optimization of the culturing process, the yield of BMP reached 10 mg/L in the clarified broth. The results of mass spectrometry/mass spectrometry (MS/MS) showed that recombinant BMP secreted from P. pastoris had a molecular weight of 873.7 Da rather than the expected 847 Da. We speculate that the last amino acid, Ala, of the BMP sequence was replaced with Pro in the BMP fragment.
Key words: beefy meaty peptide, Pichia pastoris, peptide expression, HPLC-MS.