Hydrolyze Technology Of Royal Jelly Protein And Recombinant Expression Of AccMRJP5 Gene From Apis Cerana Cerana In Pichia Pastoris And Its Activity

Royal jelly (RJ), a principal food of the honeybee queen and larvae of worker bees and drones within three days of emergence, is secreted from the hypopharyngeal and mandibular glands of nurse honeybees. RJ has anti-fatigue, prevention of cancer, immune function, and other nutritional and physiological activities which are closely related to its components. Protein is one of the most important components of RJ. RJ proteins were divided into two categories, water-soluble portion and water-insoluble portion. The water-soluble protein was also named as the major royal jelly proteins (MRJPs). It is believed that MRJPs plays an important role in the royal jelly's physiological activities according to many researches.Firstly, we study the effect on the hydrolyzed characters of pH, hydrolyzed time, protease concentration and other parameters. Results showed that the optimum condition was that MRJPs hydrolyzed at 37℃, with pepsin at pH 2.0 with 1%(w/w) enzyme dose, followed by trypsin at pH 7.5 with 1%(w/w) enzyme dose for 4 h. In this condition, the degree of hydrolysis was 28.7%, total nitrogen recovery was 35.5%. The hydrolysates of MRJPs (H-MRJPs) was separated and purified by ultrafiltrattion, we found that the antioxidant and antihypertensive activity of the less than 1 kDa peptide fraction was the highest part, which showed that peptides with molecular weight lower than 1 kDa posseses powerful inhibition of angiotensin-Ⅰconverting enzyme(ACE) with the half inhibitory concentration (IC50) to 0.67 mg pro/mL.In addition, the AccMRTP5 gene was cloned into eukaryotic expression vector pPIC 9K, and the shuttle vector pPIC 9K-AccMRJP5 was transformed into yeast, Pichia pastoris GS115. Analysis result of both SDS-PAGE and Western blot showed that the expression products contained a specific band with about 100 kDa band in size, which confirmed the successful expression of AccMRJP5 in yeast. The molecular weight of the recombinant AccMRJP5 was decreased from 100 kDa to 69 kDa, indicating that AccMRJP5 expreesed in yeast was glycosylate. The recombinant AccMRJP5 was purified through affinity chromatography, and the SDS-PAGE analysis showed that the the protein was purified successed. At last, we studied the effect on the growth of Bm-17 cell of recombinant AccMRJP5 in the serum-free medium. The results show that the AccMRJP5 has a nutritional function in cell growth. AccMRJP5 may replace FBS in cell cultures which may has important research value and application prospect.Peptides with anti-oxidative and anti-hypertensive activity were successfully prepared by hydrolysis of MRJPs. AccMRJP5 gene was cloned into the eukaryotic expression vector and expressed in pichia pastoris successfully. The study also shows that AccMRJP5 could stimulate the Bm-17 cell growth in serum-free medium.