Expression Of Tryoptophan Hydroxylase-2 In E.Coli And Construction Of Expression Vector In Pichia Pastoris Expression System

5-hydroxy tryptophan (5-HTP) is the precursor of 5-hydroxytryptamine (5-HT), which is an important inhibitory neurotransmitter, and is an important chemical substance that controls emotions, behavior, sleep, appetite in the brain.5-HTP is not only successfully used in anti-depression, insomnia treatment, but also can help produce satiety, thereby reducing appetite, and has some effect on weight loss.Tryptophan hydroxylase-2 (TPH-2) is the rate-limiting enzyme in 5-HT biosynthesis which directly catalyses tryptophan to 5-HTP. In this study TPH-2 protein was obtained by exogenous expression of TPH2 gene, which has an important academic value and may provide a new important way for industrial production of 5-HTP.The dissertation focuses on the expression of the human TPH-2 gene in E. coli expression system and in Pichia pastoris expression system. In this study the prokaryotic expression vectors pET32a-TPH and PGEX-4T-1-TPH,Pichia pastoris expression vectors pPICZa-TPH and pPIC3.5k-TPH were constructed. The expression of recombinant protein in E. coli BL21(DE3) and Pichia expression system was studied, respectively. In E. coli expression system, we successfully expressed the fusion protein with an his tag and Gst tag by the two vectors, respectively. In Pichia pastoris expression system, the target gene also had been successfully integrated into the yeast genome and a stable engineered strain for foreign protein expression was constructed for the first time.In the study of 5-HTP production from tryptophan by biotransformation, 40 strains of fungi, more than ten strains of actinomycetes and several unknown strains isolated from Griffonia simplicifolia seed were screened. Firstly, substrate was added to the fermentation broth in the early log phase for biotransformation, and in the mean time, to prevent microbial utilization of substrate tryptophan, biotransformation was studied using the method of resting cells. That is, gathering the thalli by centrifugation and dispersing it in physiological saline containing substrate for the conversion of the substrate. Through the study of microbial transformation of 5-HTP with two methods, the feasibility was preliminarily explored for 5-HTP production by microbial conversion and set up foundations for future studies.In order to study the physiological function of 5-HTP, the experiment on appetite suppression of 5-HTP was carried out in mice. By comparison of the positive control with blank control, We feed the mices with 0.5mL 0.1mg/mL 5-HTP by twice a day for 7d. Through observation and statistics data, the weight of mice fed with physiological saline increased by 7.4%, but the weight of mice fed with 5-HTP decreased by 2.5%. Finally, the results proved that 5-HTP has some effect on appetite suppression and weight lost.