Preliminary Research Of A New Pyridoxal-5'-phosphate Hydrolase From Bombyx Mori

Vitamin B6(VB6)is a group of pyridine compounds existing widely in nature,including pyridoxine(PN),pyridoxamine(PM),pyridoxal(PL),and their related phosphorylated forms: pyridoxine-5'-phosphate(PNP),pyridoxamine-5'-phosphate(PMP),pyridoxal-5'-phosphate(PLP).They may transform to each other,but PLP is the catalytically active form of vitamin B6.The main physiological function of PLP is to participate in the reaction of amino acid metabolism as the cofactor of various enzymes in the cell,they can also have important physiological functions in other biology generation and metabolism.In the structure of PLP,there exists a active aldehyde group,it's easy to combine with other amino compound in the biont,such as aldimine.This may lead to disorder of normal matter in the organism.Banlanced surveyance of PLP is the key to maintain healthy biological state.There were many researches of the hydrolysis of PLP in mammal,it was not clear how to maintain homeostasis of PLP.The factors of PLP homeostasis involved PLP phosphatase,PL kinase,and PNP oxidase,the degree of protein binding of synthesized coenzyme,and transport.The hydrolysis of PLP was a improtant mechamism for the level of intracellular PLP.Cloning PLP related genes was the basis of these studies,and provided reference material to study the evolution of human-specific genes PLPP.As a model organism,silkworm had a large number of protein synthesis due to its special abnormal physiological,and the amino acid metabolized very actively in the body.Maintaining homeostasis of PLP concentration in vivo was more prominent in Silkworm.The study of vitamin B6 metabolism of silkworn played an important referential role to understand the physiological metabolism of human and even other organisms.In this study,we used molecular biological technique to clone BmPLPP gene in bombyx mori,its ORF was 1032 bp.It encoded 343 amino acids,the molecular weight was37.9 KD,and its PI was 8.83.There was no signal peptide sequence,it was belong to a non-secretory protein.The analysis of BmPLPP protein indicated it contained hydrolase domais It belonged to HAD protein superfamily as the same as human specific PLP phosphatase Multiple sequence alignment results showed BmPLPP was different from the known alkaline phosphatase of bombyx mori in gene and protein sequences,it was not their isozyme.The prokaryotic expression carrier of pET24b(+)-BmPLPP recombinant plasmid was constructed with correct sequence and direction,and transformed to E.coli BL21(DE3)processing prokaryotic induce expression,and optimized the terms of expression.The result of SDS-PAGE indicated that there existed soluble protein in supernatant.In order to separate the protein we used Ni-NTA affinity chromatography to purify the enzyme,and then we analysised the activity of enzyme by UV spectrophotometric method.The study of enzymatic properties suggested it was a new alkaline phosphatase,may hydrolyzed PLP.It had an optimal pH at 7.5(the activity of enzyme was high and stable when pH=7-7.5)and optimal temperature at 50?(under the conditions of high temerayure,the enzyme was very unstable;the enzyme can be better stable under 30 ?-40 ?)?Mg2+ was a catalyst for this enzyme,and the catalyst would be stronger when concentration increased.Zn2+had a little inhibiting effect on its activity,but the inhibiting effects of Mn2+?Ca2+?Cu2+?EDTA were very strong,and the inhibiting effect would be stronger when concentration increased.Mg2+ could relieve the part of inhibition EDTA.Under optimal conditions,the Km values of PLP and PMP were 0.368 and 0.404mmol/L,the Vmax of both were 0.907 and 0.394 umol/min/ug protein respectively.This indicated that PLP had better affinity than PMP.Under the physiological conditions,the enzyme also had some activity other phosphate substrates,but had the most activity to PLP.