Screening Of Mehninproducing Strain And Studies On Tyrosinase Gene

Melanin is a complex polymer that is widely distributed in plants,animals and microorganisms.It is a complex polymer made from phenolic compounds.We can use natural melanin to produce microorganisms and the products can replace traditional chemical synthesis melanin in the food,chemical industry and medical and pharmaceutical industries.And tyrosinase is a key enzyme in melanin anabolic pathway.The study of tyrosinase not only lays a foundation for the regulation of microbial melanin synthesis,but also has important significance for the research and development of tyrosinase function.In this paper,a high yield melanin strain of XH2 was selected from Xinghai sea area of Dalian,and the physic-biochemical identification and 16S rDNA identification showed that XH2 belongs to Pseudomonas sp.and its melanin production and growth condition were also optimized.XH2 grew well at 20?,pH value was 7,NaCl content was 1%g/L,L-Tyr content was 0.5 g/L,CaCl₂ content was 0.1 g/L;in this condition,the melanin production of XH2 could reach 1.164 g/L after cultured for 72 hours,2.6 times as much as that of melanin without L-Tyr.A 580 bp tyrosinase gene sequence was amplified from the XH2 genome DNA,ligated with pMD19-T then transformed into E.coli DH5?competent cells.The recombinant plasmid was sequenced and the cloned sequence was analyzed by bioinformatics.the results showed that and.The gene sequence was 580 base pairs and inserted into the pMD19-T vector with positive direct.The target gene fragment encoded 162 amino acids;its molecular formula was C₇₉₄H₁₂₄₉N₂₀₃O₂₃₆S with a molecular weight of 17607.23Da,its isoelectric point was 5.47.The protein had 7 potential phosphorylation sites,so it was an unstable hydrophilic protein without signal peptide and transmembrane domain.Its secondary structure and tertiary structure showed it mainly contained?-helix and random coil structure.Sequence alignments showed that it had high similarity to known tyrosinase sequences.The target gene was transformed into BL21 expression bacterium using pET32a vector,and the optimal conditions for induction were optimized.The optimal concentration for IPTG induction was 1.0 mmol.The expressed fusion protein had a molecular weight of17.6 kDa and the maximum expression was reached after induced for 3 h.The discovery of XH2 has provided a new research object for the production of melanin by microorganisms.The study of its gene level has laid a foundation for further research and development of microbial production of natural melanin.