The Comparative Analysis Methods Of Spectinomycin And Protoplast Formation Of Streptomyces Spectabilis

Spectinomycin is a broad-spectrum antibiotic which belongs to aminoglycosides and show great efficiency against a broad range of microorganisms including G~+ and G~- germ and have a wide application future. Therefore, it will be of great social and economic value for us to screen a Spectinomycin-overproducing strain.In this paper, as an initial strain, whose potency is as high as 2000 U/mL. This strain was studied at variety of condition including cell culture time, lysozyme concentration, enzymatic hydrolysis time, hydrolysis temperature, pretreatment methods to judge facts effecting protoplast formation and regeneration of Streptomyces spectabilis so as to formate protoplasts. And the best conditions was concluded as following: the strain was incubated in medium S adding 0.5% glycine for 36 h at the constant temperature 30℃, 220 r / min, washing mycelium with 0.3 mol / L sucrose as a pretreating method before got hydrolysed, then hydrolysing for 120 min with a lysozyme concentration of 2 mg / mL at 37℃. The technique mentioned above could lay the foundation for exogenous DNA transducting protoplast, protoplast mutation and fusion.In addition to the study on protoplast formation and regeneration, the comparison was carried out as well between HPLC, GC, cup-plate method and turbidimetric method which were for the use of detecting spectinomycin. And turbidimetric method was considered as the best one owing to its accurency, high effection and short-time consuming.Besides, spcS2 coding a glutamine-aminocyclitol transferase was amplificated with PCR technique using the spcS2 nucleic acid sequence from NCBI database as a template and applying primer premier 5.0 software as primers designing, and then sequenced to be 1460 bps long and blasted with spcS2 from NCBI to get an identity of 95%.After that, the cloned spcS2 was conjucted to Escherichia coli-Streptomyces shuttle expression vector pHZ1272 formming a recombinant plasmid pHZ1272-spcS2, then transtucted E.coliDH5αand growen on LB adding 100μg/mLAmp.Unluckily no bateria of positive clone was picked from a number of clones.