Alkaline Pectinase Producing Strain Breeding By Genome Shuffling

Microbial pectinases account for 25%of the global enzymes sales.Alkaline pectinase not only broadens the pH range,but also broadens the application range,it has a significant value in the textile industry,plant flax,cotton refining,paper making and so on.The research chose Bacillus subtilis ZGL14 as the starting strain,using a variety of mutation methods to improve its production of alkaline pectinase combined with inactivated protoplast and Genome shuffling technology,and the fermentation conditions were optimized,which would lay a foundation for the research and industrial production of alkaline pectinase.Breeding high-yielding strains of alkaline pectinase by the traditional mutagenic methods.ZGL14 cells were mutagenized with ultraviolet irradiation and three antibiotics(ampicillin sodium,chloramphenicol and streptomycin).The optimal mutagenic conditions were as follows:UV lamp power 20w,vertical distance 30cm,irradiation time lOminutes;the best mutagenic concentration:amp icillin sodium 0.03?g/mL;chloramphenicol 3?g/mL;streptomycin 20?g/mL.Seven strains with high enzyme activity were screened by the method of transparent circle(H/C),and the enzyme activity of UV12 was increased by 21.83%compared with the original strain.By UV-streptomycin2O and 60Co-?-streptomycin20 compound mutation,screening of strain UV-45 and C-S50,its enzyme activity increased by 24.7%and 25.6%,The conditions of Bacillus subtilis ZGL14 protoplast preparation,regeneration and inactivation were studied.The optimized conditions for protoplast preparation were as follows:seeds solution cultured for 9h to collect cells,0.2mg/mL lysozyme enzyme at 32? and pH 7.0 for 10minutes to allow cell wall lysis.Protoplast regeneration medium with sodium succinate as the osmotic pressure stabilizer combined with Ca2+ regeneration is better,monolayer culture is conducive to regeneration.The best conditions of UV-inactivated and heat-inactivated were:UV lamp irradiation time 70minutes;70? water bath for 20minutes.Protoplast fusion was induced by polyethylene glycol,and the fusion was chosen using inactivated protoplast fusion.Fusion strains were selected Bacillus subtilis ZGL14?UV-S45?C-S50?UV10?UV12 and S9 as parental strains.After two rounds of genome shuffling,a high-yielding recombinant 105 was obtained,the alkaline pectinase yield is 1.63 times that of the Bacillus subtilis ZGL14.The fermentation medium and fermentation conditions of alkaline pectinase produced by the strain 105 were optimized.The operating factors,including the nitrogen sources,carbon sources,inorganic salt,metal ion,inoculation amount and initial pH were studied.The optimized fermentation medium by single factors and response surface analysis experiments was:pectin 1.9%,peptone 1%,K2HP040.26%,KH2PO4 0.16%,inoculation amount 9%and initial pH 9.0 at culture temperature 40? for 24h.Alkaline pectinase activity reached 1030.71U/mL,which was 115.85%higher than that of the recombinant strain 105 and 3.4 times that of the original strain ZGL14.Through rpsL gene analysis,the changes occurred G to A?G to A and C to G transversions changes occurred at nucleotide pair 52?408 and 409,which resulted in the changes of Aspartic acid(18)to Asparagine and Proline(137)to Alanine.