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Study of Mutation and Breeding of Griseofulvin Produccing Strain FS80

Author: ZhangJianQing
Tutor: MaoNing
School: Fujian Normal University
Course: Microbiology
Keywords: mutation laser griseofulvin titer fermentation
CLC: TQ927
Type: Master's thesis
Year: 2011
Downloads: 16
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Abstract

Griseofulvin producing strain Penicillium patulum FS80-1 was taken as the original strain,combined treatment of UV and LiCl, the high-yield mutant GM120-43 was obtained after three generations of selection,which producing griseofulvin capacity could be 89496μg/g using diazonium salt color detection, compared with the original strain FS80-1 which increased by 80.04%. Mutant strain GM120-43 genetic stability was well.And then, mutant GM120-43 was taken as the original strain, mutant LD100-1 and C1448-1 were respective obtained by semiconductor and CO2 laser. After genetic stability test,the titer of mutant LD100-1 producing griseofulvin was 119766μg/g, improved 22.58%than the original strain GM120-43. The mutant C1448-1 obtained by the CO2 laser tested by genetic stability,which production of griseofulvin was lower than strain GM 120-43. The solid culture conditions of mutant LD 100-1 were researched, the results show that:the optimum carbon source substrate was Wanxian Mi; the optimum nitrogen was NaNO3;the suitable radio of medium solid to water was 1:0.75;the suitable precursors chloride for the synthesis of griseofulvin was single adding NaCl,and the concentration of chloride was 0.188 mol/L was more appropriate.; Seeds were inoculated with liquid,the suitable age of inoculated liquid seed was 44 h, inoculum was 10%. After optimization of culture conditions, the titer of griseofulvin producted by strain LD100-1 was 162233μg/g, which increased by 39.16% before optimization.The culture risce sproe of strain original strain FS80-1 and mutant GM 120-43, LD100-1 were extracted with ethanol.The content of griseofulvin in extracts was detected by HPLC, compared with the difference detection methods of HPLC and diazonium salt color, the results showed that:the capacity of strain FS80-1, GM120-43 and LD100-1 producing griseofulvin were 43511μg/g,91858μg/g and 105423μg/g, which were lower than the diazonium salt color method.Extract the genomic DNA of mutant GM120-43, LD100-1 and original strain FS80-1.,and take genomic DNA as template, using 18S universal primers for PCR amplification, sequencing the amplified 18S rDNA and compared each other. The results showed that the differences between mutant GM 120-43 and LD 100-1 ware little,just individual bases different,but it existed multiple bases differences compared with the strain FS80-1,of which there were three large regional differences in sequence.The liquid volume experiment and activity ofα-amylas were studied on the original strain and mutant GM 120-43,LD100-1 liquid fermentation.The results showed that griseofulvin producing strain Penicillium patulum was a strain needs great demand for oxygen,30 mL of 250 mL flask for fermentation was appropriate. And the detection showed that the a-amylase activity of mutant GM 120-43 was the highest, the enzyme activity up to 82.3 u/mL when fermentated by 3 d;the production of conidia was less than strain FS80-1 and GM 120-43, and the griseofulvin produced by strain GM 120-43 fermentation was higher than strain FS80-1 and LD100-1.

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