| The ?-glucosidase inhibitors are a kind of oral anti-diabetic medications that can retard the absorption of intestinal carbohydrate.Acarbose is widely used in medicine for the treatment of diabetes and obesity.Soil samples were dried at room temperature for 5 days,then the soil dilutions were heated in water bath at 60°C for 20 min.To inhibit the growth of bacteria,yeasts and moulds,K2Cr2O7(100 mg/L)was added to the solid medium.A simple thin layer chromatography(TLC)-scanning technique was developed for the rapid and accurate analysis of acarbose in a large number of fermentation broths of actinomycetes for the screening of acarbose producer.The linearity of the acarbose in this way was excellent within the range from 2 to 10 ?g(r2 = 0.9997).This technique didn't need expensive instrument and complex procedure for the detection of acarbose in the fermentation broths.Streptomyces JN537 with acarbose yield of 1830 mg/L was obtained.Atmospheric and room temperature plasma(ARTP)was first employed to generate mutants of Streptomyces JN537 for improving acarbose production.The input voltage,the gas flow,the distance,and the temperature of the plasma jet were 112 V,2.0 L/min,2.0 mm,30-40°C,respectively.To obtain mutant strain with higher acarbose production,the method of screening the strains for susceptibility to penicillin was used after treatment with ARTP.Acarbose yield of the mutant strain Streptomyces M37 increased by 62.5% than that of the original strain.The contents of monosaccharides and amino acids of the cell wall of Streptomyces M37 were lower than that of the original strain.The acarbose production ability in mutant strain remained relatively stable after 10 generations.The effect of pH on Streptomyces M37 growth and the acarbose biosynthesis ability was studied.Results revealed that neutral pH was beneficial for cell growth,whereas alkaline pH favored acarbose synthesis.Moreover,addition of glucose and maltose to the fermentation medium after 72 h of cultivation promoted acarbose production.Based on these results,a two-stage fermentation strategy was developed to improve acarbose production.Accordingly,pH was kept at 7.0 during the first 72 h and switched to 8.0 thereafter.At the same time,NH4 Cl,glucose and maltose were fed to increase acarbose accumulation.With this strategy,an acarbose titer of 6210 mg/L was obtained,representing an 85.7% increase over traditional batch fermentation without pH control.Finally,it was determined that the increased acarbose production was related to the high activity of glutamate dehydrogenase and glucose 6-phosphate dehydrogenase.The sodium alginate gel was modified with powdered activated carbon(PAC).The acarbose production by entrapped Streptomyces M37 in sodium alginate and PAC gel matrix was investigated.Acarbose production increased by 23.9% compared to free cell fermentation under optimum conditions(1.5%(w/v)PAC,2%(w/v)sodium alginate).Scanning electron microscopy(SEM)showed that the porous gel bead was obtained with the addition of PAC.Meanwhile,cell leakage was reduced by 61.3% and the fermentation time was reduced to 96 h.According to the biosynthesis pathway of acarbose,iodoacetic acid and sodium fluoride were added at different stages of the acarbose fermentation.Results demonstrated that the acarbose yield was increased by 12.1% when 2 mg/L iodoacetic acid was added at 72 h.And the productivity of acarbose was increased by 14.6% when 15 mg/L sodium fluoride was fed at 96 h.The productivity of acarbose was increased by 21.5% when 1.5 mg/L iodoacetic acid was added at 72 h and 10 mg/L sodium fluoride was fed at 96 h,respectively.Meanwhile,the specific activities of 3-glyceraldehydes phosphate dehydrogenase and enolase were decreased by 19.6% and 21.0%,respectively.By contrast the activity of 6-phosphoglucose dehydrogenase was increased by 34.5%.The change of enzymes revealed that the EMP pathway was reduced and HMP pathway was increased,which promoted the fermentation of acarbose. |
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