Mutation Breeding And Optimization Of Fermentation Conditions Of Actinoplanes Utahensis

Alpha-glucosidase inhibitors have been widely used in pharmaceutical and agricultural fields as they could inhibit the hydrolysis of carbohydrate.Acarbose,one of the a-glucosidase inhibitors,has been widely used in the therapy of diabetes mellitus type II since 1990's in many countries due to its high efficacy and safety.It is believed that acarbose can reduce postprandial hyperglycemia effectively by competitively combining to glycosidase in small intestine.Nowadays,commercial production of acarbose was mainly performed by Actinoplanes sp.In this paper,a microtiter plate-based screening method was established.With this method,mutants originated from A.utahensis ZJB08196 treated with various mutation methods were screened.A mutant A.utahensis DG628-6-12 with improved acarbose productivity was obtained.Then the fermentation conditions were preliminary optimized,and the yield of acarbose of the mutant was further increased.Firstly,a colorimetric screening method was established based on the inhibition of a-amylase-catalyzed hydrolysis of 2-Chloro-4-nitrophenyl-4-O-?-D-galactopyranosyl-maltoside(Gal-G2-a-CNP)by a-glucosidase inhibitors.Gal-G2-a-CNP could be hydrolyzed to chromophore 2-chloro-4-nitro-phenol(CNP),which has a characteristic absorbance at 402 nm.Hence,this method relies on the absorbance change of the assay at 402 nm.The effect of factors such as temperature and pH on the reaction was investigated.The data showed that the optimal reaction assay system was 200 ?l reaction mixtures consisting of 40 ?l 5 mmol/L Gal-G2-a-CNP,30 ?l ?-amylase,100 ?l 50 mmol/L pH 6.0 Na2HP04-citric acid buffer and 30 ?l samples from mutants at 37?.The screening of mutants with higher acarbose productivity was greatly facilitated by this method.By different mutation methods such as UV-LiCl,?-ray irradiation and N+ low-energy ion implantation,A.utahensis was mutated and mutants were screened by the above method.The production of acarbose was improved by 30.18%,47.87%and 51.33%,respectively.To avoid the"fatigue effect" induced by repeated single mutagenic factor,different factors were combined to further improve the production of acarbose.As a result,a positive mutant A.utahensis DG628-6-12 was obtained with acarbose yield of 4099 ?g/ml,increasing by 103.12%.The fermentation medium and conditions were optimized to further improve the acarbose productivity of A.utahensis DG628-6-12.The data showed that the optimal fermentation medium was as follows(in g/L):glucose 30,maltose 60,sodium glutamate 4,glycerin 4,soybean meal 17,CaCl2 2.0,CaC03 4.0 and K2HP04 1.0.The optimal fermentation conditions was:inoculation size 9%(V/V),initial pH 7.0,50 ml culture broth in 500 ml flask and at 28 ??180 rpm for 168h.Under the optimal conditions,the yield of acarbose was further improved to 4470 ?g/ml.Finally,the utilization of glucose,maltose,sodium glutamate and changes in biomass,acarbose and osmolarity during the fermentation process were determined.The results showed that acarbose was a growth-related metabolite of A.utahensis.In the later stage of fermentation,the synthesis of acarbose might be blocked due to the low maltose concentration.Therefore,addition of maltose may be helpful for the acarbose synthesis.