| R-(+)-2-(4-hydroxyphenoxy)propionic acid(D-HPPA)is an important chiral intermediate for the synthesis of aryloxyphenoxypropionate(APP)herbicides,which has huge yearly demand.Beauveria bassiana is one of the whole-cell biocatalysts that can carry out a variety of biocatalytic reactions.The hydroxylation reaction of Beauveria bassiana is used to synthesize D-HPPA.This method has obvious advantages than traditional chemical methods and fermentation processes,because of its environmental safety,high efficiency,simplicity and convenience.In this paper,the mutant strains of Beauveria bassiana were mutagenized by high substrate tolerance and high conversion rate,and then the mutagenic mutants were embedded.The embedding and reaction conditions were determined and optimized to improve the reaction rate of biotransformation.Research contents and conclusion with main this text are as follows:(1)Original bacteria Beauveria bassiana B2660 was treated by UV mutagenesis(The UV lamp power is 30 W)and plasma mutagenesis(The working gas is helium;the instrument power is 100 W;the mutagenic distance is 2 mm;the gas helium flow rate is 10 L/min).The lethal rate was 91.1% under UV light for 25 min.After identification and genetic stability analysis,a high resistant mutant U07 was obtained.The substrate tolerance of this mutant increased from 10 g/L to 100 g/L.Then U07 was further treated by plasma mutagenesis.The mutagenesis time was 120 s,and the lethality was 92.8%.After identification and genetic stability analysis,a mutant U07D63 was obtained.It has high conversion rate 98.6% with a substrate concentration of 50 g/L-solution was bio-transformed to synthesize D-HPPA.The conversion rate and the average production intensity was 7.04 g/(L·d),which was 7.65 times higher than the average production intensity of Beauveria bassiana B2660.(2)In order to minify agglutination of the strains and reduce bad impact of external environment on the bacteria,sodium alginate and polyvinyl alcohol were used to embed Beauveria bassiana.The embedding agent concentration,crosslinking agent concentration and the cross-linking time were optimized.After the optimization experiment,the optimal embedding conditions for Beauveria bassiana were obtained:4% sodium alginate and 4.5% polyvinyl alcohol were mixed and used to make gel beads,and then placed in 2.5% calcium chloride solution for cross-linking for 6 h.Under this embedding condition,biotransformation was carried out at a substrate concentration of 60 g/L.The reaction time was 6 days,the product concentration was56.7 g/L,the conversion rate was 94.5%,and the average production intensity was 9.45g/(L· d).Compared with not embedded strain,the reaction time of biotransformation of mutant Beauveria bassiana was reduced by 25%,the average production intensity was increased by 42.3%,and the conversion rate was increased by 6.8%.(3)In order to further improve the reaction rate and conversion efficiency of the mutant U07D63 biotransformation to synthesize D-HPPA,the initial substrate concentration,temperature,p H and stirring speed were optimized.After the optimization experiment,the optimal reaction conditions of gel beads by Beauveria bassiana were as follows:the substrate concentration was 70 g/L,the reaction temperature was 28?,the reaction p H was 6.5,and the stirring speed was 200 r/min.After reaction for 6 days,the product concentration is 69.1 g/L,the conversion rate is98.7%,and the average production intensity is 11.52 g/(L·d).The gel beads can be reused four times.The product concentration was increased by 21.8%,and the average production intensity was increased by 21.9%.Through mutagenesis and immobilization of Beauveria bassiana,it not only solves the actual problems in the biotransformation process,improves the reaction rate and conversion efficiency of biotransformation,but also provides for the synthesis of D-HPPA by the biotransformation method.The research direction and theoretical basis are discussed. |
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