| Illicium verum is the characteristic spice plant of Guangxi province,and star anise is used widely in the fields of food and medicine.In this project,the endophytic fungi of Illicium verum were isolated and purified from the healthy leaves,twigs and fruits of the plant,and biotransformation activities to trans-anethole,cinnamaldehyde,and acetophenone of the endophytic fungi were studied.The main research contents and results are shown as follows:1.Isolation,screening and identification of the endophytic fungi with the biotransformation activity to trans-anethole.Used the method of plant tissue culturing on PDA medium,a total of 79 strains of endophytic fungi were isolated from the Illicium verum plant,including 24 strains of leaves,32 strains of twigs and 23 strains of fruits,respectively.By whole cell biotransformation method,two strains,BGEF13 and BJEF32,which may transform trans-anethole into anisic alcohol and isovanillic acid respectively,were screened from the 79 endophytic strains by the substrate tolerance tests,TLC and HPLC analysis.By morphological traits and fungal ITS1-5.8S-ITS2 region sequence analysis,the strains of BGEF13 was classified as Nigrospora oryzae and BJEF32 was classified as Lasiodiplodia pseudotheobromae.2.Biotransformation of trans-anethole into isovanillic acid by the endophytic fungus L.pseudotheobromae BJEF32.The biotransformation conditions and microbial culture conditions for the production of isovanillic acid were studied by the single-factor experiments.Results revealed that the optimal biotransformation conditions were as follows:0.05 mol·L-1 Na2HPO4-KH2PO4 buffer(pH 7.0)as reaction medium,20 mL of liquid volume(in a 100 mL flask),cell concentration of 50 g·L-1,traps-anethole concentration of 1.48 g·L-1,10 g·L-1 glucose as co-substrate,shaking speed of 150 rpm,reaction temperature of 30 ?,and the reaction time of 48 h.And the optimal culture conditions for the L.pseudotheobromae BJEF32 cells were as follows:potato extract concentration of 250 g·L-1,glucose concentration of 20 g-L-1,0.1 g·L-1 trans-anethole as inducer,initial pH value of 7.0,100 mL of liquid volume(in a 250 mL flask),inoculum dose of 10 pieces of mycelial cake(?6mm),culture temperature of 28 ?,shaking speed of 150 rpm and culture time of 96 h.Under the optimal biotransformation and culture conditions,the yield of isovanillic acid was about 62%with a concentration of 1.04 g·L-1.Results of enzymatic tests and intermediates transformation experiments showed that,the postulated biotransformation pathway of trans-anethole by L.pseudotheobromae BJEF32 may be as follows:trans-anethole ? anethole epoxide ? anethyl diol ? anisaldehyde/anisic alcohol ? anisic acid?isovanillic acid.3.Biotransformation of trans-anethole into anisic alcohol by the endophytic fungus N.oryzae BGEF13.The biotransformation conditions and microbial culture conditions for the production of anisic alcohol were optimized by the single-factor experiments.Results revealed that the optimal biotransformation conditions were as follows:0.05 mol·L-1 Na2HPO4-KH2PO4 buffer(pH 6.0)as reaction medium,40 mL of liquid volume(in a 100 mL flask),cell concentration of 50 g·L-1,traps-anethole concentration of 2.96 g·L-1,10g·L-1 glucose as co-substrate,shaking speed of 120 rpm,reaction temperature of 30 ?,and the reaction time of 24 h.And the optimal culture conditions for the N.oryzae BGEF13 cells were as follows:potato extract concentration of 200 g·L-1,glucose concentration of 20 g·L-1,peptone concentration of 2 g·L-*1,0.1 g·L-1 trans-anethole as inducer,initial pH value of 6.5,100 mL of liquid volume(in a 250 mL flask),inoculum dose of 10 pieces of mycelial cake((p6mm),culture temperature of 28 ?,shaking speed of 120 rpm and culture time of 96 h.Under the optimal biotransformation and culture conditions,the yield of anisic alcohol was about 42%with a concentration of 1.16 g· L-1.Results of enzymatic tests and intermediates transformation experiments showed that,the postulated biotransformation pathway of trans-anethole by N.oryzae BGEF13 may be as follows:trans-anethole? anethole epoxide?anethyl diol ? anisaldehyde ?anisic alcohol/anisic acid.4.Reduction of acetophenone into chiral compound R-(+)-1-phenylethanol catalyzed by the endophytic fungi isolated from Illicium verum.A strain Neofusicoccum parvurum BYEF07,with the catalytic activity of the reduction of acetophenone to 1-phenylethanol,was screened from the 79 endophytic strains and the main product was confirmed to be(R)-(+)-1-phenylethanol.The optimal bio-reduction conditions were as follows:0.05 mol·L-1 Na2HPO4-KH2PO4 buffer(pH 7.5)as reaction medium,40 mL of liquid volume(in a 100 mL flask),cell concentration of 100 g·L-1,acetophenone concentration of 1.8 g·L-1,10 g·L-1 glucose as co-substrate,shaking speed of 150 rpm,reaction temperature of 30 ?,and the reaction time of 48 h.And the optimal culture conditions for the N.parvum BYEF07 cells were as follows:potato extract concentration of 250 g·L-1,sucrose concentration of 20 g·L-1,initial pH value of 7.5,100 mL of liquid volume(in a 250 mL flask),inoculum dose of 12 pieces of mycelial cake(?6mm),culture temperature of 28 ?,shaking speed of 120 rpm and culture time of 120 h.Under the optimal bio-reduction and culture conditions,the yield of 1-phenylethanol was 85%with a concentration of 1.55 g·L-1 and the enantiomeric excess value of(R)-(+)-1-phenylethanol was 99%.Substrate inhibition and product inhibition phenomena were founded in the reduction of acetophenone into R-(+)-1-phenylethanol by N.parvum BYEF07 cells.And the Han-Levenspiel model were used for described the effects of substrate and product inhibition.The simulation parameters of substrate inhibition model were as follows:Vsm of 0.082 mmol·L-1·g-1·h-1,Ks of 4.24 mmol·L-1,Sc of 95 mmol·L-1 and n value of 1.45.And the simulation parameters of product inhibition model were as follows:Vpm of 0.051 mmol·L-1·g-1·h-1,Pc of 146 mmol·L-1 and m value of 1.28.5.Hydrogenation of cinnamaldehyde into cinnamic alcohol and 3-phenylpropanol by the endophytic fungi isolated from Illicium verum.Two strains,Aspergillus tubingensis BGEF03 and Botryosphaeria dothidea BGEF10,with the catalytic activity of hydrogenation of cinnamaldehyde into cinnamic alcohol and 3-phenylpropanol were screened from the 79 endophytic strains.And the biotransformation conditions were studied detailed by the single-factor experiments.The optimal conditions for the production of cinnamic alcohol by the strain Aspergillus tubingensis BGEF03 were as follows:0.05 mol·L-1 Na2HP04-KH2PO4 buffer(pH 6.5)as reaction medium,40 mL of liquid volume(in a 100 mL flask),cell concentration of 50 g·L-1,cinnamaldehyde concentration of 1.5 g·L-1,10 g·L-1 glucose as co-substrate,shaking speed of 120 rpm,reaction temperature of 25 ?,and the reaction time of 24 h.Under the optimal conditions,the yield of cinnamic alcohol was 58.5%with a concentration of 0.89 g·L-1 and no 3-phenylpropanol were detected in the reaction.The optimal conditions for the production of 3-phenylpropanol by strain Botryosphaeria dothidea BGEF 10 were as follows:0.05 mol·L-1 Na2HPO4-KH2PO4 buffer(pH 7.0)as reaction medium,40 mL of liquid volume(in a 100 mL flask),cell concentration of 75 g·L-1,cinnamaldehyde concentration of 1.0 g·L-1,15 g·L-1 glucose as co-substrate,shaking speed of 150 rpm,reaction temperature of 30 ?,and the reaction time of 72 h.Under the optimal conditions,the yield of 3-phenylpropanol and cinnamic alcohol were 78.9%and 5.4%,with the concentration of 0.81 g·L-1,and 0.06 g·L-1,respectively. |
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