Screening And Fermentation Optimization Of D-Arabitol Producing Strains And Enzymatic Properties Of D-arabitol 2-dehydrogenase

D-arabitol is an important functional polyol,which can be used in the fields of food,chemical industry,and medicine.In view of this,it has been included on the list of 12 building block compounds and earmarked for further biotechnological research.D-arabitol can be produced by chemo-catalytic direct extraction and microbial biosynthesis methods,where the latter is considered to be more advantageous over the former two methods for its green,economic,eco-friendliness,and having potential for large-scale exploitation.In this study,D-arabitol producing osmophilic yeast strains were isolated from various plant sources and M.reukaufii PT-19 was finally selected based on the results,followed by the optimization of the fermentation conditions for this newly isolated yeast strain.The exogenous expression of the key enzyme,D-arabitol 2-dehydrogenase（2-ArDH）,was performed in Escherichia coli Rosetta（DE3）to study the enzymatic properties of 2-ArDH.This study provides a reference for the production of D-arabitol by biological method.The main research contents and results are as follows:（1）A total of 113 strains with good growth were isolated from the grape epidermis,soy sauce,soybean paste,honey,and pollen,and then 79 strains were found to have the capability of producing D-arabitol as determined by paper chromatography（PC）.Based on the depth and size of the color spots on the chromatogram,20 strains with relatively high yields of D-arabitol were selected and further screened by conducting fermentation and analyzing the metabolite in the high performance liquid chromatography（HPLC）.Among the strains,JY-15 and PT-19have outstanding ability to produce D-arabitol,which were identified as Zygosaccharomyces rouxii and Metschnikowia reukaufii,respectively based on the results of colony morphology,cell morphology,physiological,biochemical characterization and 18S r DNA sequencing.The identified strains were named Z.rouxii JY-15 and M.reukaufii PT-19.（2）The culture conditions and medium composition were optimized for M.reukaufii PT-19 by the one-factor-at-a-time（OFAT）and Box-Behnken Design（BBD）methods.Firstly,OFAT was used to optimize the temperature,inoculation amount,rotating speed,and fermentation time.The optimal results were 30℃,3%（v/v）,220rpm and 6 days,respectively.Secondly,the effects of medium components on the cell metabolites were explored.Maximum 76.32 g/L D-arabitol was achieved under 200g/L glucose as the sole carbon source,7.5 g/L peptone and 1 g/L ammonium sulfate as the nitrogen source,KH₂PO₄2 g/L and MgSO₄·7H₂O 2 g/L as the inorganic salts,and 7.5 g/L fumaric acid as the exogenous electron receptor.Finally,the BBD experiment was performed to explore the effects of the interaction of fumaric acid,KH₂PO₄and MgSO₄·7H₂O on the D-arabitol yield.The optimum conditions of fumaric acid,KH₂PO₄and MgSO₄·7H₂O were adjusted from 7.50 g/L,2 g/L and 2 g/L to 4.70 g/L,1.55 g/L and 1.53 g/L respectively.Under these final optimum conditions,the titer of D-arabitol further increased to 89.55 g/L.（3）The 2-ardh gene fragments with the size of 1053 bp（Zardh）and 850 bp（Mardh）were cloned from Z.rouxii JY-15 and M.reukaufii PT-19 respectively.Two engineering strains E.coli Rosetta/p ET-Zardh and E.coli Rosetta/p ET-Mardh were successfully constructed by plasmid p ET-30a and expression strain E.coli Rosetta（DE3）.The molecular weights of Z.rouxii-2-Ard H and M.reukaufii-2-ArDH proteins encoded by Zardh and Mardh genes were 42.8 kDa and 34.8 kDa,respectively.The optimum conditions of enzyme reaction were temperature 30℃,pH 9,Fe²⁺（5 mM）,and the optimum substrate for 2-ArDH is D-arabitol.When D-arabitol was used as the substrate under the optimum conditions,the K_mand V_maxof Z.rouxii-2-ArDH and M.reukaufii-2-ArDH were 45.63 mM,20.36 U/mg,52.23 mM and 26.18 U/mg,respectively.