| Difructose anhydride ?(DFA ?)is a novel functional sweetener.This rare and natural trisaccharide is a kind of indigestible saccharide,with low-calorie.It has the functions of stimulating the absorption of mineral elements,promoting bone growth and improving constipation,etc.With its good physical and chemical properties,it is suitable for use in foods and pharmaceutical preparations.However,the yield of DFA ? is low in the nature.It will cost too much to product the DFA ? by extraction and separation.When DFA ? was preparaed by chemical methods,there were some isomers which could cause trouble for purification.In addition,production with chemical methods was not unfriendly to the environment.Therefore,recently DFA ? was mainly preparaed by biological methods.Inulin Fructotransferase(IFTase)can convert inulin into DFA ?,having a good conversion rate and potential practical application.In addition,the rich resources of inulin are helpful for the industrial production,and inulin-based method for production of DFA ? has become more and more popular in recent years.However,the existing IFTases generally has problems such as low enzyme activities,low conversion rate and high production cost.Therefore,using modern molecular biology methods to obtain new IFTases,improving catalytic efficiency and reducing conversion cost should be very necessary for promoting the application of DFA ?.In this study,through bioinformatics analysis,Kp-IFTase from Klebsiella sp have been cloned and expressed.After analysis,the Kp-IFTase was found to have function of transferring inulin into DFA ? and other fructo-oligosaccharides.The Kp-IFTase had very high thermostability.In addition this enzyme showed excellent p H stability which could maintain more than 80% of residual enzyme activity at p H 3.0-10.0,and was highly resistant to extreme acid conditions.Additionally,Kp-IFTase had very high storage stability and could maintain more than 50% residual enzyme activity after storage for 35 days at 4 °C.Moreover,the enzyme showed strong tolerance to organic solvents,metal ions and some protein denaturants.These excellent properties of Kp-IFTase predicted pertancial application of this enzyme in industrial production.In addition,the gene encoding IFTase from Arthrobacter aurescens(Aa-IFTase)have been cloned and expressed.During cloning of Aa-IFTase and other genes,there are some problems of low efficiency,inconvenience of selecting cloning sites,and low levels of protein expression.To overcome these disadvantages,we have constructed a novel mini-vector p ANY1,which has been submitted to Addgene(Addgene No.110949).The p ANY1 has two multiple cloning sites(MCSs),which can simultaneously meet the requirements of sticky-end ligation-based cloning,blunt-end ligation-based cloning ligase-independent cloning and TA cloning with the digestion of Ahd I.In addition,p ANY1 contains a ccd B cassette between multiple cloning sites,which can efficiently avoid false positives and introduce zero-background cloning.Additionally,the vector also contains both T7 promoter and T7 terminator,which permits inducible protein expression,and the hexahistidine tag(Histag)site was used for purification of expressed proteins.To further increase cloing efficiency,an annealing of PCR products(APP)-based sticky-end cloning strategy was introduced to avoid the difficulty in selection of appropriate restriction digestion sites in some cases because of the presence of internal sites.Therefore,zero-background cloning and very efficient expression of Aa-IFTase was realized based on p ANY1.This valuable vector can provide a new choice for more efficient molecular cloning and protein expression for other laboratories.On this basis,the zero-background cloning and high expression of Aa-IFTase were successfully realized,which also laid the foundation for the construction of libraries.In order to further increase activity of Aa-IFTase and productivity of DFA ?,a rignion shuffling method based on metagenomic libraries to realize molecular modification of Aa-IFTase was designed.The degenerate primers were designed and synthesized using Code Hope method based on DNA sequence alignment against IFTases.Then the degenerate primers were used for amplification of homologous DNA fragments of IFTases from metagenomic DNAs isolated from five soil samples.The obtained omologous DNA fragments were used for replace the corresponding region of DNA sequence of Aa-IFTase through homologous recombination,and the resulting genes were performed function analysis.To obtain high-quality metagenomics DNAs with richness and representative,a novel silica sands-based method have been developed for isolating high quality genomic DNAs.And DNA extraction from various soil samples was applied by it.This method and a commercially available kit were compared in analysis of microbial communities using high-throughput 16 s r DNA sequencing.As a result,the silica sands-based method was found to be even more efficient in isolating genomic DNA from gram-positive bacteria than the kit,indicating that it would become a very valuable choice to faithfully reflect the composition of microbial communities.To further improve activity of Aa-IFTase for more efficient production of DFA ?,in this study a novel strategy was used to produce expressed Aa-IFTase embedded in curdlan-based mesoporous silica microspheres(i.e.,CMSi M-Aa-IFTase).CMSi M-Aa-IFTase was constructed by co-entrapping cross-linked Aa-IFTase aggregates and curdlan into biomemitic silica.During this process,the curdlan served as an agent to introduce pores in silica microspheres.The resulting m CLEAs-Aa-IFTase showed higher activity than CLEAs.The resulting CMSi M-Aa-IFTase showed higher stability and activity,because of hard shell of mesoporous silica microspheres.In addition,the mesopores on silica microspheres could improve interaction beteween Aa-IFTase and substrate inulin.Then the optimum immobilized conditions were explored,such as curdlan concentration,saturation degree of ammonium sulfate,vortexing time and so on.As a result,the optimum reaction temperature of embedded enzyme was increased by 10 °C.Furthermore,the activity of CMSi M-Aa-IFTase was more stable on a wide range of temperature and p H,and retained more than 75% of its initial activity even after 10 cycles of reuse.The resulting CMSi M-Aa-IFTase also displayed good storage stability and excellent reusability.Interestingly,the immobilized enzyme displayed much higher enzyme activity during vortex treatment,while the activity of free Aa-IFTase was decreased.Therefore,this method should have reference value for constructing various porous composites to improve catalytic performance of enzymes.In addition,the attempt for production of DFA ? used immobilization inulin fructotransferase,which should lay the foundation for more efficient production of DFA ?. |
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