Molecular Identification And Modification Of Levansucrase And Its Application For Levan Production

Levan and inulin are two important?-frutan in nature.For levan,the repeat fructosyl unit is linked by?-?2,6?,whereas for inulin,the repeat unit is?-?2,1?linked fructosyl.Recently,the ability of levan in improving the intestinal microflora,reducing the fat accumulation and regulating the blood sugar level has been validated.As a result,attempts have been made to employ levan as emulsifier,stabilizer,and surfactant and flavor carrier in the food,medicinal and chemical industry.The plant-derived levan is limited to its content as well as the polymerization degree as a compared result of inulin.The biological production of levan is primarily implemented by microbial fermentation and enzymatic synthesis.Microbial levansucrase?LSase,EC 2.1.4.10?catalyzes the biosynthesis of the levan from sucrose by transferring the fructosyl group to the polyfructose?fructan?chain,which is the key enzyme in the industrial preparation of levan.However,the microbial resources of LSase showed an impact in the product distribution,with a molecular weight varying from 10³to 10⁸Da.It is well known that the physicochemical property as well as the practical application of levan largelyd depends on its molecular weight.For instance,the low-molecular weight?LMW?levan has been focused on their prebiotic property,while for the high-molecular weight?HMW?levan,the potential application in the hydrosol field was evaluated.Using the basic local alignment search tool in National Center for Biotechnology Information?NCBI?,we acquired the LSase-encoding gene from Brenneria sp.Eni D312.The specific gene was cloned in Escherichia coli and recombinant enzyme was intracellularly expressed.The recombinant enzyme was characterized and the physicochemical property of levan product was also determined.The thermostability of LSase was enhanced by site-directed mutagenesis as well as the PEG modification.Additionally,the crystal structure of LSase was resolved and the product length distribution was regulated by two kinds of mutants.Detailed information was shown below:?1?The locus?tag of LSase-encoding gene from Brenneria sp.Eni D312 is Br E312?3941.The Gen Bank accession number for protein is EHD23269.1.The gene was consisted of 1314 nucleotides,encoding 438 amino acids with a calculated molecular mass of 48,530 Da.The plasmid was constructed by inserting the LSase gene into p ET-22b?+?vector and then transformed into E.coli BL21?DE3?.IPTG with a 1m M final concentration was used to induce the protein expression.The recombinant enzyme was first purified by Nickel affinity chromatography and then size-exclusion chromatograpy.SDS-PAGE showed a band arounding 46,000 Da,indicating the protein was purified smoothly.The optimal temperature and p H of LSase for hydrolysis was determined at 50°C and p H at 7.5,but the optimal temperature and p H for transfructosylation was 45°C and 6.5.The recombinant enzyme had a relative weak thermostability and the half-life(t_1/2)at 35,45 and 55°C was determined as 4.6 h,2.1 h and 42 min.Divalent metal ions including Ni²⁺,Co²⁺,Zn²⁺,Ca²⁺,Mn²⁺and Mg²⁺could hardly impact the activity of LSase,suggesting Brsp-LSase was not strictly metal-stimulated or metal-dependent.The specific activity of transfructosylation and hydrolysis was determined as 414 U/mg and 153 U/mg from 200 g/L sucrose,indicating the transfructosylation was more dominant when Brsp-LSase catalyzed sucrose?The K_m,V_max and k_cat/K_m of Brsp-LSase against sucrose was calculated as 367 mmol/L,20.12 mmol/min,and 290.17/min,respectively.The product was verified as?-?2,6?linked levan by NMR analysis.The enzyme amount and substrate concentarion of LSase for levan production was optimized as 2.5 U/m L?250 g/L,and under this optimal condition,85 g/L could be produced by Brsp-LSase after 6 h reaction with a34%conversion ratio.?2?The weight-average molecular weight of levan was determined to be 1.41×10⁸ Da.The Polydispersity Index?PD?was determined as 1.28,indicating a centralized distribution.The microscope observation of levan by scanning electron microscopy?SEM?and atomic force microscopy?AFM?showed a poroid microstructure as well as series of individual ellipsoidal and spheroidal particles.The decomposition temperature,melting temperatue and enthalpy of melting was determined as 216.67°C,147.41°C and 76.9 J/g.At 3%concentration,levan aqueous solution showed an essentially Newtonian behavior;whereas the 6,9 and 12%solution showed a non-Newtonian pseudoplastic behaviour?shear thinning?at shear rates from 0.01 to 100 1/s,and the viscosity decreased with increasing shear rate.In addition,the gel strength was increased significantly when the concentration of levan increased from 6%to 8%.Meanwhile,the gel strength of 10%Levan exhibited a comparable level to that of 1%xanthan and 0.5%carrageenan.?3?The residue of glutamate?Glu?in postion 404 was found important for the thersmotability by multiple sequence alignment as well as the B-factor analysis.Site-directed mutagenesis was implemented by substituting the other 19 amino acids with the original Glu.As a result,five mutants including E404F,E404I,E404W,E404V and E404L showed a prominent increase in the thermostability as a compared result with that of wild-type,without a big difference in their K_m.The T_m of variant E404L was increased by 2.8°C,and the thermostability at 35 and 45°C was also enhanced by12.5and 1.3-fold,respectively.The improvement of thermostability through mutation in Glu404 could be ascribed to the change of microenvironment in the LSase structure.The change mainly included the enhanced structural stability between two?-hairpins?14th and 15th?-hairpin?,the enhanced hydrophobic interaction in the vicinity of active pocket and the potential“clamp effect”formed between N and C-terminal.?4?The effect of polyethylene glycol?PEG?on the thermostability of Brsp-LSase was evaluated for the first time.PEGs with different molecular weight as well as the concentration were introduced and the effect was also compared in detail.Firstly,after incubated with 10%?w/v?PEG 4000,the enzyme activity was increased by 1.24-fold.Secondly,after pre-incubation with 5%PEG 4000 for 6 h,the residual activity of Brsp-LSase at 35 and 45°C was decreased to 55%and 60%,with an increase of 1.2-and3.3-fold than that of the wild-type enzyme.However,the effect of 10%PEG 4000 on the thermostability was found similar to that of 5%PEG 4000.A kind of“protective layer”was found outside of Brsp-LSase when incubated with PEG 4000 by AFM observation.Besides,the random coil content of enzyme was decreased from 53%of the wild-type enzyme to 33.9%of the PEG pre-incubated enzyme.Additionally,the maximal fluorescence intensity?FI?was increased while the surface hydrophobicity?S₀?was decreased from 117 to 69 after incubation by 5%PEG 4000.?5?The high-resolution crystal structures of the wild-type Brsp-LSase enzyme,D68N-sucrose complexed,and A154S and H327A mutants were determined.Compared with the wild-type enzyme,the mutant A154S kept the total activity more or less,but the ratio of transglycosylation to hydrolysis?T/H?changed from 2.7 to 1.4;and mutant H327A could no longer synthetic high molecular weight levan,but only generate a series of oligosaccharides with a low degree of polymerization?DP?6?.The oligosaccharides formed by the H327A mutant enzyme were purified and determined as 6-kestose and a small portion of 1-kesotse.The yield and conversion ratio of 6-kestose is 18.4 g/L and 9.2%from 200 g/L sucrose.The histidine at position 327 has a spatial stacking effect with the tyrosine at position 374;at the same time,it has a hydrogen bond interaction with the 2-OH of glucosyl.When it was mutated to alanine,the originla stacking effect would be totally lost,resulting in a significant decrease in its activity towards sucrose.Meanwhile,the histidine resides in loop7 of Brsp-LSase,wherein loop7 has been presumed to function as a binding site for oligosaccharides.When it was substituted with alanine,the binding sites for other oligosaccharides are weakened,which leads to a formation of oligosaccharides with low degree.In the case of A154S mutant,the substituted serine would interact the D68 via a hydrogen bond.However,the D68 acts as a nucleophile and needs to attact the sucrose,so the interaction rises from the serine would lower the efficiency of nucleophilic attack,resulting in a decrease in the ratio of T/H.