Functional Identification And Modification Of Putative Sucrose Phosphorylase From Pseudoalteromonas Etraodonis GXQ-1

Sucrose phosphorylase(EC 2.4.1.7,sucrose phosphorylase)belongs to the GH13 family,which can reversibly catalyze the conversion of sucrose(alpha-D-glucopyranosyl-1,2-beta-D-fructofuranoside)and phosphate into alpha-aldehyde phosphate(Glc-1-P)and D-fructose.The enzyme can use high-yield and low-cost sucrose as substrate for transglucosylation.The glycosylation product can be widely used in food,medicine,cosmetics and other industries.It has become the main research content to obtain a sucrose phosphorylase with high catalytic activity and stability through mutation transformation.In this study,the genomic sequence of Pseudoalteromonas tetraodonis GXQ-1 has been done by whole genome high-throughput sequencing.Nine nucleotide sequences annotated as alpha-amylase family were found,including two putative sucrose phosphorylase genes Pasp and Pgsp.The two genes were cloned into the expression vector p QE30 to construct recombinant E.coli strains,respectively.The proteins Pasp and Pgsp were purified by nickel affinity chromatography.Then the optimal substrates of Pasp and Pgsp were analyzed.The recombinant enzyme Pasp has no reaction to the substrates corresponding to the enzymes in the glycoside hydrolase family 13 and the common disaccharide substrates.While,the optimal substrate of Pgsp was sucrose.It was determined as sucrose phosphorylase.The basic enzymatic properties and transglycosidation function of Pgsp were determined.The optimum p H and temperature were 6.5 and 40℃,respectively;Km and Vmax were 10.44±1.579 mmol/L and 93.88±3.480 μmol/(min*mg),respectively;The enzyme activity could be increased by certain concentrations of chemical reagents DTT,imidazole,Tween-80 and mercaptoethanol.When 15%glucose-1-phosphate was used as donor,Pgsp had transglucoside activity on L-arabinose、D-fructose、L-sorbose and xylitol;when 4% sucrose was used as donor,Pgsp had transglycoside activity on D-arabinose、L-arabinose、maltose、xylitol and mannitol.Then,Pgsp was subjected to modification.First,the relevant biological software of Pgsp was analyzed.According to the bioinformatics data provided by Func Lib for sequence space optimization,the whole plasmid PCR was used for site-directed mutagenesis,and a total of 10 single-point mutation mutants were obtained.The 10 mutant proteins were purified by nickel and chromatography,whose SDS-PAGE detection results showed that the molecular weight of all mutant proteins was consistent with the expected results.Preliminary enzymatic properties test results showed that mutants H89 V,P135K and P135 Q had enzymatic activity,and no enzymatic activity was detected in other mutants;the optimal p H of these three mutants is 7,and the optimal p H is higher than that of wild enzyme increased by 0.5;the optimum temperature is 35℃、40℃ and 40℃,respectively,the optimum temperature of P135 K and P135 Q is consistent with the wild enzyme,H89 V is decreased 5℃.Under optimal reaction conditions,the Km of mutants H89 V and P135 K are higher than wild-type Pgsp,divided into 2 fold and 4 fold,while mutant P135 Q is 1.5 fold lower.The transglycosidation function of mutants H89V、P135K and P135 Q was tested,No obvious transglycoside activity was detected by HPLC with glucose-1-phosphate was used as the glycosyl donor,sugars and sugar alcohols were used as the glycosyl acceptor.The substrate channel analysis of all mutants showed that the difference substrate channel is the key factor leading to the loss of enzyme activity and transglycosidation.It is preliminarily confirmed that site D194 is a nucleophilic attack site and site Q344 has a certain effect on the glycosylation of polyphenols.