A Preliminary Study Of Welan Gum Lyase In Sphingomonas Sp.WG

Welan gum is a class of acidic extracellular heterpolysaccharide produced by Sphingomonas sp.WG.Because of its excellent physic-chemical properties and environmentally friendly characteristics,welan gum has great commercial potential in oil drilling,concrete,food,pharmaceuticals and so on.A putative welan gum lyase gene welR was cloned from genomic DNA based on genome sequencing results of Sphingomonas sp.WG and expressed in Escherichia coli.The enzyme WelR was then purified and characterized,and biological functions were analyzed.This work will lay a theoretical foundation for establishing new strategies for fermentation regulation of welan gum.The main contents are as follows:First,the amino acids sequence of welR was analyzed by a series of bioinformatics softwares.The results showed that WelR was a hydrophilic protein consisting of 675 amino acids.The predicted molecular weight and theoretical isoelectric point of WelR were 71.044 kDa and 6.17,respectively.The predicted key amino acid sites were L19,F352,N382,M383,N494,Q558,E568.The software Phyre2 predicted that there was an opening central cavity structure in WelR.The inner diameter of the cavity at the C-terminus was large,which may be a substrate-binding pocket.Subsequently,the heterologous expression strain of WelR was constructed.The welR gene was amplified using the genomic DNA of Sphingomonas sp.WG as the template and ligated to the vector pET-28a(+)to construct the recombinant plasmid pET-28a(+)-welR.The plasmid was transformed into E.coli BL21(DE3)cells to construct the recombinant expression strain.And IPTG inducer was used to induce the expression of WelR.The optimal induction conditions were as follows:cultivated at 37? until OD600?0.6,then added 0.4 mM IPTG and cultivated at 16? for 20 h.Besides,the recombinant protein was purified by nickel ion affinity chromatography and the enzymatic properties were characterized.Viscosity analysis demonstrated that WelR can degrade welan gum effectively.The dinitrosalicylic acid(DNS)method was applied to determine the activity of welan gum lyase.WelR was most active at 25? and pH 7.4,and the enzyme was stable when the temperature was<40? and the pH was ranged 5.8-11.0.The activity of the enzyme was enhanced in the presence of K+,Mg2+,Mn2+,Ca2+ and EDTA,Zn2+significantly inhibited enzyme activity.The Vmax and Km towards welan gum were 1.55 ?mol min-1 mg-1 and 9.71 mg mL-1,respectively.To study the effects of C-terminal residues on WelR,72 amino acids were truncated by molecular cloning to obtain WelR-C72.The circular dichroism(CD)spectrum and dynamic light scattering(DLS)showed that the C-terminal amino acids could maintain an ordered structure of WelR and stabilize the interaction between proteins.Seven protein mutants of WelR were successfully constructed using alanine-scanning site-directed mutagenesis based on the prediction.The Q558A could not bind to the nickel column due to changes in the folding pattern.The UV spectroscopy and fluorescence spectroscopy showed that the spatial structures of N382A,M383A,N494A,and E568A have changed significantly compared with the wild protein.Therefore,N382,M383,N494,Q558 and E568 were the key amino acid residues of WelR,which played a critical role in maintaining the correct catalytic conformation of WelR.Finally,the function of WelR in Sphingomonas sp.WG was also investigated.The relative expression of the welR gene under the conditions of sufficient and lack of carbon source was determined by qRT-PCR.The results showed that the expression levels of welR at 48 h and 53 h were 5.99 and 2.79 times higher than that of 12 h,respectively.Coupled with the change in glucose concentration and growth profile,the WelR was proved to degrade welan gum existing in the culture to maintain bacterial metabolism when glucose was depleted.