| Alginate lyases have great potential applications,such as the development of biomass energy,the preparation of biological oligosaccharides,the preparation of seaweed protoplast,the treatment of cystic fibrosis,and so on.Therefore,it has important theoretical significance and application value to analysis the characteristics of the different enzymes and clarify the degradation and metabolism mechanism of alginate by microbiology.This article reseached the different enzymes from of anaerobes Sunxiuqinia sp.SH-52 and the key enzymes in the metabolism of alginic acid,what’s more,analyzed the degradation mechanism of alginate.The results were shown as blew:1.Heterologous recombinant expression and characteristics research of thealginate lyase from Sunxiuqinia sp.SH-52Four alginate lyases were cloned from Sunxiuqinia sp.SH-52 genome by PCR,which were named SHA-2,SHA-3,SHA-4 and SHA-5.They were induced by IPTG to express in E.coli BL21,the results showed that all these four expression vectors could be expressed in E.coli BL21.After purification by GST-Sefinose Resin,the activity of SHA-2,SHA-3,SHA-4 and SHA-5 were 5.2 U/mg,12 U/mg,21 U/mg and 20 U/mg,respectively.The optimum temperature of alginate lyase SHA-2,SHA-3,SHA-4 and SHA-5 were 65℃,55℃,37℃ and 55℃,respectively.The optimum pH of alginate lyase SHA-2,SHA-3,SHA-4 and SHA-5 were all 7.5.These four alginate lyases,which were purified from recombinant E.coli cells,were all have activity for both PolyG and PolyM,but SHA-2 and SHA-3 both have preference for PolyG,SHA-4 and SHA-5 both have preference for PolyM.The Km values of SHA-2,SHA-3,SHA-4 and SHA-5 for alginate were 4.3 mg/mL,0.55 mg/mL,2.5 mg/mL and 3.6 mg/mL,respectively.The Vmax values of SHA-2,SHA-3,SHA-4 and SHA-5 for alginate were 13.46 U/mL,3.1 U/mL,8.7 U/mL and 7.8 U/mL,respectively.The results of TLC showed that SHA-1(It has been expressed by other student)and SHA-5 were endo-alginate lyases,SHA-2,SHA-3 and SHA-4 were exo-alginate lyases.2.The research about the degradation mechanism of alginic acidThe four recombinant alginate lyases from Sphingomonas sp.ZHO and the five recombinant alginate lyases from Sunxiuqinia sp.SH-52 were used to research the synergy of alginate degradation.For the four alginate lyases from Sphingomonas sp.ZHO,whatever their substrates were alginic acid,PolyG or PolyM,they all have synergy between any two endo-alginate lyases,and the synergy between ZHO-I and ZHO-III was.the strongest.If the combination is one endo-alginate lyase and one exo-alginate lyase,when PolyG is as substrate,all the combinations have synergy except for the combination of ZHO-II and ZHO-III;when PolyM is as substrate,the synergy between ZHO-Ⅲ and ZHO-IV reached the highest value in all combinations,and its synergy degree is 4.2.For the five alginate lyases from Sunxiuqinia sp.SH-52,when alginate is as substrate,the synergy degree between SHA-4 and SHA-5 up to 1.9;when PolyM is as substrate,it has no synergy between two endo-alginate lyases,but has synergy between any two exo-alginate lyases,what’s more,the combinations that included one endo-alginate lyase and one exo-alginate lyase all have synergy except for the combinations of SHA-1 with SHA-3 and SHA-3 with SHA-5;when PolyG is as substrate,the synergy degree between two endo-alginate lyases is up to 1.6;the synergy degree between SHA-3 and SHA-4 is highest(up to 2.1)in all combinations that included any two exo-alginate lyases;the combinations that included one endo-alginate lyase and one exo-alginate lyase all have synergy,and among them,the synergy degree of SHA-1 and SHA-2 reached the maximum 2.2.Overall,when the substrates were PolyG and PolyM,the synergy degree would be higher.Therefore,we could consider to pretreat the-alginate to PolyG or PolyM,and then digested by alginate lyases,and in this way to improve the efficiency of the monomer which was prepared.3.Recombinant expression and characteristic study of key enzymes in alginatemetabolismSelecting the optimum combinations to digest PolyG and PolyM,and the monomer obtained up to 30%(m/m).Otherwise,the key enzymes DEH reductase named dehR and KDG kinase named kdgK in alginate metabolism were cloned from Sunxiuqinia sp.SH-52 genome by PCR and expressed in E.coli BL21.The SDS-PAGE experiment confirmed that the molecular mass of enzymes dehR and kdgK were 53 kDa and 64 kDa respectively.In addition,the amount of protein of dehR and kdgK could reach the maximum(1623 mg/L and 873.5 mg/L)after induced for 6 hours and 8 hours respectively.After purification,the activity of dehR and kdgK were 10.3 U/mg and 11 U/mg.At last,the recombinant plasmid pGEX-4T-1-dehR-KdgK was expressed successfully and both enzymes have considerable activity. |
