Alginate　oligosaccharides　are　enzymolysis　products　of　alginate　with　versatile　bioactivities　and　their　industrial　preparation　was　limited　by　the　insufficient　activity　and　unsatisfying　thermostability　of　alginate　lyases.　The　structure-function　information　about　PL18　alginate　lyases　was　seldom　reported　since　which　few　positive　mutants　of　PL18　alginate　lyases　were　generated.　In　present　study,　a　mutant　of　PL18　alginate　lyase　E226K　was　expressed　intracellularly　and　taken　as　parent　for　further　modification.　Site　I211　at　the　lid　loop　1　and　sites　E276,　Y292　and　R294　at　the　predicted　entrance　were　chosen　as　engineering　targets　based　on　the　E226K-PM4　binding　mode　in　prereaction-state　MD　simulation　and　29　mutants　were　constructed,　from　those,　the　variant　E226K/I211T/R294V　was　screened　out　as　the　best　mutant　(showing　4.78-fold　increased　catalytic　efficiency　and　the　half-time　t1/245celcius　increased　up　to　557　min　from　89　min).　MD　simulations　indicated　that　the　affinity　of　E226K/I211T/R294V　towards　alginate　was　improved　due　to　the　optimized　energy　distribution　of　active　center,　more　flexible　loops　around　catalytic　cleft　and　larger　substrate　entrance.　The　more　efficient　proton　transmitting　endowed　E226K/　I211T/R294V　higher　activity　and　the　more　complicated　intraprotein　interactions　together　with　stronger　backbone　rigidity　were　responsible　for　the　improved　thermostability　of　E226K/I211T/R294V　than　E226K.　The　success　in　this　study　enriches　the　structure-function　information　of　PL18　alginate　lyases　and　provides　hints　for　their　further　design.