Expression And Activity Assay Of Amylomaltase In Pichia Pastoris

Amylomaltase is a member of 4-α-Glucanotransferase family, catalyzing intramolecular transglycosylation ofα-1,4-glucans to produce cyclicα-1,4 glucans (cycloamyloses). Cyclodextrin with the structure of a hydrophobic inner cavity and a hydrophilic out surface that looks like a column. These properties are currently used in numerous applications in the food, pharmaceutical and chemical industries. But its low content in nature resources cannot meet the need of lib research or prodution of cycloamyloses.Gene MalQ was cloned from E.coli BL21(DE3) plysS, constructed expression plasmid pPIC9K-MalQ. The recombinant plasmid bearing MalQ gene was lincarized with Sal I and transformed into Pichia pastoris strain GS115. The positive clones were induced with methanol to express amylomaltase. The crude enzyme was demonstrated having 4-α-glucanotransferase activity with TLC. The amylomaltase also produced cycloamyloses with amylase. The main results are described as follows:(1) The gene MalQ was amplified from E.coli BL21(DE3) plysS by PCR, then constructed expression plasmid pPIC9K-MalQ. MalQ sequence was analyzed by BLAST and it displayed high similarity(100%) to gene MalQ of E.coli BL21(DE3) plysS reported in the GenBank.(2) The recombinant plasmid bearing MalQ gene was lincarized with Sal I and transformed into Pichia pastoris strain GS115, constructed the recombinant strain pPIC9K-MalQ/GS115. The positive clones were induced with 0.8% methanol to express amylomaltase. TLC showed the the crude enzyme had 4-α-glucanotransferase activity. SDS-PAGE showed a clear 78.5 kDa band. The enzyme activity which is 118 U. The optimal temperature and pH was 30℃,6.0, respectively.(3) The crude enzyme was used to treat amylase.The resulting mixture solution was treated by glucoamylase. TLC showed that there were existance of large cycloamylose. These all above indicated that the crude enzyme could catalyzed cyclization of amylose to produce cycloamylose.