Sequence Analysis of the Type I Pullulanase from Fervidobacterium pennavorans strain Ven5 (DSM 9078) and its Overexpression in E. coli
Fiona DUFFNER*a, Costanzo BERTOLDOb, Per Lina JORGENSENc, Garabed ANTRANIKIANb, and Carsten SJOHOLMa
a Screening Biotechnology, Novo Nordisk A/S, Novo Alle, 2889 Bagsvaerd, Denmark
b Department of Biotechnology I, Technical University Hamburg-Harburg, Denickestr. 15, 21073 Hamburg, Germany.
c Molecular Biotechnology, Novo Nordisk A/S, Novo Alle, 2889 Bagsvaerd, Denmark
We have sequenced a debranching enzyme Type I pullulanase from Fervidobacterium pennavorans strain Ven5 (1) that is cloned and expressed in Escherichia coli. The enzyme breaks down α-1,6 glucosidic linkages in pullulan to produce maltotriose exclusively and no panose or isopanose. The enzyme slightly hydrolyzesα-1,6 linkages of starch, amylopectin, glycogen andα-β-limited dextrin while no action on amylose has been detected. The enzyme is most active at pH 5.5 and the optimum temperature for activity is 85℃ which makes this enzyme interesting for commercial applications such as starch conversion (2).
The plasmid, pPL2125, conferring thermostable pullulanase activity on the E. coli clone, contains an 8.1 kb insert which was subcloned into pUC18 for sequencing. Sequencing of the entire insert revealed the presence of three large open reading frames (ORF's). Pullulanase activity was located both experimentally and using sequence homologies to a 3.9 kb fragment which contained ORF1. The presence of the N-terminal sequence of the pullulanase purified from E. coli in the deduced amino acid sequence verifies the correct translation of the ORF. Significant homology (60% amino acid) was observed with the pullulanase recently published in the databank from Thermotoga maritima (GP_new:TMPULA_1). The Shine Dalgarno sequence AGGAGG is present 10-15 bases upstream from the start site. Assuming the N- terminal sequence is at the beginning of the mature protein the predicted molecular mass of the pullulanase is 83 kDa. This is in excellent agreement with the result from the purified pullulanase from the E. coli clone which has a molecular mass of 160±6 kDa and is composed of 2 apparently identical subunits of 83±1 kDa, as estimated by native and SDS-PAGE, respectively. The pullulanase expressed in E. coli exhibits temperature and pH activity profiles as well as a substrate hydrolysis pattern characteristic of the original enzyme from F. pennavorans Ven5.
The source of the insert DNA was confirmed as being from a member of the Thermotoga group due to the presence on the 8.1 kb insert of an ORF which has most
