Abstract
The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.
Highlights
Since the open reading frame of 5.3-kbp Hind1111 XbaI fragment of the apu gene is in thecorrect orientation to the lac Z promoter of the pUC119 vector, and in thecorrect reading frame, amylopullulanase was expressed by E. coli harboring the pAPZ119 or harboring mutants constructed by deletion from the 3' end of the gene
The predicted primary sequence encoded by the open reading frame corresponds to 1450 amino acids with an estimated molecular weight of 162,780, whichagrees closely with the M, of 160,000 obtained by SDS-PAGE analysis for the enzyme expressed in E. coli, it is higher than the M, 140,000 determined for the native enzyme (Saha etal., 1988).The GTG initiation codon is preceded with a spacing of 5 bp, by a putative ribosomal binding site (5'-AAAGGGGG-3') exhibiting strong similarity to the 16 S rRNA of B. subtilis (McConnell et al, 1986).The structuralgene is preceded by a putativepromoter sequence, similar to theconsensus promoter sequences of E. coli and B. subtilis (Rosenburg and Court, 1979; McConnell et al, 1986)
Analysis of the 5’ region of apu gene enabled the identification of a sequence motif for binding of the CAMP-CAPcomplex of E. coli (Crombruggheet al., 1984)for regulation of gene expression, which may be involved icnataboliterepression of pullulanase activity, reported previously for T. ethunolicus 39E (Hyun and Zeikus,1985)
Summary
Into phagemid vector pUC119 linearized with XbaI and transformed into E. coli DH5aF' competent cells. For assay of a-amylase activity, 160 pl of1.25% (w/v) soluble pAPZ72 starch in 50 mM acetate buffer (pH 6.0) containing 5 mM CaClZand pl of enzyme solution were mixed and incubated at 60 "C for 30 min. MgSOd, and 20 mM glucose (Hanahan, 1983) was added and the transformation mixture incubateda t 37 "C for 1 h in a rotary shaker a t 225 rpm.Transformants harboring recombinant pUC 119plasmids were selected by plating the transformation sample onto 2 X YT agar (Sambrook et al.,1989) containing 50 pg/ml ampicillin, 5-bromo-4- FIG. Was used in constructing single point base mutants (Nakamaye and Eckstein, 1986).E. coli TG-1 was transformed with a nested deletion mutant, containing an upu gene fragment of 2.9 kb, denoted pAPZ 12-3 (in pUC 119),which maintained both a-amylase and pullulanase activities, as well as the~ostability. Cells were harvested by centri~gationin a microcentri~ge(14,000 rpm X 1min) and thecell pellet sonicated in 1.0 ml of 50 mM acetate buffer
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
