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TK-PUL, a novel thermo-acidophilic pullulanase from Thermococcus kodakarensis and a unique member of glycoside hydrolase family GH13 was successfully produced in Escherichia coli grown by fed batch culture in a fermenter and partially purified by simple heat treatment. Specific activity of partially purified TK-PUL was 28 U/mg. Corn starch was successfully liquefied and saccharified using this single enzyme at pH 4.2. Simultaneous liquefaction and saccharification of corn starch by TK-PUL was comparable to Termamyl, a commercially available starch-hydrolyzing industrial enzyme. Both enzymes efficiently hydrolysed corn starch into sugar syrups having major proportions of maltose. TK-PUL performs efficiently at the natural pH of starch (~4.5) in the absence of any metal ions, hence is a potential candidate for starch industry.

In order to elucidate if Escherichia coli secretion system recognizes the N-terminally truncated signal sequence of an archaeal α-amylase from Thermococcus kodakarensis (Tk1884) and secretes the recombinant protein to the extracellular medium, we have cloned Tk1884 with the deletion of the sixteen N-terminal amino acids and produced the recombinant protein Tk1884Δ16 in E. coli. Analysis of the intracellular, membranous and extracellular fractions demonstrated the presence of Tk1884Δ16 in all the three fractions. The intracellular α-amylase activity, similar to the membranous fraction, increased with the passage of time till 8 h of induction and then decreased. In contrast, the extracellular α-amylase activity slowly increased with the passage of time after induction. The extracellular amylase activity was purified and determination of the molecular mass by electrospray ionization mass spectrometry demonstrated that Tk1884Δ16 was secreted to the extracellular medium without cleavage of the signal peptide. To the best of our knowledge, this is the first report on recognition of N-terminally truncated signal peptide of archaeal origin by E. coli.