PulA, a thermostable pullulanase from an extreme thermophile Caldocellum saccharolyticum

Abstract

The pullulanase gene from Caldocellum saccharolyticum, an obligate thermophilic anaerobe, was sequenced and expressed in E. coli. Expression and substrate induction studies in E. coli showed that while gene expression was substrate inducible and the enzyme was exported into the growth medium in C. saccharolyticum, expression was non-inducible in E. coli and the enzyme remained in the cytoplasm. The nucleotide sequence of the pulA gene was shown to be 2478 basepairs (bp) in length, coding for a protein of 96 kDa. The proposed promoter sequences showed homology to both the standard E. coli sequences and the consensus sequences obtained from other C. saccharolyticum genes. The enzyme from the native organism was purified from the growth medium and shown to have a molecular mass of approximately 120 kDa. Periodic acid-Schiffs staining showed that this enzyme was glycosylated and substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but hydrolysed amylopectin, amylose and β-limit dextran to produce a number of smaller oligosaccharides. The enzyme was expressed in E. coli from its own promoters and was purified from the cytoplasmic fraction. Substrate characterisation revealed that the enzyme debranched pullulan to produce only maltotriose, but had only limited activity on β-limit dextran and amylopectin, and no activity on amylose. The pullulanase gene was also expressed under the control of a heat-inducible overexpression system in E. coli and a copper-inducible expression system in yeast. Amino acid homology comparisons of the pullulanase to other pullulanase sequences and related enzymes revealed a high degree of homology, particularly around three highly conserved regions. In α-amylases amino acids in these regions are involved in catalytic activity, substrate binding and metal ion binding.