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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.

We have cloned Tk0522, a carbohydrate esterase homologue from Thermococcus kodakarensis, with the deletion of a signal sequence of nineteen amino acids at the N-terminal and produced the recombinant Tk0522 in Escherichia coli. Recombinant Tk0522 was produced in soluble and enzymatically active form. Analysis of the cytosolic, periplasmic and extracellular fractions demonstrated the presence of Tk0522 in cytosolic and periplasmic fractions. The esterase activity in the periplasmic fraction, similar to the cytosolic fraction, increased with the passage of time after induction. No significant amount of esterase activity could be detected in the extracellular fraction. To the best of our knowledge, this is the first report on translocation of a mature archaeal esterase from cytosolic to periplasmic space in E. coli.

Pullulan-hydrolysing enzymes, more commonly known as debranching enzymes for starch and other polysaccharides, are of great interest and have been widely used in the starch-saccharification industry. Type III pullulan hydrolase fromThermococcus kodakarensis(TK-PUL) possesses both pullulanase and α-amylase activities. Until now, only two enzymes in this class, which are capable of hydrolysing both α-1,4- and α-1,6-glycosidic bonds in pullulan to produce a mixture of maltose, panose and maltotriose, have been described. TK-PUL shows highest activity in the temperature range 95–100°C and has a pH optimum in the range 3.5–4.2. Its unique ability to hydrolyse maltotriose into maltose and glucose has not been reported for other homologous enzymes. The crystal structure of TK-PUL has been determined at a resolution of 2.8 Å and represents the first analysis of a type III pullulan hydrolyse. The structure reveals that the last part of the N-terminal domain and the C-terminal domain are significantly different from homologous structures. In addition, the loop regions at the active-site end of the central catalytic domain are quite different. The enzyme has a well defined calcium-binding site and possesses a rare vicinal disulfide bridge. The thermostability of TK-PUL and its homologues may be attributable to several factors, including the increased content of salt bridges, helical segments, Pro, Arg and Tyr residues and the decreased content of serine.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_1032, annotated as glucokinase. Amino acid sequence analysis showed that Pcal_1032 belonged to ROK (repressor, open reading frame, and kinase) family of sugar kinases. To examine the properties of Pcal_1032, the coding gene was cloned and expressed in Escherichia coli. However, expression of the gene was low resulting in a poor yield of the recombinant protein. A single site directed mutation in Pcal_1032 gene, without altering the amino acid sequence, resulted in approximately tenfold higher expression. Purified recombinant Pcal_1032 efficiently phosphorylated various hexoses with a marked preference for glucose. ATP was the most preferred phosphoryl group donor. Optimum temperature and pH for the glucokinase activity of Pcal_1032 were 95 °C and 8.5, respectively. Catalytic efficiency (k cat/K m) towards glucose was 437 mM−1 s−1. The recombinant enzyme was highly stable against temperature with a half-life of 25 min at 100 °C. In addition, Pcal_1032 was highly stable in the presence of denaturants. There was no significant change in the CD spectra and enzyme activity of Pcal_1032 even after overnight incubation in the presence of 8 M urea. To the best of our knowledge, Pcal_1032 is the most active and highly stable glucokinase characterized to date from archaea, and this is the first description of the characterization of a glucokinase from genus Pyrobaculum.

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.

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.

The chaperonin genes encoding GroELGt (ESU72018) and GroESGt (ESU72017), homologues of bacterial GroEL and GroES, from Geobacillus thermopakistaniensis were cloned and expressed in Escherichia coli. The purified gene products possessed the ATPase activity similar to other bacterial and eukaryal counterparts. Recombinant GroELGt and GroESGt were able to refold the denatured insoluble aggregates of α-amylase from Bacillus licheniformis into soluble and active form. Furthermore, GroELGt and GroESGt successfully enhanced the thermostability of porcine heart malate dehydrogenase. Expression of GroELGt gene in E. coli cells enhanced the thermotolerance of the host. Furthermore, soluble production of recombinant alcohol dehydrogenase from Bacillus subtilis strain R5 in E. coli, initially produced as insoluble aggregates, was achieved by co-expressing the gene with GroELGt. Our results implied that GroELGt could assist folding of nascent protein in E. coli with the help of host co-chaperonin without requiring additional ATP. This system can be used for soluble production of recombinant proteins which otherwise are produced in insoluble form in E. coli. To the best of our knowledge this is the first report on functional characterization and applications of chaperonins from genus Geobacillus.