Pyrococcus woesei is known as an ultra-thermophilic marine archaebacterium that is sulfur-reducing and capable of growing at between 100°C and 103°C. Its cells have a roughly spherical, elongated, and constricted appearance, similar to Thermococcus celer, and frequently occur as diploforms. Cells grown on solid supports have dense tufts of flagellae or pili attached to one pole.

The α-amylase is an enzyme that catalyzes the hydrolysis of α-l,4 glucosidic linkages in polysaccharides of three or more α-1,4-linked d-glucose units to yield glucose and maltose [1]. The α-amylase isolated from the ubiquitous mesophilic soil bacterium Bacillus licheniformis 2, 3, 4 operates optimally at 90°C and pH 6, and requires the addition of calcium (Ca²⁺) to maintain thermostability [5]. The ideal conditions for starch hydrolysis are pH ∼4.5 and a temperature of 105°C. Given that this enzyme is unstable under these conditions, the pH must be increased to 5.7–6.0 and Ca²⁺ added [6]. These pH adjustments increase the energetic costs associated with the process. Furthermore, the Ca²⁺ requirement creates the need for ion-exchange refining to remove added Ca²⁺ that has a potent inhibitory effect on xylose isomerase, which is used in processing starch hydrolysate to fructose syrup [7]. Given that the majority of industrial processing of starch gelatinization is completed near 100°C, thermostable α-amylases are used. Hyperthermophilic archaeon Pyrococcus woesei producing α-amylase is a good source of such an enzyme, which displays a temperature optimum of 100°C and retains ∼60% of maximal activity in pH ranges of 4.5 to 7.0. Moreover, this enzyme precludes the necessity to add Ca²⁺ salt to the substrate [7]. In view of the industrial importance of P. woesei α-amylase, we cloned, expressed, and purified P. woesei α-amylase in Escherichia coli.

P. woesei (DSM 3773, Braunschweig, Germany) was cultivated in artificial seawater supplemented with 0.25% soluble starch, 2.5% tryptone, 2% yeast extract, and 0.1% elemental sulfur. The pH of the medium was adjusted to 7.0 with 1M NaOH. Extraction of P. woesei chromosomal DNA and genomic library construction were performed as described previously [8]. E. coli BL21 DE3 plysS cells (Stratagene, La Jolla, CA, USA) transformed with the ligation mixture were plated on Luria-Bertani (LB) agar–ampicillin (100 mg/mL) plates. After 16–20 h of incubation at 37°C, colonies were transferred to a new set of LB-ampicillin plates.

α-Amylases are enzymes that hydrolyze starch by cleaving α-l,4-glucosidic bonds. They are among the most important commercial enzymes, having broad applications in starch processing, textiles, brewing and alcohol manufacturing, and other industries. Several α-amylases have been characterized and their genes cloned from microorganisms, plants, and animals. With the exception of two enzymes obtained from eubacteria and archaea [12], α-amylases are considered members of the same α-amylase family, sharing a comparable structure, similar catalytic site, and identical catalytic mechanism [13]. Because starch is soluble beginning at ≥100°C, the only α-amylases used in industry are those that are active at temperatures ≤110°C [14]. The majority of thermostable α-amylases used in industry are purified from B. licheniformis, with optimal temperatures of 90°C and requiring the addition of Ca²⁺ for thermostability [5]. Hyperthermophilic archaea are increasingly attractive to applied research, given that their enzymes show extreme thermostability 15, 16. Recently, numerous amylolytic enzymes have been obtained from Pyrococcus furiosus 17, 18, 19, 20, P. woesei 9, 18, and Thermococcus profundus [21]. Only the P. furiosus and P. woesei intracellular α-amylase genes were cloned and expressed in E. coli [22] and their sequences shared minimal homology with other α-amylase sequences. Starch hydrolysis at high temperatures requires an α-amylase with improved thermostable characteristics that does not require Ca²⁺. Expression of the enzyme in E. coli transformed with pTY2-α-amyl led to high levels of soluble enzyme [9]. In this study, we cloned the α-amylase gene of P. woesei in E. coli with two changes in amino acids in comparison of previous work [9].

The activity of the soluble enzyme obtained here (185,000 ± 8,000 U/L of growth medium) was higher that previously reported for native α-amylase from P. woesei and the yield achieved during expression of the recombinant enzyme from P. furious in E. coli (10 g, 1,000 U/L, and 38,000 U/L of induced culture ) 18, 19. The amount of soluble enzyme obtained was similar to that reported by Grzybowska et al (195,000 ± 11,000 U/L of the growth medium) [9]. Our result emphasized that the pTYB2-α-amyl plasmid can be used as an expression system for the production of catalytically active recombinant fusion proteins of thermostable α-amylase and intein in E. coli. In regard to other systems used for P. woesei α-amylase expression, the inclusion bodies described here displayed 85% of total enzyme activity [9]. The soluble form of the active enzyme suggested that this expression system is adequate for the production of thermostable α-amylase, which can be separated from the fusion protein in the presence of thiol compounds by heat treatment at ∼85°C.

In summary, this study emphasized that the pTYB2α-amyl plasmid can be used as an expression system for the production of catalytically active, recombinant, thermostable α-amylase in E. coli, with no impact to enzyme solubility by two amino acid mutations in the C- and N-terminal regions.

The authors declare no conflicts of interest.