Stability, refolding and Ca²⁺ binding of pullulanase from the hyperthermophilic archaeon Pyrococcus woesei

Abstract

The unfolding and refolding of the extremely heat-stable pullulanase from Pyrococcus woesei has been investigated using guanidinium chloride as denaturant. The monomeric enzyme (90 kDa) was found to be very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 4.86^0.29 m for intrinsic fluorescence and 4.90^0.31 m for far-UV CD changes. The unfolding process was reversible. Reactivation of the completely denatured enzyme (in 7.8 m guanidinium chloride) was obtained upon removal of the denaturant by stepwise dilution; 100% reactivation was observed when refolding was carried out via a guanidinium chloride concentration of 4 m in the first dilution step. Particular attention has been paid to the role of Ca 2+ which activates and stabilizes this archaeal pullulanase against thermal inactivation. The enzyme binds two Ca 2+ ions with a K d of 0.080^0.010 mm and a Hill coefficient H of 1.00^0.10. This cation enhances significantly the stability of the pullulanase against guanidinium chloride-induced unfolding and the DG H 2 O D increased from 6.83^0.43 to 8.42^0.55 kcal´mol 21 . The refolding of the pullulanase, on the other hand, was not affected by Ca 2+ .Keywords: pullulanase; archaea; thermostable proteins; stability; calcium binding.A number of thermophilic, extreme thermophilic and hyperthermophilic microorganisms belonging to bacteria and archaea have been found to be capable of degrading starch and other biopolymers such as hemicellulose, cellulose and proteins [1,2]. In addition to the industrial relevance of heatstable amylolytic enzymes, the existence of these enzymes in all kingdoms of life provides the possibility of comparative studies on (thermo)stability and folding of proteins from mesophilic and thermophilic microorganisms.Pyrococcus woesei, a hyperthermophilic archaeon of the order Thermococcales grows optimally at 100±103 8C on a complex medium under strictly anaerobic conditions [3]. This archaeon is equipped with a polysaccharide-degrading enzyme system which is composed of an a-amylase [4], a pullulanase [5] and an a-glucosidase [6].The pullulanase from P. woesei belongs to pullulanase of type II (also named amylopullulanase) and it hydrolyzes the a-1,6 glycosidic linkages in pullulan and branched oligosaccharides as well as a-1,4 glycosidic linkages in linear and branched polysaccharides such as amylose and amylopectin. Many pullulanases of type II have been purified and characterized from anaerobic thermophilic bacteria and archaea [7±14]. On the other hand, type I pullulanases which attack specifically a-1,6 glycosidic linkages have been generally found in mesophilic, aerobic microorganisms [1,2,15]. Only recently a heat-stable pullulanase of type I has been detected in the newly isolated thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 [16].The pullulanase from P. woesei is a monomeric enzyme with a molecular mass of 90 kDa. Its gene has been cloned and expressed in Escherichia coli. The enzyme is opti...

Biotechnological applications of archaeal enzymes from extreme environments

“…These enzymes are important in starch processing industry due to their specific debranching capacity. They have been reported in bacteria and hyper/thermophilic archaea belonging to the genera Pyrococcus [ 74 ], Thermococcus [ 75 , 76 ], Desulfurococcus [ 78 ], Staphylothermus [ 79 ], and in the halophilic archaeon Halorubrum [ 56 ] (see Table 5 ). Most of amylopullulanases from hyper/thermophilic archaea are active in the absence of calcium, which is a required for their industrial use.…”

Marine enzymes and food industry: insight on existing and potential interactions

“…Thus, it allowed for the enzyme to retain about 92% of the initial activity after 4 h of incubation at 95 • C, whereas in the absence of the cation the half-life under the same temperature was of 10 min. Calcium has been shown to bind with high affinity to pullulananes from archea, leading to the formation of a compact form, highly resistant to thermal denaturation (Schwerdtfeger et al, 1999). Despite of the remarkable thermal stability, the hydrolytic activity of the pullulanase is relatively low (under 50% of the maximal activity) at temperatures around 55-65 • C, where most starch saccharification processes are performed due to the thermal endurance of α-and β-amylases commonly used (Fernandes, 2010b).…”

An investigation on acarbose inhibition and the number of active sites in an amylopullulanase (L14-APU) from an Iranian Bacillus sp.

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