Recombinant Cyclodextrinase fromThermococcus kodakarensisKOD1: Expression, Purification, and Enzymatic Characterization

Abstract

A gene encoding a cyclodextrinase from Thermococcus kodakarensis KOD1 (CDase-Tk) was identified and characterized. The gene encodes a protein of 656 amino acid residues with a molecular mass of 76.4 kDa harboring four conserved regions found in all members of the α-amylase family. A recombinant form of the enzyme was purified by ion-exchange chromatography, and its catalytic properties were examined. The enzyme was active in a broad range of pH conditions (pHs 4.0–10.0), with an optimal pH of 7.5 and a temperature optimum of 65°C. The purified enzyme preferred to hydrolyze β-cyclodextrin (CD) but not α- or γ-CD, soluble starch, or pullulan. The final product from β-CD was glucose. The V max and K m values were 3.13 ± 0.47 U mg−1 and 2.94 ± 0.16 mg mL−1 for β-CD. The unique characteristics of CDase-Tk with a low catalytic temperature and substrate specificity are discussed, and the starch utilization pathway in a broad range of temperatures is also proposed.

Genetic and Biochemical Characterization of a Novel Thermostable Cyclomaltodextrinase From Anoxybacillus flavithermus

“…[3] According to this study, the optimum temperature and pH for CDase are 65 C and 7.0, respectively. They are similar to those for CDase-Tk obtained from T. kodakarensis KOD1, [14] while they are different from CDase-Tc (from Thermococcus species CL1) [11] and CDase-Tp (Thermofilum pendens Hrk 5) [52] in which their optimum temperature and pH are 85 and 95 C and 5.0 and 5.5, respectively. CDase-ZNU had also high activity (>60%) at pHs ranging from 6.5 to 8.0 (compared to 100% at pH 7.0) showing that the enzyme can hydrolyze its substrates approximately in neutral pH.…”

“…[48] According to other studies, the MW for CDase has been reported to be in the range of 62-90 kDa. [12][13][14]19] As a result of overexpression of recombinant enzymes and accumulation of high concentration of folding intermediates, molecular aggregates were formed in bacterial cell which could not be recovered. [49] Therefore, soluble proteins were purified for further studies.…”

“…[21] Despite some similarity among different CDases isolated from various types of bacteria, they have different properties considering their thermal stability, response to temperature, pH and substrate specificity. [8,[10][11][12][13][14] In this study; for the first time, a new strain of A. flavithermus isolated from the mineral water of Gaynarge Nir hot spring, Ardabil, Iran and has bacteriologically and molecularly been identified. Cloning, gene expression, purification, and biochemical characterization of the CDase then were carried out.…”

“…[8] Cyclodextrinase from Bacillus macerans (CDase; EC 3.2.1.54) was reported in 1968 and has proven to catalyze efficiently the hydrolysis of CDs to create linear oligosaccharides of α-1,4-linkages. [9] Many CDases including those from Thermoanaerobacter ethanolicus strain 39E, [10] Klebsiella oxytoca strain M5a1 [11] Bacillus species, [8] Flavobacterium species, [12] Paenibacillus species, [13] and Thermococcus kodakarensis KOD1 [14] have been isolated and characterized. According to their substrate specificity, they have been recommended for application in pharmaceutical and food industries including the production of amylose-carried or amylose-free starch.…”

Thermococcus kodakarensis : the key to affordable biohydrogen production

“…The extracellular hydrolysis of starch is realized by secreting various enzymes that degrade the available starch polymers synergistically, such as amylases, pullulanase, amylopullulanase and cyclomaltodextrin glucanotransferase [59,[72][73][74][75][76]. Once imported in the cell, these maltooligosaccharides are further hydrolyzed intracellularly to either glucose or, via glucose-1-phosphate, to glucose-6-phosphate were they enter a modified Embden-Meyerhof (EM) pathway (Figure 1.3, box A-C) [59,76,77].…”