Tae-Wha Moon

Enzymatic Analysis of an Amylolytic Enzyme from the Hyperthermophilic Archaeon Pyrococcus furiosus Reveals Its Novel Catalytic Properties as both an α-Amylase and a Cyclodextrin-Hydrolyzing Enzyme

ABSTRACT Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme.

Properties of a Novel Thermostable Glucoamylase from the Hyperthermophilic Archaeon Sulfolobus solfataricus in Relation to Starch Processing

ABSTRACT A gene (ssg) encoding a putative glucoamylase in a hyperthermophilic archaeon, Sulfolobus solfataricus, was cloned and expressed in Escherichia coli, and the properties of the recombinant protein were examined in relation to the glucose production process. The recombinant glucoamylase was extremely thermostable, with an optimal temperature at 90°C. The enzyme was most active in the pH range from 5.5 to 6.0. The enzyme liberated β-d-glucose from the substrate maltotriose, and the substrate preference for maltotriose distinguished this enzyme from fungal glucoamylases. Gel permeation chromatography and sedimentation equilibrium analytical ultracentrifugation analysis revealed that the enzyme exists as a tetramer. The reverse reaction of the glucoamylase from S. solfataricus produced significantly less isomaltose than did that of industrial fungal glucoamylase. The glucoamylase from S. solfataricus has excellent potential for improving industrial starch processing by eliminating the need to adjust both pH and temperature.

A novel amylolytic enzyme from Thermotoga maritima, resembling cyclodextrinase and α-glucosidase, that liberates glucose from the reducing end of the substrates☆

The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli. The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined. The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose. Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose. Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases. Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity. These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase.

Maltosyl-erythritol, a Major Transglycosylation Product of Erythritol by Bacillus stearothermophilus Maltogenic Amylase

This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-α-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol.

Determining the gelation temperature of β-lactoglobulin using in situ microscopic imaging.

Evolution of microstructure during heat-induced gelation of β-lactoglobulin (β-LG) was investigated in situ using confocal laser scanning microscopy at various gel-preparation conditions: pH=2, 5, and 7; protein content=5, 10, and 15%; and salt (NaCl) content=0, 0.1, and 0.3 M. The number and area of evolving β-LG clusters were observed as a function of time and temperature and the data were fitted to a log-normal model and sigmoid model, respectively. The gelation temperature (Tgel) of the β-LG system was determined from both the number (Tgel/N) and total area (Tgel/A) of β-LG clusters versus temperature data. The range of Tgel/N and Tgel/A values for all the cases was 68 to 87°C. The effect of pH was the most dominant on Tgel/N and Tgel/A, whereas the effects of β-LG and salt contents were also statistically significant. Therefore, the combined effect of protein concentration, pH, and salt content is critical to determine the overall gel microstructure and Tgel. The Tgel/N and Tgel/A generally agreed well with Tgel determined by dynamic rheometry (Tgel/R). The correlations between Tgel/N and Tgel/A versus Tgel/R were 0.85 and 0.72, respectively. In addition, Tgel/N and Tgel/A values compared well with Tgel/R values reported in the literature. Based on these results, Tgel/N determined via in situ microscopy appears to be a fairly good representative of the traditionally measured gelation temperature, Tgel/R.