Role of C-terminal domain in a manganese-catalase from Geobacillus thermopakistaniensis.

Catalases catalyze the decomposition of hydrogen peroxide into water and oxygen. We have characterized two manganese-catalases from Geobacillus thermopakistaniensis, CatGt and Cat-IIGt, which exhibited significant variation in their sequence, structure and properties. There was only 23% sequence identity between the two. The striking structural difference was the presence of an extended C-terminal domain in CatGt. Molecular modelling and docking studies revealed that deletion of the C-terminal domain removes non-specific binding, which results in increased substrate affinity. To verify experimentally, a C-terminal truncated version of CatGt, named as CatGt-ΔC, was produced in Escherichia coli and effects of deletion were analyzed. There was no significant difference in optimal pH, optimal temperature and substrate specificity of CatGt and CatGt-ΔC. However, Km value was reduced from 259 to 157 mM and CatGt-ΔC exhibited ∼1.5-fold higher catalytic efficiency as compared to CatGt. Furthermore, removal of the C-terminal domain converted the tetrameric nature to monomeric, and reduced the thermostability of the truncated protein. These results demonstrate that C-terminal domain of CatGt might have little role in maintaining enzyme function but provides additional structural stability to the protein, which is a desired property for industrial applications.

Investigating the role of carbohydrate-binding module 34 in cyclomaltodextrinase from Geobacillus thermopakistaniensis: structural and functional analyses.

Carbohydrate-binding modules (CBMs) are noncatalytic regions found in several enzymes of glycoside hydrolase family 13 and are proposed to orient substrates to the catalytic site. In this study, a substantial information on the conserved aromatic residues in CBM34 regions of characterized bacterial cyclolmaltodextrinases (CDases) has been presented. Molecular modeling of CDase from Geobacillus thermopakistaniensis (CDase Gt ) revealed a change in the active site geometry due to CBM34 truncation. The binding energies of full-length (CDase Gt ) and CBM34 truncated (CDase Gt -ΔN) models showed opposite trends. The least preferred substrate molecule by the full-length model was the most preferred by the CBM34 truncated one. These exciting in silico findings were experimentally verified by recombinant production and characterization of the full-length and the CBM34 truncated proteins. Both the enzymes showed similar optimum pH and temperature. However, substrate specificity was in the reverse order. These experimental verifications matched the homology modeling and docking predictions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03089-9.

Looking into a highly thermostable and efficient recombinant manganese-catalase from Geobacillusthermopakistaniensis.

Catalases, heme or non-heme, are catalysts that decompose hydrogen peroxide. Among them, non-heme or manganese-catalases have been studied from limited organisms. We report here heterologous production of a manganese-catalase, Cat-IIGt, previously annotated as a hypothetical protein, from a thermophilic bacterium Geobacillus thermopakistaniensis. Recombinant Cat-IIGt, produced as inactive inclusion bodies in Escherichia coli, was solubilized and refolded into a soluble and highly active form. Sequence homology, absorption spectra, resistance to sodium azide inhibition and activation by Mn2+ indicated that it was a manganese-catalase. Metal analysis revealed the presence of ∼2 Mn2+ and ∼2 Ca2+ per subunit of Cat-IIGt. Recombinant Cat-IIGt exhibited highest activity at pH 10.0 and 70°C. The enzyme was highly active with a specific activity of 40,529 µmol min-1 mg-1. The apparent Km and kcat values were 75 mM and 1.5 × 104 s-1 subunit-1, respectively. Recombinant Cat-IIGt was highly thermostable with a half-life of 30 min at 100°C. The structural attributes of Cat-IIGt, including the metal and substrate binding residues, were predicted by homology modeling and molecular docking studies. High activity and thermostability and alkaline nature make Cat-IIGt a potential candidate for textile and paper processing industries.

A highly active α-cyclodextrin preferring cyclomaltodextrinase from Geobacillus thermopakistaniensis.

Cyclomaltodextrinases show diverse hydrolyzing and/or transglycosylation activities against cyclodextrins, starch and pullulan. A gene annotated as cyclomaltodextrinase from Geobacillus thermopakistaniensis was cloned and overexpressed in Escherichia coli. The gene product, CDaseGt, was purified and biochemically characterized. The recombinant enzyme exhibited highest activity with α-cyclodextrin at 55 °C and pH 6.0. Specific hydrolytic activities towards α-, ß- and γ-cyclodextrin were 1200, 735 and 360 µmol min-1 mg-1, respectively. To the best of our knowledge, the activity against α-cyclodextrin is the highest among the reported enzymes. Next to cyclodextrins, pullulan was the most preferred substrate with a specific activity of 105 µmol min-1 mg-1. CDaseGt was capable of hydrolysis of maltotriose and acarbose as well as transglycosylation of their hydrolytic products. At 65 °C, there was no significant loss in enzyme activity even after overnight incubation. Activity of CDaseGt was not metal ions dependent, however, the presence of Mn2+ significantly enhanced the α-CDase activity. EDTA had no significant effect on the CDaseGt activity, however, it enhanced the thermostability of the enzyme. CDaseGt existed in monomeric as well as dimeric form in solution. Dimeric form is more active compared to the monomeric one. Equilibrium between the two forms seems to be concentration dependent.