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

Abstract

A strain of thermophilic Anoxybacillus flavithermus is characterized bacteriologically and biochemically. Then, the gene responsible for encoding cyclomaltodextrinase (CDase) in this bacterium is isolated, cloned, and overexpressed. The sequence of the CDase is recorded in GenBank with accession number KT633577.1. Biochemical and structural characterization of the enzyme shows that CDase with molecular weight of 72 kDa is active in the optimum temperature and pH of 65 °C and 7.0, respectively and it exists in oligomeric forms. It is observed that α‐cyclodextrin (α‐CD) has higher kcat/km value as compared to starch, glycogen, β‐ and γ‐CD and is therefore a more specific substrate for the enzyme. Also, the addition of metal ions such as K1+ and Na1+ can help to improve the catalytic activity, while Fe2+ and Cu+2 have inhibitory effects on the activity of the enzyme. It is also found that the catalytic activity of the enzyme increases in the presence of 5 mM β‐mercaptoethanol, EDTA, DTT, and PMSF. Thin layer chromatography (TLC) shows that glucose and maltose are the main products of CDase‐catalyzed reaction of α‐, β‐, and γ‐CD hydrolysis. Considering the modeling results, the CDase consists of three domains in which the central domain is the catalytic core containing active site residues. Based on its specificity for various substrates, the new homologue of CDase can be considered as a suitable candidate for industrial applications involving production of maltose and glucose from α‐, β‐, and γ‐CD or a mixture of these compounds in extreme conditions of temperature.

Enhancement of the Structure and Biochemical Function of Cyclomaltodextrinase from the Anoxybacillus flavithermus ZNU-NGA with Site-directed Mutagenesis

“…Also, the activity of enzymes was measured in the presence of metal ions, and chemical compounds like Betamericaptaethanol, Ethylenediaminetetraacetic acid (EDTA), Dithiothreitol (DTT), and Phenylmethanesulfonyl uoride (PMSF) in 5 mM and 10 mM concentrations for 30 min at 65℃. The activity was measured using a spectrophotometer (T80 + PG Instrument UV/Vis) according to the Bernfeld method [13].…”

“…This enzyme works mainly as a dimer and can become an octamer . [13] H403R mutation was aimed at increasing protein stability [22]. The second mutation, L309V, which is located near the active site, was applied to the rst mutant for the purpose of decreasing the hydrophobicity around the active site by replacing leucine with valine.…”

“…Recently, a new strain of temperature-resistant bacterium called Anoxybacillus avithermus ZNU-NGA (GenBank registration number: KT633577.1) has been reported with an optimal temperature of 65℃ [13] whose preferred substrate is alpha-cyclodextrin. Hence, regarding the signi cance of cyclomaltodextrinase in the industry and the high activity of wild-type enzyme as well as high-throughput protein expression, the present study was performed on cyclomaltodextrinase extracted from the new strain Anoxybacillus avithermus ZNU-NGA.…”

A novel thermostable and processive reverse transcriptase from a group II intron ofAnoxybacillus flavithermus

“…α-amylase [20], cyclomaltodextrinase [21], xylanases [22] with a temperature optimum in the range of 50-65 °C. Therefore, it was legitimate to assume the same thermostability of an RT from the same bacterium.…”

“…The host was found in hot spring in New Zealand with an optimal temperature for growth around 60°C [18]. Previously, several thermostable enzymes were isolated from A. flavithermus, including lipase [19], α-amylase [20], cyclomaltodextrinase [21], xylanases [22] with a temperature optimum in the range of 50-65 °C. Therefore, it was legitimate to assume the same thermostability of an RT from the same bacterium.…”