Exploring a novel GH13_5 α-amylase from Jeotgalibacillus malaysiensis D5T for raw starch hydrolysis

Abstract

α-Amylase plays a crucial role in the industrial degradation of starch. The genus Jeotgalibacillus of the underexplored marine bacteria family Caryophanaceae has not been investigated in terms of α-amylase production. Herein, we report the comprehensive analysis of an α-amylase (AmyJM) from Jeotgalibacillus malaysiensis D5^T (= DSM28777^T = KCTC33550^T). Protein phylogenetic analysis indicated that AmyJM belongs to glycoside hydrolase family 13 subfamily 5 (GH13_5) and exhibits low sequence identity with known α-amylases, with its closest counterpart being the GH13_5 α-amylase from Bacillus sp. KSM-K38 (51.05% identity). Purified AmyJM (molecular mass of 70 kDa) is stable at a pH range of 5.5-9.0 and optimally active at pH 7.5. The optimum temperature for AmyJM is 40 °C, where the enzyme is reasonably stable at this temperature. Similar to other α-amylases, the presence of CaCl₂ enhanced both the activity and stability of AmyJM. AmyJM exhibited activity toward raw and gelatinized forms of starches and related α-glucans, generating a mixture of reducing sugars, such as glucose, maltose, maltotriose, maltotetraose, and maltopentaose. In raw starch hydrolysis, AmyJM exhibited its highest efficiency (51.10% degradation) in hydrolyzing raw wheat starch after 3-h incubation at 40 °C. Under the same conditions, AmyJM also hydrolyzed tapioca, sago, potato, rice, and corn raw starches, yielding 16.01-30.05%. These findings highlight the potential of AmyJM as a biocatalyst for the saccharification of raw starches, particularly those derived from wheat.

Keywords: Caryophanaceae; Jeotgalibacillus; Glycoside hydrolase family 13 subfamily 5; Marine bacteria; Raw starch hydrolysis; α-Amylase.

Fig. 1

A Evolutionary tree of GH13 family bacterial α-amylases. The α-amylase from Jeotgalibacillus malaysiensis D5^T (AmyJM) was clustered in subfamily GH13_5. The α-amylase sequences were aligned using the Clustal Omega web server (Madeira et al. 2022). The tree was constructed using the neighbor-joining method in the Molecular Evolutionary Genetic Analysis (MEGA v.11.0.13) software (Tamura et al. 2021), with 1000 bootstrap replicates. The scale bar represents 0.8 nucleotide substitution per site. ^TType strain. B Protein relationship tree of AmyJM and all 27 well-characterized bacterial α-amylases from subfamily GH13_5. Schematic representations of domain arrangements for the α-amylases are shown in the box. The NCBI accession numbers or PDB IDs (labeled in blue) are indicated in parentheses. The α-amylase from Thermobifida fusca belonging to subfamily GH13_32 was used as an out-group. Sequence identity (%) refers to amino acid sequence identity (%) of AmyJM with other GH13_5 bacterial α-amylases. The scale bar represents 0.2 amino acid substitution per site. ^TType strain

Fig. 2

3D homology model of AmyJM. Putative catalytic sites (D233, E263, and D330) of AmyJM are shown in orange. The calcium ion is shown as a sphere. Putative calcium-interacting residues (N104 and D196) are shown in brown. The putative surface-binding site (residue pair Y259 and Y360) of AmyJM is shown in green. The glucose (G1) molecule in AmyJM is shown in pink

Fig. 3

A SDS-PAGE (12% v/v) of AmyJM. B Zymogram of the amylolytic activity of AmyJM. Lane 1: molecular mass protein marker (BenchMark™ Protein Ladder), lane 2: crude enzyme, and lane 3: purified AmyJM

Fig. 4

Biochemical characterization of AmyJM. A Effects of pH on AmyJM activity and stability. B Effects of buffer (100 mM each, pH 7.5) on AmyJM activity. C Effects of temperature on AmyJM activity and stability. D Thermostability of AmyJM at 40–50 °C (in the presence or absence of 5 mM calcium chloride). Data are shown as means ± standard errors of triplicate analyses

Fig. 5

HPLC-ELSD analysis of reaction products of AmyJM with different A gelatinized substrates and B raw substrates. Data are shown as means ± standard errors of triplicate analyses. G1 glucose, G2 maltose, G3 maltotriose, G4 maltotetraose, G5 maltopentaose, G6 maltohexaose, G7 maltoheptaose

Fig. 6

FESEM images of untreated and AmyJM-hydrolyzed raw starches. A Wheat starch. B Tapioca starch. C Sago starch. D Potato starch. E Rice starch. F Corn starch