Characterization of a Thermostable α-Amylase from Bacillus licheniformis 104.K for Industrial Applications

Abstract

This study describes the characterization of a novel thermostable α-amylase from a Bacillus licheniformis 104.K strain isolated from the Kashkadarya region of Uzbekistan. Phylogenetic analysis revealed that the thermostable α-amylase belongs to glycoside hydrolase family 13 subfamily 5 (GH13_5) and shares high sequence similarity with known α-amylases. Our results demonstrate that the recombinant α-amylase exhibits optimal activity at pH 6.0 and 90 °C, retaining full activity after 30 min at 60 °C. The addition of CaCl₂ significantly enhanced thermostability, with the enzyme retaining more than 95% of its initial activity at 70 °C after 30 min. Our findings indicate that α-amylase from B. licheniformis 104.K is a functional, thermostable enzyme with potential industrial applications. This study highlights the commercial significance of thermostable amylases and the need to identify novel, cost-effective, and sustainable sources. The results of this study will contribute to the fields of enzyme applications, stabilizing additives, and genetic engineering of thermostable genes.

Keywords: Bacillus licheniformis; GH13_5 subfamily; affinity chromatography; recombinant enzyme; thermostability; α-amylase.

Figure 1

Mechanism of starch hydrolysis by α-amylase. The enzyme cleaves internal α-1,4-glycosidic linkages in polysaccharide chains, producing glucose, maltose, and maltotriose.

Figure 2

The phylogenetic tree of B. licheniformis 104.K (GenBank: OR781719.1) and related strains was constructed using MEGA 11 software. The yellow-framed accession OR781719.1 is the query sequence, phylogenetically grouped within Bacillus species, closely related to B. licheniformis. Multiple sequence alignment was performed using the MUSCLE algorithm, and the phylogenetic tree was generated using the neighbor-joining method. Bootstrap analysis with 1000 replicates was conducted to evaluate the reliability of the tree topology.

Figure 3

(A) Image of the 12% SDS-PAGE gel containing the recombinant α-amylase of B. licheniformis 104.K purified by affinity chromatography. Lane M: protein marker (Sigma-Aldrich, BLUeye Prestained Protein Ladder). Lanes 1 and 2: E. coli BL21 before and after induction at 16 °C with overnight incubation. Lanes 3 and 4: E. coli BL21 before and after induction at 30 °C with 4 h of incubation. Lanes 5 and 6: E. coli Rosetta before and after induction at 16 °C with overnight incubation. Lanes 7 and 8: E. coli Rosetta before and after induction at 30 °C with 4 h of incubation. (B) Lane M: protein marker (PageRuler Unstained Protein Ladder). Lane 1: Ni-NTA affinity-purified recombinant α-amylase expressed in E. coli Rosetta cells.

Figure 4

B. licheniformis 104.K α-amylase enzyme activity and stability are affected by different factors. (A) Effects of temperature on α-amylase activity. (B) α-Amylase thermostability in the presence or absence of 5 mM calcium chloride. (C) Effects of pH on α-amylase activity at 90 °C. (D) Effects of pH on α-amylase stability. The pH stability was determined at 90 °C in Na-phosphate buffer (100 mM) by varying the pH values. The enzyme was preincubated in the buffer at 40 °C for 1 h, and residual activity was determined. The activity of the enzyme before incubation was taken as 100%. The average and standard deviation of the measured data of three parallel measurements are shown, with trend lines connecting the data points.

Figure 5

Sequence alignment of the thermostable α-amylase (Amylase-104.K) from B. licheniformis 104.K with sequences from the NCBI protein database. A key mutation at position 349 is highlighted in blue, showcasing the amino acid variation unique to the amylase from the 104.K strain (“*”—identical residues; “:”—strongly similar residues; “.”—weakly similar residues). The alignment was generated using Clustal Omega software.

Figure 6

AlphaFold structure of B. licheniformis 104.K α-amylase. (A) Overall structure of processed B. licheniformis 104.K α-amylase. The structure is shown as a cartoon and a partially transparent surface; the three main domains are highlighted in three shades of green. (B) Structural comparison with active α-amylase (PDB ID: 1BLI), shown as cartoon colored in various shades of purple. Catalytic metal cofactors are shown as lime and brown spheres for Na⁺ and Ca²⁺, respectively. The enzyme active site is designated with superimposed acarbose (PDB ID: 1E3Z) shown as sticks with atomic coloring (C, yellow; O, red; N, blue) and an auxiliary starch binding site by a superimposed maltotriose (PDB ID: 1E40) also shown as sticks with atomic coloring (C, orange; O, red; N, blue). (C) Predicted structure B. licheniformis 104.K α-amylase highlighting the residue differences compared to B. licheniformis α-amylase (UniProt ID: P06278). The different residues are shown as sticks, and potential interactions contributing to better thermostability are indicated as black dashed lines.