Structural analysis and engineering of industrial alpha-amylases
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Sepideh Bourenjan Shirazi,1 Neda Soleimani,2 Amir Rahimi,3,*
1. Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
2. Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran.
3. Bioinformatics and Computational Biology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
- Introduction: Enzymes are a suitable alternative to catalysts in various industries due to their specificity, performance under milder conditions, and reduced production of by-products as a result of the reaction. The carbohydrases, as enzymes that catalyze the conversion of carbohydrates into simple sugars, are a pioneer in the global industrial market. The alpha-amylase enzyme is the most widely used carbohydrate in various industries such as detergent, food, poultry feed, textile, paper, and fuel production. Bacterial- and the fungal-derived Alpha-amylases are widely used in industry due to their high efficiency, and pH, and thermal stability. four important characteristics of alpha-amylases in the industry are calcium dependency, thermostability, pH profile, and oxidative resistance of enzyme. Extensive efforts have been made to optimize the activity of the enzyme in industrial conditions. But, none of the known wild-type alpha-amylase has been in the optimal state for all the mentioned above features together so far. Therefore, in recent years, many attempts have been made to produce the ideal alpha-amylase through protein engineering methods. Optimization of enzyme activity conditions and also enzyme engineering often requires accurate knowledge of the structural origin of the desired properties. Therefore, In the present study, the sequence alignment between 6 industrially important alpha-amylases was performed. Sequence alignment results were used for molecular analysis and investigation of the structural origin of their industrially important features.
- Methods: In this review, we summarize the findings from various studies related to structure, enzyme engineering, and industrial application of alpha-amylase through a comprehensive PubMed, Science Direct, and Google Scholar search. The keywords "industrial enzyme"," microbial enzyme", "alpha-amylase", "enzyme crystallography", "calcium dependency", "thermostability", "pH profile" and "oxidative resistance", "enzyme engineering "were entered into the search field and 50 articles were investigated between the years 1990-2021. Also, in another part of this study, we explored the relation of sequences and structure with four important features of alpha-amylase via bioinformatics approaches. The amino acid sequences of six-industrially important alpha-amylase were obtained from NCBI and UniProt database. The sequence alignment was performed by Clustalω software. Then, the ESPript3.0 program was used to visualize the alignment for the publication purpose. Sequence alignment was analyzed by Jalview software. The 3D structures of the enzymes were obtained from the PDB database and visualized by PyMol and Chimera software. the atomic distances were measured using Chimera software.
- Results: The overall structure of alpha-amylases consists of three domains A, B, and C. Domain A is the most conserved domain among all alpha-amylases, consisting of three catalytic residues and a fully conserved calcium-binding site which is essential for maintaining the function and stability of the enzyme structure. Domain B is the least conserved domain and this variation is one of the factors that develop unique properties in thermostable alpha-amylase. The second calcium ion together with the other calcium and a sodium ion forms a metal-triad that plays an important role in the thermostability of the alpha alpha-amylase of B.licheniformis, B.stearothermophilus, and B.amyloliquefacions. However, the binding site of the second calcium ion in A. niger and A. oryzae alpha-amylases is catalytic residues, and the presence of excess amounts of this ion will lead to a significant decrease or loss of enzyme activity. The binding site of the third calcium ion with the least conservation plays a mainly structural role. In the thermostable alpha-amylases a loop containing 21 residues, is one of the main factors besides metal-triad in developing thermal stability. Factors determining the pH profile of alpha-amylase mainly affect the pKa of catalytic residues by changing the electrostatic field. However, the dynamics of catalytic residues also play a role in determining their pH profile, which is more complicated. Under oxidative conditions, the oxidation of the amino acids methionine and cysteine present at the surface of the enzyme structure, especially near or inside the active site cleft, increases the volume of these residues, resulting in a dramatic decrease in enzyme activity.
- Conclusion: Understanding these structural factors leads to the optimal use and engineering of alpha-amylase for industrial application. This knowledge will help researchers with efficient enzyme engineering and developing optimized industrial alpha-amylases and thus enhancement of efficiency which leads to the reduction of additional costs in the industry.
- Keywords: alpha-amylase, industrial enzyme, microbial enzyme, enzyme engineering
