Native to designed: microbial α-amylases for industrial applications

Abstract

Background α-amylases catalyze the endo-hydrolysis of α-1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, α-amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial α-amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work. Survey methodology and objectives A traditional review approach was taken to focus on the engineering of microbial α-amylases to enhance industrially favoured characteristics. The action mechanisms of α- and β-amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial α-amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100 °C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial α-amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries. Conclusions Microbial α-amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial α-amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

α‐Amylase Inhibitors Based on Thiazolidinone Skeleton: A Promising Approach in Diabetes Management

“…α-Amylase (α-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) is one of the most important industrial endoamylases that belongs to family 13 of the glycoside hydrolase group of enzymes. [35] It is capable of hydrolyzing the internal α-1,4glycosidic bonds in polysaccharides with the retention of αanomeric configuration in low molecular weight products, such as glucose, maltose, and maltotriose units. Most of the αamylases are metalloenzymes, requiring calcium ions (Ca 2 + ) for their activity, structural integrity, and stability.…”

“…Docking studies revealed the potential interaction of compound 11 i within the active site of α-amylase (PDB ID: 7TAA, Aspergillus oryzae). The compound 11 i formed significant bonding with amino acids GLN 35 , TYR 79 , TRP 83 , and HIS 210 . Compound 11 i formed conventional hydrogen bonding with GLU 276 and ASP 349 within the active site of the α-glucosidase enzyme.…”

Upscaling of alkaline pea protein extraction from dry milled and pre-treated peas from laboratory to pilot scale: Optimization of process parameters for higher protein yields

“…The dry matter of the supernatant and the pellet of the alkaline extraction step in lab scale and pilot scale are shown in Table 2. As no starch was quantifiable, it was concluded that the dry matter of the supernatants was composed of proteins, minerals, and minor components such as mono-, di-and oligosaccharides, as native pea amylases could start to degrade starch during the soaking and extraction step [24,25]. The mineral content of the supernatants is composed of native minerals from the pea and sodium from the added NaOH.…”

Improving Thermostability of Chimeric Enzymes Generated by Domain Shuffling Between Two Different Original Glucoamylases

“…Although glucoamylases can be produced by many fungal species ( Norouzian et al, 2006 ), commercial or industrial glucoamylases with moderate thermostability and high activity are mainly derived from Aspergillus niger ( Norouzian et al, 2006 ), Rhizopus oryzae ( Wang et al, 2020 ), and Talaromyces emersonii ( Nielsen et al, 2002 ) due to the conversion of starch to glucose ( Zong et al, 2022 ). Because the saccharification processes are usually followed by a liquefaction process of starch and are performed at 60°C for 48–72 h, the glucoamylases required in starch industrials have to possess good thermostability and catalytic activities ( Lim and Oslan, 2021 ; Tong et al, 2021 ). So, searching for a new source of glucoamylase with potentially applicable properties encompassing elevated temperature, extreme pH, high salinity, organic solvents, surfactants, and specificities (substrate and product) is still of considerable importance ( Schmidt et al, 2019 ).…”

“…In addition to exploring novel enzymes with desirable properties in nature, attempts are being made to improve the properties of the existing enzymes by protein engineering techniques to make them suitable for industrial applications ( Parashar and Satyanarayana, 2016 ; Sharma et al, 2019 ). These techniques mainly include rational design, semi-rational design, directed evolution (error prone PCR and DNA shuffling), and fusion ( Schmidt et al, 2019 ; Sharma et al, 2019 ; Lim and Oslan, 2021 ; Tong et al, 2021 ). However, the design of chimeric enzymes by fusing different domains from native enzymes is considered to be a straightforward method for generating a novel enzyme with improved catalytic properties ( Parashar and Satyanarayana, 2016 ; Ali et al, 2020 ).…”