Topology of a-Amylases

The interesting feature of the a-amylase family is the presence of four highly conserved regions in their internal structure. They are present in the TIM-barrel structure of all a-amylases. The four conserved arrangements (I—IV) of a-amylases are found in the C-terminal end of (3-strand 3, (3-strands 4,5 and in the loop connecting (3-strand 7 to a-helix 7 (Figure 2.2). These regions are essential for the formation of the active centre and the substrate binding site. The first region carries histidine residue, which interacts with the glucose residue of the substrate; nucleophilic Asp is present in the second region; proton donating Glu residue has been observed in the third region; a histidine residue and an Asp residue are found in the fourth region that may form hydrogen bonds with glucose residue of the substrate (Prakash and Jaiswal 2010). Acidic amino acids Asp, Glu, and Asp residues are present in the catalytic site and their positions are in the core of the TIM-barrel. The comparative study of nucleotide sequences of different microbial a-amylases has shown that a greater number of base pairs exist in bacterial amylase than in TAKA amylase from Aspergillus oryzae, while the numbers of amino acid residues of these a-amylases are varied among the organisms. Alternatively, amino acids of the catalytic triad are always present in the

FIGURE 2.2 Topology of a-amylases. The positions of four conserved sequences (I—IV) are indicated with dashed boxes.

TABLE 2.3

Number of Base Pair and Amino Acid Residues, and Four (I—IV) Conserved Regions (CR) of Alpha Amylase from Mostly Exploited Four Different Microbes

Source: Yamamoto. T., Catalytic mechanisms of amylases and related enzymes, in Enzyme Chemistry and Molecular Biology of Amylases and Related Enzymes, ed. The Amylase Research Society of Japan, Vol. 84. CRC Press. 1995; Huma. T. et al„ Phylogenetic and comparative sequence analysis of thermostable alpha amylases of kingdom archea, prokaryotes and eukaryotes, Bioinformation, 10,443-448, 2014.

Single letter indicates the letter code of respective amino acid.

■ Indicates Catalytic Amino Acids.

conserved regions II, III and IV, but their position number is different. The number of bases, amino acids and the sequences of conserved regions of a-amylase of the most commonly used microbes are given in Table 2.3. The catalytic residues Asp231, Glu261 and Asp328 in amylase of Bacillus licheniformis (BLA) are equivalent to Asp206, Glu230 and Asp297 of TAKA amylase of Aspergillus oryzae. These conserved residues of the a-amylase family show their similar functional activity and also contribute to substrate and calcium binding (Kumari et al. 2012).

Mechanism of Action of a-Amylase

a-amylase reacts through the а-retaining double displacement mechanism (van der Maarel et al. 2002). The active site cleft is located in the interface between domain A and domain В at the C-terminal end of the (3-strands in the TIM-barrel (Nielsen and Borchert 2000). The substrate binding cleft can accommodate four to ten glucose units of starch, a-amylase has four highly conserved regions for substrate binding and catalysis. The substrate binding residues form the substrate binding subsite. The location of the scissile bond is present in between negative and positive subsite. The non-reducing side of the scissile bond is projected on negative subsite numbers while the reducing side of the scissile bond is fitted on the positive subsite numbers (Figure 2.3). Two aspartic acids and one glutamic acid are responsible for conducting the catalytic activity. Glutamic acid acts as acid/base catalyst and aspartic acid acts as nucleophile during the formation of enzyme substrate complex. After binding of starch to the active site, glutamic acid donates the proton to the glucosidic oxygen, whereas aspartate exerts a nucleophilic attack to the C( l) of glucose at subsite -1. These two events finally cleave the glycosidic bond. The protonated glucose molecule of 4-1 subsite moves away from the active site, and then water enters into the reaction system. Glutamate pulls off the proton from the water and the oxygen forms a hydroxyl group at Cl (Figure 2.4).

FIGURE 2.3 The pattern of subsite position, nomenclature and presentation of scissible bond for glycosyl hydrolases.

FIGURE 2.4 The а-retaining double displacement mechanism of an a-amylase catalytic reaction system.

The second aspartate residue plays an indirect role in the catalysis by holding the substrate in proper position through bonding with OH(2) and OH(3) of glucose residue of the substrate (Uitdehaag et al. 1999). The chemical modification of aspartic (nucleophile) and glutamic acid (proton donor) residues by Woodward’s reagent К (WRK) inhibits the a-amylase activity, and thus, establishes their role (Sharma and Satyanarayana 2010). Instead of these three residues (Asp-Glu-Asp) of the active site, other conserved residues like histidine, arginine and tyrosine participate to arrange the substrate in proper orientation into the active site (van der Maarel et al. 2002).

Alpha-Amylase Engineering

Protein engineering is mostly applied in industrially important proteins (enzyme) to improve the functional characteristics. The idea of protein engineering is successfully achieved by making precise changes in the amino acid sequence, based on mutation, in its encoded genes. Site-directed mutagenesis is one of the successful techniques to achieve protein engineering, which makes specific changes in a DNA sequence. This technique lowers the hazards of crude mutagenesis in cells or organisms and makes it easier to isolate the desire mutants from thousands or millions of offspring..

Engineering of a-amylase has been required to improve the characteristics like specific activity (activity/mg of protein), kinetic parameters (substrate affinity, catalytic efficiency), pH profile, thermostability, oxidation resistance capacity against oxidizing agents (iodate, heavy metals, sodium hypochlorite), activity against chelators [EDTA, EGTA, DTPA (diethylene triaminepentaacetic acid)], zeolites, and half-life (indicator of stability) by altering the amino acids in specific positions. The modi- fied/engineered enzymes are suitable for their exploitation in textile, paper, detergent and food industry. Several strategies have been adopted to make the better quality of enzyme. Alterations of some oxidation prone amino acids (methionine, tryptophan, cysteine, and histidine) by other amino acids (not affected by oxidizing agents) are beneficial to improve the oxidation resistance capacity (Kumari et al. 2012). Chi et al. (2010) reported that replacement of Met231 by leucine in a-amylase of Bacillus sp. strain TS-23 can improve the oxidative stabilities. An engineered form of a-amylase was also constructed from wild type a-amylase of Geobacillus stearothermophilus US100 by substituting Metl97 into Ala, which showed a significantly high resistance to chelator agents and chemical oxidation. Improvement of these two properties increases the possibility of its use in detergent industry (Khemakhem et al. 2009). Most of the chelator agents react with sulphur molecules and make inactive protein. However, alanine is least hydrophobic among the non-polar amino acids and promotes the stability of protein structure.

Introduction of disulphide bonds in the enzyme leads to improved stability of the protein. Disulphide bonds have more bond energy than the non-covalent interactions. Naturally, disulphide bonds form between two cysteine molecules and cross-linking is possible with the distance part of the protein chain, which improves the stability. However, formation of cysteine disulphide bonds requires a significant distance with dihedral angle constraints between two cysteine molecules. Sometimes incorporation of non-canonical amino acids with long side-chain thiol group creates an excellent position to form disulphide bonds as well as protein stability. Introduction of proline in loop regions can stabilize the proteins by lowering the entropy level of the unfolded state, as the a-amylase is present in a folded or partially unfolded state in various conditions, especially at high temperature. The insertion of proline residues depends on its accommodation in protein structure, and arginine is preferably selected for replacement by proline. Replacement of Argl24 of an a-amylase of alkalophilic Bacillus species by proline improves the stability of the enzyme (Nielsen and Borchert 2000).

Production of hybrid enzyme by using separate homologous amylase genes of different bacteria is essential to increase the thermostability and catalytic efficiency. Sequence alignment study has revealed that a-amylase from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) share more than 70% identity (Conrad et al. 1995). These two organisms were used for synthesis of hybrid enzyme. In comparison with BAA, two regions were identified in BLA, responsible for higher thermostability. Later on, three subsequent mutations were done in BAA by the deletion of Argl76 and Gly 177, and the substitution of Lys269 by Ala. These mutations imposed additional thermostability in BAA. Similar effects on improvement of thermostability were also observed in other a-amylases of various Bacillus species (Nielsen and Borchert 2000; Mehta and Satyanarayana 2016). Amylase is also engineered by random mutagenesis coupled with high-throughput screening by using error-prone PCR. A report showed that beneficial mutants (Met 15 by Thr and Asnl88 by Ser) of B. licheniformis showed improved pH profile (23 times) and higher thermostability (Shaw et al. 1999).

The uses of a-amylase in industrial sectors are carried out at diverse pH. Nielsen and Borchert (2000) also reported that several techniques can be applied to improve the pH profiles for optimum activity. These are helix capping, removal of deami- dating residues and cavity-filling. Mutation of Asn329 to Lys or Asp in amylase of Bacillus stearothermophilus (BLA Asn326) can significantly change the pH profile. Asparagine is recognized as a deaminating amino acid, which destabilizes the protein structure. Substitution by Lys or Asp increases the possibilities of formation of salt bridge and interaction with solvent, which improve stability of the enzyme. Helix capping is primarily done by formation of hydrogen bond at the end of the helices present in the protein structure. Negatively charged amino acid is more favourable at the N-cap, which can neutralize the partial dipolar charge of the unpaired amide protons. Glycine is present at the C-cap, which has the ability to present as a-L conformation for its stability. Cavity is formed during folding of protein mostly at the (3-strand present at the turn and loops and also at the protrusion. Cavity-filling is most important to increase the thermostability of the protein. This process increases the hydrophobic core of the protein as well as stabilizing energy. Incorporation of non-polar, long aliphatic, hydrocarbon chain bearing amino acids like leucine and isoleucine gives the best result instead of alanine and valine.