Fusion of an Oligopeptide to the N Terminus of an Alkaline α-Amylase from Alkalimonas amylolytica Simultaneously Improves the Enzyme's Catalytic Efficiency, Thermal Stability, and Resistance to Oxidation

Abstract

In this study, we constructed and expressed six fusion proteins composed of oligopeptides attached to the N terminus of the alkaline ␣-amylase (AmyK) from Alkalimonas amylolytica. The oligopeptides had various effects on the functional and structural characteristics of AmyK. AmyK-p1, the fusion protein containing peptide 1 (AEAEAKAKAEAEAKAK), exhibited improved specific activity, catalytic efficiency, alkaline stability, thermal stability, and oxidative stability compared with AmyK. Compared with AmyK, the specific activity and catalytic constant (k cat ) of AmyK-p1 were increased by 4.1-fold and 3.5-fold, respectively. The following properties were also improved in AmyK-p1 compared with AmyK: k cat /K m increased from 1.8 liter/(g·min) to 9.7 liter/(g·min), stable pH range was extended from 7.0 to 11.0 to 7.0 to 12.0, optimal temperature increased from 50°C to 55°C, and the half-life at 60°C increased by ϳ2-fold. Moreover, AmyK-p1 showed improved resistance to oxidation and retained 54% of its activity after incubation with H 2 O 2 , compared with 20% activity retained by AmyK. Finally, AmyK-p1 was more compatible than AmyK with the commercial solid detergents tested. The mechanisms responsible for these changes were analyzed by comparing the three-dimensional (3-D) structural models of AmyK and AmyK-p1. The significantly enhanced catalytic efficiency and stability of AmyK-p1 suggests its potential as a detergent ingredient. In addition, the oligopeptide fusion strategy described here may be useful for improving the catalytic efficiency and stability of other industrial enzymes. Most industrial enzymes are obtained from the natural environment. However, these enzymes are often used under conditions drastically different from their natural environment, in which the catalytic performance of the enzymes decreases, restricting their applications. Consequently, increasing attention is being paid to improving the catalytic performance of enzymes under extreme but application-relevant conditions such as high temperature, strong acid and alkali, and oxidative stress (1).Protein engineering has emerged as an important tool to overcome the limitations of natural enzymes (2). Common strategies to alter proteins include site-directed mutagenesis and directed evolution (e.g., error-prone PCR). However, site-directed mutagenesis requires a clear understanding of the relationship between enzyme structure and function, and directed evolution requires a straightforward and efficient high-throughput screening method (3-5).Alkaline ␣-amylases have high catalytic efficiency and stability at an alkaline pH range between 9 and 11 (6, 7) and are widely used in the detergent and textile industries for starch hydrolysis under alkaline conditions (8-10). This application requires high catalytic efficiency, high alkaline stability, and high oxidative stability (11). Although site-directed mutagenesis has been used to improve the oxidative stability of ␣-amylases, this was accompanied by decreased catalytic efficiency, alkaline stability,...

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

“…Melting temperature (T m ) was detected on a Q2000 differential scanning calorimeter (TA, New Castle, DE) as described in our previous study (7). To eliminate signal baseline drift of the equipment, we warmed up the calorimeter for 10 min.…”

“…AmyK activity was characterized as the release of reducing sugar during hydrolysis of soluble starch and measured using a modified dinitrosalicylic acid method (7). One unit of AmyK activity was defined as the amount of enzyme that released 1 mol of reducing sugar as glucose per minute under these assay conditions.…”

“…The wild-type AmyK and its mutants were purified with an Akta purifier system using a Ni 2ϩ -charged 1-ml HisTrap FF crude column at a flow rate of 1 ml/min as described in our previous study (7). The purified enzymes were stored in Tris-HCl buffer (pH 9.0, 20 mM) at 4°C and diluted to 16 U/ml with glycine-NaOH buffer (pH 9.5, 20 mM) for activity assay.…”

“…In our recent work, an alkaline ␣-amylase from alkaliphilic Alkalimonas amylolytica N10 (AmyK) was heterologously expressed in various hosts (5), and the oxidative stability and catalytic efficiency were increased significantly through site-directed mutagenesis of oxidation-prone residues in the catalytic domain (6) and the fusion of an oligopeptide at the N terminus (7). However, both AmyK and its mutants remained unstable at temperatures above 60°C (5)(6)(7). Thus, the thermostability of AmyK must be improved to make the enzyme useful in the textile industry.…”

Integrating Terminal Truncation and Oligopeptide Fusion for a Novel Protein Engineering Strategy To Improve Specific Activity and Catalytic Efficiency: Alkaline α-Amylase as a Case Study

“…In previous work, we conducted systemslevel molecular engineering of a novel alkaline ␣-amylase (AmyK) from Alkalimonas amylolytica to improve its oxidative stability by replacing five methionine residues around the active site with oxidation-resistant amino acids (17). In our recent work, an oligopeptide (AEAEAKAKAEAEAKAK) obtained from Zuotin protein sequence in Saccharomyces cerevisiae was fused at the N terminus of AmyK, and the catalytic efficiency was enhanced 3.5-fold compared to that of wild-type AmyK (18).…”