Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

Abstract

High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center.

A general and efficient strategy for generating the stable enzymes

“…7B,C ). These intramolecular interactions play important roles in LIP1 stability by rigidifying the active center 43 .…”

Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase

“…The folding or anchoring of terminal tails is typically achieved through ion pairing, hydrogen bonds or hydrophobic interactions [ 35 ]. These interactions anchor the flexible terminal tail, consequently reducing the probabilities of the overall structure from unfolding since it typically begins at the terminal tails of protein [ 35 , 36 ]. Further analysis of the N -terminal tail of the mutant lipases reveals hydrogen bonds and hydrophobic interactions between the residues of N -terminal with the C -terminal residues and surrounding residues that are responsible for the anchoring ( Table 3 ).…”

The response to selection in Glycoside Hydrolase Family 13 structures: A comparative quantitative genetics approach

“…Part of the interest in researching this family lies in its industrial importance [ 25 , 26 ]. The GH13 family is the target of engineering efforts, focusing on factors such as thermal and alkaline stability [ 27 – 30 ], specific activity [ 27 , 31 ], and other diverse biochemical properties that are important to the industrial context [ 28 , 32 , 33 ]. Many strategies have been used to engineer this family including different “rational design” approaches [ 34 ] such as B-fitter [ 35 ], proline theory [ 36 ], PoPMuSiC-2.1 [ 37 ], and sequence consensus [ 34 ].…”