Question

There is an enzyme “amylase” used in Bioethanol production at large scale. The enzyme has excellent Kcat and Km values but there is issue of thermostability. The enzyme cannot work beyond 50oC temperature which is not optimum for industry. We have been giving a target to increase its thermostability by using Rational Design. Can you please let us know that which region (Helix, Sheet, Loops) will be targeted to increase thermostability of enzyme and why?

Answer 1

Ans. -

The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications.

Applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes’ performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications. Recombinant DNA
techniques have allowed the isolation and cloning of genes encoding for enzymes from all possible sources, including microbes and other microorganisms that are particularly difficult to
manipulate, and high-yield heterologous protein expression.

In the starch processing industry, a number of other end product-specific amylases are
commonly used for the synthesis of different maltooligosaccharides. Finally, several actinomy-
cetes are the source of cold-active a-amylases that find application in textile, detergent, and bioethanol industries.  rational design meth-
ods need to be able to predict stabilizing mutations in the context of a given fold and, at the same time, minimize any change in the backbone conformation that might disrupt the structure of the active site or reduce its flexibility. The techniques used to increase protein stability with a rational approach are often based on one or multiple methods including phylogenetic analysis, comparison to homologous proteins, optimization of
charged interactions, optimization of residues and loops showing unfavorable Ramachandran angles and high B-factors, methods based on the calculation of free energies, and structure-based computational design.

Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model.