Stabilizing biocatalysts

Abstract

The area of biocatalysis itself is in rapid development, fueled by both an enhanced repertoire of protein engineering tools and an increasing list of solved problems. Biocatalysts, however, are delicate materials that hover close to the thermodynamic limit of stability. In many cases, they need to be stabilized to survive a range of challenges regarding temperature, pH value, salt type and concentration, co-solvents, as well as shear and surface forces. Biocatalysts may be delicate proteins, however, once stabilized, they are efficiently active enzymes. Kinetic stability must be achieved to a level satisfactory for large-scale process application. Kinetic stability evokes resistance to degradation and maintained or increased catalytic efficiency of the enzyme in which the desired reaction is accomplished at an increased rate. However, beyond these limitations, stable biocatalysts can be operated at higher temperatures or co-solvent concentrations, with ensuing reduction in microbial contamination, better solubility, as well as in many cases more favorable equilibrium, and can serve as more effective templates for combinatorial and data-driven protein engineering. To increase thermodynamic and kinetic stability, immobilization, protein engineering, and medium engineering of biocatalysts are available, the main focus of this work. In the case of protein engineering, there are three main approaches to enhancing the stability of protein biocatalysts: (i) rational design, based on knowledge of the 3D-structure and the catalytic mechanism, (ii) combinatorial design, requiring a protocol to generate diversity at the genetic level, a large, often high throughput, screening capacity to distinguish 'hits' from 'misses', and (iii) data-driven design, fueled by the increased availability of nucleotide and amino acid sequences of equivalent functionality.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…Methods capable of revealing the conformation of proteins in solution and on solid surface would therefore be required to test and eventually establish correlations between the degree of conformational distortion and the residual specific activity of the immobilized enzyme. In a similar manner, stability of immobilized enzymes could be analyzed where the degree of [90] stability enhancement might be related to detectable and ideally also quantifiable conformational distortions in the immobilized enzyme [19,21,91]. Problem is that only few of the various spectroscopic techniques applied routinely to the study of protein conformations in solution are suitable for analysis of proteins on surface, especially that of a porous solid support.…”

Significantly improved thermostability of a reductase CgKR1 from Candida glabrata with a key mutation at Asp 138 for enhancing bioreduction of aromatic α-keto esters

“…The commercial application of reductases for synthesis of fine chemicals is often limited by a low stability of the biocatalyst under process conditions, which determines process economic (Bommarius et al, 2006). Good thermostability has multiple benefits, such as enabling reactions at higher temperature for an increase in reaction rate or reactant solubility and increasing biocatalyst lifetime for a decrease of the cost of biocatalyst (Bommarius and Paye, 2013;Wu and Arnold, 2013).…”

Novel immobilization process of a thermophilic catalase: efficient purification by heat treatment and subsequent immobilization at high temperature

“…Thermostability is closely related to operational stability; most research on enzyme reactions has focused on thermostability because it can be conveniently tested [19,43]. Figure 5 reveals the thermal stability of the free and immobilized catalase.…”