Helping proteins come in from the cold: 5 burning questions about cold-active enzymes

Abstract

Enzymes from psychrophilic (cold-loving) organisms have attracted considerable interest over the past decades for their potential in various low-temperature industrial processes. However, we still lack large-scale commercialization of their activities. Here, we review their properties, limitations and potential. Our review is structured around answers to 5 central questions: 1. How do cold-active enzymes achieve high catalytic rates at low temperatures? 2. How is protein flexibility connected to cold-activity? 3. What are the sequence-based and structural determinants for cold-activity? 4. How does the thermodynamic stability of psychrophilic enzymes reflect their cold-active capabilities? 5. How do we effectively identify novel cold-active enzymes, and can we apply them in an industrial context? We conclude that emerging screening technologies combined with big-data handling and analysis make it reasonable to expect a bright future for our understanding and exploitation of cold-active enzymes.

Keywords: Bioprospecting; Enzyme catalysis; Protein dynamics; Psychrophilicity; Thermodynamic stability.

Fig. 1

Thermodynamic aspects of enzyme catalysis. (a) Schematic of activation energy (E_A) as a barrier between the reactant and the products. Enzymes catalyze reactions by lowering E_A. (b) Activity versus temperature (upper box) of two structurally homologous psychrophilic (blue) and mesophilic (red) enzymes and a comparison with structural unfolding (lower box) (J.S.N. and D.E.O., unpublished data). t_opt is represented by dotted lines, while t_m is shown as a stippled line. (c) Eyring plot of the catalytic rates in the range 5-25°C of structurally homologous psychrophilic (blue) and mesophilic (red) enzymes from panel b. The slope of the mesophilic enzyme is 13% steeper than the psychrophilic, while the secondary axis intercept is 7% higher for the psychrophilic enzyme. (d) Transition state diagram according to Eyring-Polanyi. A reduction of the stability of ES (and possibly an increase in the stability of ES^‡) in a psychrophile can lead to a lower ΔG^‡.

Fig. 2

Stability profiles of different temperature classes of enzymes. (a) Chemical unfolding of a psychro-, meso- and thermophilic amylase using the denaturant guanidinium chloride (GdmCl). Data adapted from ref. 24 and fit to a two-state unfolding model (psychro- and mesophile) and a three-state model (thermophile). (b) - Thermal unfolding of a psychro- (blue circles), meso- (orange triangles), and thermophilic (red squares) α-amylase measured by a shift in Trp fluorescence. The relative activity of the enzymes is shown as semitransparent fill, colour-coded in the same way as unfolding data. Data adapted from ref. 24 and fit to a two-state thermal unfolding model. (c) Conformational stability curves of a psychro- (blue), meso- (orange), and thermophilic (red) α-amylase measured by DSC. All 3 panels based on data from ref. 25 using digitization, normalization and replotting in GraphPad Prism 9.

Fig. 3

Potential ways to isolate and identify novel psychrophilic enzymes. Created with BioRender.com