A practical approach to steady-state kinetic analysis of cellulases acting on their natural insoluble substrate

Kari, Jeppe; Christensen, Stefan Jarl; Andersen, Morten; Baiget, Selene Sellés; Borch, Kim; Westh, Peter

doi:10.1016/j.ab.2019.113411

Cited by 12 publications

(8 citation statements)

References 69 publications

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“…The resulting kinetic parameters (K M and k cat ) are listed in supplementary Table 4. Previous studies have identified practical procedures for measuring the quasi-steady-state rate for this type of system 23 and shown that Eq. 1 is valid and applicable even though the substrate is solid and specified by its mass load (S 0 ) in units of g/L 24,25 .…”

Section: Resultsmentioning

confidence: 99%

Physical constraints and functional plasticity of cellulases

et al. 2021

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Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes.

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Section: Resultsmentioning

confidence: 99%

Physical constraints and functional plasticity of cellulases

et al. 2021

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“…Despite much progress in the enzyme area, further improvements still seem possible. For example, it is still not fully clear how processive cellulases work and how the interplay of these essential but rather slow enzymes with other enzymes could be optimized [57,169,302,306,362]. Recent insights concerning the role of H 2 O 2 and enzyme inactivation suggest that so far, we have not harnessed the full potential of LPMOs.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

Østby

Hansen

Horn

et al. 2020

Journal of Industrial Microbiology and Biotechnology

129

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Efficient saccharification of lignocellulosic biomass requires concerted development of a pretreatment method, an enzyme cocktail and an enzymatic process, all of which are adapted to the feedstock. Recent years have shown great progress in most aspects of the overall process. In particular, increased insights into the contributions of a wide variety of cellulolytic and hemicellulolytic enzymes have improved the enzymatic processing step and brought down costs. Here, we review major pretreatment technologies and different enzyme process setups and present an in-depth discussion of the various enzyme types that are currently in use. We pay ample attention to the role of the recently discovered lytic polysaccharide monooxygenases (LPMOs), which have led to renewed interest in the role of redox enzyme systems in lignocellulose processing. Better understanding of the interplay between the various enzyme types, as they may occur in a commercial enzyme cocktail, is likely key to further process improvements.

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“…Furthermore, for conversion of solid substrates (either those above saturation concentration, where high concentrations are required or those, which at the conversion temperature, have a low solubility in the reaction medium (for example cellulose in water)) the rate law needs adjusting to account for surface catalysis. 72 In all cases, the sensitivity of the observed reaction rate to the concentration of all reactants and products emphasizes the need to obtain the rate law, with the estimated kinetic constants. Several procedures are now in existence for the collection of such data, both at a more global level to establish rate limiting effects, 73 as well as to obtain kinetic constants.…”

Section: Towards a Systematic Approach To Reaction Engineeringmentioning

confidence: 99%