Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site

Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

doi:10.1074/jbc.m113.536045

Cited by 226 publications

(182 citation statements)

References 44 publications

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“…One of the mutants mCALB168 with three additional hydrogen bonds displayed on yeast showed 14-fold improvement in t 1/2 -value at 60°C compared to the wild type and also had higher thermo stability than a commercially available lipase (Peng 2013). Xie et al (2014) reported the use of iterative saturation mutagenesis on the structurally flexible residues of the active site of lipase B from C. antartica to improve the thermal and active site stability. One of the mutants (D223G/L278M) exhibited a 13-fold increase t 1/2 at 48°C compared to the native enzyme (Xie et al 2014).…”

Section: Development Of Thermostable Lipasesmentioning

confidence: 98%

“…However, at a higher temperature thermal denaturation of lipase usually takes place. Directed evolution and rational design have been used to create a permanent solution to this problem (Hwang et al 2014;Korman et al 2013;Peng 2013;Xie et al 2014;Yu et al 2012a). Rational design increased the thermostability of lipase from Rhizopus chinensis by incorporating a disulphide bond in the hinge region of the lipase lid domain based on the prediction of ''Disulfide by Design'' algorithm and 3D structural model (Hwang and Kim 2004;Yu et al 2012a).…”

Section: Development Of Thermostable Lipasesmentioning

confidence: 99%

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Recent developments in biocatalysis beyond the laboratory

et al. 2015

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Recent developments in biocatalysis, where implementation beyond the laboratory has been demonstrated, are explored: the use of transglutaminases to modify foods, reduce allergenicity and produce advanced materials, lipases for biodiesel production, and transaminases for biochemical production. The availability and application of enzymes at pilot and larger scale opens up possibilities for further improvements of biocatalyst-based processes and the development of new processes. Enzyme production, stability, activity, re-use, and product retrieval are common challenges for biocatalytic processes. We explore recent advances in biocatalysis within the process chain, such as protein engineering, enzyme expression, and biocatalyst immobilization, in the context of these challenges.

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Section: Development Of Thermostable Lipasesmentioning

confidence: 98%

Section: Development Of Thermostable Lipasesmentioning

confidence: 99%

Recent developments in biocatalysis beyond the laboratory

et al. 2015

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“…This enhancement in low-temperature activity will then be accompanied by an increase in enzyme thermostability if the structural elements (e.g., Asn136 in S41) contribute to enzymatic activity by stabilizing key components that are involved in the catalytic cycle (e.g., the catalytic triad). Recently, lipase variants with an increase in both thermostability and activity have been engineered by rigidifying the catalytic residues (59,60). In this context, stabilizing the geometry of catalytic residues is a valuable strategy to engineer enzymes that couple high thermostability with high activity.…”

Section: Discussionmentioning

confidence: 99%

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

Bin

Dai

Chen

et al. 2015

Appl Environ Microbiol

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bThe incorporation of the structural elements of thermostable enzymes into their less stable counterparts is generally used to improve enzyme thermostability. However, the process of engineering enzymes with both high thermostability and high activity remains an important challenge. Here, we report that the thermostability and activity of a thermophilic subtilase were simultaneously improved by incorporating structural elements of a psychrophilic subtilase. There were 64 variable regions/residues (VRs) in the alignment of the thermophilic WF146 protease, mesophilic sphericase, and psychrophilic S41. The WF146 protease was subjected to systematic mutagenesis, in which each of its VRs was replaced with those from S41 and sphericase. After successive rounds of combination and screening, we constructed the variant PBL5X with eight amino acid residues from S41. The halflife of PBL5X at 85°C (57.1 min) was approximately 9-fold longer than that of the wild-type (WT) WF146 protease (6.3 min). The substitutions also led to an increase in the apparent thermal denaturation midpoint temperature (T m ) of the enzyme by 5.5°C, as determined by differential scanning calorimetry. Compared to the WT, PBL5X exhibited high caseinolytic activity (25 to 95°C) and high values of K m and k cat (25 to 80°C). Our study may provide a rational basis for developing highly stable and active enzymes, which are highly desired in industrial applications. Many microorganisms have successfully colonized extreme temperature environments ranging from Ϫ20°C to 122°C (1, 2). Enzymes from psychrophiles, mesophiles, and (hyper)thermophiles usually perform efficient catalysis at low, moderate, and high temperatures, respectively. Psychrophilic enzymes are characterized as cold active but heat labile, and these characteristics may arise from an increase in either the global or localized flexibility of enzyme structure (3, 4). Compared to their psychrophilic and mesophilic counterparts, (hyper)thermophilic enzymes generally exhibit enhanced conformational rigidity (5-7). However, some (hyper)thermophilic enzymes may combine local flexibility in their active site with high overall rigidity, thus making them more thermostable and cold active than their mesophilic counterparts (5-7).Accumulating evidence suggests that the cumulative effect of minor improvements of local interactions enhances the intrinsic stability of (hyper)thermophilic enzymes (5, 8). Identification of protein stabilization mechanisms is normally based on comparative studies of homologous enzymes that are adapted to different temperatures, on mutational analyses, on directed evolution and on computational methods (6, 9). The results of these studies have provided a rational basis for improving enzyme stability by sitedirected mutagenesis (SDM) (10). Nevertheless, the process of engineering enzymes for higher thermostability and activity remains important and difficult. One reason for this problem is that structural differences between homologous enzymes that are adapted to different tempe...

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“…In case of conjugated AuNPs-NH 2 -lipase, nanoparticles act as a support that either minimize the denaturation process or act as a scaffold to speed up the renaturation of protein. 46 Along with the maintenance of active open conformation as mentioned earlier, immobilization generally increases the rigidity of the protein and thus increases the thermal stability of the immobilized enzyme.…”

Section: Enzyme Kinetics Measurementmentioning

confidence: 99%

Facile fabrication of lipase to amine functionalized gold nanoparticles to enhance stability and activity

2017

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Among various techniques of immobilization, EDC/NHS cross linking is a simple and single step process for covalent coupling between enzymes and nanoparticles. Here we describe immobilization of lipase on amine functionalized gold nanoparticles (AuNPs-NH 2 ) to attain enhanced activity and stability. To achieve a suitable orientation, it is necessary to understand the contribution of different functional groups on the enzyme's surface. Therefore, the crystal structure of lipase was analyzed using a computational method (PyMOL) to find the exposed acidic amino acid residues that can be exploited for conjugation. Confirmation of conjugation (AuNP-NH 2 -lipase) was determined by various techniques such as agarose gel electrophoresis, zeta measurement, FTIR-spectroscopy and TEM. Further, catalytic parameters (V max , K M,app , K cat , and K cat /K M,app ) have been studied to establish activity enhancement upon immobilization. The data also suggested that, AuNP-NH 2 -lipase has desirable improved parameters such as temperature and storage stability. The thermodynamic parameters for the kinetics of deactivation IntroductionEnzymes have wide application in food, detergent, medicine, diagnostics, energy and many other industrial sectors due to their catalytic activity under mild conditions, specicity, costeffectiveness and greener production conditions. 1,2 However industrial application of free enzymes is restricted due to high lability, poor stability and problems in their recovery and reuse. 3Immobilization is the effective way to overcome these limitations and extend the horizon of their applications.4 Various methods of immobilization include physical adsorption, ionic interaction, covalent bonding between support and enzyme or entrapment within the different matrices.5 Hydrophobic interactions, ionic interactions, hydrogen bonds, van der Waals interactions and solvation are the major forces responsible for non-covalent interactions. Except for covalent attachment, the above mentioned techniques mainly rely on weak interactions, therefore enzymes are prone to detachment from the support surface with time. Covalent bonding mediated immobilization helps in improving enzyme activity at the cost of loss of conformational integrity.6 A very simple method of crosslinking biomolecules to nanoparticles relies on EDC/NHS coupling in which carbodiimides mediate formation of amide bond between carboxy and amine groups.7 High solubility in water, ease to remove byproducts, zero length cross linking molecule and single step reaction provides an edge over other methods in EDC/NHS coupling.8 Several enzymes have been conjugated using this strategy, but they lack in their structure based consideration prior to immobilization. Improper orientation aer immobilization ultimately affects activity; therefore, it is highly essential to understand the structure of protein before immobilization. One of the rationale approaches is to exploit specic surface amino acids for conjugating enzymes with the nanosurface so that the catalytic p...

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Enhanced Enzyme Kinetic Stability by Increasing Rigidity within the Active Site

Cited by 226 publications

References 44 publications

Recent developments in biocatalysis beyond the laboratory

Recent developments in biocatalysis beyond the laboratory

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

Facile fabrication of lipase to amine functionalized gold nanoparticles to enhance stability and activity

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