Rational design of a Bacillus circulans xylanase by introducing charged residue to shift the pH optimum

Pokhrel, Subarna; Joo, Jeong Chan; Kim, Yong Hwan; Yoo, Young Je

doi:10.1016/j.procbio.2012.10.011

Cited by 24 publications

(7 citation statements)

References 42 publications

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“…YM55-1 based on surface charge rational design [ 35 ]. The shift in pH optimum was probably due to charge repulsion, as explained by Pokhrel et al [ 33 ], the charge repulsion may be direct, between the Asp/Glu inserted and the catalytic sites, or it may be indirect.…”

Section: Discussionmentioning

confidence: 90%

“…Kim et al [ 32 ] achieved a shift in the pH optima of Aspergillus niger PhyA Phytase by substituting amino acids in the substrate-binding site with different charges and polarities. Pokhrel et al [ 33 ] successfully shifted the pH optimum of a B. circulans xylanase by introducing charged residue. In a previous study, we also successfully modified the optimal pH of an aspartase from Bacillus sp.…”

Section: Discussionmentioning

confidence: 99%

“…Kim et al [ 32 ] achieved to shift of the pH optima of Aspergillus niger PhyA Phytase to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Pokhrel et al [ 33 ] successfully shifted the pH optimum of a Bacillus circulans xylanase by introducing charged residue. Ma et al [ 34 ] constructed an elaborate GH11 xylanase database with pH annotation, and developed a data driven protein engineering strategy to redesign the pH adaptation of xylanase.…”

Section: Introductionmentioning

confidence: 99%

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N20D/N116E Combined Mutant Downward Shifted the pH Optimum of Bacillus subtilis NADH Oxidase

Yang

Pan

et al. 2023

Biology

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Cofactor regeneration is indispensable to avoid the addition of large quantities of cofactor NADH or NAD+ in oxidation-reduction reactions. Water-forming NADH oxidase (Nox) has attracted substantive attention as it can oxidize cytosolic NADH to NAD+ without concomitant accumulation of by-products. However, its applications have some limitations in some oxidation-reduction processes when its optimum pH is different from its coupled enzymes. In this study, to modify the optimum pH of BsNox, fifteen relevant candidates of site-directed mutations were selected based on surface charge rational design. As predicted, the substitution of this asparagine residue with an aspartic acid residue (N22D) or with a glutamic acid residue (N116E) shifts its pH optimum from 9.0 to 7.0. Subsequently, N20D/N116E combined mutant could not only downshift the pH optimum of BsNox but also significantly increase its specific activity, which was about 2.9-fold at pH 7.0, 2.2-fold at pH 8.0 and 1.2-fold at pH 9.0 that of the wild-type. The double mutant N20D/N116E displays a higher activity within a wide range of pH from 6 to 9, which is wider than the wide type. The usability of the BsNox and its variations for NAD+ regeneration in a neutral environment was demonstrated by coupling with a glutamate dehydrogenase for α-ketoglutaric acid (α-KG) production from L-glutamic acid (L-Glu) at pH 7.0. Employing the variation N20D/N116E as an NAD+ regeneration coenzyme could shorten the process duration; 90% of L-Glu were transformed into α-KG within 40 min vs. 70 min with the wild-type BsNox for NAD+ regeneration. The results obtained in this work suggest the promising properties of the BsNox variation N20D/N116E are competent in NAD+ regeneration applications under a neutral environment.

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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N20D/N116E Combined Mutant Downward Shifted the pH Optimum of Bacillus subtilis NADH Oxidase

Yang

Pan

et al. 2023

Biology

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“…In many of these applications extremes of pH are encountered yet, interestingly, while much is already known of this important industrial enzyme, little is known of its relationship to pH and of the pH dependence of its activity and stability. In fact, GH8 also contains many other industrially important enzymes such as cellulases, licheninases and chitosanases yet it appears that, in contrast to family 10 and 11 enzymes where a large number of studies have been carried out [5,9,10,[22][23][24][25][26], pH adaptation in GH8 enzymes has not been studied. Furthermore, in contrast to adaptation of enzymes to temperature, and in particular high temperatures, much less is currently known of adaptation to pH, with variable and sometimes conflicting observations on adaptation strategies being reported [5,9,10,[22][23][24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Deciphering the factors defining the pH-dependence of a commercial glycoside hydrolase family 8 enzyme

Barroca

Santos

Johansson

et al. 2017

Enzyme and Microbial Technology

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A prerequisite to the use of any enzyme in any industrial process is an understanding of its activity and stability under process conditions. Glycoside hydrolase family 8 enzymes include many important biotechnological biocatalysts yet little is known of the performance of these with respect to pH. A better understanding of this parameter and its relationship to structure and function in these enzymes will allow for an improved use of these in industry as well as an enhanced ability in their engineering and optimisation for a particular application. An in-depth analysis of the pH induced changes in activity, irreversible inactivation, conformation, stability and solubility of a commercial glycoside hydrolase family 8 xylanase was carried out with the aim of identifying the factors determining the pH dependence of this enzyme. Our study showed that different phenomena play different roles at the various pHs examined. Both reversible and irreversible processes are involved at acidic pHs, with the irreversible processes dominating and being due to protein aggregation and precipitation. At basic pHs, loss of activity is principally due to reversible processes, possibly deprotonation of an essential catalytic residue, but at higher pHs, near the pI of the protein, precipitation again dominates while structure unfolding was discerned at the higher pHs investigated. Such insights demonstrate the complexity of factors involved in the pH dependence of proteins and advances our knowledge on design principles and concepts for engineering proteins. Our results highlight the major role of protein precipitation in activity and stability losses at both low and high pHs but it is proposed that different strategies be used in tailoring the enzyme to overcome this in each case. Indeed the detailed understanding obtained here will allow for a more focused, informed and hence successful tailoring of glycoside hydrolase family 8 proteins for a specific pH and process application.

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“…Each enzyme includes a suitable or optimal pH stability range [30,32]. Apart from temperature and pH, ionic strength can also affect the enzymatic reaction [33]. For more accurate and consistent results, each of these physical and chemical parameters must be considered and optimized accordingly [34].…”

Section: Introductionmentioning

confidence: 99%

Analytical modelling of monolayer graphene-based ion-sensitive FET to pH changes

et al. 2013

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Graphene has attracted great interest because of unique properties such as high sensitivity, high mobility, and biocompatibility. It is also known as a superior candidate for pH sensing. Graphene-based ion-sensitive field-effect transistor (ISFET) is currently getting much attention as a novel material with organic nature and ionic liquid gate that is intrinsically sensitive to pH changes. pH is an important factor in enzyme stabilities which can affect the enzymatic reaction and broaden the number of enzyme applications. More accurate and consistent results of enzymes must be optimized to realize their full potential as catalysts accordingly. In this paper, a monolayer graphene-based ISFET pH sensor is studied by simulating its electrical measurement of buffer solutions for different pH values. Electrical detection model of each pH value is suggested by conductance modelling of monolayer graphene. Hydrogen ion (H+) concentration as a function of carrier concentration is proposed, and the control parameter (Ƥ) is defined based on the electro-active ions absorbed by the surface of the graphene with different pH values. Finally, the proposed new analytical model is compared with experimental data and shows good overall agreement.

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Rational design of a Bacillus circulans xylanase by introducing charged residue to shift the pH optimum

Cited by 24 publications

References 42 publications

N20D/N116E Combined Mutant Downward Shifted the pH Optimum of Bacillus subtilis NADH Oxidase

N20D/N116E Combined Mutant Downward Shifted the pH Optimum of Bacillus subtilis NADH Oxidase

Deciphering the factors defining the pH-dependence of a commercial glycoside hydrolase family 8 enzyme

Analytical modelling of monolayer graphene-based ion-sensitive FET to pH changes

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