Protein‐engineering of chitosanase from <i>Bacillus</i> sp. MN to alter its substrate specificity

Regel, Eva K.; Weikert, Tobias; Niehues, Anna; Moerschbacher, Bruno M.; Singh, Ratna

doi:10.1002/bit.26533

Cited by 20 publications

(17 citation statements)

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“…To obtain muteins of CSN that display reduced activity on D 4 but retain high activity on longer substrates, we generated CSN-E59A, CSN-W118A, and CSN-Y270A. In the active center of CSN, D 4 is forced into a “V-shape” conformation and is symmetrically positioned between the subsites (− 2) and (+ 2) before hydrolysis [35, 36]. By disrupting the substrate binding at either subsite (− 2) or subsite (+ 2), the formation of the “V-shape” and a subsequent cleavage of the substrate should no longer be possible.…”

Section: Resultsmentioning

confidence: 99%

“…While we had to use a higher concentration of CSN-W118A compared to CSN-E59A, the former was clearly more suitable for efficient production of D 4 . Using a different approach for engineering, Regel et al recently published the generation of a mutein of CSN named CSN-VRE which exhibited a strongly altered subsite specificity [35]. This enzyme could be utilized to produce oligomer mixtures containing D 4 as well, but a production of pure D 4 at a larger scale was not demonstrated.…”

Section: Discussionmentioning

confidence: 99%

“…This enzyme can be classified as GH 8, former class III and current class A [22]. CSN contains seven subsites in its active center [34], and functional roles for a variety of residues have been identified [35]. Substrate binding first involves the substrate forming a “V-shape” conformation before hydrolysis, a process that is aided by the residues E59, W118, and Y270, among others [34, 36].…”

Section: Introductionmentioning

confidence: 99%

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Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers

et al. 2019

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BackgroundPartially acetylated chito-oligosaccharides (paCOS) have a variety of potential applications in different fields, but to harness their benefits, pure paCOS or well-defined paCOS mixtures are essential. For example, if one could produce fully acetylated (A4) and fully deacetylated (D4) tetramers in abundance, all possible variants of tetrameric paCOS could be generated reliably from them. A promising approach for generating defined paCOS is by enzymatic depolymerization of chitosan polymers using chitosanases, since these enzymes’ subsite specificities directly influence the composition of the paCOS produced; however, enzymatic production of e.g. D4 is challenging because the substrate is generally hydrolyzed further by most chitosanases. To overcome this, chitosanases could potentially be engineered so that upon hydrolyzing chitosan, they are unable to hydrolyze certain substrates, leaving well-defined oligomers intact in the hydrolysate.ResultsFor this purpose, we performed rational protein engineering on the extensively studied GH 8 chitosanase CSN from Bacillus sp. MN. By specifically targeting residues with a predicted function in substrate binding, we created new muteins incapable of efficiently hydrolyzing the fully deacetylated tetramer D4, and we were able to demonstrate efficient large-scale production of D4 with an altered version of CSN. Furthermore, we were able to uncover differences in the substrate positioning and subsite specificities of the muteins, which result in altered paCOS mixtures produced from partially acetylated chitosan polymers, with possibly altered bioactivities.ConclusionThe value of protein engineering as a tool for the more efficient production of pure oligomers and potentially bioactive paCOS mixtures was demonstrated by the results and the suitability of specific muteins for the large-scale production of strictly defined, pure paCOS in a batch process was shown using the example of D4.Electronic supplementary materialThe online version of this article (10.1186/s13036-019-0152-9) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers

et al. 2019

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“…Altered substrate specificity was also obtained by site directed mutagenesis of residues involved in substrate binding of the Bacillus sp. MN chitosanase (E309R and N319E), leading to muteins able to bind N-acetyl- d -glucosamine [ 98 ] and unable to hydrolyze the fully deacetylated chito-oligosaccharide tetramer [ 99 ]. Moreover, a series of seven mutations (V119D, S262K, N291D, D293T, G319S, D358G, and D368H) induced in alpha-gliadin peptidase by a computational protein design approach that enhanced the number of hydrogen bonds within substrate binding pockets led to an increased specificity for the immunogenic fragments of gluten peptides by 877-fold, with putative application in biosensing [ 100 ].…”

Section: The Innovative Use Of Enzyme Kinetic Particularities To Improve the Selectivitymentioning

confidence: 99%

Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives

Bucur

Purcarea²,

Andreescu

et al. 2021

Sensors

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Enzymatic biosensors enjoy commercial success and are the subject of continued research efforts to widen their range of practical application. For these biosensors to reach their full potential, their selectivity challenges need to be addressed by comprehensive, solid approaches. This review discusses the status of enzymatic biosensors in achieving accurate and selective measurements via direct biocatalytic and inhibition-based detection, with a focus on electrochemical enzyme biosensors. Examples of practical solutions for tackling the activity and selectivity problems and preventing interferences from co-existing electroactive compounds in the samples are provided such as the use of permselective membranes, sentinel sensors and coupled multi-enzyme systems. The effect of activators, inhibitors or enzymatic substrates are also addressed by coupled enzymatic reactions and multi-sensor arrays combined with data interpretation via chemometrics. In addition to these more traditional approaches, the review discusses some ingenious recent approaches, detailing also on possible solutions involving the use of nanomaterials to ensuring the biosensors’ selectivity. Overall, the examples presented illustrate the various tools available when developing enzyme biosensors for new applications and stress the necessity to more comprehensively investigate their selectivity and validate the biosensors versus standard analytical methods.

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“…Hence, the size of chitinase products increases, while that of chitosanase products decreases with decreasing F A of the substrate. While the DP range of the products is more difficult to predict compared to acid hydrolysis, unless the subsite specificity of the enzyme used is precisely known, enzymatic hydrolysis can more easily yield a broader range of products, as enzymes with different levels of subsite specificities are available [ 17 , 32 , 54 , 55 , 56 ]. Clearly, the more specific the enzyme, the larger its products.…”

Section: Preparation Of the Substrates (An And Dn)mentioning

confidence: 99%

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

Bonin

Sreekumar

Cord‐Landwehr

et al. 2020

IJMS

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During the past decade, detailed studies using well-defined ‘second generation’ chitosans have amply proved that both their material properties and their biological activities are dependent on their molecular structure, in particular on their degree of polymerisation (DP) and their fraction of acetylation (FA). Recent evidence suggests that the pattern of acetylation (PA), i.e., the sequence of acetylated and non-acetylated residues along the linear polymer, is equally important, but chitosan polymers with defined, non-random PA are not yet available. One way in which the PA will influence the bioactivities of chitosan polymers is their enzymatic degradation by sequence-dependent chitosan hydrolases present in the target tissues. The PA of the polymer substrates in conjunction with the subsite preferences of the hydrolases determine the type of oligomeric products and the kinetics of their production and further degradation. Thus, the bioactivities of chitosan polymers will at least in part be carried by the chitosan oligomers produced from them, possibly through their interaction with pattern recognition receptors in target cells. In contrast to polymers, partially acetylated chitosan oligosaccharides (paCOS) can be fully characterised concerning their DP, FA, and PA, and chitin deacetylases (CDAs) with different and known regio-selectivities are currently emerging as efficient tools to produce fully defined paCOS in quantities sufficient to probe their bioactivities. In this review, we describe the current state of the art on how CDAs can be used in forward and reverse mode to produce all of the possible paCOS dimers, trimers, and tetramers, most of the pentamers and many of the hexamers. In addition, we describe the biotechnological production of the required fully acetylated and fully deacetylated oligomer substrates, as well as the purification and characterisation of the paCOS products.

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Protein‐engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity

Cited by 20 publications

References 47 publications

Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers

Rational protein design of Bacillus sp. MN chitosanase for altered substrate binding and production of specific chitosan oligomers

Addressing the Selectivity of Enzyme Biosensors: Solutions and Perspectives

Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

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