Alkalophilic adaptation of XynB endoxylanase from Aspergillus niger via rational design of pKa of catalytic residues

Xu, Hui; Zhang, Feiwei; Shang, Huiyi; Li, Xinran; Wang, Jing; Qiao, Dan; Cao, Yi

doi:10.1016/j.jbiosc.2012.12.006

Cited by 16 publications

(8 citation statements)

References 24 publications

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“…Previous studies have reported that a high content of arginine increases the activity of xylanases under alkaline conditions 36 , 37 . For example, the arginine content of GH11 Xyn11-1 xylanase from saline-alkali soil is 6.9%, much like the alkali-tolerant XynG1-3 xylanase, and much higher than 3.0% in acidic xylanases 36 .…”

Section: Discussionmentioning

confidence: 95%

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicola grisea xylanase

Oliveira

Garay

Souza

et al. 2022

Sci Rep

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Humicolagrisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichiapastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40–50 °C and pH 5.0–9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.

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Section: Discussionmentioning

confidence: 95%

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicola grisea xylanase

Oliveira

Garay

Souza

et al. 2022

Sci Rep

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“…Cell-free synthetic biology systems often involve non-physiological pH conditions, therefore, reprograming pH adaption of enzymes has always been an important goal. Since pH optimum is mostly governed by the ionization states of the side chains of the catalytic residues, common strategies for changing pH-activity profiles was done by introducing mutations around the active site based on structural analysis [15] , [35] , [51] , [52] and computational predictions [33] , [53] . Although these studies have successfully identified some mutations that shifting enzymatic pH optima, there is risky of losing catalytic activity to introduce mutations near the catalytic residue.…”

Section: Discussionmentioning

confidence: 99%

Sequence homolog-based molecular engineering for shifting the enzymatic pH optimum

Xie

Luo

et al. 2016

Synthetic and Systems Biotechnology

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Cell-free synthetic biology system organizes multiple enzymes (parts) from different sources to implement unnatural catalytic functions. Highly adaption between the catalytic parts is crucial for building up efficient artificial biosynthetic systems. Protein engineering is a powerful technology to tailor various enzymatic properties including catalytic efficiency, substrate specificity, temperature adaptation and even achieve new catalytic functions. However, altering enzymatic pH optimum still remains a challenging task. In this study, we proposed a novel sequence homolog-based protein engineering strategy for shifting the enzymatic pH optimum based on statistical analyses of sequence-function relationship data of enzyme family. By two statistical procedures, artificial neural networks (ANNs) and least absolute shrinkage and selection operator (Lasso), five amino acids in GH11 xylanase family were identified to be related to the evolution of enzymatic pH optimum. Site-directed mutagenesis of a thermophilic xylanase from Caldicellulosiruptor bescii revealed that four out of five mutations could alter the enzymatic pH optima toward acidic condition without compromising the catalytic activity and thermostability. Combination of the positive mutants resulted in the best mutant M31 that decreased its pH optimum for 1.5 units and showed increased catalytic activity at pH < 5.0 compared to the wild-type enzyme. Structure analysis revealed that all the mutations are distant from the active center, which may be difficult to be identified by conventional rational design strategy. Interestingly, the four mutation sites are clustered at a certain region of the enzyme, suggesting a potential “hot zone” for regulating the pH optima of xylanases. This study provides an efficient method of modulating enzymatic pH optima based on statistical sequence analyses, which can facilitate the design and optimization of suitable catalytic parts for the construction of complicated cell-free synthetic biology systems.

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“…Protein engineering is powerful in improving various enzymatic catalytic features such as substrate promiscuity, catalytic activity and selectivity, enzymatic stability and pH optimum [ 19 , 20 , 21 ]. Generally, the most universal and effective method of modifying pH optimum is to change the electrostatic environment of the active site by protein engineering to alter the p K a values of amino acid side chains in the protein [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. So far, a series of enzymes such as serine proteases [ 22 , 23 , 24 ], xylanases [ 25 , 26 , 27 , 28 ], phytases [ 29 , 30 ], DNase I [ 31 ], phospholipase [ 32 ] or an aspartase [ 33 ], have been successfully engineered for optimal pH values through modifying their surface charge or mutating crucial residues adjacent to their active site.…”

Section: Introductionmentioning

confidence: 99%

“…Generally, the most universal and effective method of modifying pH optimum is to change the electrostatic environment of the active site by protein engineering to alter the p K a values of amino acid side chains in the protein [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 ]. So far, a series of enzymes such as serine proteases [ 22 , 23 , 24 ], xylanases [ 25 , 26 , 27 , 28 ], phytases [ 29 , 30 ], DNase I [ 31 ], phospholipase [ 32 ] or an aspartase [ 33 ], have been successfully engineered for optimal pH values through modifying their surface charge or mutating crucial residues adjacent to their active site. Most of these enzymes have a Ser or Glu residue in the active site and follow a general acid-base catalytic mechanism.…”

Section: Introductionmentioning

confidence: 99%

Shifting the pH Optima of (R)-Selective Transaminases by Protein Engineering

Xiang

Höhne

et al. 2022

IJMS

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Amine transaminases (ATAs) are powerful biocatalysts for the stereoselective synthesis of chiral amines. However, wild-type ATAs usually show pH optima at slightly alkaline values and exhibit low catalytic activity under physiological conditions. For efficient asymmetric synthesis ATAs are commonly used in combination with lactate dehydrogenase (LDH, optimal pH: 7.5) and glucose dehydrogenase (GDH, optimal pH: 7.75) to shift the equilibrium towards the synthesis of the target chiral amine and hence their pH optima should fit to each other. Based on a protein structure alignment, variants of (R)-selective transaminases were rationally designed, produced in E. coli, purified and subjected to biochemical characterization. This resulted in the discovery of the variant E49Q of the ATA from Aspergillus fumigatus, for which the pH optimum was successfully shifted from pH 8.5 to 7.5 and this variant furthermore had a two times higher specific activity than the wild-type protein at pH 7.5. A possible mechanism for this shift of the optimal pH is proposed. Asymmetric synthesis of (R)-1-phenylethylamine from acetophenone in combination with LDH and GDH confirmed that the variant E49Q shows superior performance at pH 7.5 compared to the wild-type enzyme.

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Alkalophilic adaptation of XynB endoxylanase from Aspergillus niger via rational design of pKa of catalytic residues

Cited by 16 publications

References 24 publications

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicola grisea xylanase

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicola grisea xylanase

Sequence homolog-based molecular engineering for shifting the enzymatic pH optimum

Shifting the pH Optima of (R)-Selective Transaminases by Protein Engineering

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