C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Irfan, Muhammad; Güler, Halil İbrahim; Özer, Ayşegül; Sapmaz, Merve Tuncel; Beldüz, Ali Osman; Hasan, Fariha; Shah, Aamer Ali

doi:10.1016/j.enzmictec.2016.05.012

Cited by 12 publications

(7 citation statements)

References 45 publications

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“…Interestingly, arginine often appears in the proline-rich linker, which has a stronger ability to form hydrogen bonds and salt bridges [ 22 ]. Thus, the enriched proline and arginine residues of C60 might result in the better thermostability of XynA compared to XynA-Tr, mentioned previously [ 23 ]. Glycine is usually involved in flexible linkers, and the “GGGGS” motif is a common flexible unit that appears in linkers.…”

Section: Resultsmentioning

confidence: 78%

“…In addition to the catalytic domain, it has been well documented that N- and C-terminal regions are also crucial factors for enzyme properties [ 13 ]. For example, proline-rich sequence broadens the optimal pH and temperature ranges of xylanase from Geobacillus thermodenitrificans C5 [ 23 ], the chaperone-like ability of artemin is reduced by deletion of its extra C-terminal 39 residues [ 24 ], and the thermostability and optimal temperature of GH11 xylanase increased after C-terminal region deletion [ 25 ].…”

Section: Resultsmentioning

confidence: 99%

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Crucial Residues of C-Terminal Oligopeptide C60 to Improve the Yield of Prebiotic Xylooligosaccharides by Truncated Mutation

Pan

Jin

Wang

et al. 2022

Foods

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Increasing the yields of short xylooligosaccharides by enzymatic production is efficient to improve prebiotic effects. Previously, C-terminal oligopeptide C60 was found to accelerate short xylooligosaccharides. Herein, in order to further understand the molecular mechanism of C60, the sequence analysis firstly showed that C60 displays typical properties of a linker (rich in proline/alanine/glycine/glutamine/arginine, 8.33–20.00%). C60 shared the highest identity with the N-terminal region of esterase (98.33%) and high identity with the linker between xylanase and esterase from Prevotella sp. (56.50%), it is speculated to originate from an early linker between XynA and another domain. Besides, structure simulation showed that C60 enhances the molecular interactions between substrate and active residues to improve catalytic efficiency. Moreover, three truncated variants with different lengths of C-terminal regions were successfully generated in Escherichia coli. The specific activities of variants were 6.44–10.24 fold of that of XynA-Tr, and their optimal temperature and pH were the same as XynA-Tr. Three truncated variants released more xylooligosaccharides, especially xylobiose (46.33, 43.41, and 49.60%), than XynA-Tr (32.43%). These results are helpful to understand the molecular mechanism of C60, and also provide new insight to improve the yields of short xylooligosaccharides by molecular modification at the terminal of xylanases.

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Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

Crucial Residues of C-Terminal Oligopeptide C60 to Improve the Yield of Prebiotic Xylooligosaccharides by Truncated Mutation

Pan

Jin

Wang

et al. 2022

Foods

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“…Bioinformatic analysis predicted the proline-rich, glycoside hydrolase family 5, and X8 domains in the exo-1,3-β-glucanase (Exo1) of P. insidiosum (Figure 2A). The proline-rich domain involves proteinprotein interaction, signal transduction, ligand binding, regulation of biological structures, and enhancing catalytic efficiency (Ball et al, 2005;Irfan et al, 2016;Li et al, 2014;Williamson, 1994). Based on the Carbohydrate-Active Enzymes database (CAZy), the presence of glycoside hydrolase family 5 is a characteristic of some carbohydrate-metabolizing enzymes, including exo-1,3-β-glucanase, cellulase, β-glucosidase, chitosanase, and endo-1,3(4)-β-glucanase (Lombard et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

Expression, purification, and characterization of the recombinant exo-1,3-β-glucanase (Exo1) of the pathogenic oomycete Pythium insidiosum

Rotchanapreeda

Kumsang

Sae‐Chew

et al. 2020

Heliyon

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Pythiosis is a deadly infectious disease of humans and animals living in tropical and subtropical countries. The causative agent is the oomycete Pythium insidiosum. Treatment of pythiosis is challenging. The use of antimicrobial agents usually fails in the treatment of pythiosis. Many patients undergo surgical removal of an infected organ (i.e., eye, arm, and leg). The immunotherapeutic vaccine, prepared from the crude extract of P. insidiosum, shows limited efficacy against pythiosis. The fatal outcome occurs in patients with advanced disease. There are urgent needs for an effective therapeutic modality for pythiosis. Recently, the exo-1,3-β-glucanase (Exo1) has been identified as a conserve immunoreactive protein of P. insidiosum. Exo1 was predicted to reside at the cell membrane and hydrolyze cell wall β-glucan during cell growth. An Exo1 ortholog is absent in the human genome, making it an appealing target for drug or vaccine development. We attempted to clone and express the codonoptimized exo1 gene of P. insidiosum in E. coli. To solve the inclusion body formation, expression and purification of Exo1 were achievable in the denaturing condition using SDS-and urea-based buffers. Exo1 lacked hydrolytic activity due to the absence of proper protein folding and post-translational modifications. ELISA and Western blot analyses demonstrated the immunoreactivity of Exo1 against pythiosis sera. In conclusion, we successfully expressed and purified the immunoreactive Exo1 protein of P. insidiosum. The recombinant Exo1 can be produced at an unlimited amount and could serve as an extra protein to enhance the effectiveness of the current form of the vaccine against pythiosis.

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“…Several of the mutants were reported to show higher rate of hydrolysis of cellulosic substrates but no effect on thermostability was reported . Irfan et al fused proline rich sequence with C‐terminal of xylanase gene from Geobacillus thermodenitrificans C5; the modified enzyme was reported to have a higher temperature optimum (70 °C as opposed to 60 °C of native enzyme) and pH optimum (8.0 as opposed to 6.0 of native enzyme). Further, the enzyme was active across a broader range of temperature and pH …”

Section: Stabilization Of Cellulolytic Enzymesmentioning

confidence: 99%

Stable Cellulolytic Enzymes and Their Application in Hydrolysis of Lignocellulosic Biomass

2018

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The effective utilization of lignocellulosic biomass for production of valueadded products in bio-refineries mainly depends on their hydrolysis into constituent soluble sugars. Despite the commercial availability of lignocellulolytic enzymes, there are certain challenges in using them at an industrial scale which arise from their inherent characteristics. Hence, development of industrially potent and stable cellulolytic enzymes is of great importance. While a lot of emphasis has been given to produce the enzymes with high titre and productivity, the properties of these enzymes, which affect their performance in hydrolyzing lignocellulosic residues, have largely been ignored. The various properties that affect the hydrolysis are thermal stability, catalytic efficiency, non-specific adsorption, end-product inhibition resistance, and shear inactivation. This review discusses several approaches such as finding new enzymes from extremophiles, recombinant production of cellulolytic enzymes in industrial strains, directed evolution and mutagenesis, and formation of the enzyme aggregates that are being investigated to develop stable cellulase enzymes. The authors also suggest that for a meaningful information on the modified cellulolytic enzymes and their application in cellulose hydrolysis, they should be evaluated under practical process condition.

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C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Cited by 12 publications

References 45 publications

Crucial Residues of C-Terminal Oligopeptide C60 to Improve the Yield of Prebiotic Xylooligosaccharides by Truncated Mutation

Crucial Residues of C-Terminal Oligopeptide C60 to Improve the Yield of Prebiotic Xylooligosaccharides by Truncated Mutation

Expression, purification, and characterization of the recombinant exo-1,3-β-glucanase (Exo1) of the pathogenic oomycete Pythium insidiosum

Stable Cellulolytic Enzymes and Their Application in Hydrolysis of Lignocellulosic Biomass

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