Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity

Qin, Yu; Qu, Yin Bo

doi:10.5483/bmbrep.2014.47.5.166

Cited by 12 publications

(5 citation statements)

References 44 publications

(52 reference statements)

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“…To investigate whether the mannosyltransferase deletion also affect the secretion of non-glycosylated protein, we expressed a non-glycosylated protein, a N -glycosylation site mutation rCel5A (rN124D) of Trichoderma reesei endoglucanase Cel5A in the mannosyltransferase deletion strains 29 . As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The Cel3A gene with its native signal sequence and FLAG-tagged sequence was amplified from the plasmid pTH-BGL, the CelA gene with an INU1 signal sequence and myc-tagged sequence was amplified from pTH-CEL (37) and each gene was then ligated into the plasmid PYX242WS 42 under the control of the TPI1 promoter and the PGK1 terminator. The gene of Cel5A from T. reesei with a site mutation (rN124D) was amplified from recombinant plasmid pAJ401-cel5A-N124D 29 and inserted into plasmid PYX242WS. The yeast centromere plasmid pJFE1 containing the URA3 gene as a marker was used to over-express the genes RHO1 and PKC1 .…”

Section: Methodsmentioning

confidence: 99%

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N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae

Tang

Wang

et al. 2016

Sci Rep

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Saccharomyces cerevisiae is a robust host for heterologous protein expression. The efficient expression of cellulases in S. cerevisiae is important for the consolidated bioprocess that directly converts lignocellulose into valuable products. However, heterologous proteins are often N-hyperglycosylated in S. cerevisiae, which may affect protein activity. In this study, the expression of three heterologous proteins, β-glucosidase, endoglucanase and cellobiohydrolase, was found to be N-hyperglycosylated in S. cerevisiae. To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Och1p, Mnn9p and Mnn1p), respectively. Their extracellular activities improved markedly in the OCH1 and MNN9 deletion strains. Interestingly, truncation of the N-hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield. Further analysis showed OCH1 and MNN9 deletion up-regulated genes in the secretory pathway, such as protein folding and vesicular trafficking, but did not induce the unfolded protein response. The cell wall integrity was also affected by OCH1 and MNN9 deletion, which contributed to the release of secretory protein extracellularly. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae

Tang

Wang

et al. 2016

Sci Rep

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“…The removal of N-glycan at the N88 position of β-1,4-endoglucanase CTendo45 increased its catalytic activity toward CMC-Na and β- d -glucan; however, the addition of polysaccharides at the N65 position increased its catalytic activity [ 13 ]. In most cases, the lack of N-glycosylation reduces enzyme activity, which may be caused by changes in the secondary structure of the protein, as detected by circular dichroism analysis [ 14 ]. Conversely, recent studies have reported that the introduction of glycosylation sites increased enzyme activity.…”

Section: Introductionmentioning

confidence: 99%

Improved methanol tolerance of Rhizomucor miehei lipase based on N‑glycosylation within the α-helix region and its application in biodiesel production

Tian

Yang

Wang

et al. 2021

Biotechnol Biofuels

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Background Liquid lipases are widely used to convert oil into biodiesel. Methanol-resistant lipases with high catalytic activity are the first choice for practical production. Rhizomucor miehei lipase (RML) is a single-chain α/β-type protein that is widely used in biodiesel preparation. Improving the catalytic activity and methanol tolerance of RML is necessary to realise the industrial production of biodiesel. Results In this study, a semi-rational design method was used to optimise the catalytic activity and methanol tolerance of ProRML. After N-glycosylation modification of the α-helix of the mature peptide in ProRML, the resulting mutants N218, N93, N115, N260, and N183 increased enzyme activity by 66.81, 13.54, 10.33, 3.69, and 2.39 times than that of WT, respectively. The residual activities of N218 and N260 were 88.78% and 86.08% after incubation in 50% methanol for 2.5 h, respectively. In addition, the biodiesel yield of all mutants was improved when methanol was added once and reacted for 24 h with colza oil as the raw material. N260 and N218 increased the biodiesel yield from 9.49% to 88.75% and 90.46%, respectively. Conclusions These results indicate that optimising N-glycosylation modification in the α-helix structure is an effective strategy for improving the performance of ProRML. This study provides an effective approach to improve the design of the enzyme and the properties of lipase mutants, thereby rendering them suitable for industrial biomass conversion.

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“…Meanwhile the deletion of N -linked glycans at the positions locating at the “bottom” of their catalytic domains resulted in a partial decrease of enzyme activity, because the glycan chains attaching to these residues could help the enzyme orient appropriately to the surface of cellulose microfibril. In most cases, the loss of N -linked glycosylation would reduce the enzyme activity, which was probably caused by the change of secondary structure via circular dichroism spectroscopy analysis [23]. Meanwhile it has been reported that the enzyme activity is increased by introducing glycosylation sites recently [18, 24].…”

Section: Introductionmentioning

confidence: 99%

Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability

Tan

et al. 2019

J Biol Eng

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BackgroundInulinase can hydrolyze polyfructan into high-fructose syrups and fructoligosaccharides, which are widely used in food, the medical industry and the biorefinery of Jerusalem artichoke. In the present study, a recombinant exo-inulinase (rKcINU1), derived from Kluyveromyces cicerisporus CBS4857, was proven as an N-linked glycoprotein, and the removal of N-linked glycan chains led to reduced activity.ResultsFive N-glycosylation sites with variable high mannose-type oligosaccharides (Man3–9GlcNAc2) were confirmed in the rKcINU1. The structural modeling showed that all five glycosylation sites (Asn-362, Asn-370, Asn-399, Asn-467 and Asn-526) were located at the C-terminus β-sandwich domain, which has been proven to be more conducive to the occurrence of glycosylation modification than the N-terminus domain. Single-site N-glycosylation mutants with Asn substituted by Gln were obtained, and the Mut with all five N-glycosylation sites removed was constructed, which resulted in the loss of all enzyme activity. Interestingly, the N362Q led to an 18% increase in the specific activity against inulin, while a significant decrease in thermostability (2.91 °C decrease in Tm) occurred, and other single mutations resulted in the decrease in the specific activity to various extents, among which N467Q demonstrated the lowest enzyme activity.ConclusionThe increased enzyme activity in N362Q, combined with thermostability testing, 3D modeling, kinetics data and secondary structure analysis, implied that the N-linked glycan chains at the Asn-362 position functioned negatively, mainly as a type of steric hindrance toward its adjacent N-glycans to bring rigidity. Meanwhile, the N-glycosylation at the other four sites positively regulated enzyme activity caused by altered substrate affinity by means of fine-tuning the β-sandwich domain configuration. This may have facilitated the capture and transfer of substrates to the enzyme active cavity, in a manner quite similar to that of carbohydrate binding modules (CBMs), i.e. the chains endowed the β-sandwich domain with the functions of CBM. This study discovered a unique C-terminal sequence which is more favorable to glycosylation, thereby casting a novel view for glycoengineering of enzymes from fungi via redesigning the amino acid sequence at the C-terminal domain, so as to optimize the enzymatic properties.

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Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity

Cited by 12 publications

References 44 publications

N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae

N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae

Improved methanol tolerance of Rhizomucor miehei lipase based on N‑glycosylation within the α-helix region and its application in biodiesel production

Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability

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