Modified pPIC9K vector-mediated expression of a family 11 xylanase gene, Aoxyn11A, from Aspergillus oryzae in Pichia pastoris

Li, Jianfang; Gao, Siyu; Liu, Xiaotong; Yan-Yan, Gong; Chen, Zhongfa; Wei, Xi-Huan; Zhang, Huimin; Wu, Minchen

doi:10.1007/s13213-012-0568-7

Cited by 5 publications

(10 citation statements)

References 24 publications

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“…Its activity was not significantly affected by Ca 2+ , Mg 2+ , Fe 2+ , Fe 3+ , Al 3+ , Ba 2+ , Zn 2+ , Sn 2+ , Co 2+ , Li + , Cu 2+ and EDTA, but strongly inhibited by Mn 2+ and Ag + with only 38.1% and 29.5% of its original activity, respectively. The experimental results showed that the re‐NhXyn11 57 had similar pH and metal ion characteristics to those of re‐AoXyn11 …”

Section: Resultsmentioning

confidence: 93%

“…The pPIC9K N was originally constructed in our laboratory from a parent pPIC9K vector (Invitrogen, San Diego, CA, USA). The recombinant proteins or enzymes expressed in P. pastoris GS115 mediated by the pPIC9K N could retain the N ‐termini of native proteins or enzymes expressed and modified in their own cells . E. coli JM109 and DH5α were cultured in a Luria‐Bertani medium containing 10 g L −1 tryptone, 5 g L −1 yeast extract and 10 g L −1 NaCl, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

“…12 A mesophilic xylanase-encoding gene from Aspergillus oryzae CICC40186, Aoxyn11, was cloned in our previous work, encoding a 188-amino acid mature peptide, namely AoXyn11. 13 The amino acid sequence of AoXyn11 shared 66.5% identity with that of EvXyn11 TS , but the identity of N-terminal 62 residues (numbered by EvXyn11 TS ) between them was only 43.5%, which, to some extent, implied the significance of the N-terminus to thermostability. An NhXyn11 57 with high thermostability, through substituting the most appropriate N-terminal segment of AoXyn11 with the corresponding one of EvXyn11 TS , was predicted by MD simulation, and an Nhxyn11 57 was constructed as designed theoretically in this work.…”

Section: Introductionmentioning

confidence: 97%

“…The Ev Xyn11 TS was the most thermotolerant family 11 xylanase, retaining about 100% and 85% of its original activity after incubation at 80 and 85 °C, respectively, for 15 min . A mesophilic xylanase‐encoding gene from Aspergillus oryzae CICC40186, Aoxyn11 , was cloned in our previous work, encoding a 188‐amino acid mature peptide, namely AoXyn11 . The amino acid sequence of AoXyn11 shared 66.5% identity with that of Ev Xyn11 TS , but the identity of N ‐terminal 62 residues (numbered by Ev Xyn11 TS ) between them was only 43.5%, which, to some extent, implied the significance of the N ‐terminus to thermostability.…”

Section: Introductionmentioning

confidence: 99%

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Enhanced thermostability of a mesophilic xylanase byN-terminal replacement designed by molecular dynamics simulation

Yin

Wang

et al. 2013

J. Sci. Food Agric.

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Section: Resultsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Enhanced thermostability of a mesophilic xylanase byN-terminal replacement designed by molecular dynamics simulation

Yin

Wang

et al. 2013

J. Sci. Food Agric.

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“…The vectors pET-28a- Aoxyn11A and pPIC9K- Aoxyn11A , and transformants E. coli / Aoxyn11A and P. pastoris / Aoxyn11A were constructed in our lab (Li et al 2013). The genes, Aoxyn11A and its variants, were expressed in P. pastoris GS115.…”

Section: Methodsmentioning

confidence: 99%

Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design

et al. 2017

AMB Expr

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To improve the temperature characteristics and catalytic efficiency of a glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae (AoXyn11A), its variants were predicted based on in silico design. Firstly, Gly21 with the maximum B-factor value, which was confirmed by molecular dynamics (MD) simulation on the three-dimensional structure of AoXyn11A, was subjected to site-saturation mutagenesis. Thus, one variant with the highest thermostability, AoXyn11AG21I, was selected from the mutagenesis library, E. coli/Aoxyn11A G21X (X: any one of 20 amino acids). Secondly, based on the primary structure multiple alignment of AoXyn11A with seven thermophilic GHF11 xylanases, AoXyn11AY13F or AoXyn11AG21I–Y13F, was designed by replacing Tyr13 in AoXyn11A or AoXyn11AG21I with Phe. Finally, three variant-encoding genes, Aoxyn11A G21I, Aoxyn11A Y13F and Aoxyn11A G21I–Y13F, were constructed by two-stage whole-plasmid PCR method, and expressed in Pichia pastoris GS115, respectively. The temperature optimum (T opt) of recombinant (re) AoXyn11AG21I–Y13F was 60 °C, being 5 °C higher than that of reAoXyn11AG21I or reAoXyn11AY13F, and 10 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t 1/2) of reAoXyn11AG21I–Y13F at 50 °C was 240 min, being 40-, 3.4- and 2.5-fold longer than those of reAoXyn11A, reAoXyn11AG21I and reAoXyn11AY13F. The melting temperature (T m) values of reAoXyn11A, reAoXyn11AG21I, reAoXyn11AY13F and reAoXyn11AG21I–Y13F were 52.3, 56.5, 58.6 and 61.3 °C, respectively. These findings indicated that the iterative mutagenesis of both Gly21Ile and Tyr13Phe improved the temperature characteristics of AoXyn11A in a synergistic mode. Besides those, the catalytic efficiency (k cat/K m) of reAoXyn11AG21I–Y13F was 473.1 mL mg−1 s−1, which was 1.65-fold higher than that of reAoXyn11A.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0399-9) contains supplementary material, which is available to authorized users.

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Substituting Both the N-Terminal and “Cord” Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics

Wang

et al. 2018

Appl Biochem Biotechnol

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To improve the temperature characteristics of AoXyn11A, a mesophilic glycoside hydrolase family (GHF) 11 xylanase from Aspergillus oryzae CICC40186, its N-terminal and "cord" regions were selected to be substituted by means of the computer-aided analysis and calculation. In brief, one mutant, named ATX11A, possessing the lowest root-mean-square deviation (RMSD) value was designed based on the molecular dynamics (MD) simulation by substituting the N-terminal 41 amino acids of AoXyn11A with the corresponding 42 ones of pXYL11, a thermophilic GHF11 xylanase from Thermobifida fusca. On the basis of the primary structure alignment of pXYL11 with ATX11A (or AoXyn11A), another mutant, named ATX11A, was designed by substituting the cord region (GTYNPGSGG) of ATX11A with the corresponding one (GTYRPTG) of pXYL11. Both mutant-encoding genes, ATx11A and ATx11A, were constructed as designed theoretically by a megaprimer PCR technique and were expressed in Pichia pastoris GS115. The specific activities of recombinant (re) AoXyn11A, ATX11A, and ATX11A were 2916.7, 2667.6, and 2457.0 U/mg, respectively. The analysis of temperature characteristics displayed that the temperature optimum (T) of reATX11A or reATX11A was 65 °C, which was 15 °C higher than that of reAoXyn11A. The thermal inactivation half-life (t) values of reATX11A and reATX11A at 60 °C were 55 and 83 min, respectively, whereas that of reAoXyn11A was only 18 min at 50 °C. The melting temperature (T) values of reAoXyn11A, reATX11A, and reATX11A were 54.2, 66.7, and 71.9 °C, respectively. In conclusion, the above findings indicated that the substitution of both the N-terminal and cord regions of a mesophilic AoXyn11A greatly contributed to its improved temperature characteristics.

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Modified pPIC9K vector-mediated expression of a family 11 xylanase gene, Aoxyn11A, from Aspergillus oryzae in Pichia pastoris

Cited by 5 publications

References 24 publications

Enhanced thermostability of a mesophilic xylanase byN-terminal replacement designed by molecular dynamics simulation

Enhanced thermostability of a mesophilic xylanase byN-terminal replacement designed by molecular dynamics simulation

Improving the temperature characteristics and catalytic efficiency of a mesophilic xylanase from Aspergillus oryzae, AoXyn11A, by iterative mutagenesis based on in silico design

Substituting Both the N-Terminal and “Cord” Regions of a Xylanase from Aspergillus oryzae to Improve Its Temperature Characteristics

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