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

Abstract

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.

Improvement of thermostability and catalytic efficiency of xylanase from Myceliophthora thermophilar by N-terminal and C-terminal truncation

“…Compared to Mtxylan2, 28N, 28C, and 28NC showed 2.4-fold, 4.1-fold, and 9.3-fold higher specific activities for beechwood xylan, 7.1-fold, 26.7-fold, and 10.4-fold higher specific activities for birchwood xylan, and 4.5-fold, 13.4-fold, and 7.9-fold higher specific activities for oat spelling, respectively ( Table 1 ). These results suggested that truncation of the N-terminus, C-terminus, and both the N-terminus and C-terminus of Mtxylan2 enhanced its specific enzyme activity, which was in agreement with the findings of Kang and Ishikawa (2007) , Li et al (2018) , Liu et al (2011) and Pan et al (2022) . The kinetic parameters of the enzymes with beechwood xylan are listed in Table 2 .…”

“…Among them, GH11 xylanase has a conserved β-jelly roll structure with a right-handed partially closed conformation ( Paes et al, 2012 ). GH11 xylanase has a small molecular weight, fast catalytic efficiency, and excellent substrate selectivity ( Li C. et al, 2018 ; Teng et al, 2019 ; Wang et al, 2021 ). These properties make GH11 xylanase promising for a wide range of applications in industries such as animal feeding, food baking, and pulp bleaching.…”

Simultaneously Enhanced Thermostability and Catalytic Activity of Xylanase from Streptomyces rameus L2001 by Rigidifying Flexible Regions in Loop Regions of the N-Terminus

“…The flexibility of the GH11 xylanase N-terminus is closely associated with its thermal stability . Rigidification of the flexible regions of xylanase, particularly the N-terminus, is an effective strategy to improve thermostability. − In the present study, MD simulations and multiple sequence alignments were used to localize five loop regions with greater flexibility in the N-terminus of S. rameus L2001 xylanase XynA, comprising loop1- 5 TTN 7 , loop2- 11 TDNGFY 16 , loop3- 23 AQ 24 , loop4- 32 GS 33 and S 36 , and loop5- 42 RNT 44 (Figure ).…”

Purification and characteration of xylanase from Aspegillus oryzae VTCC F187

“…Xylanase or endo-1,4-beta-xylanase (EC 3.2.1.8) is a group of enzyme which plays a crucial role in the xylan hydrolysis, the most common natural hemicellulose, into short oligosacharides and xylose (He et al, 2015), (Li et al, 2018).…”

“…In particular, the species A. oryzae is a potential target of scientific researches around the world (Chipeta et al, 2005), (Golubev et al, 1993), (He et al, 2015), (Kimura et al, 2000). Besides the advantages of production of xylanase with high activity, xylanase produced from A. oryzae has some outstanding features such as specific structural regions which we can use mutation techniques, recombinant proteins, the catalyst… to significantly increases catalytic efficiency and heat resistance (Gao et al, 2012), (Li et al, 2018), Li et al, 2017).…”