Seven N-terminal Residues of a Thermophilic Xylanase Are Sufficient to Confer Hyperthermostability on Its Mesophilic Counterpart

Dong Z (2014) Seven N-terminal Residues of a Thermophilic Xylanase Are Sufficient to Confer Hyperthermostability on Its Mesophilic Counterpart. PLoS ONE 9(1): e87632. doi:10.1371/journal.pone.0087632 Seven N-terminal Residues of a Thermophilic Xylanase Are Sufficient to Confer Hyperthermostability on Its Mesophilic Counterpart Shan Zhang 0 Yongzhi He 0 Haiying Yu 0 Zhiyang Dong 0 Danilo Roccatano, Jacobs University Bremen, Germany 0 State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , Beijing , P. R. China Xylanases, and especially thermostable xylanases, are increasingly of interest for the deconstruction of lignocellulosic biomass. In this paper, the termini of a pair of xylanases, mesophilic SoxB and thermophilic TfxA, were studied. Two regions in the N-terminus of TfxA were discovered to be potentially important for the thermostability. By focusing on Region 4, it was demonstrated that only two mutations, N32G and S33P cooperated to improve the thermostability of mesophilic SoxB. By introducing two potential regions into SoxB in combination, the most thermostable mutant, M2-N32G-S33P, was obtained. The M2-N32G-S33P had a melting temperature (Tm) that was 25.6uC higher than the Tm of SoxB. Moreover, M2N32G-S33P was even three-fold more stable than TfxA and had a Tm value that was 9uC higher than the Tm of TfxA. Thus, for the first time, the mesophilic SoxB ''pupil'' outperformed its thermophilic TfxA ''master'' and acquired hyperthermostability simply by introducing seven thermostabilizing residues from the extreme N-terminus of TfxA. This work suggested that mutations in the extreme N-terminus were sufficient for the mesophilic xylanase SoxB to acquire hyperthermostability. - Funding: This work was supported by the National Basic Research Program of China (2011CB707402), the National Natural Science Foundation of China (30970073) and the National High Technology Research and Development Program of China (2012AA092103). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Xylanases are increasingly recognized to be important for the deconstruction of lignocellulosic biomass [1,2]. Xylanases not only catalyze the hydrolysis of hemicelluloses but also render cellulose more accessible to enzymatic hydrolysis [36]. Thermostable xylanases possess obvious advantages over their counterparts because of the reduced associated costs and their ability to be manipulated for the deconstruction of lignocellulosic biomass [7,8]. It is not surprising, therefore, that widespread research endeavors have focused on developing thermostable xylanases by rational design or directed evolution. The rational design strategy of replacing the N-terminus of mesophilic xylanases with the first 31 residues of the thermophilic xylanase TfxA from Thermomonospora fusca has successfully produced several thermostable hybrid xylanases, such as Stx15 [9], StxAB [10], Btx [11] and ATx [12]. These hybrid xylanases exhibited higher thermostabilities than their corresponding mesophilic parents. However, compared with TfxA, all of the hybrid xylanases displayed significantly inferior thermostabilities. Recently, another hybrid xylanase AEx11A was designed by substituting N-terminus of AoXyn11A with the corresponding region of a hyperthermostable xylanase, EvXyn11TS [13]. Likewise, the thermostability of the hybrid xylanase was higher than that of mesophilic parent AoXyn11A, but still significantly lower than that of thermophilic parent EvXyn11TS [13,14]. That is to say, all these hybrid xylanases containing the N-terminus of thermophilic xylanases displayed improved thermostabilities, but could not outperform their thermophilic parents to acquire hyperthermostabilities. Thus, a both industrially and biologically intriguing and important question arose: Are mutations in the extreme N-terminus sufficient for conferring hyperthermostability on mesophilic xylanases? Directed evolution has been successfully applied to confer thermostability (or even hyperthermostability) on mesophilic xylanases. Using this technique, Ruller et al. [15] improved the melting temperature (Tm) of xylanase A from Bacillus subtilis from 59uC to 76.5uC. This mutant xylanase contained four mutations (Q7H, G13R, S22P and S179C), of which three were in the extreme N-terminus. Palackal et al. [16] also employed directed evolution and successfully obtained a thermostable variant (9X) with a high Tm (95.6uC). The 9X enzyme harbored nine mutations, of which four (D8F, Q11H, N12L and G17I) were in the extreme Nterminus. Likewise, Miyazaki et al. [17] created one thermostable variant, Xylst, by directed evolution. This mutant contained three amino acid substitutions, of which two were in the extreme Nterminus. In addition, Dumon et al. [14] successfully engineered a hype (...truncated)