Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii

May Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii Xin Yin 0 1 2 Die Hu 0 1 2 Jian-Fang Li 0 1 2 Yao He 0 1 2 Tian-Di Zhu 0 1 2 Min-Chen Wu 0 1 2 0 1 Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University , Wuxi , China , 2 State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University , Wuxi , China , 3 Wuxi Medical School, Jiangnan University , Wuxi , China 1 Data Availability Statement: All relevant data are within the paper 2 Academic Editor: Marie-Joelle Virolle, University Paris South , FRANCE The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6C compared to that of re-AuFaeA. The thermal inactivation half-lives of reAuFaeAA126C-N152C at 55 and 60C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10C decrease in thermal stability of recombinant AuEaeA variants were also observed. - Funding: This work was financially supported by the Fundamental Research Fund for the Central Universities of China (No. JUDCF13011, JUSRP51412B) (http://yjsb.jiangnan.edu.cn/, http:// www.moe.gov.cn/) and the Postgraduate Innovation Training Project of Jiangsu (No. CXZZ13_0757) (http://www.ec.js.edu.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. A disulfide bridge is formed by the oxidation of two thiols each from two cysteines, thus linking the two cysteines and their respective main peptide chains, which can restrict the motion of the unfolded, random coil of protein or stabilize the folded state of protein [1,2]. One disulfide bridge can contribute 2.35.2 kcal/mol to the thermodynamic stability of proteins [1,3]. The contribution of disulfide bridges to the stability of proteins can be measured, to a certain extent, by the change in protein thermostability upon introduction or elimination of one or more disulfide bridges. Considerable evidence has demonstrated the thermostability effects of engineered disulfide bridges in protein. For example, upon introduction of a disulfide bridge Competing Interests: The authors have declared that no competing interests exist. A162C-K308C in the lipase B (CalB) from Candida antarctica, the half-life of the enzyme was increased by 4.5-fold at 50C [4]. The optimum temperature of xylanase (TLX) from Thermomyces lanuginosus increased approximately 10C upon introduction of a disulfide bridge Q1C-Q24C [5]. Conversely, the absence of a disulfide bridge contributed to the increased conformational flexibility and thermolability of the lipase (PFL) from Pseudomonas fragi [6,7]. Feruloyl esterases (FAEs, EC 3.1.1.73) cleave the ester bond between polysaccharides and hydroxycinnamic acid in hemicellulose networks for the subsequent hydrolysis of hemicellulose by hemicellulose-digesting enzymes. Their potential for degradation and reutilization of the natural biomass is significant [8]. Based on substrate preference and primary structure homology, FAEs have been classified into four types: type A, B, C and D [9]. Hitherto, crystallographic structure of a type A FAE from Aspergillus niger has been analyzed. The structure of this enzyme is based on an / hydrolase fold and consists of a major nine-stranded mixed sheet, two minor two-stranded -sheet arrangements and seven helixes [10,11]. Moreover, there are three disulfide bridges located in the FAE simulating three legs of a tripod [10]. FAEs that are similar to other biomass-degradation enzymes can be applied in several industries, such as animal feed preparation, papermaking, baking, biofuel, and production of bioactive phenolic components [12,13]. Unfortunately, most wild-type FAEs have poor thermostability, which lowers their tolerance to the high temperatures encountered in bioprocesses, as for example in pulp bleaching and feedstuff preparation. Only a limited number of thermostable FAEs have been reported thus far, and these FAEs are mostly from bacteria, such as type A TtFAE from Thermoanaerobacter tengcongensis, and type B Tx-Est1 from Thermobacillus xylanilyticus [14,15]. Generally, the FAEs from fungi are not thermostable, such as the AfFaeA from Aspergillus flavus and AnFaeA from A. niger [16,17]. Accordingly, it is very important to discover more thermophilic FAEs or to improve the thermostability of mesophilic enzymes and proteins by employing the promising strategy of protein engineering [18,19]. The thermostability of AnFaeA was improved through multiple amino acid substitutions by error prone PCR technique [20]. Some rational design methods have been developed and applied to increase the protein thermostability. The lipase CalB was thermally improved by rational design based on the flexibility of the amino acid residues (B-factor values) and RosettaDesign [21]. The rational engineering of disulfide bridges in protein is another promising strategy that has been used to improve the thermostability of T4 lysozyme [22], and Trichoderma reesei endo-1,4--xylanase II [23]. These rational protein engineering methods could greatly decrease the heavy workloads of the researcher. In our previous work, a gene AufaeA encoding a mesophilic type A FAE (AuFaeA) from A. usamii was cloned and expressed in Pichia pastoris [24]. In the present study, the contribution of disulfide bridges to AuFaeA thermostability was studied by introducing an extra disulfide bridge designed by computational prediction or eliminating a native one from the enzyme. This work made a first step for further studies on higher thermostability modification of type A FAEs, especially those from fungi, by other methods, such as N- or C-terminus substitution [25,26] and directed evolution [27]. Materials and Methods Strains, Plasmids and Culture Media Escherichia coli JM109 and plasmid pUCm-T (Sangon, Shanghai, China) were used for gene cloning and DNA sequencing. A recombinant T-plasmid, pUCm-T-AufaeA, was constructed and preserved in our laboratory [24]. E. coli DH5 a (...truncated)