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Thermostable enzymes have many advantages for industrial applications. Therefore, in this study, computer-aided design technology was used to improve the thermostability of a highly active endo-polygalacturonase from Talaromyces leycettanus JCM12802 at an optimal temperature of 70 °C. The melting temperature and specific activity of the obtained mutant T316C/G344C were increased by 10 °C and 36.5%, respectively, compared with the wild-type enzyme. The crystal structure of the T316C/G344C mutant showed no formation of a disulfide bond between the introduced cysteines, indicating a different mechanism than the conventional mechanism underlying improved enzyme thermostability. The cysteine substitutions directly formed a new alkyl hydrophobic interaction and caused conformational changes in the side chains of the adjacent residues Asn315 and Thr343, which in turn caused a local reconstruction of hydrogen bonds. This method greatly improved the thermostability of the enzyme without affecting its activity; thus, our findings are of great significance for both theoretical research and practical applications.
Sheng Wang; Kun Meng; Xiaoyun Su; Nina Hakulinen; Yaru Wang; Jie Zhang; Huiying Luo; Bin Yao; Huoqing Huang; Tao Tu. Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability. Journal of Agricultural and Food Chemistry 2021, 69, 6351 -6359.
AMA StyleSheng Wang, Kun Meng, Xiaoyun Su, Nina Hakulinen, Yaru Wang, Jie Zhang, Huiying Luo, Bin Yao, Huoqing Huang, Tao Tu. Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability. Journal of Agricultural and Food Chemistry. 2021; 69 (22):6351-6359.
Chicago/Turabian StyleSheng Wang; Kun Meng; Xiaoyun Su; Nina Hakulinen; Yaru Wang; Jie Zhang; Huiying Luo; Bin Yao; Huoqing Huang; Tao Tu. 2021. "Cysteine Engineering of an Endo-polygalacturonase from Talaromyces leycettanus JCM 12802 to Improve Its Thermostability." Journal of Agricultural and Food Chemistry 69, no. 22: 6351-6359.
Thermostability is a key property of industrial enzymes. Endo-polygalacturonases of the glycoside hydrolase family 28 have many practical applications, but only few of their structures have been determined, and the reasons for their stability remain unclear. We identified and characterized the Talaromyces leycettanus JCM12802 endo-polygalacturonase TlPGA, which differs from other GH28 family members because of its high catalytic activity, with an optimum temperature of 70 °C. Distinctive features were revealed by comparison of thermophilic TlPGA and all known structures of fungal endo-polygalacturonases, including a relatively large exposed polar accessible surface area in thermophilic TlPGA. By mutating potentially important residues in thermophilic TlPGA, we identified Thr284 as a critical residue. Mutant T284A was comparable to thermophilic TlPGA in melting temperature but exhibited a significantly lower half-life and half-inactivation temperature, implicating residue Thr284 in the kinetic stability of thermophilic TlPGA. Structure analysis of thermophilic TlPGA and mutant T284A revealed that a carbon–oxygen hydrogen bond between the hydroxyl group of Thr284 and the Cα atom of Gln255, and the stable conformation adopted by Gln255, contribute to its kinetic stability. Our results clarify the mechanism underlying the kinetic stability of GH28 endo-polygalacturonases and may guide the engineering of thermostable enzymes for industrial applications.
Tao Tu; Zhiyun Wang; Yan Luo; Yeqing Li; Xiaoyun Su; Yuan Wang; Jie Zhang; Juha Rouvinen; Bin Yao; Nina Hakulinen; Huiying Luo. Structural Insights into the Mechanisms Underlying the Kinetic Stability of GH28 Endo-Polygalacturonase. Journal of Agricultural and Food Chemistry 2021, 69, 815 -823.
AMA StyleTao Tu, Zhiyun Wang, Yan Luo, Yeqing Li, Xiaoyun Su, Yuan Wang, Jie Zhang, Juha Rouvinen, Bin Yao, Nina Hakulinen, Huiying Luo. Structural Insights into the Mechanisms Underlying the Kinetic Stability of GH28 Endo-Polygalacturonase. Journal of Agricultural and Food Chemistry. 2021; 69 (2):815-823.
Chicago/Turabian StyleTao Tu; Zhiyun Wang; Yan Luo; Yeqing Li; Xiaoyun Su; Yuan Wang; Jie Zhang; Juha Rouvinen; Bin Yao; Nina Hakulinen; Huiying Luo. 2021. "Structural Insights into the Mechanisms Underlying the Kinetic Stability of GH28 Endo-Polygalacturonase." Journal of Agricultural and Food Chemistry 69, no. 2: 815-823.
The strong and inducible cbh1 promoter is most widely used to express heterologous proteins useful in food and feed industries in Trichoderma reesei. Enhancing its ability to direct transcription provides a general strategy to improve protein production in T. reesei. The cbh1 promoter was engineered by replacing eight binding sites of the transcription repressor ACE1 to those of the activators ACE2, Hap2/3/5, and Xyr1, respectively. While changing ACE1 to Hap2/3/5-binding sites completely abolished the transcription ability, replacements with ACE2- and Xyr1-binding sites (designated cbh1pA and cbh1pX promoters, respectively) largely improved the promoter transcription efficiency, as reflected by expression of a reporter gene DsRed. The cbh1pA and cbh1pX promoters were applied to improve secretory expression of a codon-optimized mannanase from Aspergillus niger to 3.6- and 5.0-fold higher, respectively, which has high application potential in feed industry.
Xianhua Sun; Xuhuan Zhang; Huoqing Huang; Yuan Wang; Tao Tu; Yingguo Bai; Yaru Wang; Jie Zhang; Huiying Luo; Bin Yao; Xiaoyun Su. Engineering the cbh1 Promoter of Trichoderma reesei for Enhanced Protein Production by Replacing the Binding Sites of a Transcription Repressor ACE1 to Those of the Activators. Journal of Agricultural and Food Chemistry 2020, 68, 1337 -1346.
AMA StyleXianhua Sun, Xuhuan Zhang, Huoqing Huang, Yuan Wang, Tao Tu, Yingguo Bai, Yaru Wang, Jie Zhang, Huiying Luo, Bin Yao, Xiaoyun Su. Engineering the cbh1 Promoter of Trichoderma reesei for Enhanced Protein Production by Replacing the Binding Sites of a Transcription Repressor ACE1 to Those of the Activators. Journal of Agricultural and Food Chemistry. 2020; 68 (5):1337-1346.
Chicago/Turabian StyleXianhua Sun; Xuhuan Zhang; Huoqing Huang; Yuan Wang; Tao Tu; Yingguo Bai; Yaru Wang; Jie Zhang; Huiying Luo; Bin Yao; Xiaoyun Su. 2020. "Engineering the cbh1 Promoter of Trichoderma reesei for Enhanced Protein Production by Replacing the Binding Sites of a Transcription Repressor ACE1 to Those of the Activators." Journal of Agricultural and Food Chemistry 68, no. 5: 1337-1346.
Glucose oxidases are widely used in various industrial processes, including bread baking. In this study, a novel glucose oxidase gene, CngoxA, from Cladosporium neopsychrotolerans SL16, was cloned and expressed in Pichia pastoris. Recombinant CnGOXA exhibited maximal activity at 20 °C and pH 7.0, and was stable at 30 °C and pH 6.0–9.0 for 1 h, with a half-life of 15 min at 40 °C. Compared with CnGOXA, the half-life of its mutant CnGOXA-M1 (Y169C-A211C), at 40 °C increased approximately 48-fold, and was stable at 30 °C and pH 3.0–12.0 for 1 h. The kcat and catalytic efficiency of CnGOXA-M1 were enhanced 0.7- and 1.6-fold, respectively. Both enzymes were cold-adapted and highly resistant to SDS. Furthermore, CnGOXA-M1 had a more significant effect on bread volume than that of GOX from Aspergillus niger. These favorable enzymatic properties of CnGOXA-M1 make it a potentially useful enzyme for many industrial applications.
Jianzhong Ge; Xiao Jiang; Weina Liu; Yuan Wang; Huoqing Huang; Yingguo Bai; Xiaoyun Su; Bin Yao; Huiying Luo. Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16. Food Chemistry 2019, 310, 125970 .
AMA StyleJianzhong Ge, Xiao Jiang, Weina Liu, Yuan Wang, Huoqing Huang, Yingguo Bai, Xiaoyun Su, Bin Yao, Huiying Luo. Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16. Food Chemistry. 2019; 310 ():125970.
Chicago/Turabian StyleJianzhong Ge; Xiao Jiang; Weina Liu; Yuan Wang; Huoqing Huang; Yingguo Bai; Xiaoyun Su; Bin Yao; Huiying Luo. 2019. "Characterization, stability improvement, and bread baking applications of a novel cold-adapted glucose oxidase from Cladosporium neopsychrotolerans SL16." Food Chemistry 310, no. : 125970.
Background Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. Results In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. Conclusion This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.
Yindi Chu; Zhenzhen Hao; Kaikai Wang; Tao Tu; Huoqing Huang; Yuan Wang; Ying Guo Bai; Yaru Wang; Huiying Luo; Bin Yao; Xiaoyun Su. The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan. Biotechnology for Biofuels 2019, 12, 1 -10.
AMA StyleYindi Chu, Zhenzhen Hao, Kaikai Wang, Tao Tu, Huoqing Huang, Yuan Wang, Ying Guo Bai, Yaru Wang, Huiying Luo, Bin Yao, Xiaoyun Su. The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan. Biotechnology for Biofuels. 2019; 12 (1):1-10.
Chicago/Turabian StyleYindi Chu; Zhenzhen Hao; Kaikai Wang; Tao Tu; Huoqing Huang; Yuan Wang; Ying Guo Bai; Yaru Wang; Huiying Luo; Bin Yao; Xiaoyun Su. 2019. "The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan." Biotechnology for Biofuels 12, no. 1: 1-10.
Background Xylanase is one of the most extensively used biocatalysts for biomass degradation. However, its low catalytic efficiency and poor thermostability limit its applications. Therefore, improving the properties of xylanases to enable synergistic degradation of lignocellulosic biomass with cellulase is of considerable significance in the field of bioenergy. Results Using fragment replacement, we improved the catalytic performance and thermostability of a GH10 xylanase, XylE. Of the ten hybrid enzymes obtained, seven showed xylanase activity. Substitution of fragments, M3, M6, M9, and their combinations enhanced the catalytic efficiency (by 2.4- to fourfold) as well as the specific activity (by 1.2- to 3.3-fold) of XylE. The hybrids, XylE-M3, XylE-M3/M6, XylE-M3/M9, and XylE-M3/M6/M9, showed enhanced thermostability, as observed by the increase in the T50 (3–4.7 °C) and Tm (1.1–4.7 °C), and extended t1/2 (by 1.8–2.3 h). In addition, the synergistic effect of the mutant xylanase and cellulase on the degradation of mulberry bark showed that treatment with both XylE-M3/M6 and cellulase exhibited the highest synergistic effect. In this case, the degree of synergy reached 1.3, and the reducing sugar production and dry matter reduction increased by 148% and 185%, respectively, compared to treatment with only cellulase. Conclusions This study provides a successful strategy to improve the catalytic properties and thermostability of enzymes. We identified several xylanase candidates for applications in bioenergy and biorefinery. Synergistic degradation experiments elucidated a possible mechanism of cellulase inhibition by xylan and xylo-oligomers.
Shuai You; Chen Xie; Rui Ma; Huo-Qing Huang; Richard Ansah Herman; Xiao-Yun Su; Yan Ge; Hui-Yi Cai; Bin Yao; Jun Wang; Hui-Ying Luo. Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase. Biotechnology for Biofuels 2019, 12, 1 -15.
AMA StyleShuai You, Chen Xie, Rui Ma, Huo-Qing Huang, Richard Ansah Herman, Xiao-Yun Su, Yan Ge, Hui-Yi Cai, Bin Yao, Jun Wang, Hui-Ying Luo. Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase. Biotechnology for Biofuels. 2019; 12 (1):1-15.
Chicago/Turabian StyleShuai You; Chen Xie; Rui Ma; Huo-Qing Huang; Richard Ansah Herman; Xiao-Yun Su; Yan Ge; Hui-Yi Cai; Bin Yao; Jun Wang; Hui-Ying Luo. 2019. "Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase." Biotechnology for Biofuels 12, no. 1: 1-15.
The mycotoxin zearalenone (ZEN) is a secondary metabolite produced mainly by Fusarium species. ZEN poses health hazards both for humans and animals, as a major contaminant in the food and feed industries. Currently, there is no effective technique for degrading ZEN during industrial processes. In this study, we isolated and biochemically characterized a novel lactone hydrolase, ZHD607, isolated from Phialophora americana, cloned, and exogenously expressed in Pichia pastoris. ZHD607 was characterized as a mesophilic lactone hydrolase having a neutral pH, and showing optimal activity at 35 °C and pH 8.0. Two mutants, ZHDM1 and I160Y, generated from ZHD607 based on structure and sequence alignment analyses, exhibited 2.9- and 3.4-fold higher activity towards ZEN than did ZHD607. Molecular dynamics simulation revealed diverse mechanisms driving this improved catalytic activity. These findings enrich our knowledge about ZHD enzyme family and represent an important step toward industrialization of ZEN-detoxifying lactone hydrolases.
Xinrui Yu; Tao Tu; Huiying Luo; Huoqing Huang; Xiaoyun Su; Yuan Wang; Yaru Wang; Jie Zhang; Yingguo Bai; Bin Yao. Biochemical Characterization and Mutational Analysis of a Lactone Hydrolase from Phialophora americana. Journal of Agricultural and Food Chemistry 2019, 68, 2570 -2577.
AMA StyleXinrui Yu, Tao Tu, Huiying Luo, Huoqing Huang, Xiaoyun Su, Yuan Wang, Yaru Wang, Jie Zhang, Yingguo Bai, Bin Yao. Biochemical Characterization and Mutational Analysis of a Lactone Hydrolase from Phialophora americana. Journal of Agricultural and Food Chemistry. 2019; 68 (8):2570-2577.
Chicago/Turabian StyleXinrui Yu; Tao Tu; Huiying Luo; Huoqing Huang; Xiaoyun Su; Yuan Wang; Yaru Wang; Jie Zhang; Yingguo Bai; Bin Yao. 2019. "Biochemical Characterization and Mutational Analysis of a Lactone Hydrolase from Phialophora americana." Journal of Agricultural and Food Chemistry 68, no. 8: 2570-2577.
Aflatoxin B1 (AFB1) and zearalenone (ZEN) exert deleterious effects to human and animal health. In this study, the ability of a CotA laccase from Bacillus subtilis (BsCotA) to degrade these two mycotoxins was first investigated. Among the nine structurally defined chemical compounds, methyl syringate was the most efficient mediator assisting BsCotA to degrade AFB1 (98.0%) and ZEN (100.0%). BsCotA could also use plant extracts, including the Epimedium brevicornu, Cucumis sativus L., Lavandula angustifolia, and Schizonepeta tenuifolia extracts to degrade AFB1 and ZEN. Using hydra and BLYES as indicators, it was demonstrated that the degraded products of AFB1 and ZEN using the laccase/mediator systems were detoxified. Finally, a laccase of fungal origin was also able to degrade AFB1 and ZEN in the presence of the discovered mediators. The findings shed light on the possibility of using laccases and a mediator, particularly a natural plant-derived complex mediator, to simultaneously degrade AFB1 and ZEN contaminants in food and feed.
Xiaolu Wang; Yingguo Bai; Huoqing Huang; Tao Tu; Yuan Wang; Yaru Wang; Huiying Luo; Bin Yao; Xiaoyun Su. Degradation of Aflatoxin B1 and Zearalenone by Bacterial and Fungal Laccases in Presence of Structurally Defined Chemicals and Complex Natural Mediators. Toxins 2019, 11, 609 .
AMA StyleXiaolu Wang, Yingguo Bai, Huoqing Huang, Tao Tu, Yuan Wang, Yaru Wang, Huiying Luo, Bin Yao, Xiaoyun Su. Degradation of Aflatoxin B1 and Zearalenone by Bacterial and Fungal Laccases in Presence of Structurally Defined Chemicals and Complex Natural Mediators. Toxins. 2019; 11 (10):609.
Chicago/Turabian StyleXiaolu Wang; Yingguo Bai; Huoqing Huang; Tao Tu; Yuan Wang; Yaru Wang; Huiying Luo; Bin Yao; Xiaoyun Su. 2019. "Degradation of Aflatoxin B1 and Zearalenone by Bacterial and Fungal Laccases in Presence of Structurally Defined Chemicals and Complex Natural Mediators." Toxins 11, no. 10: 609.
Enzymatic treatment is an attractive method for mycotoxin detoxification, which ideally prefers the use of one or a few enzymes. However, this is challenged by the diverse structures and co-contamination of multiple mycotoxins in food and feed. Lignin-degrading fungi have been discovered to detoxify organics including mycotoxins. Manganese peroxidase (MnP) is a major enzyme responsible for lignin oxidative depolymerization in such fungi. Here, we demonstrate that eight MnPs from different lignocellulose-degrading fungi (five from Irpex lacteus, one from Phanerochaete chrysosporium, one from Ceriporiopsis subvermispora, and another from Nematoloma frowardii) could all degrade four major mycotoxins (aflatoxin B1, AFB1; zearalenone, ZEN; deoxynivalenol, DON; fumonisin B1, FB1) only in the presence of a dicarboxylic acid malonate, in which free radicals play an important role. The I. lacteus and C. subvermispora MnPs behaved similarly in mycotoxins transformation, outperforming the P. chrysosporium and N. frowardii MnPs. The large evolutionary diversity of these MnPs suggests that mycotoxin degradation tends to be a common feature shared by MnPs. MnP can, therefore, serve as a candidate enzyme for the degradation of multiple mycotoxins in food and feed if careful surveillance of the residual toxicity of degradation products is properly carried out.
Xiaolu Wang; Xing Qin; Zhenzhen Hao; Huiying Luo; Bin Yao; Xiaoyun Su. Degradation of Four Major Mycotoxins by Eight Manganese Peroxidases in Presence of a Dicarboxylic Acid. Toxins 2019, 11, 566 .
AMA StyleXiaolu Wang, Xing Qin, Zhenzhen Hao, Huiying Luo, Bin Yao, Xiaoyun Su. Degradation of Four Major Mycotoxins by Eight Manganese Peroxidases in Presence of a Dicarboxylic Acid. Toxins. 2019; 11 (10):566.
Chicago/Turabian StyleXiaolu Wang; Xing Qin; Zhenzhen Hao; Huiying Luo; Bin Yao; Xiaoyun Su. 2019. "Degradation of Four Major Mycotoxins by Eight Manganese Peroxidases in Presence of a Dicarboxylic Acid." Toxins 11, no. 10: 566.
An aspartic protease gene (Bsapa) was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris. The recombinant BsAPA showed maximal activity at pH 3.0 and 75 °C and remained stable at 70 °C and below, indicating the thermostable nature of BsAPA. However, heat inactivation still limits the application of BsAPA. To further improve its thermostability, an autocatalysis site (L205-F206) in BsAPA was identified and three mutants (F193W, K204P, and A371V) were generated based on structure analysis neighboring the autocatalysis site. These mutants have improved thermostability, and their half-life at 75 °C increased by 0.5-, 0.2- and 0.3-fold, respectively. A triple-site mutant (F193W/K204P/A371V) was generated, with 1.5-fold-increased half-life at 80 °C, and a 10.7 °C-increased Tm, compared with the wild-type. These results indicate that autocatalysis of aspartic protease reduces enzyme thermostability. Further, site-directed mutagenesis at regions near the autocatalysis site is an efficient approach to improve aspartic protease thermostability.
Yujie Guo; Tao Tu; Jie Zheng; Yingguo Bai; Huoqing Huang; Xiaoyun Su; Yuan Wang; Yaru Wang; Bin Yao; Huiying Luo. Improvement of BsAPA Aspartic Protease Thermostability via Autocatalysis-Resistant Mutation. Journal of Agricultural and Food Chemistry 2019, 67, 10505 -10512.
AMA StyleYujie Guo, Tao Tu, Jie Zheng, Yingguo Bai, Huoqing Huang, Xiaoyun Su, Yuan Wang, Yaru Wang, Bin Yao, Huiying Luo. Improvement of BsAPA Aspartic Protease Thermostability via Autocatalysis-Resistant Mutation. Journal of Agricultural and Food Chemistry. 2019; 67 (37):10505-10512.
Chicago/Turabian StyleYujie Guo; Tao Tu; Jie Zheng; Yingguo Bai; Huoqing Huang; Xiaoyun Su; Yuan Wang; Yaru Wang; Bin Yao; Huiying Luo. 2019. "Improvement of BsAPA Aspartic Protease Thermostability via Autocatalysis-Resistant Mutation." Journal of Agricultural and Food Chemistry 67, no. 37: 10505-10512.
Wheat bran is an effective raw material for preparation xylooligosaccharides; however, current research mainly focuses on alkali extraction and enzymatic hydrolysis methods. Since ester bonds are destroyed during the alkali extraction process, xylanase and arabinofuranosidase are mainly used to hydrolyze xylooligosaccharides. However, alkali extraction costs are very high, and the method also causes pollution. Therefore, this study focuses on elucidating a method to efficiently and directly degrade destarched wheat bran. First, an acidic acetyl xylan esterase (AXE) containing a carbohydrate-binding module-1 (CBM1) domain was cloned from Talaromyces leycettanus JCM12802 and successfully expressed in Pichia pastoris. Characterization showed that the full-length acetyl xylan esterase AXE + CBM1 was similar toe uncovered AXE with an optimum temperature and pH of 55 °C and 6.5, respectively. Testing the acetyl xylan esterase and xylanase derived from Neocallimastix patriciarum in a starch-free wheat bran cooperative experiment revealed that AXE + CBM1 and AXE produced 29% and 16% reducing sugars respectively, compared to when only NPXYN11 was used. In addition, introduced the CBM1 domain into NPXYN11, and the results indicated that the CBM1 domain showed little effect on NPXYN11 properties. Finally, the systematically synergistic effects between acetyl xylan esterase and xylanase with/without the CBM1 domain demonstrated that the combined ratio of AXE + CBM1 coming in first and NPXYN11 + CBM1 s increased reducing sugars by almost 35% with AXE and NPXYN11. Furthermore, each component’s proportion remained the same with respect to xylooligosaccharides, with the largest proportion (86%) containing of 49% xylobiose and 37% xylotriose.
Yueqi Zhang; Hong Yang; Xinrui Yu; Haiyang Kong; Jiaming Chen; Huiying Luo; Yingguo Bai; Bin Yao. Synergistic effect of acetyl xylan esterase from Talaromyces leycettanus JCM12802 and xylanase from Neocallimastix patriciarum achieved by introducing carbohydrate-binding module-1. AMB Express 2019, 9, 1 -12.
AMA StyleYueqi Zhang, Hong Yang, Xinrui Yu, Haiyang Kong, Jiaming Chen, Huiying Luo, Yingguo Bai, Bin Yao. Synergistic effect of acetyl xylan esterase from Talaromyces leycettanus JCM12802 and xylanase from Neocallimastix patriciarum achieved by introducing carbohydrate-binding module-1. AMB Express. 2019; 9 (1):1-12.
Chicago/Turabian StyleYueqi Zhang; Hong Yang; Xinrui Yu; Haiyang Kong; Jiaming Chen; Huiying Luo; Yingguo Bai; Bin Yao. 2019. "Synergistic effect of acetyl xylan esterase from Talaromyces leycettanus JCM12802 and xylanase from Neocallimastix patriciarum achieved by introducing carbohydrate-binding module-1." AMB Express 9, no. 1: 1-12.
Aspartic proteases exhibit optimum enzyme activity under acidic condition and have been extensively used in food, fermentation and leather industries. In this study, a novel aspartic protease precursor (proTlAPA1) fromTalaromyces leycettanuswas identified and successfully expressed inPichia pastoris. Subsequently, the auto-activation processing of the zymogen proTlAPA1 was studied by SDS-PAGE and N-terminal sequencing, under different processing conditions.TlAPA1 shared the highest identity of 70.3 % with the aspartic endopeptidase fromByssochlamys spectabilis(GAD91729) and was classified into a new subgroup of the aspartic protease A1 family, based on evolutionary analysis. MatureTlAPA1 protein displayed an optimal activity at 60 °C and remained stable at temperatures of 55 °C and below, indicating the thermostable nature ofTlAPA1 aspartic protease. During the auto-activation processing of proTlAPA1, a 45 kDa intermediate was identified that divided the processing mechanism into two steps: formation of intermediates, and activation of the mature protein (TlAPA1). The former step was completely induced by pH of the buffer, while the latter process depended on protease activity. The discovery of the novel aspartic proteaseTlAPA1 and study of its activation process will contribute to a better understanding of the mechanism of aspartic proteases auto-activation.IMPORTANCEThe novel aspartic proteaseTlAPA1 was identified fromT. leycettanusand expressed as a zymogen (proTlAPA1) inP. pastoris. Enzymatic characteristics of the mature protein were studied and the specific pattern of zymogen conversion was described. The auto-activation processing of proTlAPA1 proceeded in two stages and an intermediate was identified in this process. These results describe a new subgroup of aspartic protease A1 family and provide insights into a novel mode of activation processing in aspartic proteases.
Yujie Guo; Tao Tu; Yaxin Ren; Yaru Wang; Ying Guo Bai; Xiaoyun Su; Yuan Wang; Bin Yao; Huoqing Huang; Huiying Luo. A novel thermostable aspartic protease fromTalaromyces leycettanusand its specific autocatalytic activation through an intermediate transition state. 2019, 528265 .
AMA StyleYujie Guo, Tao Tu, Yaxin Ren, Yaru Wang, Ying Guo Bai, Xiaoyun Su, Yuan Wang, Bin Yao, Huoqing Huang, Huiying Luo. A novel thermostable aspartic protease fromTalaromyces leycettanusand its specific autocatalytic activation through an intermediate transition state. . 2019; ():528265.
Chicago/Turabian StyleYujie Guo; Tao Tu; Yaxin Ren; Yaru Wang; Ying Guo Bai; Xiaoyun Su; Yuan Wang; Bin Yao; Huoqing Huang; Huiying Luo. 2019. "A novel thermostable aspartic protease fromTalaromyces leycettanusand its specific autocatalytic activation through an intermediate transition state." , no. : 528265.
Aspartic proteases are promising fining agents used in the production of fruit juices. In this study, a novel aspartic protease gene (Tlap) was identified in Talaromyces leycettanus JCM12802 and heterologously expressed in Pichia pastoris. Using casein as the substrate, purified recombinant TlAP showed optimal activities at pH 3.0 and 55 °C with a specific activity of 1795.4 ± 62.8 U/mg, and remained stable over a pH range of 3.0–6.0 and at temperatures of 45 °C and below. Moreover, the enzyme was highly resistant to most metal ions and chemical reagents except for Fe3+ and β-mercaptoethanol. When added to apple, orange, grape and kiwi fruit juice, it showed excellent proteolytic activity against haze-forming proteins, decreasing the turbidity by up to 49.9 nephelometry turbidity units (NTU). These favorable enzymatic properties make TlAP attractive for potential use in the juice industry.
Yujie Guo; Tao Tu; Peng Yuan; Yaru Wang; Yaxin Ren; Bin Yao; Huiying Luo. High-level expression and characterization of a novel aspartic protease from Talaromyces leycettanus JCM12802 and its potential application in juice clarification. Food Chemistry 2019, 281, 197 -203.
AMA StyleYujie Guo, Tao Tu, Peng Yuan, Yaru Wang, Yaxin Ren, Bin Yao, Huiying Luo. High-level expression and characterization of a novel aspartic protease from Talaromyces leycettanus JCM12802 and its potential application in juice clarification. Food Chemistry. 2019; 281 ():197-203.
Chicago/Turabian StyleYujie Guo; Tao Tu; Peng Yuan; Yaru Wang; Yaxin Ren; Bin Yao; Huiying Luo. 2019. "High-level expression and characterization of a novel aspartic protease from Talaromyces leycettanus JCM12802 and its potential application in juice clarification." Food Chemistry 281, no. : 197-203.
Glucose oxidase (Gox) has many applications in numerous industries. However, thermal instability is a major drawback that prevents its broader use. Here, Gox from Aspergillus niger (GoxA) was selected for laboratory evolution for purposes of enhancing thermostability and catalytic efficiency through random and rational mutagenesis. The most active mutant, M4, accumulated six amino acid substitutions. The T50 of M4, the temperature corresponding to a 50% loss of maximal enzyme activity, increased by 7.5 °C and thermal inactivation half-lives (t1/2) at 60 °C and 70 °C increased 8.4-fold and 5.6-fold, respectively, compared to wild-type GoxA. Concomitantly, M4 demonstrated a 1.86-fold increase in kcat, resulting in a 1.78-fold increase in catalytic efficiency. Molecular dynamics simulation revealed diverse mechanisms underlying the effects of each mutation on thermostability and catalytic efficiency. These results suggest that key properties of glucose oxidase can be modified in vitro by laboratory evolution, which may have remarkable economic importance.
Tao Tu; Yuan Wang; Huoqing Huang; Yaru Wang; Xiao Jiang; Zhenxing Wang; Bin Yao; Huiying Luo. Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution. Food Chemistry 2019, 281, 163 -170.
AMA StyleTao Tu, Yuan Wang, Huoqing Huang, Yaru Wang, Xiao Jiang, Zhenxing Wang, Bin Yao, Huiying Luo. Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution. Food Chemistry. 2019; 281 ():163-170.
Chicago/Turabian StyleTao Tu; Yuan Wang; Huoqing Huang; Yaru Wang; Xiao Jiang; Zhenxing Wang; Bin Yao; Huiying Luo. 2019. "Improving the thermostability and catalytic efficiency of glucose oxidase from Aspergillus niger by molecular evolution." Food Chemistry 281, no. : 163-170.
To study the molecular basis for thermophilic β-mannanase of glycoside hydrolase family 5, two β-mannanases, TlMan5A and PMan5A, from Talaromyces leycettanus JCM12802 and Penicillium sp. WN1 were used as models. The four residues, His112 and Phe113 located near the antiparallel β-sheet at the barrel bottom and Leu375 and Ala408 from loop 7 and loop 8 of PMan5A, were inferred to be key thermostability contributors through module substitution, truncation, and site-directed mutagenesis. The effects of these four residues on the thermal properties followed the order H112Y>A408P>L375H>F113Y and were strongly synergetic. These results were interpreted structurally using molecular dynamics (MD) simulations, which showed that improved hydrophobic interactions in the inner wall of the β-barrel and the rigidity of loop 8 were caused by the outside domain of the barrel bottom and proline, respectively. The TIM barrel bottom and four specific residues responsible for the thermostability of GH5 β-mannanases were elucidated.
Weina Liu; Tao Tu; Yuan Gu; Yuan Wang; Fei Zheng; Jie Zheng; Yaru Wang; Xiaoyun Su; Bin Yao; Huiying Luo. Insight into the Thermophilic Mechanism of a Glycoside Hydrolase Family 5 β-Mannanase. Journal of Agricultural and Food Chemistry 2018, 67, 473 -483.
AMA StyleWeina Liu, Tao Tu, Yuan Gu, Yuan Wang, Fei Zheng, Jie Zheng, Yaru Wang, Xiaoyun Su, Bin Yao, Huiying Luo. Insight into the Thermophilic Mechanism of a Glycoside Hydrolase Family 5 β-Mannanase. Journal of Agricultural and Food Chemistry. 2018; 67 (1):473-483.
Chicago/Turabian StyleWeina Liu; Tao Tu; Yuan Gu; Yuan Wang; Fei Zheng; Jie Zheng; Yaru Wang; Xiaoyun Su; Bin Yao; Huiying Luo. 2018. "Insight into the Thermophilic Mechanism of a Glycoside Hydrolase Family 5 β-Mannanase." Journal of Agricultural and Food Chemistry 67, no. 1: 473-483.
The white rot fungus Irpex lacteus exhibits a great potential in biopretreatment of lignocellulose as well as in biodegradation of xenobiotic compounds by extracellular ligninolytic enzymes. Among these enzymes, the possible involvement of dye-decolorizing peroxidase (DyP) in lignin degradation is not clear yet. Based on the extracellular enzyme activities and secretome analysis, I. lacteus CD2 produced DyPs as the main ligninolytic enzymes when grown in Kirk’s medium supplemented with lignin. Further transcriptome analysis revealed that induced transcription of genes encoding DyPs was accompanied by the increased expression of transcripts for H2O2-generating enzymes such as alcohol oxidase, pyranose 2-oxidase, and glyoxal oxidases. Meanwhile, accumulation of transcripts for glycoside hydrolase and protease was observed, in agreement with abundant proteins. Moreover, the biochemical analysis of IlDyP2 and IlDyP1 confirmed that DyPs were able to catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound, veratryl alcohol. More importantly, IlDyP1 enhanced catalytic activity for veratryl alcohol oxidation in the presence of mediator 1-hydroxybenzotriazole, which was similar to the laccase/1-hydroxybenzotriazole system. The results proved for the first time that DyPs depolymerized lignin individually, combining catalytic features of different peroxidases on the functional level. Therefore, DyPs may be considered an important part of ligninolytic system in wood-decaying fungi.
Xing Qin; Huiying Luo; Xiaoyu Zhang; Bin Yao; Fuying Ma; Xiaoyun Su. Dye-decolorizing peroxidases in Irpex lacteus combining the catalytic properties of heme peroxidases and laccase play important roles in ligninolytic system. Biotechnology for Biofuels 2018, 11, 1 -11.
AMA StyleXing Qin, Huiying Luo, Xiaoyu Zhang, Bin Yao, Fuying Ma, Xiaoyun Su. Dye-decolorizing peroxidases in Irpex lacteus combining the catalytic properties of heme peroxidases and laccase play important roles in ligninolytic system. Biotechnology for Biofuels. 2018; 11 (1):1-11.
Chicago/Turabian StyleXing Qin; Huiying Luo; Xiaoyu Zhang; Bin Yao; Fuying Ma; Xiaoyun Su. 2018. "Dye-decolorizing peroxidases in Irpex lacteus combining the catalytic properties of heme peroxidases and laccase play important roles in ligninolytic system." Biotechnology for Biofuels 11, no. 1: 1-11.
Bacillus amyloliquefaciens K11 is a hyperproducer of extracellular neutral protease, which can produce recombinant homologous protein steadily and is amenable to scale up to high-cell density fermentation. The present study aims to genetically modify strain K11 as a highly efficient secretory expression system for high-level production of heterologous proteins. Using B. amyloliquefaciens K11 and alkaline protease gene BcaprE as the expression host and model gene, the gene expression levels mediated by combinations of promoters PamyQ, PaprE and Pnpr and signal peptides SPamyQ, SPaprE and SPnpr were assessed on shake flask level. The PamyQ-SPaprE was found to be the best secretory expression cassette, giving the highest enzyme activities of extracellular BcaprE (13,800 ± 308 U/mL). Using the same expression system, the maltogenic α-amylase Gs-MAase and neutral protease BaNPR were successfully produced with the enzyme activities of 19. ± 0.2 U/mL and 17,495 ± 417 U/mL, respectively. After knocking out the endogenous neutral protease-encoding gene Banpr, the enzyme activities of BcaprE and Gs-MAase were further improved by 25.4% and 19.4%, respectively. Moreover, the enzyme activities of BcaprE were further improved to 30,200 ± 312 U/mL in a 15 L fermenter following optimization of the fermentation conditions. In the present study, the genetically engineered B. amyloliquefaciens strain 7-6 containing PamyQ-SPaprE as the secretory expression cassette was developed. This efficient expression system shows general applicability and represents an excellent industrial strain for the production of heterologous proteins.
Hui Wang; Xin Zhang; Jin Qiu; Kaikai Wang; Kun Meng; Huiying Luo; Xiaoyun Su; Rui Ma; Huoqing Huang; Bin Yao. Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system. Journal of Industrial Microbiology and Biotechnology 2018, 46, 113 -123.
AMA StyleHui Wang, Xin Zhang, Jin Qiu, Kaikai Wang, Kun Meng, Huiying Luo, Xiaoyun Su, Rui Ma, Huoqing Huang, Bin Yao. Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system. Journal of Industrial Microbiology and Biotechnology. 2018; 46 (1):113-123.
Chicago/Turabian StyleHui Wang; Xin Zhang; Jin Qiu; Kaikai Wang; Kun Meng; Huiying Luo; Xiaoyun Su; Rui Ma; Huoqing Huang; Bin Yao. 2018. "Development of Bacillus amyloliquefaciens as a high-level recombinant protein expression system." Journal of Industrial Microbiology and Biotechnology 46, no. 1: 113-123.
The major enzymes involved in lignin degradation are laccase, class II peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase) and dye peroxidase, which use an oxidative or peroxidative mechanism to deconstruct the complex and recalcitrant lignin. Laccase and manganese peroxidase directly oxidize phenolic lignin components, while lignin peroxidase and versatile peroxidase can act on the more recalcitrant non-phenolic lignin compounds. Mediators or co-oxidants not only increase the catalytic ability of these enzymes, but also largely expand their substrate scope to those with higher redox potential or more complicated structures. Neither laccase nor the peroxidases are stringently selective of substrates. The promiscuous nature in substrate preference can be employed in detoxification of a range of organics.
Xiaolu Wang; Bin Yao; Xiaoyun Su. Linking Enzymatic Oxidative Degradation of Lignin to Organics Detoxification. International Journal of Molecular Sciences 2018, 19, 3373 .
AMA StyleXiaolu Wang, Bin Yao, Xiaoyun Su. Linking Enzymatic Oxidative Degradation of Lignin to Organics Detoxification. International Journal of Molecular Sciences. 2018; 19 (11):3373.
Chicago/Turabian StyleXiaolu Wang; Bin Yao; Xiaoyun Su. 2018. "Linking Enzymatic Oxidative Degradation of Lignin to Organics Detoxification." International Journal of Molecular Sciences 19, no. 11: 3373.
In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive. Herein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library. The coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.
Fei Gao; Zhenzhen Hao; Xianhua Sun; Lina Qin; Tong Zhao; Weiquan Liu; Huiying Luo; Bin Yao; Xiaoyun Su. A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting. Biotechnology for Biofuels 2018, 11, 1 -13.
AMA StyleFei Gao, Zhenzhen Hao, Xianhua Sun, Lina Qin, Tong Zhao, Weiquan Liu, Huiying Luo, Bin Yao, Xiaoyun Su. A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting. Biotechnology for Biofuels. 2018; 11 (1):1-13.
Chicago/Turabian StyleFei Gao; Zhenzhen Hao; Xianhua Sun; Lina Qin; Tong Zhao; Weiquan Liu; Huiying Luo; Bin Yao; Xiaoyun Su. 2018. "A versatile system for fast screening and isolation of Trichoderma reesei cellulase hyperproducers based on DsRed and fluorescence-assisted cell sorting." Biotechnology for Biofuels 11, no. 1: 1-13.
A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley β-glucan were 769, 498, and 486 U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0–6.0 and 60–65 °C with barley β-glucan and CMC-Na as substrates and had a melting temperature (Tm) of 68.4 °C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 °C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and Tm values of 50 and 60.2 °C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.
Hong Yang; Yueqi Zhang; Xinxin Li; Yingguo Bai; Wei Xia; Rui Ma; Huiying Luo; Pengjun Shi; Bin Yao. Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase. Applied Microbiology and Biotechnology 2018, 102, 9183 -9192.
AMA StyleHong Yang, Yueqi Zhang, Xinxin Li, Yingguo Bai, Wei Xia, Rui Ma, Huiying Luo, Pengjun Shi, Bin Yao. Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase. Applied Microbiology and Biotechnology. 2018; 102 (21):9183-9192.
Chicago/Turabian StyleHong Yang; Yueqi Zhang; Xinxin Li; Yingguo Bai; Wei Xia; Rui Ma; Huiying Luo; Pengjun Shi; Bin Yao. 2018. "Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase." Applied Microbiology and Biotechnology 102, no. 21: 9183-9192.