Determinants for the improved thermostability of a mesophilic family 11 xylanase predicted by computational methods

Biotechnology for Biofuels

Huimin Zhang³  Jianfang Li³  Junqing Wang²  Yanjun Yang³  Minchen Wu¹ 

[1] Wuxi Medical School, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China

[2] School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China

[3] State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, China

关键词: Xylooligosaccharides;    Site-directed mutagenesis;    N-terminus replacement;    Computational method;    Thermostability;    Xylanase;   

Others  :  794120
DOI  :  10.1186/1754-6834-7-3

 received in 2013-09-09, accepted in 2013-12-17,  发布年份 2014

【 摘 要 】

Background

Xylanases have drawn much attention owing to possessing great potential in various industrial applications. However, the applicability of xylanases, exemplified by the production of bioethanol and xylooligosaccharides (XOSs), was bottlenecked by their low stabilities at higher temperatures. The main purpose of this work was to improve the thermostability of AuXyn11A, a mesophilic glycoside hydrolase (GH) family 11 xylanase from Aspergillus usamii E001, by N-terminus replacement.

Results

A hybrid xylanase with high thermostability, named AEXynM, was predicted by computational methods, and constructed by substituting the N-terminal 33 amino acids of AuXyn11A with the corresponding 38 ones of EvXyn11^TS, a hyperthermostable family 11 xylanase. Two AuXyn11A- and AEXynM-encoding genes, Auxyn11A and AExynM, were then highly expressed in Pichia pastoris GS115, respectively. The specific activities of two recombinant xylanases (reAuXyn11A and reAEXynM) were 10,437 and 9,529 U mg^-1. The temperature optimum and stability of reAEXynM reached 70 and 75°C, respectively, much higher than those (50 and 45°C) of reAuXyn11A. The melting temperature (T_m) of reAEXynM, measured using the Protein Thermal Shift (PTS) method, increased by 34.0°C as compared with that of reAuXyn11A. Analyzed by HPLC, xylobiose and xylotriose as the major hydrolytic products were excised from corncob xylan by reAEXynM. Additionally, three single mutant genes from AExynM (AExynM^C5T, AExynM^P9S, and AExynM^H14N) were constructed by site-directed mutagenesis as designed theoretically, and expressed in P. pastoris GS115, respectively. The thermostabilities of three recombinant mutants clearly decreased as compared with that of reAEXynM, which demonstrated that the three amino acids (Cys⁵, Pro⁹, and His¹⁴) in the replaced N-terminus contributed mainly to the high thermostability of AEXynM.

Conclusions

This work highly enhanced the thermostability of AuXyn11A by N-terminus replacement, and further verified, by site-directed mutagenesis, that Cys⁵, Pro⁹, and His¹⁴ contributed mainly to the improved thermostability. It will provide an effective strategy for improving the thermostabilities of other enzymes.

【 授权许可】

   
2014 Zhang et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Collins T, Gerday C, Feller G: Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 2005, 29(1):3-23.
• [2]Singh S, Madlala AM, Prior BA: Thermomyces lanuginosus: properties of strains and their hemicellulases. FEMS Microbiol Rev 2003, 27(1):3-16.
• [3]Taibi Z, Saoudi B, Boudelaa M, Trigui H, Belghith H, Gargouri A, Ladjama A: Purification and biochemical characterization of a highly thermostable xylanase from Actinomadura sp. strain Cpt20 isolated from poultry compost. Appl Biochem Biotechnol 2012, 166(3):663-679.
• [4]Sriprang R, Asano K, Gobsuk J, Tanapongpipat S, Champreda V, Eurwilaichitr L: Improvement of thermostability of fungal xylanase by using site-directed mutagenesis. J Biotechnol 2006, 126(4):454-462.
• [5]Zhang J, Siika-aho M, Puranen T, Tang M, Tenkanen M, Viikari L: Thermostable recombinant xylanases from Nonomuraea flexuosa and Thermoascus aurantiacus show distinct properties in the hydrolysis of xylans and pretreated wheat straw. Biotechnol Biofuels 2011, 4:12. BioMed Central Full Text
• [6]Cai H, Shi P, Bai Y, Huang H, Yuan T, Yang P, Luo H, Meng K, Yao B: A novel thermoacidophilic family 10 xylanase from Penicillium pinophilum C1. Process Biochem 2011, 46(12):2341-2346.
• [7]Yoon HS, Han NS, Kim CH: Expression of Thermotoga maritima endo-β-1,4-xylanase gene in E. coli and characterization of the recombinant enzyme. Agric Chem Biotechnol 2004, 47(3):157-160.
• [8]Sunna A, Bergquist PL: A gene encoding a novel extremely Thermostable 1,4-β-xylanase isolated directly from an environmental DNA sample. Extremophiles 2003, 7(1):63-70.
• [9]Hakulinen N, Turunen O, Janis J, Leisola M, Rouvinen J: Three-dimensional structures of thermophilic β-1,4-xylanases from Chaetomium thermophilum and Nonomuraea flexuosa: Comparison of twelve xylanases in relation to their thermal stability. Eur J Biochem 2003, 270(7):1399-1412.
• [10]Kumar S, Tsai CJ, Nussinov R: Factors enhancing protein thermostability. Protein Eng 2000, 13(3):179-191.
• [11]Wang Y, Fu Z, Huang H, Zhang H, Yao B, Xiong H, Turunen O: Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge. Bioresour Technol 2012, 112:275-279.
• [12]He J, Yu B, Zhang K, Ding X, Chen D: Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2. New Biotechnol 2009, 26(1–2):53-59.
• [13]Dumon C, Varvak A, Wall MA, Flint JE, Lewis RJ, Lakey JH, Morland C, Luginbuhl P, Healey S, Todaro T, et al.: Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure. J Biol Chem 2008, 283(33):22557-22564.
• [14]Wu M, Fu D, Zhu J, Xia M: Purification and some properties of two xylanases from Aspergillus usamii. J Food Sci Biotechnol 2005, 24(6):29-33.
• [15]Wu M, Wang J, Zhang H, Tang C, Gao J, Tan Z: Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from Aspergillus usamii E001. World J Microbiol Biotechnol 2011, 27(4):831-839.
• [16]Jaenicke R, Bohm G: The stability of proteins in extreme environments. Curr Opin Struct Biol 1998, 8(6):738-748.
• [17]Reetz MT, Carballeira JD, Vogel A: Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew Chem Int Ed 2006, 45(46):7745-7751.
• [18]Radestock S, Gohlke H: Exploiting the link between protein rigidity and thermostability for data-driven protein engineering. Eng Life Sci 2008, 8(5):507-522.
• [19]Tan Z, Li J, Wu M, Tang C, Zhang H, Wang J: High-level heterologous expression of an alkaline lipase gene from Penicillium cyclopium PG37 in Pichia pastoris. World J Microbiol Biotechnol 2011, 27(12):2767-2774.
• [20]Zhang H, Wu M, Li J, Gao S, Yang Y: Cloning and expression of a novel xylanase gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris. Appl Biochem Biotechnol 2012, 167(8):2198-2211.
• [21]Sun JY, Liu MQ, Weng XY, Qian LC, Gu SH: Expression of recombinant Thermomonospora fusca xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Food Chem 2007, 104(3):1055-1064.
• [22]Niesen FH, Berglund H, Vedadi M: The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2007, 2(9):2212-2221.
• [23]Liu MQ, Liu GF: Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it. Protein Expres Purif 2008, 57(2):101-107.
• [24]Brienzo M, Carvalho W, Milagres AMF: Xylooligosaccharides production from alkali-pretreated sugarcane bagasse using xylanases from Thermoascus aurantiacus. Appl Biochem Biotechnol 2010, 162(4):1195-1205.
• [25]Potapov V, Cohen M, Schreiber G: Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details. Protein Eng Des Sel 2009, 22(9):553-560.
• [26]Tina KG, Bhadra R, Srinivasan N: PIC: Protein interactions calculator. Nucleic Acids Res 2007, 35:W473-W476.
• [27]Le QAT, Joo JC, Yoo YJ, Kim YH: Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol Bioeng 2012, 109(4):867-876.
• [28]Turunen O, Etuaho K, Fenel F, Vehmaanpera J, Wu XY, Rouvinen J, Leisola M: A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism. J Biotechnol 2001, 88(1):37-46.
• [29]Reetz MT, Carballeira JD: Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat Protoc 2007, 2(4):891-903.
• [30]Badieyan S, Bevan DR, Zhang C: Study and design of stability in GH5 cellulases. Biotechnol Bioeng 2012, 109(1):31-44.
• [31]Xie ZH, Shi XJ: Fast and almost 100% efficiency site-directed mutagenesis by the megaprimer PCR method. Prog Biochem Biophys 2009, 36(11):1490-1494.
• [32]Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227(5259):680-685.
• [33]Jang MK, Lee SW, Lee DG, Kim NY, Yu KH, Jang HJ, Kim S, Kim A, Lee SH: Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis. Biotechnol Lett 2010, 32(7):943-949.
• [34]Lin YS, Tseng MJ, Lee WC: Production of xylooligosaccharides using immobilized endo-xylanase of Bacillus halodurans. Process Biochem 2011, 46(11):2117-2121.