11家族木聚糖酶的热稳定性与结构特征的关联研究
摘要
蛋白质的三维结构决定其功能,二硫键,离子对,疏水作用等结构因素都会影响蛋白质的热稳定性。大量的晶体结构研究表明,嗜温性和嗜热性的蛋白质内部疏水区域的序列和结构很保守,这表明嗜温性的蛋白质内部疏水核心的氨基酸同样堆积得非常有效,以保证其结构的稳定及功能的行使。在蛋白质内部疏水区域的点突变常常会破坏这种堆积效果,从而导致蛋白质热稳定的降低。因此目前对于蛋白质热稳定性提高的点突变大多数都是发生在蛋白质的表面。
本论文第一次报道了在蛋白质内部疏水区域的两个半胱氨酸之间的疏水结合提高了蛋白质的热稳定性。本论文通过定向进化和定点突变鉴定了来源于Neocallimastix patriciarum的木聚糖酶C(XynC)内部疏水核心的一个点突变(G201C),该点突变导致该突变体的热稳定性明显提高,同时酶的比活也显著提高。在该突变体中,201位和50位的半胱氨酸所形成的疏水结合是导致热稳定性提高的主要原因。突变体的SDS-PAGE和自由巯基的测定均表明50,201位的半胱氨酸之间并不形成二硫键。这表明半胱氨酸不但可以通过形成二硫键,还能通过其本身的强疏水性质提高蛋白质的稳定性。在50和201位点上为半胱氨酸的G201C和C60A-G201C两个突变体比本研究中其余的木聚糖酶突变体的热稳定性都要高。通过圆二色光谱分析,G201C的T_m值比野生型提高了9度,C60A-G201C提高了12度。经过酶活测定,G201C和C60A-G201C在所有的温度和pH值所测定的比活都要比野生型高,在72度,C60A-G201C的酶活是野生型的6倍。这表示C60A-G201C突变体酶在工业应用中具有比野生型更加广阔的应用前景。该实验结果提示我们可以通过改变蛋白质内部氨基酸的疏水性从而改变蛋白质的热稳定性,为后来的蛋白质理性设计提供新的思路和方向。
另一方面,一些外界因素同样有可能导致酶的热稳定性发生变化,在这些外界因素中,底物一直被认为能够通过稳定酶的活性中心而使酶在高温下稳定。虽然底物对酶的热稳定作用是被报道得较多的,但通过点突变提高底物对蛋白质的热稳定作用却很少被报道。本研究中,我们将XynC活性中心的57位的Asp突变为Asn。在底物不存在的情况下,XynC和D57N突变体的热稳定性是相同的;但是在底物存在的情况下,底物却能更好的稳定D57N突变体,使其表现出比野生型更高的热稳定性。突变体中的Asn57,Glu202与底物分子之间可能存在的氢键导致底物能更好的稳定突变体。该结果表明,酶在底物存在时的热稳定性同样可以改造的,这就为以后通过定向进化或理论设计提高工业用酶的热稳定性指出了另一种方向:通过增强底物对酶的稳定作用而使酶的热稳定性提高。这种突变体的获得一方面可以满足于工业生产的需要,另一方面为了解底物和酶的结合机制提供了理论模型。
The protein structure determintes its function.Some structural basis like disulfide bond,ion pairs,hydrophobic effect may affect the protein thermostability.As the protein interior hydrophobic region of thermostable proteins and mesostable homologues are both packed almost as efficiently as possible,mutation in the protein interior hydrophobic region are often destabilizing,most current stabilization strategies are directed on the protein surface.In the presented researches,by using directed evolution in combination with site-directed mutagenesis,we identified a point-mutation G201C taken place in protein interior hydrophobic region in a family 11 xylanase(XynC) from Neocallimastix patriciarum.G201C is hydrophobically associated with C50 rather than the formation of a new disulfide bond between C50 and C201.RosettaDesign and point mutation using residues with different hydrophobic nature demonstrate that the thermostability of the variant is correlation with hydrophobicity of the residues in site 50 and 201.Two mutants,G201C and C60A-G201C,were identifed greatly increased thermostability than the wild-type. Both in site 50 and 201 of these two mutants are cys,which is considered as the most hydrophobicity residue.The far-ultraviolet circular dichroism signal showed that the transition temperature(T_m) of the mutant G201C is about 9℃higher than that of the wild-type,while the C60A-G201C about 12℃higher.This paper first found a hydrophobic interaction between two cysteines,this interaction stabilizes the protein by decreasing the entropy of the unfolded state.Accompanied with the increased thermostability,these two mutants also possess higher special activity than their parent at all tested pHs and temperatures.This suggests that cysteine could stabilize the protein not only by the formation of disulfide bond,but also for its strong hydrophobicity in the protein interior hydrophobic region.The obtained mutants with higher thermostability and special activity are implied their potential applications in industrial processes.Our results suggest that we could improve the protein thermostability through modifing the residues in protein inner hydrophobic region.
On the other hand,extrinsic factors such as substrate molecules have long been known to stabilize the enzymes.Many thennostable enzymes have been improved intrisincally by site-directed mutagenesis,but increasing the thermostability of the enzymes in the presence of substrate by such mutation method is rarely been reported. Here,we report a mutant(D57N) of XynC in the active site could be stabilized by the substrates while the wild-type not.Despite the thermostability of these two enzymes are almost identical in the absence of the substrate,the mutant enzyme displays an increased optimal temperature.The potential hydrogen bond between N57.E202 of the mutant and the substrate molecule may account for this stabilization.These results suggest that enzyme thermostablity engineering processes should consider the stabilization effect by the substrate.The 'thermophilic' mutants which could be stabilized by the substrate from such engineering processes will deepen our understanding about the binding mechanism between the enzyme and the substrate.
本论文第一次报道了在蛋白质内部疏水区域的两个半胱氨酸之间的疏水结合提高了蛋白质的热稳定性。本论文通过定向进化和定点突变鉴定了来源于Neocallimastix patriciarum的木聚糖酶C(XynC)内部疏水核心的一个点突变(G201C),该点突变导致该突变体的热稳定性明显提高,同时酶的比活也显著提高。在该突变体中,201位和50位的半胱氨酸所形成的疏水结合是导致热稳定性提高的主要原因。突变体的SDS-PAGE和自由巯基的测定均表明50,201位的半胱氨酸之间并不形成二硫键。这表明半胱氨酸不但可以通过形成二硫键,还能通过其本身的强疏水性质提高蛋白质的稳定性。在50和201位点上为半胱氨酸的G201C和C60A-G201C两个突变体比本研究中其余的木聚糖酶突变体的热稳定性都要高。通过圆二色光谱分析,G201C的T_m值比野生型提高了9度,C60A-G201C提高了12度。经过酶活测定,G201C和C60A-G201C在所有的温度和pH值所测定的比活都要比野生型高,在72度,C60A-G201C的酶活是野生型的6倍。这表示C60A-G201C突变体酶在工业应用中具有比野生型更加广阔的应用前景。该实验结果提示我们可以通过改变蛋白质内部氨基酸的疏水性从而改变蛋白质的热稳定性,为后来的蛋白质理性设计提供新的思路和方向。
另一方面,一些外界因素同样有可能导致酶的热稳定性发生变化,在这些外界因素中,底物一直被认为能够通过稳定酶的活性中心而使酶在高温下稳定。虽然底物对酶的热稳定作用是被报道得较多的,但通过点突变提高底物对蛋白质的热稳定作用却很少被报道。本研究中,我们将XynC活性中心的57位的Asp突变为Asn。在底物不存在的情况下,XynC和D57N突变体的热稳定性是相同的;但是在底物存在的情况下,底物却能更好的稳定D57N突变体,使其表现出比野生型更高的热稳定性。突变体中的Asn57,Glu202与底物分子之间可能存在的氢键导致底物能更好的稳定突变体。该结果表明,酶在底物存在时的热稳定性同样可以改造的,这就为以后通过定向进化或理论设计提高工业用酶的热稳定性指出了另一种方向:通过增强底物对酶的稳定作用而使酶的热稳定性提高。这种突变体的获得一方面可以满足于工业生产的需要,另一方面为了解底物和酶的结合机制提供了理论模型。
The protein structure determintes its function.Some structural basis like disulfide bond,ion pairs,hydrophobic effect may affect the protein thermostability.As the protein interior hydrophobic region of thermostable proteins and mesostable homologues are both packed almost as efficiently as possible,mutation in the protein interior hydrophobic region are often destabilizing,most current stabilization strategies are directed on the protein surface.In the presented researches,by using directed evolution in combination with site-directed mutagenesis,we identified a point-mutation G201C taken place in protein interior hydrophobic region in a family 11 xylanase(XynC) from Neocallimastix patriciarum.G201C is hydrophobically associated with C50 rather than the formation of a new disulfide bond between C50 and C201.RosettaDesign and point mutation using residues with different hydrophobic nature demonstrate that the thermostability of the variant is correlation with hydrophobicity of the residues in site 50 and 201.Two mutants,G201C and C60A-G201C,were identifed greatly increased thermostability than the wild-type. Both in site 50 and 201 of these two mutants are cys,which is considered as the most hydrophobicity residue.The far-ultraviolet circular dichroism signal showed that the transition temperature(T_m) of the mutant G201C is about 9℃higher than that of the wild-type,while the C60A-G201C about 12℃higher.This paper first found a hydrophobic interaction between two cysteines,this interaction stabilizes the protein by decreasing the entropy of the unfolded state.Accompanied with the increased thermostability,these two mutants also possess higher special activity than their parent at all tested pHs and temperatures.This suggests that cysteine could stabilize the protein not only by the formation of disulfide bond,but also for its strong hydrophobicity in the protein interior hydrophobic region.The obtained mutants with higher thermostability and special activity are implied their potential applications in industrial processes.Our results suggest that we could improve the protein thermostability through modifing the residues in protein inner hydrophobic region.
On the other hand,extrinsic factors such as substrate molecules have long been known to stabilize the enzymes.Many thennostable enzymes have been improved intrisincally by site-directed mutagenesis,but increasing the thermostability of the enzymes in the presence of substrate by such mutation method is rarely been reported. Here,we report a mutant(D57N) of XynC in the active site could be stabilized by the substrates while the wild-type not.Despite the thermostability of these two enzymes are almost identical in the absence of the substrate,the mutant enzyme displays an increased optimal temperature.The potential hydrogen bond between N57.E202 of the mutant and the substrate molecule may account for this stabilization.These results suggest that enzyme thermostablity engineering processes should consider the stabilization effect by the substrate.The 'thermophilic' mutants which could be stabilized by the substrate from such engineering processes will deepen our understanding about the binding mechanism between the enzyme and the substrate.
引文
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[1] Viikari, L., Kantelinen, A. and Sundquist, J.L., M. (1994). Xylanases in bleaching: from an idea to the industry. FEMS Microbiol. Rev. 13, 335-350.
[2] Kulkarni, N., Shendye, A. and Rao, M. (1999). Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23,411-456.
[3] Polizeli, M.L.T.M., Rizzatti, A.C.S., Monti. R., Terenzi, H.F., Jorge, J.A. and Amorim, D.S. (2005). Xylanases from fungi: properties and industrial applications. Appl. Microbiol. Biotechnol. 67, 577-591.
[4] Miyazaki, K., Takenouchi, M., Kondo, H., Noro, N., Suzuki. M. and Tsuda, S. (2006). Thermal stabilization of Bacillus subtilis family-11 xylanase by directed evolution. J.Biol.Chem. 281, 10236-10242.
[5] Jeong, M.Y., Kim, S., Yun, C.W., Choi, Y.J. and Cho, S.G. (2007). Engineering a de novo internal disulfide bridge to improve the thermal stability of xylanase from Bacillus stearothermophilus No. 236. J.Biotechnol. 127, 300-309.
[6] Stephens, D.E., Rumbold, K., Permaul, K., Prior. B.A. and Singh, S. (2007). Directed evolution of the thermostable xylanase from Thermomyces lanuginosus. J.Biotechnol. 127, 348-354.
[7] Dumon, C. et al. (2008). Engineering hyperthermostability into a GH11 xylanase is mediated by subtle changes to protein structure. J.Biol.Chem. 283, 22557-22564.
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