毕赤酵母表达的两种植酸酶的分离纯化与表征及其N-糖基化研究
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摘要
本文是关于重组毕赤酵母（Pichia pastoris）高密度发酵的发酵液中两种植酸酶的分离纯化与表征、糖基化研究、结晶实验及其变性与复性的研究。使用CM-Sephadex C-50弱阳离子交换色谱分离技术，从基因重组的植酸酶发酵液中分离纯化出两种植酸酶，CMⅠ和CMⅡ，经SDS-PAGE和N－末端氨基酸测序鉴定，均为单一组分。
植酸酶CMⅠ和CMⅡ分子量分别为55.3kD和53.1kD，二者作用的最适温度均为55℃，最适pH都是5.0，在37℃下CMⅠ的Km是0.42mmol/L，CMⅡ的Km是0.79mmol/L, Cu2+，Fe2+，Al3+和Zn2+对两种酶活性有较强的抑制作用，CMⅡ的酶比活力明显高于CMⅠ的酶比活力，两种植酸酶具有相同的N－端序列。
用N-糖基化酶Endo H对两种植酸酶进行N-糖基化分析，CMⅠ相对含糖量为13.1%，CMⅡ相对含糖量为10.9%（分别以未去糖基的两种植酸酶分子量为对照），证实是糖基化的不同引起植酸酶CMⅠ和CMⅡ分子量的差异。分别测定去糖基后的酶活性，CMⅠ比活力降低为原来的81％，而CMⅡ比活力基本不变，CMⅡ比活力仍比CMⅠ高，表明植酸酶肽链糖基化会影响植酸酶活性，在一定糖基化程度下对植酸酶的活力起稳定甚至提高的作用。
根据Crystal Screen配制了38种结晶条件进行两种植酸酶结晶尝试，确定了Crystal Screen中的第12号条件，即：30％ 2－丙醇，0.1mol/L NaHepes，0.2mol/L MgCl2，pH7.5作为结晶条件，其中CMⅡ得到晶体。
改进H.D.Bae等报道用氯化钴做染色剂对植酸酶SDS-PAGE电泳胶染色，建立一种可以通过SDS-PAGE对比染色法特异检测植酸酶酶活性的新方法：SDS-PAGE结合硫酸亚铁－钼酸铵对比染色法。
植酸酶在经过热、SDS-PAGE电泳完全变性后，仍能部分复性。利用SDS-PAGE对比染色法研究植酸酶的复性机理，发现Triton X-100和低温是植酸酶复性的关键，为蛋白质重折叠机制和蛋白质一级结构决定蛋白质高级结构提供了又一个证据。
Two types of ecto-phytase, which were isolated and characterized from recombinant Pichia pastoris yeast, were studied on their N-glycosylation, crystal birth and renaturation. The two phytases, CMⅠand CMⅡ, were isolated from the fermentation of phytase by gene recombinant with one step of CM-Sephadex C-50 chromatography. They were identified to be homogeneous with SDS-PAGE and the partial N-terminal amino acid sequences.
The molecular weight of CMⅠand CMⅡ were 55.3kD and 53.1kD respectively, and their optimum temperature and pH were the same as 55℃ and 5.0. The Km values of CMⅠunder 37℃ was 0.42mmol/L, and the Km values of CMⅡunder 37℃ was 0.79mmol/L. The activity of CMⅠand CMⅡ could be inhibited by Cu2+, Fe2+, Al3+, and Zn2+ . The specific activity of CMⅡ were higher than that of CMⅠ. The partial N-terminal amino acid sequence of two phytases were identical .
The N-glycosylation analysis was performed with Endoglycosidase H, CMⅠ and CMⅡwere about 13.1% and 10.9% glycosylated respectively (calculations were made on the basis of original molecular weight before deglycosylation). It was testified that the difference between their molecular weight were induced by the different extents of N-glycosylation. After deglycosylation, it was found that the specific activity of CMⅠwas reduced to 81 percent of original specific activity of CMⅠ, while deglycosylation had little effect on the specific activity of CMⅡ. And the specific activity of CMⅡ was still higher than that of CMⅠ after deglycosylation. It showed that N-glycosylation of the phytases could stabilitate, even enhance the specific activity of the two phytases.
According to the Crystal Screen, crystal birth was tested with 38 kinds of crystal conditions. Under the 12nd condition (i.e. 30% 2-Propanol, 0.1mol/L NaHepes pH7.5, and 0.2 mol/L MgCl2), the crystal of CMⅡ was obtained.
Bae et al reported a staining method for detecting phytase activity. Here the method was improved, and a novel SDS-PAGE counterstaining, which was SDS-PAGE and ferrous sulfate-ammonium molybdate counterstaining, was established to detect phytase activity specially.
After thermal denaturation and SDS-PAGE, the phytases could recover their activity partially. The mechanism of renaturation was studied with SDS-PAGE counterstaining. It
showed that Triton X-100 and low temperature had played key roles in refolding the phytases. This provided a new evidence for understanding protein refolding mechanism and the theory of the primary structure of proteins determining the advanced structure.
植酸酶CMⅠ和CMⅡ分子量分别为55.3kD和53.1kD，二者作用的最适温度均为55℃，最适pH都是5.0，在37℃下CMⅠ的Km是0.42mmol/L，CMⅡ的Km是0.79mmol/L, Cu2+，Fe2+，Al3+和Zn2+对两种酶活性有较强的抑制作用，CMⅡ的酶比活力明显高于CMⅠ的酶比活力，两种植酸酶具有相同的N－端序列。
用N-糖基化酶Endo H对两种植酸酶进行N-糖基化分析，CMⅠ相对含糖量为13.1%，CMⅡ相对含糖量为10.9%（分别以未去糖基的两种植酸酶分子量为对照），证实是糖基化的不同引起植酸酶CMⅠ和CMⅡ分子量的差异。分别测定去糖基后的酶活性，CMⅠ比活力降低为原来的81％，而CMⅡ比活力基本不变，CMⅡ比活力仍比CMⅠ高，表明植酸酶肽链糖基化会影响植酸酶活性，在一定糖基化程度下对植酸酶的活力起稳定甚至提高的作用。
根据Crystal Screen配制了38种结晶条件进行两种植酸酶结晶尝试，确定了Crystal Screen中的第12号条件，即：30％ 2－丙醇，0.1mol/L NaHepes，0.2mol/L MgCl2，pH7.5作为结晶条件，其中CMⅡ得到晶体。
改进H.D.Bae等报道用氯化钴做染色剂对植酸酶SDS-PAGE电泳胶染色，建立一种可以通过SDS-PAGE对比染色法特异检测植酸酶酶活性的新方法：SDS-PAGE结合硫酸亚铁－钼酸铵对比染色法。
植酸酶在经过热、SDS-PAGE电泳完全变性后，仍能部分复性。利用SDS-PAGE对比染色法研究植酸酶的复性机理，发现Triton X-100和低温是植酸酶复性的关键，为蛋白质重折叠机制和蛋白质一级结构决定蛋白质高级结构提供了又一个证据。
Two types of ecto-phytase, which were isolated and characterized from recombinant Pichia pastoris yeast, were studied on their N-glycosylation, crystal birth and renaturation. The two phytases, CMⅠand CMⅡ, were isolated from the fermentation of phytase by gene recombinant with one step of CM-Sephadex C-50 chromatography. They were identified to be homogeneous with SDS-PAGE and the partial N-terminal amino acid sequences.
The molecular weight of CMⅠand CMⅡ were 55.3kD and 53.1kD respectively, and their optimum temperature and pH were the same as 55℃ and 5.0. The Km values of CMⅠunder 37℃ was 0.42mmol/L, and the Km values of CMⅡunder 37℃ was 0.79mmol/L. The activity of CMⅠand CMⅡ could be inhibited by Cu2+, Fe2+, Al3+, and Zn2+ . The specific activity of CMⅡ were higher than that of CMⅠ. The partial N-terminal amino acid sequence of two phytases were identical .
The N-glycosylation analysis was performed with Endoglycosidase H, CMⅠ and CMⅡwere about 13.1% and 10.9% glycosylated respectively (calculations were made on the basis of original molecular weight before deglycosylation). It was testified that the difference between their molecular weight were induced by the different extents of N-glycosylation. After deglycosylation, it was found that the specific activity of CMⅠwas reduced to 81 percent of original specific activity of CMⅠ, while deglycosylation had little effect on the specific activity of CMⅡ. And the specific activity of CMⅡ was still higher than that of CMⅠ after deglycosylation. It showed that N-glycosylation of the phytases could stabilitate, even enhance the specific activity of the two phytases.
According to the Crystal Screen, crystal birth was tested with 38 kinds of crystal conditions. Under the 12nd condition (i.e. 30% 2-Propanol, 0.1mol/L NaHepes pH7.5, and 0.2 mol/L MgCl2), the crystal of CMⅡ was obtained.
Bae et al reported a staining method for detecting phytase activity. Here the method was improved, and a novel SDS-PAGE counterstaining, which was SDS-PAGE and ferrous sulfate-ammonium molybdate counterstaining, was established to detect phytase activity specially.
After thermal denaturation and SDS-PAGE, the phytases could recover their activity partially. The mechanism of renaturation was studied with SDS-PAGE counterstaining. It
showed that Triton X-100 and low temperature had played key roles in refolding the phytases. This provided a new evidence for understanding protein refolding mechanism and the theory of the primary structure of proteins determining the advanced structure.
引文
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[4]马玺,单安山.植酸酶研究进展及其在饲料工业中的应用. 粮食与饲料工业，2001，4：27-30.
[5]朱靖环,杨永红,毛华明. 植酸酶的研究与应用进展. 微生物学杂志，2002，22(1)：43-46.
[6]黄遵锡，章克昌. 植酸酶基础与应用研究概况. 食品与发酵工业，1998，25(2)：54-59.
[7]黄遵锡，章克昌. 植酸酶作用机理初探. 工业微生物，1999，29(4)：12-14.
[8]Young-OK Kim, Hyung-Kwoun Kim, Kyung-Sook Bae, et al.Purification and properties of a thermostable phytase from Bacillus sp.DS11. Enzyme and Microbial Technology，1998，22：2-7.
[9]苗雪霞，李喜梅，王金荣，等.根霉植酸酶的分离纯化和酶学性质研究.农业大学学报，1998，21(3)：53-58.
[10]王亚茹，姚斌，曹虹，等.枯草芽孢杆菌中植酸酶的纯化和酶学性质.微生物学报，2001，41(2)：198-203.
[11]Anne Casey, Gary Walsh . Purification and characterization of extrecelluar phytase from Aspergillus niger ATCC 9142. Bioresource Technology，2003，86：183-188.
[12]李明刚，郝亚恒，但野利秋.番茄植酸酶分离提纯及其特性研究.烟台大学学报，1998，11(4)：258-266.
[13]陈惠，王鸿宁，谢晶.黑曲霉N25的分离纯化及酶学性质研究.四川农业大学学报，2001，19(1)：1-3.
[14]Raf Greiner, Edith Haller, Ursula Konietzny, et al.Purification and characterization of phytase from Klebsiella terrigena. ARCHIVES OF BIOCHEMISTRY AND BIOPHYNICS，1997，341（2）： 201-206.
[15]Vosilios M.E.Andriotis, James D.Ross. Isolation and characterization of phytase from domant Corylus avellana seeds. Phytochemistry，2003，64：689-699.
[16]Maria De Angelis, Giovanna Gallo, Maria Rosaria Corbo, et al. Phytase activity in sourdough lactic acid bacteria：purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1. International Journal of Food Microbiology，2003，87：259-270.
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