CN111197038A - 一种壳聚糖酶的晶体及其制备方法 - Google Patents
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Abstract
本发明涉及一种壳聚糖酶的晶体及其制备方法。采用本发明所述晶体制备方法,可以获得分辨率达到1.6Å的壳聚糖酶晶体。该晶体为棱形形貌,其空间结构为精细三维结构,结构中含有271个氨基酸,含有6个底物结合位点,分别为谷氨酸(E48),谷氨酸(E60),精氨酸(R61),甘氨酸(G82),谷氨酸(E84),缬氨酸(V142),其中谷氨酸(E48)‑天冬氨酸(D64)之间的序列构成了酶的催化结构域。该壳聚糖酶是一类重要的生物催化剂,50℃下半衰期达到4h,在pH 5的醋酸溶液下孵育120min,酶活力保留率在85%以上。该酶可高效的降解壳聚糖,产物为聚合度2‑8的壳寡糖,可被广泛用于日化、食品加工及医药等催化领域。
Description
技术领域
本发明涉结构生物学领域,特别是涉及一种壳聚糖酶的晶体及其制备方法。
背景技术
我国是甲壳素生产大国,甲壳素资源十分丰富,目前每年约生产2000~3000 吨。但我国的甲壳素精深加工水平不高,产品大多仅限于甲壳素和壳聚糖等用于大量出口到国外的初级加工原料,产品的附加值低,行业污染较严重。开发低分子量水溶性壳寡糖是甲壳素和壳聚糖天然资源开发应用的核心问题。低分子量壳寡糖(聚合度2-8)作为壳聚糖的降解产物之一几乎涵盖了壳聚糖的所有优点。科学研究表明,壳寡糖的功能作用和生物活性比壳聚糖高数十倍,它水溶性较好,易于被人体吸收(人体吸收率近100%,壳聚糖吸收率6.48%),具有抗氧化、抑菌、抗肿瘤、抗炎、调节血脂和血糖、增强免疫、活化肠道菌群等多种生理作用,被发达国家称为“软黄金”。
壳聚糖酶是来自于芽孢杆菌的一种重要的生物酶,pH4-6下它能将脱乙酰度90%的壳聚糖完全水解为聚合度2-8的壳寡糖,它与钙、锌、铁等金属离子及表面活性剂具有良好的配伍性,可广泛应用于壳寡糖等海洋甲壳多糖衍生物产品的制备。然而该酶的温度性不理想,45℃下半衰期为72min,限制了其在壳寡糖工业上规模化生产中的应用。
现今壳寡糖主要制备方法有化学法、物理法和生物酶法。其中,化学方法是最早用于工业化生产的制备方法,但是由于甲壳质化学性质的特殊性使其在应用酸解工艺时氨糖分子极易破坏,导致产物中杂质含量升高,难以达到医药级产品的要求,而且废水量较多,处理成本较高。物理法与化学法相比,虽然其可控性好,污染小,但该方法普遍存在的问题是降解初期的速率较快,后期较缓慢,这意味着降解成更小的壳寡糖分子需要耗费很长的时间和较高的能量,而且设备投资较大。生物酶法(壳聚糖酶)降解条件温和,产物较纯,工艺条件可控,易于得到低分子量壳寡糖,且环保,是目前较理想的制备方法。
壳聚糖酶分布广泛,目前已报道产壳聚糖酶的微生物有:细菌:芽孢杆菌(Bacillus
circulans,B subtilis,B.megaterium,B.ehimensis,B cereus,B.pumilus),假单胞菌(Psuedomanas),粘细菌(Myxobacter),肠杆菌(Enterobacter),唐营蒲伯克霍尔德氏菌(Burkholderia gladioli),拟无枝杆菌(Amycolatopsis),金杆菌 (Aureobacterium)等。但是大多数的壳聚糖酶存在活力水平低,高温下不稳定等问题。伴随着新的壳聚糖酶基因的筛选与克隆的困难不断增加,如何在现有基因资源基础之上,运用现代分子生物学技术对已有的壳聚糖基因进行有目的的改造成为当前研究的热点。而获取高质量的壳聚糖酶晶体,确定其精细三维空间结构,找到其催化活性中心与底物结合位点是蛋白分子改造过程中的瓶颈问题之一。
发明内容
本发明的目的在于克服上述现有技术中存在的不足,发明人长期从事微生物酶、蛋白结构及蛋白功能的研究与开发。本发明的目的是提出一种壳聚糖酶的晶体培养方法及通过该方法获得的蛋白晶体的结构数据。
一种壳聚糖酶晶体,为棱形形貌,结构中含有271个氨基酸。酶分子中共含 52.4%α-螺旋,5.1%β折叠与42.4%其它结构(图3)。该酶含有6个底物结合位点,分别为谷氨酸(E48),谷氨酸(E60),精氨酸(R61),甘氨酸(G82),谷氨酸(E84)缬氨酸(V142)。其中谷氨酸(E48)-天冬氨酸(D64)之间的序列构成了酶的催化结构域。该壳聚糖酶晶体中具有 α=β=γ=90°晶包元。
本发明的壳聚糖酶的晶体培养方法如下:
1)结晶池液配制
结晶池液由沉淀剂聚乙二醇8000和pH值缓冲液组成。所述沉淀剂为聚乙二醇8000,质量百分含量为7-11%。pH值缓冲液为磷酸盐缓冲液,缓冲液的摩尔浓度为0.1-0.5M,pH值4.0-5.2;
2)壳聚糖酶蛋白溶液的配制
称取酶活力为30000U/g的壳聚糖酶溶解于lmLMilliporeQ超纯水中,4℃下12000rpm离心10min,除去不溶物质,配制成浓度为10-20毫克/毫升的水溶液;
3)晶体培养
结晶方法采用气相扩散法,具体过程如下:将步骤1所述的结晶缓冲液作为池液加入16孔的Linbro组织培养板内,将1μl步骤2所述的的蛋白溶液与1 μl步骤1所述的池液混合点在硅化好的玻璃片上,将玻璃片翻过来后盖在池液加样孔的上方,二者接触的空间用真空脂密闭。晶体板在16℃严格温控晶体室内进行晶体生长。48h后在冷光源显微镜下观察晶体形态(图1)。
本发明的有益效果:
本发明提供培养获得一种壳聚糖酶晶体的方法,对于明确其三维空间结构,确定它的催化中心与底物结合位点,对有目的的对该壳聚糖酶进行分子改造,提高其温度稳定性有积极的意义。
附图说明
图1壳聚糖酶晶体冷光源显微镜照片;
图2壳聚糖酶晶体三维空间结构;
图3壳聚糖酶的结构域分析。
具体实施方式
本发明用下列实施例来进一步说明本发明,但本发明的保护范围并不限于下列实施例。
实施例1——壳聚糖酶的晶体的制备
1)结晶(生长)池液的配制:配制0.5M、pH为5.0的磷酸缓冲液,其中含沉淀剂聚乙二醇8000的质量百分含量为8%。磷酸盐缓冲液由磷酸氢二钠与磷酸二氢钾组成。
2)壳聚糖酶蛋白溶液的配制
称取酶活力为30000U/g的壳聚糖酶溶解于lmL MilliporeQ超纯水中,4℃下12000rpm离心10min,除去不溶物质,配制成浓度为12毫克/毫升的水溶液;
3)结晶液的配制:
结晶方法采用气相扩散法,具体过程如下:将步骤1所述的结晶缓冲液作为池液加入16孔的Linbro组织培养板内,将1μl步骤2所述的的蛋白溶液与1 μl步骤1所述的池液混合点在硅化好的玻璃片上,将玻璃片翻过来后盖在池液加样孔的上方,二者接触的空间用真空脂密闭。晶体板在16℃严格温控晶体室内进行晶体生长。48h后在冷光源显微镜下观察晶体形态,如图1所示,所得晶体为短棱柱状形貌。
实施例2、3—壳聚糖酶晶体的制备
实施例2、3的制备步骤与实施例1相同,不同是:其中配制0.4M,0.1M、pH 为4.8的磷酸盐缓冲液,含沉淀剂聚乙二醇8000的百分含量分别为12%与14%;配制的壳聚糖酶水溶液分别为10、15mg/ml。
实施例4——壳聚糖酶晶体的结构解析
首先使用HKL2000等软件对上一步骤中收集得到的衍射数据进行处理,获得完整的数据文件;其次,使用CCP4程序包中的Phaser、Molrep等软件,利用分子置换(MR,Molecular Replacement)方法,得到壳聚糖的结构域(图2) 与精细三维结构(图3)。
本发明提供的壳聚糖酶晶体结构衍射数据采集在光源实验室进行。步骤如下:培养得到的壳聚糖酶晶体浸没在30%(v/v)的甘油保护剂中,然后用毛细尼龙环将壳聚糖酶晶体迅速浸入中液氮中进行冷却。当X射线通过晶体后,利用旋进法收集X射线衍射数据(表1)。
表1壳聚糖酶晶体X射线衍射数据;
检测器为日本尼康Q-315CCD检测器,波长收集到晶体的X射线衍射数据后,按照下述步骤进行相应的数据处理:首先使用HKL2000等软件对上一步骤中收集得到的衍射数据进行处理,获得完整的数据文件;其次,使用 CCP4程序包中的Phaser、Molrep等软件,利用分子置换(MR,Molecular Replacement)方法,得到壳聚糖酶的精细三维结构(图2)。
Claims (2)
1.一种壳聚糖酶的晶体,其特征在于该酶晶体为棱形形貌,结构中含有271个氨基酸;酶分子中共含52.4% α-螺旋,5.1% β折叠与42.4%其它结构;该酶含有6个底物结合位点,分别为谷氨酸(E48),谷氨酸(E60),精氨酸(R61),甘氨酸(G82),谷氨酸(E84)缬氨酸(V142);其中谷氨酸(E48)-天冬氨酸(D64)之间的序列构成了酶的催化结构域;该壳聚糖酶晶体中具有a=128.87Å , b=117.35Å, c=96.17Å, α = β = γ = 90˚晶包元。
2.一种权利要求1的壳聚糖酶晶体的制备方法,包括下列步骤:
1)结晶池液配制
结晶池液由沉淀剂聚乙二醇8000和pH 值缓冲液组成;所述沉淀剂为聚乙二醇8000,质量百分含量为7-11%;pH 值缓冲液为磷酸盐缓冲液,缓冲液的摩尔浓度为0.1-0.5M, pH值4.0-5.2;
2)壳聚糖酶蛋白溶液的配制
称取酶活力为30000U/g的壳聚糖酶溶解于lmL MilliporeQ超纯水中,4℃下12000rpm离心10min,除去不溶物质,配制成蛋白浓度为10-20毫克/毫升的水溶液;
3)晶体培养
结晶方法采用气相扩散法,具体过程如下:将步骤1所述的结晶缓冲液作为池液加入16孔的Linbro组织培养板内,将1μl 步骤2所述的的蛋白溶液与1μl 步骤1所述的池液混合点在硅化好的玻璃片上,将玻璃片翻过来后盖在池液加样孔的上方,二者接触的空间用真空脂密闭;晶体板在 16°C 严格温控晶体室内进行晶体生长。
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