CN116640745B - 透明质酸酶突变体及其在水解硫酸软骨素中的应用 - Google Patents
透明质酸酶突变体及其在水解硫酸软骨素中的应用 Download PDFInfo
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Abstract
本发明涉及透明质酸酶突变体及其在水解硫酸软骨素中的应用,属于生物工程技术领域。本发明的透明质酸酶突变体是将位于水蛭来源透明质酸酶的底物结合口袋中的氨基酸残基进行突变后得到的蛋白质,具体是以氨基酸序列SEQ ID NO.1所示的透明质酸酶为出发序列,将第65位的苯丙氨酸突变为丝氨酸;和/或将第180位苏氨酸突变为精氨酸。本发明应用定向进化技术和方法对透明质酸酶进行了改造,其中,所得双突变体对于硫酸软骨素的催化效率提高了15.9倍,并将其应用于低分子量硫酸软骨素的水解催化,具有重要的应用前景。
Description
技术领域
本发明涉及生物工程技术领域,尤其是指透明质酸酶突变体及其在水解硫酸软骨素中的应用。
背景技术
硫酸软骨素(Chondroitin Sulfate),由葡萄糖醛酸和N-乙酰氨基半乳糖通过β-1,3和β-1,4糖苷键交替连接,并经过不同位点的磺酸化修饰而成。高分子量硫酸软骨素目前被广泛用于治疗骨关节炎、抗血栓生成以及促进伤口愈合。天然来源的硫酸软骨素具有高度的多分散性,即使从单一来源分离出来,链长也存在显著差异。此外,高分子量硫酸软骨素溶解度较低,不利于消化系统的吸收,还会导致FXII因子的激活和血小板聚集。与高分子量硫酸软骨素相比,低分子量硫酸软骨素具有更好的生物相容性与生物活性。口服低分子量硫酸软骨素更容易被胃肠道吸收,可比天然硫酸软骨素更有效地到达关节软骨中用于治疗关节炎。同样地,使用低分子量硫酸软骨素可防止在肝脏的积聚并提高其肾滤过的效率。
低分子量硫酸软骨素的制备方法主要分为两类:化学降解法和生物酶解法。化学降解法是目前商业化生产低分子量硫酸软骨素的主要方式,其中NaNO2是最常用的解聚剂,其在酸性溶液中具有强氧化性。尽管化学法生产低分子量硫酸软骨素的工艺已经十分成熟,但也存在比如脱硫酸基破坏结构、产品组分单一性差、环境污染等问题。相比于化学降解法,生物酶解法具有反应条件温和、无污染、工艺简单、产品分子量易于控制、不会破坏硫酸活性基团等优势。
除了用于酶法制备低分子量硫酸软骨素,硫酸软骨素降解酶也被用于治疗脊髓损伤、抑制黑色素瘤增殖、缓解椎间盘突出等。目前对于硫酸软骨素降解酶的研究集中在细菌来源的硫酸软骨素裂解酶,它们通过β-消除反应裂解硫酸软骨素中的β-1,4糖苷键。然而,它们的临床应用仍面临着存在免疫原性、稳定性差和体内活性低等诸多问题。因此,开发更多具有特定活性的硫酸软骨素降解酶对于研究硫酸软骨素的功能与应用,以及其本身在医学上的作用具有重要的科学价值。
发明内容
为了解决上述问题,本发明提供了透明质酸酶突变体及其在水解硫酸软骨素中的应用,本发明的透明质酸酶突变体是以水蛭来源的透明质酸酶为亲本,将亲本的第65位苯丙氨酸和/或180位苏氨酸进行突变得到的。该透明质酸酶突变体可高效水解硫酸软骨素成低分子量的硫酸软骨素。
本发明通过以下技术方案实现:
本发明第一个目的是提供透明质酸酶突变体,以氨基酸序列SEQ ID NO.1所示的透明质酸酶为出发序列,将第65位的苯丙氨酸突变为丝氨酸;和/或将第180位苏氨酸突变为精氨酸。
本发明第二个目的是提供编码所述透明质酸酶突变体的基因。
本发明第三个目的是提供携带所述基因的重组表达载体。
在本发明的一个实施例中,所述质粒包括但不限于pGAPm-sp23。
本发明第四个目的是提供表达所述透明质酸酶突变体或含有所述基因的微生物细胞。
在本发明的一个实施例中,以大肠杆菌、酿酒酵母、毕赤酵母为宿主细胞。
在本发明的一个实施例中,所述宿主细胞为毕赤酵母GS115。
本发明第五个目的是提供一种水解硫酸软骨素的方法,以所述透明质酸酶突变体为催化剂,将硫酸软骨素转化为低分子量硫酸软骨素。
在本发明的一个实施例中,所述硫酸软骨素底物浓度为10g/L~40g/L;所述透明质酸酶突变体的添加量为5.0×103U/L~3.0×104U/L。
在本发明的一个实施例中,所述转化是在30℃~40℃条件下进行1h~6h。
在本发明的一个实施例中,所述低分子量硫酸软骨素不含双键;所述低分子量硫酸软骨素的分子量为3.6kDa~21.3kDa。
本发明第六个目的是提供所述的透明质酸酶突变体,或所述的基因,或所述的重组表达载体,或所述微生物细胞在制备低分子量硫酸软骨素的产品中的应用。
在本发明的一种实施方式中,将第65位的苯丙氨酸突变为丝氨酸,并将第180位苏氨酸突变为精氨酸的透明质酸酶突变体的氨基酸序列如SEQ IDNo.2所示;编码该透明质酸酶突变体的基因的核苷酸序列如SEQ ID NO.3所示。
将第65位的苯丙氨酸突变为丝氨酸的透明质酸酶突变体的氨基酸序列如SEQ IDNo.4所示;
将第180位苏氨酸突变为精氨酸的透明质酸酶突变体的氨基酸序列如SEQ IDNo.5所示;
本发明的上述技术方案相比现有技术具有以下优点:
本发明提供了水蛭透明质酸酶3种突变体F65S、F65S/T180R、T180R,相比野生型,突变体的催化效率kcat/Km分别提高了11.3倍、15.9倍、10.1倍。用获得的突变体制备低分子量硫酸软骨素,发现有良好的效果,控制添加的酶量和反应的时间,即可得到不同分子量的低分子量硫酸软骨素。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中,
图1是本发明实施例2中SDS-PAGE分析单突变体蛋白纯化图;
图2是本发明实施例2中SDS-PAGE分析野生型及突变体蛋白纯化图;
图3是本发明实施例3中野生型透明质酸酶对底物硫酸软骨素的动力学参数;
图4是本发明实施例3中透明质酸酶突变体F65S对底物硫酸软骨素的动力学参数;
图5是本发明实施例3中透明质酸酶突变体T180R对底物硫酸软骨素的动力学参数;
图6是本发明实施例3中透明质酸酶突变体F65S/T180R对底物硫酸软骨素的动力学参数;
图7是本发明实施例4中不同酶活条件下野生型WT对CS水解的时间过程;
图8是本发明实施例4中不同酶活条件下F65S/T180R突变体对CS水解的时间过程;
图9本发明实施例4中不同酶活条件下F65S突变体对CS水解的时间过程;
图10本发明实施例4中不同酶活条件下T180R突变体对CS水解的时间过程。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。
材料与方法如下:
LB培养基(g·L-1):酵母提取物5、胰蛋白胨10、氯化钠10,固体培养基中添加2%(w/w)琼脂粉。
YPD培养基(g·L-1):酵母提取物10、胰蛋白胨20、葡萄糖20;其中葡萄糖配成10×母液单独灭菌,固体培养基中添加2%(w/w)琼脂粉。
改良型BMGY培养基(g·L-1):酵母提取物10.0、胰蛋白胨20.0、甘油40.0、酵母无氨基酸氮源(YNB)13.4、D-生物素4.0×10-4、100mM K2HPO4-KH2PO4缓冲液(pH 6.0),其中D-生物素配成500×母液过滤除菌,YNB配成10×母液单独灭菌后加入。
动力学参数的测定:测定一定浓度的透明质酸酶野生型或突变体在不同硫酸软骨素底物浓度下的初始反应速率,底物为40μL硫酸软骨素(2.0-20.0mg·mL-1,50mM柠檬酸-磷酸氢二钠,pH 5.0),加入10μL适当浓度的纯化蛋白,37℃反应30min,加入100μL DNS试剂终止反应,PCR仪中98℃反应10min,96孔酶标板中测定540nm下吸光值。使用GraphPadPrism7软件进行Michaelis-Menten方程非线性拟合,得到Km及Vmax值,Vmax/[E]为kcat值,[E]代表蛋白的摩尔浓度。
硫酸软骨素分子量测定:使用凝胶排阻色谱检测硫酸软骨素的降解以及测定硫酸软骨素寡糖的分子量。测定分子量时,使用50mM柠檬酸-磷酸氢二钠缓冲液(pH 5.0)溶解硫酸软骨素,底物浓度为20mg·mL-1,加入不同终酶活的突变体F65S/T180蛋白,37℃反应,反应过程中定时取样,煮沸样品使蛋白灭活,12000rpm离心10min,上清液使用0.22μm水系滤膜去除杂质后测定样品分子量。高效液相色谱条件:色谱柱为G2000SWxl(7.8×300mm,5μm),柱温为25℃;以浓度0.1M的NaNO3溶液为流动相,设定流速0.8mL·min-1;进样量为20μL;使用示差检测器检测。以不同分子量的葡聚糖作为标准样品,制作不同标样洗脱时间与分子量之间的标准曲线。
实施例1:透明质酸酶突变体质粒的构建
(1)、以之前构建的质粒pPIC9K-His-HaseA3887(专利申请号:201310597818.1)为模板,设计引物对LHyal-F/R,PCR扩增得到野生型透明质酸酶基因片段。以之前构建的质粒pGAP(m)-sp23-HAase(专利申请号:201811172714.5)为模板,设计引物对GAP(m)-sp23-F/R,进行PCR扩增,得到载体骨架片段。利用重组克隆试剂盒,一步法无缝连接载体骨架片段及野生型透明质酸酶基因片段,获得pGAP(m)-sp23-LHyal。
(2)、以含有野生型透明质酸酶基因的重组质粒pGAP(m)-sp23-LHyal为模板,对氨基酸序列的第65位的苯丙氨酸、第180位的苏氨酸进行定点突变,以及对氨基酸序列的第65位的苯丙氨酸和第180位的苏氨酸进行定点突变,分别得到三种基因突变F65S、T180R及F65S/T180R,引物序列见表1。使用限制性内切酶DpnI消化模板质粒后柱纯化回收,将纯化的片段使用平末端磷酸化连接酶试剂盒(Blunting Kination LigationKit)进行连接,接着转化E.coli JM109感受态细胞并涂布含有卡那霉素的LB平板,单克隆子提质粒测序验证。
表1实施例1中所用引物
实施例2:透明质酸酶野生型及突变体蛋白的制备
将实施例1中含有野生型(未定点突变)或三种突变体基因的整合载体用限制性内切酶SalI单酶切线性化,电转化表达宿主Pichiapastoris GS115,转化后涂布于G418抗生素筛选平板,挑选阳性克隆,提取基因组进行PCR验证。挑取单菌落于含有30mLYPD液体培养基的250mL摇瓶,使用恒温振荡培养箱30℃,220rpm培养24h。然后按照10%(v/v)的接种量将种子转接至含有40mL改良型BMGY发酵培养基的250mL摇瓶中,使用恒温振荡培养箱30℃,220rpm继续培养84h。
重组菌培养结束后,将发酵液10000×g,4℃离心30min,收集上清液,然后使用0.22μm水系滤膜过滤进一步除去杂质,使用Ni柱纯化重组蛋白。缓冲液A:20mM PB缓冲液,500mM NaCl,pH 7.4;缓冲液B:20mM PB缓冲液,500mM NaCl,500mM咪唑,pH 7.4。首先使用缓冲液A平衡预装纯化柱HisTrap HP column(GE healthcare),接着上样过膜后的样品,使用缓冲液A冲洗柱子,然后按照缓冲液B浓度4%,8%进行梯度洗脱洗去杂质,目的蛋白在60%的缓冲液B浓度洗脱。将收集的目的蛋白使用Millipore 10kDa浓缩管进行超滤浓缩,然后使用凝胶色谱柱HiLoad 16/600Superdex 200pg column(GE healthcare)进行分子筛纯化。凝胶色谱柱使用缓冲液C平衡(25mM Tris-HCl,150mM NaCl,pH 7.4),上样后,再使用缓冲液C进行洗脱,280nm下监测洗脱过程,收集目的样品,分别得到三种基因突变蛋白F65S、F65S/T180R及T180R,其中图1为F65S和T180R突变体蛋白纯化图;图2为野生型和F65S/T180R突变体蛋白纯化图。
实施例3:透明质酸酶野生型及突变体蛋白
对透明质酸酶野生型WT及突变体进行蛋白纯化后测定了它们的动力学参数。如图3-6所示,相较于WT的Km值(10.9±1.5mg·mL-1),F65S、F65S/T180R及T180R的Km值分别降低至7.2±0.8mg·mL-1、6.1±0.5mg·mL-1和8.1±1.0mg·mL-1,表明突变体对于硫酸软骨素的底物亲和力提高了。此外,F65S及F65S/T180R的催化转换数值(kcat)分别为802.3±42.3min-1和957.5±34.4min-1,是野生型kcat值(106.9±7.8min-1)的7.5倍及9.0倍。转换数以及底物亲和力的提高导致突变体的催化效率(kcat/Km)得到明显提升,F65S和F65S/T180R的kcat/Km值分别是野生型的11.3倍及15.9倍。
实施例4:制备特定分子量分布的低分子量硫酸软骨素
为了考察突变体水解制备低分子量硫酸软骨素的效率,向20g·L-1的硫酸软骨素溶液中加入不同酶活的野生型WT和三种突变体蛋白,并在降解过程中定时取样分析反应体系中硫酸软骨素分子量的变化情况。由图7和图8可知,在反应的初期,与野生型WT对比,F65S/T180R突变体水解的底物的分子量迅速降低,之后逐渐趋于平缓。在1h时,分别添加酶活为5.0×103U·L-1、1.0×104U·L-1和3.0×104U·L-1的F65S/T180R突变体,水解产物的分子量分别是25.1kDa、18.6kDa和7.3kDa。而在相同条件下,野生型WT的分子量分别38.1kDa、35.8kDa和30.1kDa。此外,随着反应时间的延长,水解的反应速率逐渐降低。最终,当添加酶活为5.0×103U·L-1时,野生型WT和F65S/T180R突变体终产物的分子量分别为35.1kDa和21.3kDa;当添加的终酶活为1.0×104U·L-1时,野生型WT和F65S/T180R突变体终产物的分子量为33.2kDa和13.6kDa;当添加酶活为3.0×104U·L-1时,野生型WT和F65S/T180R突变体终产物的分子量为30.1kDa和3.6kDa。
图9为不同酶活条件下F65S突变体对CS水解的时间过程;图10为不同酶活条件下T180R突变体对CS水解的时间过程;F65S和T180R的水解过程与F65S/T180R突变体相似,都随着反应时间延长,分子量逐渐降低。在1h时,分别添加酶活为5.0×103U·L-1、1.0×104U·L-1和3.0×104U·L-1的F65S突变体,水解产物的分子量分别是32.1kDa、24.6kDa和10.3kDa。在1h时,分别添加酶活为5.0×103U·L-1、1.0×104U·L-1和3.0×104U·L-1的F65S突变体,水解产物的分子量分别是30.1kDa、25.6kDa和12.3kDa。最终,当添加酶活为3.0×104U·L-1时,F65S和T180R突变体终产物的分子量为5.6kDa和4.5kDa。
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
1.透明质酸酶突变体,其特征在于,以氨基酸序列SEQ ID NO .1所示的透明质酸酶为出发序列,将第65位的苯丙氨酸突变为丝氨酸;和/或将第180位苏氨酸突变为精氨酸。
2.编码权利要求1所述透明质酸酶突变体的基因。
3.携带权利要求2所述基因的重组表达载体。
4.表达权利要求1所述透明质酸酶突变体或含有权利要求2所述基因的微生物细胞。
5.根据权利要求4所述的微生物细胞,其特征在于,以大肠杆菌、酿酒酵母或毕赤酵母为宿主细胞。
6.一种水解硫酸软骨素的方法,其特征在于,以权利要求1所述透明质酸酶突变体为催化剂,将硫酸软骨素转化为低分子量硫酸软骨素。
7.根据权利要求6所述的方法,其特征在于,所述硫酸软骨素底物浓度为10 g/L~40 g/L;所述透明质酸酶突变体的添加量为5.0 × 103 U/L~3.0 × 104 U/L。
8.根据权利要求6所述的方法,其特征在于,所述转化是在30℃~40℃条件下进行1 h~6h。
9.根据权利要求6所述的方法,其特征在于,所述低分子量硫酸软骨素不含双键;所述低分子量硫酸软骨素的分子量为3.6 kDa~21.3 kDa。
10.权利要求1所述的透明质酸酶突变体,或权利要求2所述的基因,或权利要求3所述的重组表达载体,或权利要求4或5所述微生物细胞在制备低分子量硫酸软骨素的产品中的应用。
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