CN114410619B - 一种固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法 - Google Patents
一种固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法 Download PDFInfo
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- CN114410619B CN114410619B CN202210140440.1A CN202210140440A CN114410619B CN 114410619 B CN114410619 B CN 114410619B CN 202210140440 A CN202210140440 A CN 202210140440A CN 114410619 B CN114410619 B CN 114410619B
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- hydroxypiperidine
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Abstract
本发明公开了一种固定化生物催化剂合成(S)‑N‑Boc‑羟基哌啶的方法,该方法包括以下步骤:(1)以ZnO纳米线/介孔二氧化硅复合物为载体,同时固定游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH得到固定化的生物催化剂;(2)以N‑Boc‑3‑哌啶酮为底物,加入步骤(1)中得到的固定化的生物催化剂于恒温摇床中反应制备(S)‑N‑Boc‑羟基哌啶,反应结束后过滤回收固定化的生物催化剂重复使用。本发明方法中固定化的醇脱氢酶突变体L114V以及葡萄糖脱氢酶BmGDH的酶共同负载量可达到220mg/g载体,当N‑Boc‑3‑哌啶酮的浓度为500g/l时,转化率高达99.4%,产物的对映体过量值为99.6%。固定化的生物催化剂温度及pH稳定性好,可重复性能好,有效降低工业化生产成本。
Description
技术领域
本发明属于生物技术领域,具体涉及一种高活性及稳定性的固定化生物催化剂制备(S)-N-Boc-羟基哌啶的方法。
背景技术
(S)-N-boc-羟基哌啶((S)-NBHP)是合成Ibrutinib(依鲁替尼)的关键手性中间体,依鲁替尼是经美国食品药品管理局(FDA)突破性药物通道批准的新药,2013年11月13日,FDA批准了Imbruvica可用于套细胞淋巴瘤(MCL)的治疗。此外,(S)-NBHP还可以用于合成一种非天然药物抗充血性心力衰竭药卡莫瑞林、天然物质异白刺啉淋胺(isonitramine)和小果白刺碱(sibirine)等,因此该化合物具有广泛的应用前景。由于化学法的种种弊端,人们寄希望于反应条件温和,反应装置简单,产物对映选择性高的酶催化法,因此开发出一种高效而稳定的酶是目前研究的重点。
目前已报道的酶法制备(S)-N-Boc-羟基哌啶文献较多,其中可最终实现底物N-Boc-3-哌啶酮(NBPO)浓度较高的生物转化有如下:
2016年ZHONG-LIU WU等人从Chryseobacterium sp.CA49基因组中调取27个酮还原酶并筛选获得CHKRED03,将CHKRED03与GDH偶联实现辅因子再循环系统的生物合成方法,最终在加入甲醇助溶的反应体系中实现了底物浓度200g/l的生物转化。(ProcessBiochemistry,2016,51(7):881-885.)
2017年MENGYANH等人通过筛选酮还原酶库,获得来源于(Therm-otogamaritima)的耐高温酮还原酶AKR-43,其催化过程也是利用GDH进行辅酶再生循环利用,并且在加入异丙醇助溶剂的水相体系中实现了底物浓度200g/l的生物转化。(Applied Biochemistryand Biotechnology,2017,181(4):1304-1313.)
2017年LI-FENGCHEN等人分离来自马克斯克鲁维酵母ATCC 748(Kluyveromycesmarxianus ATCC 748)的NADPH依赖性还原酶(YGL039W)生产(R)-N-Boc-3-羟基哌啶显示出优异的催化活性,同样利用GDH进行循环辅酶再生,在反应体系中添加助溶剂异丙醇实现了底物浓度400g/l的生物转化。(Catalysis Communications,2017,97:5-9.)
2017年LI-FENG CHEN等人分离获得来自酿酒酵母(Saccharomy-cescerevisiae)的NADPH依赖性还原酶(YDR541C),采用GDH构建辅酶再生循环,但是在单水相反应体系中发现有严重的产物抑制现象,最终引入1:1(V/V)辛酸乙酯和水的两相体系减轻了产物抑制,实现了底物浓度240g/l的生物转化。(Tetrahedronletters,2017,58(16):1644-1650.)
2018年XIANGXIAN YING等人通过基因组挖掘获得能够催化手性酮的还原酶RECR,其来源于红平红球菌WZ010(Rhodococcuserythropoliswz010),并且作者探索了RECR在手性醇合成中的应用。最后,作者利用RECR突变体Y54F,以异辛醇为共底物构建辅酶循环,在异辛醇两相体系中实现了底物浓度300g/l的生物转化。(Molecules,2018,23(3117):2-13.)
专利CN201310054684.9公开了一种利用醇脱氢酶PAR来不对称合成(S)-1-叔丁氧羰基-3-羟基哌啶,但是利用了有机试剂异丙醇来进行辅酶循环,有机试剂对酶活力有较大程度的损害,且有明显的抑制作用。专利CN201610132936.9公开了一种利用羰基还原酶RECR酶不对称还原酮还原酶(KRED),但是该酶需要经过NI-NTA纯化,且是用仲辛醇-水两相反应,不利于放大生产或生产成本比较高。专利CN108220358A报道的毕赤酵母PICHIASP.SIT2014可作为制备(S)-NBHP的生物催化剂,但是催化剂加量过多增加了生产成本。CN10822061A报道酮还原酶MT-KRED用于制备(S)-NBHP,但在反应过程中需要添加昂贵的辅酶。专利CN110777125A对醇脱氢酶KpADH进行分子改造,以葡萄糖脱氢酶BmGDH偶联实现底物浓度高达600g/l的转化。
以上报道的酮还原酶虽然可以用于制备(S)-NBHP,但是反应过程均使用的是游离酶进行催化且大部分反应过程需要添加昂贵的辅酶,较多的加酶量及有机溶剂,不利于实际工业中的放大应用,也无法进行酶的重复利用反应。
纳米催化是纳米科学和纳米化学中一个令人着迷,有广阔发展前景的领域。纳米粒子由于其尺寸小、比表面大,使其具备了作为非均相催化剂的基本条件,可以应用于能源利用、医药制造、环境保护等领域。但是,由于纳米粒子比表面积较大、表面能非常高,极容易发生团聚。在反应过程中还存在活性组分易流失、催化剂难回收等问题。如果不能解决好这些问题,将无法发挥纳米材料作为催化剂的优势。而纳米线作为一种新型的一维纳米材料,具有高度规整的表面晶体结构,纳米线与纳米粒子相比,保留了纳米粒子的小尺寸效应(直径纳米级),可以具有较高的催化反应活性:同时还引入了体相材料的宏观特性(长度微米级),可以很好地从反应过程中分离,使催化剂的有效反应活性中心得到更充分地利用。在各种一维纳米结构中,具有六角纤铮矿结构的ZnO材料引起人们格外关注。因为ZnO纳米线原材料资源丰富、价格便宜,对环境无毒无害,且其表面积巨大,使其产生了本体块状材料所不具备的表面效应、小尺寸效应和宏观量子隧道效应等。
发明内容
本发明所要解决的技术问题是提供一种高稳定性及活性的固定化生物催化剂用于催化合成(S)-N-Boc-羟基哌啶。
为解决上述技术问题,本发明所采用的的技术方案如下:
通过将来源于Bdellovibrio bacteriovorus菌的醇脱氢酶(BbADH,NCBI登录号WP_015091854.1)氨基酸序列进行三维结构模拟以及与底物的对接分析,利用理性设计的方法在该醇脱氢酶与底物结合位点选择关键位点进行定点突变,获得了该醇脱氢酶的突变体L114V。
所述醇脱氢酶的突变体L114V是在氨基酸序列如SEQ ID NO.2所示的醇脱氢酶上,将114位的亮氨酸(Leu)氨基酸突变为缬氨酸(Val)后得到,其氨基酸序列如SEQ ID NO.3所示。
一种固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,该方法包括以下步骤:
(1)以ZnO纳米线/介孔二氧化硅复合物为载体,固定上述游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH得到固定化的生物催化剂;
(2)以N-Boc-3-哌啶酮为底物,加入步骤(1)中得到的固定化的生物催化剂于恒温摇床中反应制备(S)-N-Boc-羟基哌啶,反应结束后过滤回收固定化的生物催化剂重复使用;
步骤(1)中,以ZnO纳米线/介孔二氧化硅复合物为载体,固定游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH的固定化酶的方法包括如下步骤:
(1a)载体准备:将ZnO纳米线/介孔二氧化硅复合物载体浸入到含有阴离子交联剂的水溶液中,半小时后,将样品从溶液中取出并用蒸馏水洗涤三次以除去水中的游离交联剂;
(1b)游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH共固定化:将吸附有阴离子交联剂的ZnO纳米线/介孔二氧化硅复合物载体浸泡在含有游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH的混合溶液中12~24小时,温度保持在16℃,之后,用去离子水和乙酸钠缓冲溶液洗涤样品,储存在4℃冰箱中。
步骤(1)中所述醇脱氢酶突变体L114V的氨基酸序列如SEQ ID No:3所示,所述葡萄糖脱氢酶BmGDH的氨基酸序列如SEQ ID No:4所示。
步骤(1a)中,所述的ZnO纳米线/介孔二氧化硅复合物载体为小于8mm3的颗粒状载体。
步骤(1a)中,所述的阴离子交联剂为聚乙二醇600,含有聚乙二醇600的水溶液浓度为12mg/ml。
步骤(1b)中,所述含有游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH的混合溶液为15ml~30ml、pH7.0~8.0、浓度为1~10mg/ml的酶溶液,且所述游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH酶浓度比为1:1.5。
步骤(1b)中,所述乙酸钠缓冲液为100mM、pH7.0~8.0的乙酸钠缓冲液。
步骤(2)中,所述固定化的生物催化剂的用量为0.2~1.2mg/ml反应体系,优选为1.0mg/ml。
步骤(2)中,使用的N-Boc-3-哌啶酮浓度为100~500g/l,优选为500g/l。
步骤(2)中,反应条件为pH7.0~8.0、温度30~40℃、摇床转速200~300rpm、时间1~12h,优选为pH7.0、温度37℃、转速250rpm、时间10h。
进一步地,所述固定化的生物催化剂的载体还包括ZnO纳米线/大孔二氧化硅复合载体、ZnO纳米线/介孔二氧化钛复合载体、ZnO纳米线/分子筛MCM复合载体、ZnO纳米线/分子筛SBA复合载体。
ZnO纳米线/介孔二氧化硅复合物载体的制备
采用Zn(Ac)2/聚乙二醇600/H2O三元混合溶液作为前驱物,通过100~200℃温度范围内的两阶段加热使Zn(Ac)2水解,再经过高温煅烧使ZnO晶种在介孔二氧化硅孔壁上形成。以锌氨络合物为锌源,在90℃下热分解后生成的Zn(OH)2沉积在孔道中,并在100℃下利用水热合成原位制备ZnO纳米线,通过改变三元前驱物组分用量以调节ZnO晶种的尺寸与分布,进而控制纳米线的形貌,最终获得了直径为15~20nm的ZnO纳米线,其以无规线团形貌均匀填充于三维孔道中。更详细制备步骤可参考李雪飞等人的报道(李雪飞,尚传洋,张瑞丰.ZnO纳米线/大孔SiO2复合物的制备及吸附性能[J].复合材料学报,2014,31(6):1490-1496)。
有益效果:本发明与现有技术相比,具有如下优势:
1.本发明首次将醇脱氢酶突变体L114V以及葡萄糖脱氢酶BmGDH共固定化在ZnO纳米线/介孔二氧化硅复合载体上。固定化的生物催化剂的酶负载量随着pH7.0的两种游离酶混合溶液浓度的增加而增加,当两种游离酶混合溶液浓度达到8.0mg/ml时,负载量最大值可达220mg/g。
2.经过固定化的生物催化剂中醇脱氢酶酶活可达到421.7U/mg蛋白质,并且固定化后的生物催化剂较游离酶相比展现出了更好的温度和pH稳定性。
3.固定化的生物催化剂,可以在不添加任何外源辅酶和有机助溶剂的单水相体系中将高浓度NBPO(500g/l)催化转化为(S)-NBHP,转化率在pH7.0、37℃条件下10h达到99.4%,(S)-NBHP的对映体过量值为99.6%。
4.固定化的生物催化剂具有相当好的重复性能,经过15次循环后,重复使用后(S)-NBHP转化率仍保持在78.5%。
附图说明
图1为游离酶混合液的浓度对固定化酶负载量的影响。
图2为反应温度对生物催化合成(S)-NBHP的影响。
图3为反应pH对生物催化合成(S)-NBHP的影响。
具体实施方式
根据下述实施例,可以更好地理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
使用气相(Agilent 7820A)测定底物及产物
样品预处理
1.取500μl反应液样品置于1ml离心管中,加入等体积乙酸丁酯;
2.离心管剧烈振荡混匀;
3.步骤2中离心管在12000rpm离心10min分离有机与水相;
4.小心吸取步骤3中上层乙酸丁酯过有机膜,进样分析。
N-Boc-3-哌啶酮与(S)-NBHP、(R)-NBHP手性分析条件为:HYDRODEXβ-TBDAC毛细管柱(25m×0.25mm;anpel),汽化和检测温度220℃,柱头压0.03MPa,氢气0.05MPa,空气0.1MPa,尾吹0.08MPa,柱温130℃保持20min再10℃/min升至150℃保持8min;检测器FID。
N-Boc-3-哌啶酮与NBHP的分析条件为:PEG20M毛细管柱(20m×0.32mm×0.25μm);载气为氮气,分流比1:20;汽化和检测室的温度220℃,柱头压0.03MPa,氢气0.05MPa,空气0.1MPa,尾吹0.08Mpa,柱温170℃保持13min;检测器为FID。
实施例1:醇脱氢酶突变体L114V及葡萄糖脱氢酶BmGDH的获得
亲本BbADH的基因由南京金斯瑞公司合成得到重组到表达载体pET-28a-BbADH(含酶切位点BamH I、Hind III)上,然后转化至大肠杆菌BL21中获得重组表达基因工程菌E.coli BL21-28a-BbADH。
定点突变的引物采用Primer premier 5.0设计,引物设计的原则为:正反向扩增引物5’端包含15-21bp反向互补区域,各引物非互补区域长度至少为15bp,所需引入的突变包含在互补区域内。突变引物如下:
L114V-F:5’-GAACCTTTATCGGTGTGGGACAAGACGGTGCCTAT-3’
L114V-R:5’-CCGTCTTGTCCCACACCGATAAAGGTTCTGCTCG-3’
定点突变以上述pET28a-BbADH重组质粒为模板,利用PrimerStar Mix(ABM公司)进行全质粒扩增,扩增产物经过Dpn I酶(ABM公司)消化去除PCR反应体系中的模板,然后在重组酶催化下将5’端和3’端进行同源重组,完成质粒的环化。定点突变体系如下:
PCR扩增程序:95℃预变性5min,98℃变性10s,62℃退火30s,72℃延伸5min,反应30个循环后,再72℃延伸5min,最后4℃保温。PCR反应结束后,用0.8%的琼脂糖凝胶电泳检测PCR产物。然向每个PCR管中加入1μl Dpn I轻轻混匀后置37℃金属浴2h,之后将消化好的扩增产物进行重组反应。重组反应体系如下:
200ng线性质粒 | 5μl |
5x Ligation Free Cloning(ABM公司) | 4μl |
ddH2O | 补充至20μl |
将环化的扩增产物转入E.coli DH5α感受态细胞中,并涂布在含卡那霉素的平板上,置于37℃培养箱中过夜培养。次日从平板中挑选含有不同突变质粒的E.coli DH5α菌株,用5mL含有相应抗性的液体LB培养后抽提质粒,送金斯瑞公司进行测序。最后将测序正确的突变重组质粒转化E.coli BL21菌株,获得突变体重组基因工程菌株:E.coli BL21-28a-L114Y。获得的BbADH突变体命名为L114V,其氨基酸序列为SEQ ID NO.3所示,编码核苷酸序列为SEQ ID NO.1所示。
同样由金斯瑞公司合成获得含有来源于巨大芽孢杆菌的葡萄糖脱氢酶BmGDH(NCBI登录号:WP_097824161.1)的重组表达基因工程菌E.coli BL21-28a-BbADH,BmGDH氨基酸序列如SEQ ID NO.4所示。
实施例2:游离酶的获得
将上述实施例中获得的重组表达基因工程菌株E.coli BL21-28a-BbADH、E.coliBL21-28a-L114Y及E.coli BL21-28a-BbADH分别接种到含有卡那霉素的10mL LB液体培养基(LB(g/L):蛋白胨10,氯化钠10,酵母提取物5)的50mL的摇管中,在摇床上37℃恒温培养8h,转速200rpm。再将培养菌液按2%接种量接入含有400mL诱导培养基TB中(TB(g/l):酵母粉25,胰蛋白胨15,氯化钠10,葡萄糖2,乳糖3)的1000mL摇瓶中,于200rpm,37℃培养2h,待OD600达到0.6左右时转30℃诱导24h离心收集菌体。再用适量100mM、pH7.0~8.0乙酸钠缓冲液洗菌一次后重悬菌体,超声破菌,离心取上清液为粗酶液,采用BCA试剂盒法测定蛋白质含量后置于4℃冰箱,可用于后续纯化、酶活性测定和生物催化制备(S)-NBHP。
实施例3:Ni柱纯化
采用GE公司的AKTA prime层析系统,使用5mL的HisTrap HP镍柱亲和层析。层析柱用pH 7.4,0.5M NaCl,20mM咪唑,20mM磷酸钠缓冲(缓冲液A)预平衡,使用pH 7.4,0.5MNaCl,0.5M咪唑,20mM磷酸缓冲为洗脱液(缓冲液B),采用0%-100%缓冲B梯度洗脱,将收集的活性蛋白装入透析袋,4℃下边搅拌边透析2h,每半小时更换一次透析缓冲。透析后可直接用于测定蛋白浓度和活性。
实施例4:酶活性测定
亲本醇脱氢酶BbADH以及突变体L114V的酶活测定方法如下:在96孔酶标板上设置200μl应体系包括1mM NADPH,20mM N-Boc-3-哌啶酮,100mM磷酸缓冲液(pH 7.0)。在30℃温浴5分钟后加入适量酶(或固定化生物催化剂)激活反应,使用酶标仪在30℃、波长340nm处检测吸光值变化。以每分钟催化氧化1μmol NADPH所需的酶量为一个活力单位(U)。
葡萄糖脱氢酶的酶活测定方法如下:在96孔酶标板中设置200μl反应体系包括0.5mM的NADP+,100mM葡萄糖,pH 7.0磷酸钠缓冲液,在30℃温浴5分钟后加入适当酶(或固定化生物催化剂)启动反应。于30℃下测定波长340nm处吸光值的上升率。酶活定义为每分钟内还原1μmol NADP+所需要的酶量为一个酶活单位U。
结果测得,亲本醇脱氢酶BbADH粗酶液的活性为102.3U/mg蛋白,而突变体L114V粗酶液活性高达511.6U/mg,比突变前提高了近5倍。
实施例5:固定化生物催化剂的制备
(1)将ZnO纳米线/介孔二氧化硅复合载体切成小颗粒(小于8mm3),将载体浸入含有12mg/ml阴离子交联剂(聚乙二醇600)的水溶液中,半小时后,将样品从溶液中取出并用蒸馏水洗涤三次以除去水中的游离交联剂。
(2)将实施例2中所得粗酶液按蛋白浓度比L114V:BmGDH=1:1.5混合制备得到不同浓度的游离酶混合溶液(1~10mg/ml)。
(3)将吸附有聚乙二醇600的ZnO纳米线/介孔二氧化硅复合物载体浸泡在30ml、pH7.0~8.0游离混合酶溶液中12~24小时,温度保持在16℃,之后,用去离子水和乙酸钠缓冲溶液洗涤样品,储存在4℃冰箱中供下一步实验使用。
实施例6:固定化酶负载量测定
为了测定实施例5中所述的固定化生物催化剂酶的负载量,酶液中蛋白含量用BCA试剂盒法测定,用牛血清蛋白作为标准蛋白,绘制蛋白浓度与吸光度的标准曲线;测定用于反应初始游离酶混合液蛋白浓度C1(mol/ml),固定化反应结束后体系中的游离酶混合液蛋白浓度C2(mol/ml),用于洗涤样品的缓冲溶液中的游离酶混合液蛋白浓度C3(mol/ml);反应体系初始体积V1(ml),用于洗涤样品的缓冲液体积V2(ml),所用复合载体质量为M(g),则单位载体酶负载量为:
实验结果如图1所示,在固定化反应过程中当游离酶混合液浓度为1-8mg/ml时,酶负载量随游离酶混合液浓度增加快速上升,负载量高达220mg/g载体;当游离酶混合液蛋白浓度超过8mg/ml时,酶的负载量不再继续上升基本保持稳定。
当反应体系中加入的固定化生物催化剂为酶负载量达到最大值时的固定化生物催化剂时,测得醇脱氢酶突变体L114V和葡萄糖脱氢酶酶活分别为421.7U/mg、496.1U/mg蛋白质。
实施例6:高浓度底物生物催化实验
设置20ml游离双酶偶联单一水相催化体系:含乙酸钠缓冲100mM pH7.0,N-Boc-3-哌啶酮500g/L,葡萄糖625g/L,ZnCl24mg/L,加入4mg/ml突变体L113V粗酶液以及4mg/mlBmGDH粗酶液,在37℃下250rpm恒温摇床中反应10h。产物(S)-N-boc-3-羟基哌啶的得率为75.6学纯度e.e%为99.5%。
设置20ml固定化生物催化剂单一水相催化体系:含乙酸钠缓冲100mM pH7.0,N-Boc-3-哌啶酮500g/L,葡萄糖625g/L,ZnCl2 4mg/L,加入固定化生物催化剂1.0mg/ml,在37℃下250rpm恒温摇床中反应10h。产物(S)-N-boc-3-羟基哌啶的得率为99.4%,光学纯度e.e%为99.6%。
实施列7:反应温度对生物催化合成(S)-NBHP的影响
以游离双酶偶联生物催化剂以及固定化生物催化剂为催化剂分别设置7组与实施例6中相同的催化体系,仅将恒温摇床的反应温度设置为梯度温度:20℃、25℃、30℃、35℃、40℃、45℃、50℃,10h后结束反应并分别取样检测。
图2显示了(S)-NBHP转化率随反应温度变化的情况。在所有温度范围内,由固定化生物催化剂催化合成(S)-NBHP的效率比游离双酶偶联高得多。游离生物催化剂最适反应温度为35℃,固定化生物催化剂为40℃,该固定化生物催化剂显示出比游离生物催化剂更好的热稳定性。在游离双酶偶联生物催化剂反应中,当温度高于35℃,转化率迅速下降,而固定化的生物催化剂即使在45℃时,转化率仍高达84.6%。因此,与游离生物催化剂相比,固定化生物催化剂即使在高温下活性损失较低。
实施列8:反应pH对生物催化合成(S)-NBHP的影响
以游离双酶偶联生物催化剂和固定化生物催化剂为催化剂分别设置7组与实施例6中相同的催化体系,仅将100mM缓冲溶液pH依梯度改为5.0、5.5、6.0、6.5、7.0、8.0、9.0,恒温摇床的反应温度均改为37℃,10h后结束反应并分别取样检测。
实验结果如图3所示。游离生物催化剂的最佳pH值为6.5,最高(S)-NBHP产率为79.3%,而固定的生物催化剂的最佳pH值移到7.0,(S)-NBHP转化率高达99.4%。结果还表明固定化的生物催化剂与游离生物催化剂相比,在更宽的pH范围内具有更好的适应性。
实施列9:固定化生物催化剂的重复性
将实施例6中反应结束后体系中的固定化生物催化剂过滤出来,用pH7.0乙酸钠缓冲清洗三次,加入新的pH7.0乙酸钠缓冲,N-Boc-3-哌啶酮500g/L,葡萄糖625g/L,ZnCl24mg/L进行催化,检测发现其可以在单一水相中循环使用15次仍能保持78.5%左右的转化率。
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Claims (10)
1.一种固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,该方法包括以下步骤:
(1)以ZnO纳米线/介孔二氧化硅复合物为载体,固定游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH得到固定化的生物催化剂;
(2)以N-Boc-3-哌啶酮为底物,加入步骤(1)中得到的固定化的生物催化剂于恒温摇床中反应制备(S)-N-Boc-羟基哌啶,反应结束后过滤回收固定化的生物催化剂重复使用;
步骤(1)中,以ZnO纳米线/介孔二氧化硅复合物为载体,固定游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH的固定化酶的方法包括如下步骤:
(1a)载体准备:将ZnO纳米线/介孔二氧化硅复合物载体浸入到含有阴离子交联剂的水溶液中,半小时后,将样品从溶液中取出并用蒸馏水洗涤三次以除去水中的游离交联剂;
(1b)游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH共固定化:将吸附有阴离子交联剂的ZnO纳米线/介孔二氧化硅复合物载体浸泡在含有游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH的混合溶液中12~24小时,温度保持在16℃,之后,用去离子水和乙酸钠缓冲溶液洗涤样品,储存在4℃冰箱中;
步骤(1)中所述醇脱氢酶突变体L114V的氨基酸序列如SEQ ID No:3所示。
2.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(1)中所述葡萄糖脱氢酶BmGDH的氨基酸序列如SEQ ID No:4所示。
3.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(1a)中,所述的ZnO纳米线/介孔二氧化硅复合物载体为小于8mm3的颗粒状载体。
4.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(1a)中,所述的阴离子交联剂为聚乙二醇600,含有聚乙二醇600的水溶液浓度为12mg/ml。
5.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(1b)中,所述含有游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH的混合溶液为15ml~30ml、pH7 .0~8 .0、浓度为1~10mg/ml的酶溶液,且所述游离醇脱氢酶突变体L114V以及游离葡萄糖脱氢酶BmGDH酶浓度比为1:1 .5。
6.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(1b)中,乙酸钠缓冲液为100mM、pH7 .0~8 .0的乙酸钠缓冲液。
7.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(2)中,所述固定化的生物催化剂的用量为0 .2~1 .2mg/ml反应体系。
8.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(2)中,使用的N-Boc-3-哌啶酮浓度为100~500g/l。
9.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,步骤(2)中,反应条件为pH7 .0~8 .0、温度30~40℃、摇床转速200~300rpm、时间1~12h。
10.根据权利要求1所述的固定化生物催化剂合成(S)-N-Boc-羟基哌啶的方法,其特征在于,所述固定化生物催化剂的载体还包括ZnO纳米线/大孔二氧化硅复合载体、ZnO纳米线/介孔二氧化钛复合载体、ZnO纳米线/分子筛MCM复合载体、ZnO纳米线/分子筛SBA复合载体。
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