CN113528607B - 一种化学-酶法制备螺内酯的方法 - Google Patents

一种化学-酶法制备螺内酯的方法 Download PDF

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CN113528607B
CN113528607B CN202110905013.3A CN202110905013A CN113528607B CN 113528607 B CN113528607 B CN 113528607B CN 202110905013 A CN202110905013 A CN 202110905013A CN 113528607 B CN113528607 B CN 113528607B
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王友富
邵振平
王荣
罗敏
王洪福
黄橙橙
雷灵芝
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ZHEJIANG SHENZHOU PHARMACEUTICAL CO Ltd
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Abstract

本发明公开了一种化学‑酶法制备螺内酯的方法，属于有机合成技术领域。本发明提供的化学‑酶法制备螺内酯的方法以4AD为原料，经烯醇醚化、环氧化、内酯化、酶法脱氢和硫代反应得到螺内酯。本发明螺内酯合成工艺路线采用的酶法脱氢的方法专一性好，条件温和，不需要特殊设备，催化速率高；本发明方法所涉及各步反应产物纯化容易，最终产物总质量收率高于92％，HPLC纯度大于99.8％；本发明方法成本低，适合工业化大规模生产，具有良好的经济效益。

Description

一种化学-酶法制备螺内酯的方法
【技术领域】
本发明涉及有机合成技术领域，具体涉及一种制备螺内酯的方法,尤其涉及一种化学和酶法相结合的制备螺内酯的方法。
【背景技术】
螺内酯是一种弱的保钾利尿药，又名安体舒通、螺旋内酯，为白色或类白色细微结晶性粉末，微有苦味，无臭或有轻微硫醇臭。在氯仿中极易溶解，不溶于水，溶于乙醇，易溶于苯或醋酸乙酯。化学结构与醛固酮相似，可与醛固酮竞争远曲小管及集合管细胞浆内的醛固酮受体，影响醛固酮和受体结合，从而阻碍醛固酮诱导蛋白的合成，进而抑制K+-Na+交换，钾的排出减少，起到保钾利尿作用。结构式如式I所示：
Figure BDA0003201323120000011
目前已有关于式(I)化合物螺内酯制备工艺的报道，主要有两种方法，一种是以去氢表雄酮为原料，经乙炔加成，格氏反应增碳、二氧化碳羧化、催化氢化、内酯化等反应得到五元螺环后，然后再经脱氢、加成等反应制得。该合成方法反应条件苛刻，对设备的要求高，不易操作，步骤多，试剂昂贵、总收率低，成本高。
第二种是以雄烯二酮为起始原料，经烯醚化、环氧化、内酯化得到内酯化合物，再经溴化、脱溴化氢，然后加成反应制得；该合成方法在上溴、脱溴过程中使用了溴化剂，对环境不友好，脱溴采用高温脱溴，杂质大，收率低。也有文献报道在3位酮基还是烯醇醚化时使用四氯苯醌脱氢，但是四氯苯醌及副产物四氯氢醌难处理，造成大量污染。
【发明内容】
本发明的目的是为了克服现有技术的不足，提供了一种对环境的污染小，反应选择性高，副产物少，收率高，工业化操作成熟，适合工业化大规模生产的化学-酶法制备螺内酯的方法。
本发明的目的是通过以下方式来实现的：
本发明提供了一种化学-酶法制备螺内酯的方法，该方法的合成路线如下：
Figure BDA0003201323120000021
具体包括如下步骤：
1)将4-雄烯-3,17二酮投入无水乙醇中，加入原甲酸三乙酯和对甲苯磺酸，保温40～45℃反应，待反应完成后，过滤，烘干得到3-乙氧基-雄甾-3,5-二烯-17-酮(1)；
2)投入二甲亚砜、乙醇钠和三甲基溴化锍，0～50℃搅拌半小时，投入3-乙氧基-雄甾-3,5-二烯-17-酮(1)，保温0～50℃反应，待反应完成后，加水，过滤，干燥得17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯(2)；
3)在无水乙醇中投入乙醇钠和丙二酸二乙酯，保温30～80℃反应，投入17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯(2)，保温30～80℃反应完成后，加入氢氧化钠水溶液，回流反应，待反应完成后，加水，过滤，干燥得17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯(3)；
4)将17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯(3)、二甲基四氢呋喃和碱性磷酸酶缓冲溶液构成酶反应体系，加入胆固醇氧化酶、辅酶I、辅酶再生系统和硫酸镁，在28-37℃的温度条件下进行生物酶促反应，整个反应过程控制pH在7.0～7.5，通过HPLC检测生物酶促反应进程，当转化率达到95～99％时，反应结束，经后续处理，得到17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯(4)
5)在17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯(4)中依次投入甲醇和硫代醋酸，回流2-5小时，降温至0℃，过滤，得到的固体使用甲醇结晶精制，干燥得到螺内酯；
进一步，步骤1)中所述无水乙醇的体积用量为底物4-雄烯-3,17二酮重量的0.5～10倍；所述原甲酸三乙酯的体积用量为底物4-雄烯-3,17二酮重量的0.6～3倍；所述对甲苯磺酸的重量用量为底物4-雄烯-3,17二酮重量的0.01～0.1倍。
进一步，步骤2)中所述二甲亚砜的体积用量为底物3-乙氧基-雄甾-3,5-二烯-17-酮(1)重量的2～10倍；所述乙醇钠的重量用量为底物3-乙氧基-雄甾-3,5-二烯-17-酮(1)重量的0.22～0.6倍；所述三甲基溴化锍的重量用量为底物3-乙氧基-雄甾-3,5-二烯-17-酮(1)重量的0.5～1倍。
进一步，步骤3)中所述无水乙醇的体积用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯(2)重量的2～10倍；所述乙醇钠的重量用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯(2)重量的0.21～1倍；所述丙二酸二乙酯的体积用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯(2)重量的0.5～2.3倍；所述氢氧化钠水溶液的质量浓度为10～20％，其体积用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯(2)重量的1～3倍。
进一步，步骤4)中所述生物酶为胆固醇氧化酶，所述辅酶为烟酰胺腺嘌呤二核苷酸，所述辅酶再生系统由葡萄糖和葡萄糖脱氢酶的混合物组成；
进一步，步骤4)中的各物料投料质量比为：
17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯：生物酶＝1：0.006～0.01，
17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯：辅酶I＝1：0.003～0.005，
17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯：葡萄糖：葡萄糖脱氢酶＝1：0.5～1：0.003～0.005；
进一步，所述碱性磷酸酶缓冲溶液pH为7.5；
进一步，所述胆固醇氧化酶包括如SEQ ID NO.1～12中的任一所示的氨基酸序列至少有90％同源性的氨基酸序列；
更进一步，所述胆固醇氧化酶是在非致病性微生物中的表达产物；
更进一步，所述非致病性微生物为大肠杆菌；
进一步，所述后续处理具体为：加入盐酸调节pH值<3，加入乙酸乙酯和活性炭搅拌，过滤，滤饼用乙酸乙酯淋洗，合并滤液和淋洗液，旋蒸浓缩去除有机相，水相中加入乙酸乙酯萃取，萃取液合并旋蒸浓缩至大量晶体析出，过滤，干燥，得到17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯(4)
进一步，步骤5)中所述甲醇的体积用量为底物17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯(4)重量的5～10倍；所述硫代醋酸的体积用量为底物17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯(4)重量的0.5～1倍。
所述葡萄糖脱氢酶通过市购获得。
与现有技术相比，本发明的有益效果是：
1、本发明螺内酯合成工艺路线采用胆固醇氧化酶脱氢的方法，此法专一性好，相对于传统的使用上脱溴的方法需要100℃的高温，本发明反应只需要30℃左右，条件温和，不需要特殊设备，催化速率高，脱氢工序质量收率最高达到88.7％。
2、本发明合成路线步骤3)中脱酯基使用乙醇做溶剂，碱性环境下回流的方法，避免了传统的缩合反应后需先醋酸中和，浓缩去除溶剂，再水析，过滤拿出固体，固体加入DMF和溴化锂高温回流反应，通过本发明改进后，缩短了步骤，反应温度由约150℃降低至78℃，免除了DMF回流导致的安全风险。
3、本发明方法所涉及各步反应产物纯化容易，最终产物总质量收率高于92％，HPLC纯度大于99.8％。
4、本发明方法总体生产成本低，可操作性强，有极高的商业竞争力，适合工业化大规模生产，具有良好的经济效益。
【附图说明】
图1为本发明实施例13中产物螺内酯的HPLC谱图。
图2为本发明实施例14中产物螺内酯的HPLC谱图。
图3为本发明实施例15中产物螺内酯的HPLC谱图。
【具体实施方式】
结合以下实施例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例中未说明具体实验步骤或条件者，按照本领域内的公开文本所描述的常规实验方法的操作即可进行，所用试剂或设备未注明厂商者，均为可以通过市购获得的常规产品。
实例1 3-乙氧基-雄甾-3,5-二烯-17-酮的制备1
将4-雄烯-3,17二酮(4AD)30g投入至15ml无水乙醇中，加入18ml原甲酸三乙酯，0.3g对甲苯磺酸，保温40℃反应，待反应完成后，过滤，烘干得到3-乙氧基-雄甾-3,5-二烯-17-酮31.5g。
实例2 3-乙氧基-雄甾-3,5-二烯-17-酮的制备2
将4-雄烯-3,17二酮(4AD)30g投入至150ml无水乙醇中，加入60ml原甲酸三乙酯，0.15g对甲苯磺酸，保温42℃反应，待反应完成后，过滤，烘干得到3-乙氧基-雄甾-3,5-二烯-17-酮31.3g。
实例3 3-乙氧基-雄甾-3,5-二烯-17-酮的制备3
将4-雄烯-3,17二酮(4AD)30g投入至300ml无水乙醇中，加入90ml原甲酸三乙酯，0.3g对甲苯磺酸，保温45℃反应，待反应完成后，过滤，烘干得到3-乙氧基-雄甾-3,5-二烯-17-酮31.2g。
实例4 17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯的制备1
投入60ml二甲亚砜、6.6g乙醇钠和15g三甲基溴化锍，0℃搅拌半小时，投入3-乙氧基-雄甾-3,5-二烯-17-酮30g，保温0℃反应，待反应完成后，加水，过滤，干燥得17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯30.5g。
实例5 17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯的制备2
投入150ml二甲亚砜、9g乙醇钠和22g三甲基溴化锍，30℃搅拌半小时，投入3-乙氧基-雄甾-3,5-二烯-17-酮30g，保温30℃反应，待反应完成后，加水，过滤，干燥得17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯30.4g。
实例6 17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯的制备3
投入300ml二甲亚砜、18g乙醇钠和30g三甲基溴化锍，50℃搅拌半小时，投入3-乙氧基-雄甾-3,5-二烯-17-酮30g，保温50℃反应，待反应完成后，加水，过滤，干燥得17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯30.3g。
实例7 17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯的制备1
在60ml无水乙醇中投入6.3g乙醇钠和15ml丙二酸二乙酯，保温80℃反应，投入30g17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯，保温80℃反应完成后，加入30ml 20％氢氧化钠水溶液，回流反应，待反应完成后，加水，过滤，干燥得17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯31.3g。
实例8 17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯的制备2
在150ml无水乙醇中投入15g乙醇钠和35ml丙二酸二乙酯，保温50℃反应，投入30g17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯，保温50℃反应完成后，加入60ml 13％氢氧化钠水溶液，回流反应，待反应完成后，加水，过滤，干燥得17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯31.2g。
实例9 17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯的制备3
在300ml无水乙醇中投入30g乙醇钠和69ml丙二酸二乙酯，保温30℃反应，投入30g17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯，保温30℃反应完成后，加入90ml 10％氢氧化钠水溶液，回流反应，待反应完成后，加水，过滤，干燥得17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯31.4g。
实例10 17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯的制备1
将17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯30g溶于180ml二甲基四氢呋喃中，加入180ml 100mM pH7.5的碱性磷酸酶缓冲溶液，开始搅拌，依次加入180mg由SEQ ID NO.3在大肠杆菌中表达得到的胆固醇氧化酶，15g葡萄糖，480mg硫酸镁，90mg葡萄糖脱氢酶，90mg辅酶I，28℃温度条件下进行生物酶促反应，并用40％氢氧化钠溶液控制pH值7.0。5小时后取样检测，转化率为98.2％，加入37％盐酸调节pH值<3，加入180ml乙酸乙酯和15g活性炭搅拌1小时，过滤，滤饼用120ml乙酸乙酯淋洗三遍，合并滤液和淋洗液，旋蒸浓缩去除有机相，水相中加入180ml乙酸乙酯萃取三遍，萃取液合并旋蒸浓缩至大量晶体析出，室温析晶12小时，过滤，干燥，得到17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯26.1g，HPLC纯度99％。
实例11 17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯的制备2
将17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯30g溶于180ml二甲基四氢呋喃中，加入180ml 100mM pH7.5的碱性磷酸酶缓冲溶液，开始搅拌，依次加入240mg由SEQ ID NO.8在大肠杆菌中表达得到的胆固醇氧化酶，24g葡萄糖，480mg硫酸镁，120mg葡萄糖脱氢酶，120mg辅酶I，32℃温度条件下进行生物酶促反应，并用40％氢氧化钠溶液控制pH值7.2。5小时后取样检测，转化率为97.9％，加入37％盐酸调节pH值<3，加入180ml乙酸乙酯和15g活性炭搅拌1小时，过滤，滤饼用120ml乙酸乙酯淋洗三遍，合并滤液和淋洗液，旋蒸浓缩去除有机相，水相中加入180ml乙酸乙酯萃取三遍，萃取液合并旋蒸浓缩至大量晶体析出，室温析晶12小时，过滤，干燥，得到17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯25.8g，HPLC纯度99％。
实例12 17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯的制备3
将17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯-21-羧酸-γ-内酯30g溶于180ml二甲基四氢呋喃中，加入180ml 100mM pH7.5的碱性磷酸酶缓冲溶液，开始搅拌，依次加入300mg由SEQ ID NO.11在大肠杆菌中表达得到的胆固醇氧化酶，30g葡萄糖，480mg硫酸镁，150mg葡萄糖脱氢酶，150mg辅酶I，37℃温度条件下进行生物酶促反应，并用40％氢氧化钠溶液控制pH值7.5。5小时后取样检测，转化率为98.5％，加入37％盐酸调节pH值<3，加入180ml乙酸乙酯和15g活性炭搅拌1小时，过滤，滤饼用120ml乙酸乙酯淋洗三遍，合并滤液和淋洗液，旋蒸浓缩去除有机相，水相中加入180ml乙酸乙酯萃取三遍，萃取液合并旋蒸浓缩至大量晶体析出，室温析晶12小时，过滤，干燥，得到17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯26.6g，HPLC纯度99％。
实例13螺内酯的制备1
在20g17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯中依次投入100ml甲醇和10ml硫代醋酸，回流5小时，降温至0℃，过滤，固体使用甲醇结晶精制，干燥得到螺内酯20.1g，质量收率：100.5％，HPLC纯度99.823％，如图1所示。
实例14螺内酯的制备2
在20g17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯中依次投入150ml甲醇和15ml硫代醋酸，回流4小时，降温至0℃，过滤，固体使用甲醇结晶精制，干燥得到螺内酯19.8g，质量收率：99.0％，HPLC纯度99.848％，如图2所示。
实例15螺内酯的制备3
在20g17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯中依次投入200ml甲醇和20ml硫代醋酸，回流2小时，降温至0℃，过滤，固体使用甲醇结晶精制，干燥得到螺内酯19.7g，质量收率：98.5％，HPLC纯度99.842％，如图3所示。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求的保护范围为准。
序列表
<110> 浙江神洲药业有限公司
<120> 一种化学-酶法制备螺内酯的方法
<130> HFY200350-FM
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 578
<212> PRT
<213> 来源于Mycobacterium sp.的胆固醇氧化酶氨基酸序列(Mycobacteriumsp.)
<400> 1
Met Lys Pro Asp Tyr Asp Val Leu Ile Ile Gly Ser Gly Phe Gly Gly
1 5 10 15
Ser Val Thr Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg Val Gly Val
20 25 30
Leu Glu Ala Gly Arg Arg Phe Ser Asp Glu Glu Phe Ala Lys Thr Ser
35 40 45
Trp Asp Leu Arg Lys Phe Leu Trp Ala Pro Arg Leu Gly Cys Tyr Gly
50 55 60
Ile Gln Arg Ile His Pro Leu Arg Asn Val Met Ile Leu Ala Gly Ala
65 70 75 80
Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu Tyr Val Pro
85 90 95
Pro Glu Pro Phe Phe Ala Asp Gln Gln Trp Ser His Ile Thr Asp Trp
100 105 110
Arg Gly Glu Leu Met Pro His Tyr Gln Gln Ala Gln Arg Met Leu Gly
115 120 125
Val Val Gln Asn Pro Thr Phe Thr Asp Ala Asp Arg Ile Val Lys Glu
130 135 140
Val Ala Asp Glu Met Gly Phe Gly Asp Thr Trp Val Pro Thr Pro Val
145 150 155 160
Gly Val Phe Phe Gly Pro Asp Gly Thr Lys Thr Pro Gly Lys Thr Val
165 170 175
Pro Asp Pro Tyr Phe Gly Gly Ala Gly Pro Ala Arg Thr Gly Cys Leu
180 185 190
Glu Cys Gly Cys Cys Met Thr Gly Cys Arg His Gly Ala Lys Asn Thr
195 200 205
Leu Val Lys Asn Cys Leu Gly Leu Ala Glu Ser Ala Gly Ala Gln Val
210 215 220
Ile Pro Met Thr Thr Val Lys Gly Phe Glu Arg Arg Ser Asp Gly Leu
225 230 235 240
Trp Glu Val Arg Thr Val Arg Thr Gly Ser Trp Leu Arg Arg Asp Arg
245 250 255
Arg Thr Phe Thr Ala Thr Gln Leu Val Leu Ala Ala Gly Thr Trp Gly
260 265 270
Thr Gln His Leu Leu Phe Lys Met Arg Asp Arg Gly Arg Leu Pro Gly
275 280 285
Leu Ser Lys Arg Leu Gly Val Leu Thr Arg Thr Asn Ser Glu Ser Ile
290 295 300
Val Gly Ala Ala Thr Leu Lys Val Asn Pro Asp Leu Asp Leu Thr His
305 310 315 320
Gly Val Ala Ile Thr Ser Ser Ile His Pro Thr Ala Asp Thr His Ile
325 330 335
Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ala Met Gly Leu Leu Gln
340 345 350
Thr Leu Met Thr Asp Gly Ser Gly Pro Gln Gly Thr Asp Val Pro Arg
355 360 365
Trp Arg Gln Leu Leu Gln Thr Ala Ser Gln Asp Pro Arg Gly Thr Ile
370 375 380
Arg Met Leu Asn Pro Arg Gln Trp Ser Glu Arg Thr Val Ile Ala Leu
385 390 395 400
Val Met Gln His Leu Asp Asn Ser Ile Thr Thr Phe Thr Lys Arg Gly
405 410 415
Lys Leu Gly Ile Arg Trp Tyr Ser Ser Lys Gln Gly His Gly Glu Pro
420 425 430
Asn Pro Thr Trp Ile Pro Ile Gly Asn Gln Val Thr Arg Arg Ile Ala
435 440 445
Ala Lys Ile Asp Gly Val Ala Gly Gly Thr Trp Gly Glu Leu Phe Asn
450 455 460
Ile Pro Leu Thr Ala His Phe Leu Gly Gly Ala Val Ile Gly Asp Asp
465 470 475 480
Pro Glu His Gly Val Ile Asp Pro Tyr His Arg Val Tyr Gly Tyr Pro
485 490 495
Thr Leu Tyr Val Val Asp Gly Ala Ala Ile Ser Ala Asn Leu Gly Val
500 505 510
Asn Pro Ser Leu Ser Ile Ala Ala Gln Ala Glu Arg Ala Ala Ser Leu
515 520 525
Trp Pro Asn Lys Gly Glu Thr Asp Arg Arg Pro Pro Gln Gly Glu Pro
530 535 540
Tyr Arg Arg Leu Ala Pro Ile Gln Pro Ala His Pro Val Val Pro Ala
545 550 555 560
Asp Ala Pro Gly Ala Leu Arg Trp Leu Pro Ile Asp Pro Val Ser Asn
565 570 575
Ala Gly
<210> 2
<211> 578
<212> PRT
<213> 来源于Mycobacterium sp.的胆固醇氧化酶氨基酸序列(Mycobacteriumsp.)
<400> 2
Met Lys Pro Asp Tyr Asp Val Leu Ile Ile Gly Ser Gly Phe Gly Gly
1 5 10 15
Ser Val Thr Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg Val Gly Val
20 25 30
Leu Glu Ala Gly Arg Arg Phe Ser Asp Glu Glu Phe Ala Lys Thr Ser
35 40 45
Trp Asp Leu Arg Lys Phe Leu Trp Ala Pro Arg Leu Gly Cys Tyr Gly
50 55 60
Ile Gln Arg Ile His Pro Leu Arg Asn Val Met Ile Leu Ala Gly Ala
65 70 75 80
Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu Tyr Val Pro
85 90 95
Pro Glu Pro Phe Phe Ala Asp Gln Gln Trp Ser His Ile Thr Asp Trp
100 105 110
Arg Gly Glu Leu Met Pro His Tyr Gln Gln Ala Gln Arg Met Leu Gly
115 120 125
Val Val Gln Asn Pro Thr Phe Thr Asp Ala Asp Arg Ile Val Lys Glu
130 135 140
Val Ala Asp Glu Met Gly Phe Ala Asp Thr Trp Val Pro Thr Pro Val
145 150 155 160
Gly Val Phe Phe Gly Pro Asp Gly Thr Lys Thr Pro Gly Lys Thr Val
165 170 175
Pro Asp Pro Tyr Phe Gly Gly Ala Gly Pro Ala Arg Thr Gly Cys Leu
180 185 190
Glu Cys Gly Cys Cys Met Thr Gly Cys Arg His Gly Ala Lys Asn Thr
195 200 205
Leu Val Lys Asn Cys Leu Gly Leu Ala Glu Ser Ala Gly Ala Gln Val
210 215 220
Ile Pro Met Thr Thr Val Lys Gly Phe Glu Arg Arg Ser Asp Gly Leu
225 230 235 240
Trp Glu Val Arg Thr Val Arg Thr Gly Ser Trp Leu Arg Arg Asp Arg
245 250 255
Arg Thr Phe Thr Ala Thr Gln Leu Val Leu Ala Ala Gly Thr Trp Gly
260 265 270
Thr Gln His Leu Leu Phe Lys Met Arg Asp Arg Gly Arg Leu Pro Gly
275 280 285
Leu Ser Lys Arg Leu Gly Val Leu Thr Arg Thr Asn Ser Glu Ser Ile
290 295 300
Val Gly Ala Ala Thr Leu Lys Val Asn Pro Asp Leu Asp Leu Thr His
305 310 315 320
Gly Val Ala Ile Thr Ser Ser Ile His Pro Thr Ala Asp Thr His Ile
325 330 335
Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ala Met Gly Leu Leu Gln
340 345 350
Thr Leu Met Thr Asp Gly Ser Gly Pro Gln Gly Thr Asp Val Pro Arg
355 360 365
Trp Arg Gln Leu Leu Gln Thr Ala Ser Gln Asp Pro Arg Gly Thr Ile
370 375 380
Arg Met Leu Asn Pro Arg Gln Trp Ser Glu Arg Thr Val Ile Ala Leu
385 390 395 400
Val Met Gln His Leu Asp Asn Ser Ile Thr Thr Phe Thr Lys Arg Gly
405 410 415
Lys Leu Gly Ile Arg Trp Tyr Ser Ser Lys Gln Gly His Gly Glu Pro
420 425 430
Asn Pro Thr Trp Ile Pro Ile Gly Asn Gln Val Thr Arg Arg Ile Ala
435 440 445
Ala Lys Ile Asp Gly Val Ala Gly Gly Thr Trp Gly Glu Leu Phe Asn
450 455 460
Ile Pro Leu Thr Ala His Phe Leu Gly Gly Ala Val Ile Gly Asp Asp
465 470 475 480
Pro Glu His Gly Val Ile Asp Pro Tyr His Arg Val Tyr Gly Tyr Pro
485 490 495
Thr Leu Tyr Val Val Asp Gly Ala Ala Ile Ser Ala Asn Leu Gly Val
500 505 510
Asn Pro Ser Leu Ser Ile Ala Ala Gln Ala Glu Arg Ala Ala Ser Leu
515 520 525
Trp Pro Asn Lys Gly Glu Thr Asp Arg Arg Pro Pro Gln Gly Glu Pro
530 535 540
Tyr Arg Arg Leu Ala Pro Ile Gln Pro Ala His Pro Val Val Pro Ala
545 550 555 560
Asp Ala Pro Gly Ala Leu Arg Trp Leu Pro Ile Asp Pro Val Ser Asn
565 570 575
Ala Gly
<210> 3
<211> 578
<212> PRT
<213> 人工氨基酸序列(人工序列)
<400> 3
Met Lys Pro Asp Tyr Asp Val Leu Ile Ile Gly Ser Gly Phe Gly Gly
1 5 10 15
Ser Val Thr Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg Val Gly Val
20 25 30
Leu Glu Ala Gly Arg Arg Phe Ser Asp Glu Glu Phe Ala Lys Thr Ser
35 40 45
Trp Asp Leu Arg Lys Phe Leu Trp Ala Pro Arg Leu Gly Cys Tyr Gly
50 55 60
Ile Gln Arg Ile His Pro Leu Arg Asn Val Met Ile Leu Ala Gly Ala
65 70 75 80
Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu Tyr Val Pro
85 90 95
Pro Glu Pro Phe Phe Ala Asp Gln Gln Trp Ser His Ile Thr Asp Trp
100 105 110
Arg Gly Glu Leu Met Pro His Tyr Gln Gln Ala Gln Arg Met Leu Gly
115 120 125
Val Val Gln Asn Pro Thr Phe Thr Asp Ala Asp Arg Ile Val Lys Glu
130 135 140
Val Ala Asp Glu Met Gly Phe Gly Asp Thr Trp Val Pro Thr Pro Val
145 150 155 160
Gly Val Phe Phe Gly Pro Asp Gly Thr Lys Thr Pro Gly Lys Thr Val
165 170 175
Pro Asp Pro Tyr Phe Gly Gly Ala Gly Pro Ala Arg Thr Gly Cys Leu
180 185 190
Glu Cys Gly Cys Cys Met Thr Gly Cys Arg His Gly Ala Lys Asn Thr
195 200 205
Leu Val Lys Asn Cys Leu Gly Leu Ala Glu Ser Ala Gly Ala Gln Val
210 215 220
Ile Pro Met Thr Thr Val Lys Gly Phe Glu Arg Arg Ser Asp Gly Leu
225 230 235 240
Trp Glu Val Arg Thr Val Arg Thr Gly Ser Trp Leu Arg Arg Asp Arg
245 250 255
Arg Thr Phe Thr Ala Thr Gln Leu Val Leu Ala Ala Gly Thr Trp Gly
260 265 270
Thr Gln His Leu Leu Phe Lys Met Arg Asp Arg Ala Arg Leu Pro Gly
275 280 285
Leu Ser Lys Arg Leu Gly Val Leu Thr Arg Thr Asn Ser Glu Ser Ile
290 295 300
Val Gly Ala Ala Thr Leu Lys Val Asn Pro Asp Leu Asp Leu Thr His
305 310 315 320
Gly Val Ala Ile Thr Ser Ser Ile His Pro Thr Ala Asp Thr His Ile
325 330 335
Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ala Met Gly Leu Leu Gln
340 345 350
Thr Leu Met Thr Asp Gly Ser Gly Pro Gln Gly Thr Asp Val Pro Arg
355 360 365
Trp Arg Gln Leu Leu Gln Thr Ala Ser Gln Asp Pro Arg Gly Thr Ile
370 375 380
Arg Met Leu Asn Pro Arg Gln Trp Ser Glu Arg Thr Val Ile Ala Leu
385 390 395 400
Val Met Gln His Leu Asp Asn Ser Ile Thr Thr Phe Thr Lys Arg Gly
405 410 415
Lys Leu Gly Ile Arg Trp Tyr Ser Ser Lys Gln Gly His Gly Glu Pro
420 425 430
Asn Pro Thr Trp Ile Pro Ile Gly Asn Gln Val Thr Arg Arg Ile Ala
435 440 445
Ala Lys Ile Asp Gly Val Ala Gly Gly Thr Trp Gly Glu Leu Phe Asn
450 455 460
Ile Pro Leu Thr Ala His Phe Leu Gly Gly Ala Val Ile Gly Asp Asp
465 470 475 480
Pro Glu His Gly Val Ile Asp Pro Tyr His Arg Val Tyr Gly Tyr Pro
485 490 495
Thr Leu Tyr Val Val Asp Gly Ala Ala Ile Ser Ala Asn Leu Gly Val
500 505 510
Asn Pro Ser Leu Ser Ile Ala Ala Gln Ala Glu Arg Ala Ala Ser Leu
515 520 525
Trp Pro Asn Lys Gly Glu Thr Asp Arg Arg Pro Pro Gln Gly Glu Pro
530 535 540
Tyr Arg Arg Leu Ala Pro Ile Gln Pro Ala His Pro Val Val Pro Ala
545 550 555 560
Asp Ala Pro Gly Ala Leu Arg Trp Leu Pro Ile Asp Pro Val Ser Asn
565 570 575
Ala Gly
<210> 4
<211> 578
<212> PRT
<213> 人工氨基酸序列(人工序列)
<400> 4
Met Lys Pro Asp Tyr Asp Val Leu Ile Ile Gly Ser Gly Phe Gly Gly
1 5 10 15
Ser Val Thr Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg Val Gly Val
20 25 30
Leu Glu Ala Gly Arg Arg Phe Ser Asp Glu Glu Phe Ala Lys Thr Ser
35 40 45
Trp Asp Leu Arg Lys Phe Leu Trp Ala Pro Arg Leu Gly Cys Tyr Gly
50 55 60
Ile Gln Arg Ile His Pro Leu Arg Asn Val Met Ile Leu Ala Gly Ala
65 70 75 80
Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu Tyr Val Pro
85 90 95
Pro Glu Pro Phe Phe Ala Asp Gln Gln Trp Ser His Ile Thr Asp Trp
100 105 110
Arg Gly Glu Leu Met Pro His Tyr Gln Gln Ala Gln Arg Met Leu Gly
115 120 125
Val Val Gln Asn Pro Thr Phe Thr Asp Ala Asp Arg Ile Val Lys Glu
130 135 140
Val Ala Asp Glu Met Gly Phe Gly Asp Thr Trp Val Pro Thr Pro Val
145 150 155 160
Gly Val Phe Phe Gly Pro Asp Gly Thr Lys Thr Pro Gly Lys Thr Val
165 170 175
Pro Asp Pro Tyr Phe Gly Gly Ala Gly Pro Ala Arg Thr Gly Cys Leu
180 185 190
Glu Cys Gly Cys Cys Met Thr Gly Cys Arg His Gly Ala Lys Asn Thr
195 200 205
Leu Val Lys Asn Cys Leu Gly Leu Ala Glu Ser Ala Gly Ala Gln Val
210 215 220
Ile Pro Met Thr Thr Val Lys Gly Phe Glu Arg Arg Ser Asp Gly Leu
225 230 235 240
Trp Glu Val Arg Thr Val Arg Thr Gly Ser Trp Leu Arg Arg Asp Arg
245 250 255
Arg Thr Phe Thr Ala Thr Gln Leu Val Leu Ala Ala Gly Thr Trp Gly
260 265 270
Thr Gln His Leu Leu Phe Lys Met Arg Asp Arg Gly Arg Leu Pro Gly
275 280 285
Leu Ser Lys Arg Leu Gly Val Leu Thr Arg Thr Asn Ser Glu Ser Ile
290 295 300
Val Gly Ala Ala Thr Leu Lys Val Asn Pro Asp Leu Asp Leu Thr His
305 310 315 320
Gly Val Ala Ile Thr Ser Ser Ile His Pro Thr Ala Asp Thr His Ile
325 330 335
Glu Pro Val Arg Tyr Gly Lys Ala Ser Asn Ala Met Gly Leu Leu Gln
340 345 350
Thr Leu Met Thr Asp Gly Ser Gly Pro Gln Gly Thr Asp Val Pro Arg
355 360 365
Trp Arg Gln Leu Leu Gln Thr Ala Ser Gln Asp Pro Arg Gly Thr Ile
370 375 380
Arg Met Leu Asn Pro Arg Gln Trp Ser Glu Arg Thr Val Ile Ala Leu
385 390 395 400
Val Met Gln His Leu Asp Asn Ser Ile Thr Thr Phe Thr Lys Arg Gly
405 410 415
Lys Leu Gly Ile Arg Trp Tyr Ser Ser Lys Gln Gly His Gly Glu Pro
420 425 430
Asn Pro Thr Trp Ile Pro Ile Gly Asn Gln Val Thr Arg Arg Ile Ala
435 440 445
Ala Lys Ile Asp Gly Val Ala Gly Gly Thr Trp Gly Glu Leu Phe Asn
450 455 460
Ile Pro Leu Thr Ala His Phe Leu Gly Gly Ala Val Ile Gly Asp Asp
465 470 475 480
Pro Glu His Gly Val Ile Asp Pro Tyr His Arg Val Tyr Gly Tyr Pro
485 490 495
Thr Leu Tyr Val Val Asp Gly Ala Ala Ile Ser Ala Asn Leu Gly Val
500 505 510
Asn Pro Ser Leu Ser Ile Ala Ala Gln Ala Glu Arg Ala Ala Ser Leu
515 520 525
Trp Pro Asn Lys Gly Glu Thr Asp Arg Arg Pro Pro Gln Gly Glu Pro
530 535 540
Tyr Arg Arg Leu Ala Pro Ile Gln Pro Ala His Pro Val Val Pro Ala
545 550 555 560
Asp Ala Pro Gly Ala Leu Arg Trp Leu Pro Ile Asp Pro Val Ser Asn
565 570 575
Ala Gly
<210> 5
<211> 578
<212> PRT
<213> 人工氨基酸序列(人工序列)
<400> 5
Met Lys Pro Asp Tyr Asp Val Leu Ile Ile Gly Ser Gly Phe Gly Gly
1 5 10 15
Ser Val Thr Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg Val Gly Val
20 25 30
Leu Glu Ala Gly Arg Arg Phe Ser Asp Glu Glu Phe Ala Lys Thr Ser
35 40 45
Trp Asp Leu Arg Lys Phe Leu Trp Ala Pro Arg Leu Gly Cys Tyr Gly
50 55 60
Ile Gln Arg Ile His Pro Leu Arg Asn Val Met Ile Leu Ala Gly Ala
65 70 75 80
Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu Tyr Val Pro
85 90 95
Pro Glu Pro Phe Phe Ala Asp Gln Gln Trp Ser His Ile Thr Asp Trp
100 105 110
Arg Gly Glu Leu Met Pro His Tyr Gln Gln Ala Gln Arg Met Leu Gly
115 120 125
Val Val Gln Asn Pro Thr Phe Thr Asp Ala Asp Arg Ile Val Lys Glu
130 135 140
Val Ala Asp Glu Met Gly Phe Gly Asp Thr Trp Val Pro Thr Pro Val
145 150 155 160
Gly Val Phe Phe Gly Pro Asp Gly Thr Lys Thr Pro Gly Lys Thr Val
165 170 175
Pro Asp Pro Tyr Phe Gly Gly Ala Gly Pro Ala Arg Thr Gly Cys Leu
180 185 190
Glu Cys Gly Cys Cys Met Thr Gly Cys Arg His Gly Ala Lys Asn Thr
195 200 205
Leu Val Lys Asn Cys Leu Gly Leu Ala Glu Ser Ala Gly Ala Gln Val
210 215 220
Ile Pro Met Thr Thr Val Lys Gly Phe Glu Arg Arg Ser Asp Gly Leu
225 230 235 240
Trp Glu Val Arg Thr Val Arg Thr Gly Ser Trp Leu Arg Arg Asp Arg
245 250 255
Arg Thr Phe Thr Ala Thr Gln Leu Val Leu Ala Ala Gly Thr Trp Gly
260 265 270
Thr Gln His Leu Leu Phe Lys Met Arg Asp Arg Gly Arg Leu Pro Gly
275 280 285
Leu Ser Lys Arg Leu Gly Val Leu Thr Arg Thr Asn Ser Glu Ser Ile
290 295 300
Val Gly Ala Ala Thr Leu Lys Val Asn Pro Asp Leu Asp Leu Thr His
305 310 315 320
Gly Val Ala Ile Thr Ser Ser Ile His Pro Thr Ala Asp Thr His Ile
325 330 335
Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ala Met Gly Leu Leu Gln
340 345 350
Thr Leu Met Thr Asp Gly Ser Gly Pro Gln Gly Thr Asp Val Pro Arg
355 360 365
Trp Arg Gln Leu Leu Gln Thr Ala Ser Gln Asp Pro Arg Gly Thr Ile
370 375 380
Arg Met Leu Asn Pro Arg Gln Trp Ser Glu Arg Thr Val Ile Ala Leu
385 390 395 400
Val Met Gln His Leu Asp Asn Ser Ile Thr Thr Phe Thr Lys Arg Gly
405 410 415
Lys Leu Gly Ile Arg Trp Tyr Ser Ser Lys Gln Gly His Ala Glu Pro
420 425 430
Asn Pro Thr Trp Ile Pro Ile Gly Asn Gln Val Thr Arg Arg Ile Ala
435 440 445
Ala Lys Ile Asp Gly Val Ala Gly Gly Thr Trp Gly Glu Leu Phe Asn
450 455 460
Ile Pro Leu Thr Ala His Phe Leu Gly Gly Ala Val Ile Gly Asp Asp
465 470 475 480
Pro Glu His Gly Val Ile Asp Pro Tyr His Arg Val Tyr Gly Tyr Pro
485 490 495
Thr Leu Tyr Val Val Asp Gly Ala Ala Ile Ser Ala Asn Leu Gly Val
500 505 510
Asn Pro Ser Leu Ser Ile Ala Ala Gln Ala Glu Arg Ala Ala Ser Leu
515 520 525
Trp Pro Asn Lys Gly Glu Thr Asp Arg Arg Pro Pro Gln Gly Glu Pro
530 535 540
Tyr Arg Arg Leu Ala Pro Ile Gln Pro Ala His Pro Val Val Pro Ala
545 550 555 560
Asp Ala Pro Gly Ala Leu Arg Trp Leu Pro Ile Asp Pro Val Ser Asn
565 570 575
Ala Gly
<210> 6
<211> 587
<212> PRT
<213> 来源于Rhodococcus sp. 的胆固醇氧化酶氨基酸序列(Rhodococcus sp. )
<400> 6
Met Thr Ala Gln Asp Glu Lys Phe Arg Leu Ser Arg Arg Gly Phe Met
1 5 10 15
Ala Ala Gly Ala Gly Ala Val Ala Ala Thr Ala Phe Ala Gly Trp Thr
20 25 30
Pro Ala Tyr Ala Val Pro Ala Gly Ser Ser Gly Ser Ala Gly Gly Pro
35 40 45
Val Ser Thr Leu Thr Pro Pro Pro Ala Phe Pro Glu Gly Ile Ala Leu
50 55 60
Tyr Gln Gln Ala Tyr Gln Asn Trp Ser Lys Glu Ile Met Leu Asp Ala
65 70 75 80
Ile Trp Thr Cys Ser Pro Lys Thr Pro Glu Asp Val Val Arg Leu Ala
85 90 95
Asn Trp Gly His Ala Asn Gly Tyr Thr Ile Arg Pro Arg Gly Ala Met
100 105 110
His Gly Trp Thr Pro Leu Thr Ile Val Asn Gly Ala Pro Val Asp Lys
115 120 125
Val Ile Leu Ala Asp Thr Thr Val His Leu Thr Gly Val Ser Val Asn
130 135 140
Ala Gly Gly Ser Pro Ala Thr Val Thr Ala Gly Pro Gly Ala Thr Leu
145 150 155 160
Asp Ala Ile Thr Thr Ala Leu Gln Ala Gln Gly Leu Gly Phe Ala Asn
165 170 175
Leu Pro Ala Pro Gly Val Leu Thr Ile Ala Gly Cys Leu Ala Val Asp
180 185 190
Ala His Gly Ala Ala Leu Pro Ala Glu Gly Glu Ala His Val Pro Gly
195 200 205
Gln Thr Phe Gly Ser Leu Ser Asn Leu Val Thr Ser Leu Thr Ala Val
210 215 220
Val Trp Asn Gly Ser Glu Tyr Ala Leu Glu Thr Tyr Ala Arg Ser Asp
225 230 235 240
Ala Ala Ile Lys Pro Leu Leu Thr His Leu Gly Arg Thr Phe Leu Thr
245 250 255
Ser Val Thr Leu Gln Ala Ala Pro Asn Tyr Arg Met Arg Cys Val Ser
260 265 270
His Thr Asp Ile Gly Trp Gln Glu Leu Phe Gly Ala Arg Gly Ala Ser
275 280 285
Gly Arg Thr Phe Glu Lys Phe Val Arg Glu Asn Gly Arg Ala Glu Ala
290 295 300
Ile Trp Tyr Pro Phe Thr Glu Arg Pro Trp Met Lys Val Trp Ser Leu
305 310 315 320
Ala Pro Thr Lys Pro Pro Phe Ser Arg Glu Val Thr Gly Pro Tyr Asn
325 330 335
Tyr Ile Phe Ser Asp Asn Leu Pro Glu Pro Val Thr Asp Met Ile Gly
340 345 350
Gln Ile Asn Ala Gly Asn Pro Gly Ile Ala Pro Ala Phe Gly Gln Ile
355 360 365
Met Tyr Ala Thr Thr Val Ala Gly Leu Ala Ala Thr Phe Ser Asn Asp
370 375 380
Leu Trp Gly Trp Ser Lys Asp Val Gln Phe Tyr Ile Arg Ala Thr Thr
385 390 395 400
Leu Arg Leu Thr Glu Gly Gly Gly Ala Val Ile Thr Ser Arg Ala Asn
405 410 415
Ile Gly Gln Val Ile His Asp Phe Thr Gln Trp Phe Asn Gly Arg Met
420 425 430
Glu Tyr Tyr Arg Ser Ile Gly Gln Phe Pro Leu Asn Gly Pro Val Glu
435 440 445
Ile Arg Cys Cys Gly Leu Asp Gln Pro Ser Asp Val Glu Val Asp Ser
450 455 460
Ala Gly Ala Pro Thr Ile Ser Ala Met Arg Pro Arg Pro Asp His Pro
465 470 475 480
Glu Trp Asp Thr Ala Ile Trp Leu Asn Val Leu Gly Val Pro Gly Thr
485 490 495
Pro Gly Met Phe Ala Phe Tyr Arg Glu Met Glu Gln Trp Met Arg Asn
500 505 510
His Tyr Asn Asn Asn Asp Ala Thr Phe Arg Pro Glu Trp Ser Lys Gly
515 520 525
Trp Ala Phe Gly Pro Asp Lys Pro Tyr Thr Asp Ala Pro Ile Ile Thr
530 535 540
Gln Gly Leu Pro Gln Thr Tyr Arg Asp Gly Val Pro Ser Ser Asp Asn
545 550 555 560
Trp Asp Thr Ala Asn Ala Ala Tyr Asn Ala Leu Asp Pro His Lys Val
565 570 575
Phe Ser Asn Thr Phe Leu Asp Gln Leu Leu Pro
580 585
<210> 7
<211> 587
<212> PRT
<213> 来源于Rhodococcus sp. 的胆固醇氧化酶氨基酸序列(Rhodococcus sp.)
<400> 7
Met Thr Ala Gln Asp Glu Lys Phe Arg Leu Ser Arg Arg Gly Phe Met
1 5 10 15
Ala Ala Gly Ala Gly Ala Val Ala Ala Thr Ala Phe Ala Gly Trp Thr
20 25 30
Pro Ala Tyr Ala Val Pro Ala Gly Ser Ser Gly Ser Ala Gly Gly Pro
35 40 45
Val Ser Thr Leu Thr Pro Pro Pro Ala Phe Pro Glu Gly Ile Ala Leu
50 55 60
Tyr Gln Gln Ala Tyr Gln Asn Trp Ser Lys Glu Ile Met Leu Asp Ala
65 70 75 80
Ile Trp Thr Cys Ser Pro Lys Thr Pro Glu Asp Val Val Arg Leu Ala
85 90 95
Asn Trp Gly His Ala Asn Gly Tyr Thr Ile Arg Pro Arg Ala Ala Met
100 105 110
His Gly Trp Thr Pro Leu Thr Ile Val Asn Gly Ala Pro Val Asp Lys
115 120 125
Val Ile Leu Ala Asp Thr Thr Val His Leu Thr Gly Val Ser Val Asn
130 135 140
Ala Gly Gly Ser Pro Ala Thr Val Thr Ala Gly Pro Gly Ala Thr Leu
145 150 155 160
Asp Ala Ile Thr Thr Ala Leu Gln Ala Gln Gly Leu Gly Phe Ala Asn
165 170 175
Leu Pro Ala Pro Gly Val Leu Thr Ile Ala Gly Cys Leu Ala Val Asp
180 185 190
Ala His Gly Ala Ala Leu Pro Ala Glu Gly Glu Ala His Val Pro Gly
195 200 205
Gln Thr Phe Gly Ser Leu Ser Asn Leu Val Thr Ser Leu Thr Ala Val
210 215 220
Val Trp Asn Gly Ser Glu Tyr Ala Leu Glu Thr Tyr Ala Arg Ser Asp
225 230 235 240
Ala Ala Ile Lys Pro Leu Leu Thr His Leu Gly Arg Thr Phe Leu Thr
245 250 255
Ser Val Thr Leu Gln Ala Ala Pro Asn Tyr Arg Met Arg Cys Val Ser
260 265 270
His Thr Asp Ile Gly Trp Gln Glu Leu Phe Gly Ala Arg Gly Ala Ser
275 280 285
Gly Arg Thr Phe Glu Lys Phe Val Arg Glu Asn Gly Arg Ala Glu Ala
290 295 300
Ile Trp Tyr Pro Phe Thr Glu Arg Pro Trp Met Lys Val Trp Ser Leu
305 310 315 320
Ala Pro Thr Lys Pro Pro Phe Ser Arg Glu Val Thr Gly Pro Tyr Asn
325 330 335
Tyr Ile Phe Ser Asp Asn Leu Pro Glu Pro Val Thr Asp Met Ile Gly
340 345 350
Gln Ile Asn Ala Gly Asn Pro Gly Ile Ala Pro Ala Phe Gly Gln Ile
355 360 365
Met Tyr Ala Thr Thr Val Ala Gly Leu Ala Ala Thr Phe Ser Asn Asp
370 375 380
Leu Trp Gly Trp Ser Lys Asp Val Gln Phe Tyr Ile Arg Ala Thr Thr
385 390 395 400
Leu Arg Leu Thr Glu Gly Gly Gly Ala Val Ile Thr Ser Arg Ala Asn
405 410 415
Ile Gly Gln Val Ile His Asp Phe Thr Gln Trp Phe Asn Gly Arg Met
420 425 430
Glu Tyr Tyr Arg Ser Ile Gly Gln Phe Pro Leu Asn Gly Pro Val Glu
435 440 445
Ile Arg Cys Cys Gly Leu Asp Gln Pro Ser Asp Val Glu Val Asp Ser
450 455 460
Ala Gly Ala Pro Thr Ile Ser Ala Met Arg Pro Arg Pro Asp His Pro
465 470 475 480
Glu Trp Asp Thr Ala Ile Trp Leu Asn Val Leu Gly Val Pro Gly Thr
485 490 495
Pro Gly Met Phe Ala Phe Tyr Arg Glu Met Glu Gln Trp Met Arg Asn
500 505 510
His Tyr Asn Asn Asn Asp Ala Thr Phe Arg Pro Glu Trp Ser Lys Gly
515 520 525
Trp Ala Phe Gly Pro Asp Lys Pro Tyr Thr Asp Ala Pro Ile Ile Thr
530 535 540
Gln Gly Leu Pro Gln Thr Tyr Arg Asp Gly Val Pro Ser Ser Asp Asn
545 550 555 560
Trp Asp Thr Ala Asn Ala Ala Tyr Asn Ala Leu Asp Pro His Lys Val
565 570 575
Phe Ser Asn Thr Phe Leu Asp Gln Leu Leu Pro
580 585
<210> 8
<211> 587
<212> PRT
<213> 人工氨基酸序列(人工序列)
<400> 8
Met Thr Ala Gln Asp Glu Lys Phe Arg Leu Ser Arg Arg Gly Phe Met
1 5 10 15
Ala Ala Gly Ala Gly Ala Val Ala Ala Thr Ala Phe Ala Gly Trp Thr
20 25 30
Pro Ala Tyr Ala Val Pro Ala Gly Ser Ser Gly Ser Ala Gly Gly Pro
35 40 45
Val Ser Thr Leu Thr Pro Pro Pro Ala Phe Pro Glu Gly Ile Ala Leu
50 55 60
Tyr Gln Gln Ala Tyr Gln Asn Trp Ser Lys Glu Ile Met Leu Asp Ala
65 70 75 80
Ile Trp Thr Cys Ser Pro Lys Thr Pro Glu Asp Val Val Arg Leu Ala
85 90 95
Asn Trp Gly His Ala Asn Gly Tyr Thr Ile Arg Pro Arg Gly Ala Met
100 105 110
His Gly Trp Thr Pro Leu Thr Ile Val Asn Gly Ala Pro Val Asp Lys
115 120 125
Val Ile Leu Ala Asp Thr Thr Val His Leu Thr Gly Val Ser Val Asn
130 135 140
Ala Gly Gly Ser Pro Ala Thr Val Thr Ala Gly Pro Gly Ala Thr Leu
145 150 155 160
Asp Ala Ile Thr Thr Ala Leu Gln Ala Gln Gly Leu Gly Phe Ala Asn
165 170 175
Leu Pro Ala Pro Gly Val Leu Thr Ile Ala Gly Cys Leu Ala Val Asp
180 185 190
Ala His Gly Ala Ala Leu Pro Ala Glu Gly Glu Ala His Val Pro Gly
195 200 205
Gln Thr Phe Gly Ser Leu Ser Asn Leu Val Thr Ser Leu Thr Ala Val
210 215 220
Val Trp Asn Gly Ser Glu Tyr Ala Leu Glu Thr Tyr Ala Arg Ser Asp
225 230 235 240
Ala Ala Ile Lys Pro Leu Leu Thr His Leu Gly Arg Thr Phe Leu Thr
245 250 255
Ser Val Thr Leu Gln Ala Ala Pro Asn Tyr Arg Met Arg Cys Val Ser
260 265 270
His Thr Asp Ile Gly Trp Gln Glu Leu Phe Gly Ala Arg Gly Ala Ser
275 280 285
Gly Arg Thr Phe Glu Lys Phe Val Arg Glu Asn Gly Arg Ala Glu Ala
290 295 300
Ile Trp Tyr Pro Phe Thr Glu Arg Pro Trp Met Lys Val Trp Ser Leu
305 310 315 320
Ala Pro Thr Lys Pro Pro Phe Ser Arg Glu Val Thr Gly Pro Tyr Asn
325 330 335
Tyr Ile Phe Ser Asp Asn Leu Pro Glu Pro Val Thr Asp Met Ile Gly
340 345 350
Gln Ile Asn Ala Gly Asn Pro Ala Ile Ala Pro Ala Phe Gly Gln Ile
355 360 365
Met Tyr Ala Thr Thr Val Ala Gly Leu Ala Ala Thr Phe Ser Asn Asp
370 375 380
Leu Trp Gly Trp Ser Lys Asp Val Gln Phe Tyr Ile Arg Ala Thr Thr
385 390 395 400
Leu Arg Leu Thr Glu Gly Gly Gly Ala Val Ile Thr Ser Arg Ala Asn
405 410 415
Ile Gly Gln Val Ile His Asp Phe Thr Gln Trp Phe Asn Gly Arg Met
420 425 430
Glu Tyr Tyr Arg Ser Ile Gly Gln Phe Pro Leu Asn Gly Pro Val Glu
435 440 445
Ile Arg Cys Cys Gly Leu Asp Gln Pro Ser Asp Val Glu Val Asp Ser
450 455 460
Ala Gly Ala Pro Thr Ile Ser Ala Met Arg Pro Arg Pro Asp His Pro
465 470 475 480
Glu Trp Asp Thr Ala Ile Trp Leu Asn Val Leu Gly Val Pro Gly Thr
485 490 495
Pro Gly Met Phe Ala Phe Tyr Arg Glu Met Glu Gln Trp Met Arg Asn
500 505 510
His Tyr Asn Asn Asn Asp Ala Thr Phe Arg Pro Glu Trp Ser Lys Gly
515 520 525
Trp Ala Phe Gly Pro Asp Lys Pro Tyr Thr Asp Ala Pro Ile Ile Thr
530 535 540
Gln Gly Leu Pro Gln Thr Tyr Arg Asp Gly Val Pro Ser Ser Asp Asn
545 550 555 560
Trp Asp Thr Ala Asn Ala Ala Tyr Asn Ala Leu Asp Pro His Lys Val
565 570 575
Phe Ser Asn Thr Phe Leu Asp Gln Leu Leu Pro
580 585
<210> 9
<211> 599
<212> PRT
<213> 来源于Streptomyces sp. 的胆固醇氧化酶氨基酸序列(Streptomycessp. )
<400> 9
Met Pro Gln Asp Ala Tyr Asp Tyr Asp Val Leu Ile Val Gly Ser Gly
1 5 10 15
Phe Gly Gly Ser Val Ser Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg
20 25 30
Val Gly Val Leu Glu Ala Gly Arg Arg Phe Thr Arg Glu Ser Leu Pro
35 40 45
Lys Asn Ser Trp Asp Leu Lys Asn Tyr Leu Trp Ala Pro Arg Leu Gly
50 55 60
Met Phe Gly Ile Gln Arg Ile His Leu Leu Gly Asn Val Met Val Leu
65 70 75 80
Ala Gly Ala Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu
85 90 95
Tyr Val Pro Pro Lys Pro Phe Phe Asp Asp Pro Gln Trp Arg Gly Ile
100 105 110
Thr Asp Trp His Glu Glu Leu Thr Pro Tyr Tyr Asp Gln Ala Arg Arg
115 120 125
Met Leu Gly Val Arg Leu Asn Pro Thr Met Thr Pro Ser Asp Val His
130 135 140
Leu Lys Ala Ala Ala Glu Lys Leu Gly Cys Gly Asp Thr Phe His Met
145 150 155 160
Thr Pro Val Gly Val Phe Phe Cys Asp Gly Glu Asp Ala Asp Gly Arg
165 170 175
Ala Lys Ala Gly Pro Gly Glu Gln Val Pro Asp Pro Tyr Phe Gly Gly
180 185 190
Ala Gly Pro Asp Arg Lys Ala Cys Asn Glu Cys Gly Glu Cys Met Thr
195 200 205
Gly Cys Arg His Gly Ala Lys Asn Thr Leu Asn Glu Asn Tyr Leu Tyr
210 215 220
Leu Ala Glu Lys Ala Gly Ala Val Val His Pro Met Thr Thr Val Val
225 230 235 240
Ser Val Thr Asp Asp Ser Arg Gly Gly Phe Ala Val Ala Thr Leu Pro
245 250 255
Thr Asp Arg Lys Lys Arg Gly Lys Arg Asp Ala Gly Arg Thr Phe Thr
260 265 270
Ala Arg Arg Val Val Met Ala Ala Gly Thr Tyr Gly Thr Gln Thr Leu
275 280 285
Leu His Arg Met Lys Ala Gly Gly Gln Leu Pro Tyr Leu Ser Asp Lys
290 295 300
Leu Gly Asp Leu Thr Arg Thr Asn Ser Glu Ala Leu Val Gly Ala Gln
305 310 315 320
Thr Asp Asp Arg Arg Tyr Arg Arg Ala Thr Gly Glu Ala Arg Ala Asp
325 330 335
Phe Thr Arg Gly Val Ala Ile Thr Ser Ser Val His Pro Asp Ala Asn
340 345 350
Thr His Ile Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ser Met Gly
355 360 365
Gly Leu Ser Ile Leu Gln Val Pro Tyr Ala Gly Gln Thr Ala Ser Gly
370 375 380
Ala Ser Arg Val Leu Gly Phe Leu Gly His Ala Ala Lys His Pro Leu
385 390 395 400
Leu Val Leu Arg Ser Leu Ser Asn Arg Lys Trp Ser Glu Arg Thr Ile
405 410 415
Ile Gly Leu Val Met Gln Ser Leu Asp Asn Ser Leu Ala Thr His Leu
420 425 430
Lys Pro Thr Cys Val Gly Lys Gly Leu Leu Thr Ala Arg Gln Gly His
435 440 445
Gly Ser Pro Asn Pro Lys Gln Ile Lys Ala Ala Thr Glu Gly Ala Ser
450 455 460
Ala Leu Ala Ala Glu Ile Asn Gly Phe Ala Gly Ser Asn Val Gly Glu
465 470 475 480
Leu Met Gly Thr Pro Leu Thr Ala His Phe Leu Gly Gly Cys Pro Ile
485 490 495
Gly Ala Ser Arg Glu Thr Gly Val Ile Asp Pro Tyr His Arg Leu Tyr
500 505 510
Gly His Pro Gly Ile Ser Val Val Asp Gly Ala Ala Val Ser Ala Asn
515 520 525
Leu Gly Val Asn Pro Ser Leu Thr Ile Thr Ala Gln Ala Glu Arg Ala
530 535 540
Met Ser Tyr Trp Pro Asn Lys Gly Glu Ser Asp Pro Arg Pro Ala Gln
545 550 555 560
Gly Ala Ala Tyr Ala Arg Leu Ala Ala Val Glu Pro His Ala Pro Ala
565 570 575
Val Pro Glu Lys Ala Phe Gly Ala Leu Arg Leu Pro Leu Leu Asp Val
580 585 590
Pro Ala Val Pro Pro Lys Lys
595
<210> 10
<211> 599
<212> PRT
<213> 人工氨基酸序列(人工序列)
<400> 10
Met Pro Gln Asp Ala Tyr Asp Tyr Asp Val Leu Ile Val Gly Ser Gly
1 5 10 15
Phe Gly Gly Ser Val Ser Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg
20 25 30
Val Gly Val Leu Glu Ala Gly Arg Arg Phe Thr Arg Glu Ser Leu Pro
35 40 45
Lys Asn Ser Trp Asp Leu Lys Asn Tyr Leu Trp Ala Pro Arg Leu Gly
50 55 60
Met Phe Gly Ile Gln Arg Ile His Leu Leu Gly Asn Val Met Val Leu
65 70 75 80
Ala Gly Ala Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu
85 90 95
Tyr Val Pro Pro Lys Pro Phe Phe Asp Asp Pro Gln Trp Arg Gly Ile
100 105 110
Thr Asp Trp His Glu Glu Leu Thr Pro Tyr Tyr Asp Gln Ala Arg Arg
115 120 125
Met Leu Gly Val Arg Leu Asn Pro Thr Met Thr Pro Ser Asp Val His
130 135 140
Leu Lys Ala Ala Ala Glu Lys Leu Gly Cys Gly Asp Thr Phe His Met
145 150 155 160
Thr Pro Val Gly Val Phe Phe Cys Asp Gly Glu Asp Ala Asp Gly Arg
165 170 175
Ala Lys Ala Gly Pro Gly Glu Gln Val Pro Asp Pro Tyr Phe Gly Gly
180 185 190
Ala Gly Pro Asp Arg Lys Ala Cys Asn Glu Cys Gly Glu Cys Met Thr
195 200 205
Gly Cys Arg His Gly Ala Lys Asn Thr Leu Asn Glu Asn Tyr Leu Tyr
210 215 220
Leu Ala Glu Lys Ala Gly Ala Val Val His Pro Met Thr Thr Val Val
225 230 235 240
Ser Val Thr Asp Asp Ser Arg Gly Gly Phe Ala Val Ala Thr Leu Pro
245 250 255
Thr Asp Arg Lys Lys Arg Gly Lys Arg Asp Ala Gly Arg Thr Phe Thr
260 265 270
Ala Arg Arg Val Val Met Ala Ala Gly Thr Tyr Gly Thr Gln Thr Leu
275 280 285
Leu His Arg Met Lys Ala Gly Gly Gln Leu Pro Tyr Leu Ser Asp Lys
290 295 300
Leu Gly Asp Leu Thr Arg Thr Asn Ser Glu Ala Leu Val Gly Ala Gln
305 310 315 320
Thr Asp Asp Arg Arg Tyr Arg Arg Ala Thr Gly Glu Ala Arg Ala Asp
325 330 335
Phe Thr Arg Gly Val Ala Ile Thr Ser Ser Val His Pro Asp Ala Asn
340 345 350
Thr His Ile Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ser Met Gly
355 360 365
Gly Leu Ser Ile Leu Gln Val Pro Tyr Ala Gly Gln Thr Ala Ser Gly
370 375 380
Ala Ser Arg Val Leu Gly Phe Leu Gly His Ala Ala Lys His Pro Leu
385 390 395 400
Leu Val Leu Arg Ser Leu Ser Asn Arg Lys Trp Ser Glu Arg Thr Ile
405 410 415
Ile Ala Leu Val Met Gln Ser Leu Asp Asn Ser Leu Ala Thr His Leu
420 425 430
Lys Pro Thr Cys Val Gly Lys Gly Leu Leu Thr Ala Arg Gln Gly His
435 440 445
Gly Ser Pro Asn Pro Lys Gln Ile Lys Ala Ala Thr Glu Gly Ala Ser
450 455 460
Ala Leu Ala Ala Glu Ile Asn Gly Phe Ala Gly Ser Asn Val Gly Glu
465 470 475 480
Leu Met Gly Thr Pro Leu Thr Ala His Phe Leu Gly Gly Cys Pro Ile
485 490 495
Gly Ala Ser Arg Glu Thr Gly Val Ile Asp Pro Tyr His Arg Leu Tyr
500 505 510
Gly His Pro Gly Ile Ser Val Val Asp Gly Ala Ala Val Ser Ala Asn
515 520 525
Leu Gly Val Asn Pro Ser Leu Thr Ile Thr Ala Gln Ala Glu Arg Ala
530 535 540
Met Ser Tyr Trp Pro Asn Lys Gly Glu Ser Asp Pro Arg Pro Ala Gln
545 550 555 560
Gly Ala Ala Tyr Ala Arg Leu Ala Ala Val Glu Pro His Ala Pro Ala
565 570 575
Val Pro Glu Lys Ala Phe Gly Ala Leu Arg Leu Pro Leu Leu Asp Val
580 585 590
Pro Ala Val Pro Pro Lys Lys
595
<210> 11
<211> 599
<212> PRT
<213> 人工氨基酸序列(人工序列)
<400> 11
Met Pro Gln Asp Ala Tyr Asp Tyr Asp Val Leu Ile Val Gly Ser Gly
1 5 10 15
Phe Gly Gly Ser Val Ser Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg
20 25 30
Val Gly Val Leu Glu Ala Gly Arg Arg Phe Thr Arg Glu Ser Leu Pro
35 40 45
Lys Asn Ser Trp Asp Leu Lys Asn Tyr Leu Trp Ala Pro Arg Leu Gly
50 55 60
Met Phe Gly Ile Gln Arg Ile His Leu Leu Gly Asn Val Met Val Leu
65 70 75 80
Ala Gly Ala Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu
85 90 95
Tyr Val Pro Pro Lys Pro Phe Phe Asp Asp Pro Gln Trp Arg Gly Ile
100 105 110
Thr Asp Trp His Glu Glu Leu Thr Pro Tyr Tyr Asp Gln Ala Arg Arg
115 120 125
Met Leu Gly Val Arg Leu Asn Pro Thr Met Thr Pro Ser Asp Val His
130 135 140
Leu Lys Ala Ala Ala Glu Lys Leu Gly Cys Gly Asp Thr Phe His Met
145 150 155 160
Thr Pro Val Gly Val Phe Phe Cys Asp Gly Glu Asp Ala Asp Gly Arg
165 170 175
Ala Lys Ala Gly Pro Gly Glu Gln Val Pro Asp Pro Tyr Phe Gly Gly
180 185 190
Ala Gly Pro Asp Arg Lys Ala Cys Asn Glu Cys Ala Glu Cys Met Thr
195 200 205
Gly Cys Arg His Gly Ala Lys Asn Thr Leu Asn Glu Asn Tyr Leu Tyr
210 215 220
Leu Ala Glu Lys Ala Gly Ala Val Val His Pro Met Thr Thr Val Val
225 230 235 240
Ser Val Thr Asp Asp Ser Arg Gly Gly Phe Ala Val Ala Thr Leu Pro
245 250 255
Thr Asp Arg Lys Lys Arg Gly Lys Arg Asp Ala Gly Arg Thr Phe Thr
260 265 270
Ala Arg Arg Val Val Met Ala Ala Gly Thr Tyr Gly Thr Gln Thr Leu
275 280 285
Leu His Arg Met Lys Ala Gly Gly Gln Leu Pro Tyr Leu Ser Asp Lys
290 295 300
Leu Gly Asp Leu Thr Arg Thr Asn Ser Glu Ala Leu Val Gly Ala Gln
305 310 315 320
Thr Asp Asp Arg Arg Tyr Arg Arg Ala Thr Gly Glu Ala Arg Ala Asp
325 330 335
Phe Thr Arg Gly Val Ala Ile Thr Ser Ser Val His Pro Asp Ala Asn
340 345 350
Thr His Ile Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ser Met Gly
355 360 365
Gly Leu Ser Ile Leu Gln Val Pro Tyr Ala Gly Gln Thr Ala Ser Gly
370 375 380
Ala Ser Arg Val Leu Gly Phe Leu Gly His Ala Ala Lys His Pro Leu
385 390 395 400
Leu Val Leu Arg Ser Leu Ser Asn Arg Lys Trp Ser Glu Arg Thr Ile
405 410 415
Ile Gly Leu Val Met Gln Ser Leu Asp Asn Ser Leu Ala Thr His Leu
420 425 430
Lys Pro Thr Cys Val Gly Lys Gly Leu Leu Thr Ala Arg Gln Gly His
435 440 445
Gly Ser Pro Asn Pro Lys Gln Ile Lys Ala Ala Thr Glu Gly Ala Ser
450 455 460
Ala Leu Ala Ala Glu Ile Asn Gly Phe Ala Gly Ser Asn Val Gly Glu
465 470 475 480
Leu Met Gly Thr Pro Leu Thr Ala His Phe Leu Gly Gly Cys Pro Ile
485 490 495
Gly Ala Ser Arg Glu Thr Gly Val Ile Asp Pro Tyr His Arg Leu Tyr
500 505 510
Gly His Pro Gly Ile Ser Val Val Asp Gly Ala Ala Val Ser Ala Asn
515 520 525
Leu Gly Val Asn Pro Ser Leu Thr Ile Thr Ala Gln Ala Glu Arg Ala
530 535 540
Met Ser Tyr Trp Pro Asn Lys Gly Glu Ser Asp Pro Arg Pro Ala Gln
545 550 555 560
Gly Ala Ala Tyr Ala Arg Leu Ala Ala Val Glu Pro His Ala Pro Ala
565 570 575
Val Pro Glu Lys Ala Phe Gly Ala Leu Arg Leu Pro Leu Leu Asp Val
580 585 590
Pro Ala Val Pro Pro Lys Lys
595
<210> 12
<211> 599
<212> PRT
<213> 人工氨基酸序列(人工序列)
<400> 12
Met Pro Gln Asp Ala Tyr Asp Tyr Asp Val Leu Ile Val Gly Ser Gly
1 5 10 15
Phe Gly Gly Ser Val Ser Ala Leu Arg Leu Thr Glu Lys Gly Tyr Arg
20 25 30
Val Gly Val Leu Glu Ala Gly Arg Arg Phe Thr Arg Glu Ser Leu Pro
35 40 45
Lys Asn Ser Trp Asp Leu Lys Asn Tyr Leu Trp Ala Pro Arg Leu Gly
50 55 60
Met Phe Gly Ile Gln Arg Ile His Leu Leu Gly Asn Val Met Val Leu
65 70 75 80
Ala Gly Ala Gly Val Gly Gly Gly Ser Leu Asn Tyr Ala Asn Thr Leu
85 90 95
Tyr Val Pro Pro Lys Pro Phe Phe Asp Asp Pro Gln Trp Arg Gly Ile
100 105 110
Thr Asp Trp His Glu Glu Leu Thr Pro Tyr Tyr Asp Gln Ala Arg Arg
115 120 125
Met Leu Gly Val Arg Leu Asn Pro Thr Met Thr Pro Ser Asp Val His
130 135 140
Leu Lys Ala Ala Ala Glu Lys Leu Gly Cys Gly Asp Thr Phe His Met
145 150 155 160
Thr Pro Val Gly Val Phe Phe Cys Asp Gly Glu Asp Ala Asp Gly Arg
165 170 175
Ala Lys Ala Gly Pro Gly Glu Gln Val Pro Asp Pro Tyr Phe Gly Gly
180 185 190
Ala Gly Pro Asp Arg Lys Ala Cys Asn Glu Cys Gly Glu Cys Met Thr
195 200 205
Gly Cys Arg His Gly Ala Lys Asn Thr Leu Asn Glu Asn Tyr Leu Tyr
210 215 220
Leu Ala Glu Lys Ala Gly Ala Val Val His Pro Met Thr Thr Val Val
225 230 235 240
Ser Val Thr Asp Asp Ser Arg Gly Gly Phe Ala Val Ala Thr Leu Pro
245 250 255
Thr Asp Arg Lys Lys Arg Gly Lys Arg Asp Ala Gly Arg Thr Phe Thr
260 265 270
Ala Arg Arg Val Val Met Ala Ala Gly Thr Tyr Gly Thr Gln Thr Leu
275 280 285
Leu His Arg Met Lys Ala Gly Gly Gln Leu Pro Tyr Leu Ser Asp Lys
290 295 300
Leu Gly Asp Leu Thr Arg Thr Asn Ser Glu Ala Leu Val Gly Ala Gln
305 310 315 320
Thr Asp Asp Arg Arg Tyr Arg Arg Ala Thr Gly Glu Ala Arg Ala Asp
325 330 335
Phe Thr Arg Gly Val Ala Ile Thr Ser Ser Val His Pro Asp Ala Asn
340 345 350
Thr His Ile Glu Pro Val Arg Tyr Gly Lys Gly Ser Asn Ser Met Gly
355 360 365
Gly Leu Ser Ile Leu Gln Val Pro Tyr Ala Gly Gln Thr Ala Ser Gly
370 375 380
Ala Ser Arg Val Leu Gly Phe Leu Gly His Ala Ala Lys His Pro Leu
385 390 395 400
Leu Val Leu Arg Ser Leu Ser Asn Arg Lys Trp Ser Glu Arg Thr Ile
405 410 415
Ile Gly Leu Val Met Gln Ser Leu Asp Asn Ser Leu Ala Thr His Leu
420 425 430
Lys Pro Thr Cys Val Gly Lys Gly Leu Leu Thr Ala Arg Gln Gly His
435 440 445
Ala Ser Pro Asn Pro Lys Gln Ile Lys Ala Ala Thr Glu Gly Ala Ser
450 455 460
Ala Leu Ala Ala Glu Ile Asn Gly Phe Ala Gly Ser Asn Val Gly Glu
465 470 475 480
Leu Met Gly Thr Pro Leu Thr Ala His Phe Leu Gly Gly Cys Pro Ile
485 490 495
Gly Ala Ser Arg Glu Thr Gly Val Ile Asp Pro Tyr His Arg Leu Tyr
500 505 510
Gly His Pro Gly Ile Ser Val Val Asp Gly Ala Ala Val Ser Ala Asn
515 520 525
Leu Gly Val Asn Pro Ser Leu Thr Ile Thr Ala Gln Ala Glu Arg Ala
530 535 540
Met Ser Tyr Trp Pro Asn Lys Gly Glu Ser Asp Pro Arg Pro Ala Gln
545 550 555 560
Gly Ala Ala Tyr Ala Arg Leu Ala Ala Val Glu Pro His Ala Pro Ala
565 570 575
Val Pro Glu Lys Ala Phe Gly Ala Leu Arg Leu Pro Leu Leu Asp Val
580 585 590
Pro Ala Val Pro Pro Lys Lys
595

Claims (3)

1.一种化学-酶法制备螺内酯的方法，其特征在于，该方法的合成路线如下：
Figure QLYQS_1
，
具体包括如下步骤：
1）将4-雄烯-3,17二酮投入无水乙醇中，加入原甲酸三乙酯和对甲苯磺酸，保温40～45℃反应，待反应完成后，过滤，烘干得到3-乙氧基-雄甾-3,5-二烯-17-酮（1）；
2）投入二甲亚砜、乙醇钠和三甲基溴化锍，0～50℃搅拌半小时，投入3-乙氧基-雄甾-3,5-二烯-17-酮（1），保温0～50℃反应，待反应完成后，加水，过滤，干燥得17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯（2）；
3）在无水乙醇中投入乙醇钠和丙二酸二乙酯，保温30～80℃反应，投入17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯（2），保温30～80℃反应完成后，加入氢氧化钠水溶液，回流反应，待反应完成后，加水，过滤，干燥得17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯 -21-羧酸-γ-内酯（3）；
4）将17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯 -21-羧酸-γ-内酯（3）、二甲基四氢呋喃和碱性磷酸酶缓冲溶液构成酶反应体系，加入胆固醇氧化酶、辅酶I、辅酶再生系统和硫酸镁，在28-37℃的温度条件下进行生物酶促反应，整个反应过程控制pH在7.0~7.5，通过HPLC检测生物酶促反应进程，当转化率达到95~99%时，反应结束，经过后续处理，得到17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯（4）；
5）在17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯（4）中依次投入甲醇和硫代醋酸，回流2-5小时，降温至0℃，过滤，得到的固体用甲醇结晶精制，干燥得到所述的螺内酯；
其中，步骤1）中所述无水乙醇的体积用量为底物4-雄烯-3,17二酮重量的0.5～10倍；所述原甲酸三乙酯的体积用量为底物4-雄烯-3,17二酮重量的0.6～3倍；所述对甲苯磺酸的重量用量为底物4-雄烯-3,17二酮重量的0.01～0.1倍；
步骤2）中所述二甲亚砜的体积用量为底物3-乙氧基-雄甾-3,5-二烯-17-酮（1）重量的2～10倍；所述乙醇钠的重量用量为底物3-乙氧基-雄甾-3,5-二烯-17-酮（1）重量的0.22～0.6倍；所述三甲基溴化锍的重量用量为底物3-乙氧基-雄甾-3,5-二烯-17-酮（1）重量的0.5～1倍；
步骤3）中所述无水乙醇的体积用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯（2）重量的2～10倍；所述乙醇钠的重量用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯（2）重量的0.21～1倍；所述丙二酸二乙酯的体积用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯（2）重量的0.5～2.3倍；所述氢氧化钠水溶液的质量浓度为10～20%，其体积用量为底物17β，20β-环氧-3-乙氧基-17α-孕甾-3,5-二烯（2）重量的1～3倍；
步骤4）中所述辅酶I为烟酰胺腺嘌呤二核苷酸，所述辅酶再生系统由葡萄糖和葡萄糖脱氢酶的按一定比例混合组成；上述各物料投料质量比为：
17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯 -21-羧酸-γ-内酯：胆固醇氧化酶=1：0.006~0.01，
17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯 -21-羧酸-γ-内酯：辅酶I=1：0.003~0.005，
17β-羟基-3-乙氧基-17α-孕甾-3,5-二烯 -21-羧酸-γ-内酯：葡萄糖：葡萄糖脱氢酶=1：0.5~1：0.003~0.005；
所述碱性磷酸酶缓冲溶液pH为7.5；
所述胆固醇氧化酶的氨基酸序列如SEQ ID NO .3、SEQ ID NO .8或SEQ ID NO .11，所述胆固醇氧化酶是在非致病性微生物中的表达产物；
步骤5）中所述甲醇的体积用量为底物17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯（4）重量的5～10倍；所述硫代醋酸的体积用量为底物17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯（4）重量的0.5～1倍。
2.根据权利要求1所述的化学-酶法制备螺内酯的方法，其特征在于，所述非致病性微生物为大肠杆菌。
3.根据权利要求1所述的化学-酶法制备螺内酯的方法，其特征在于，步骤4）中所述的后续处理具体为：加入盐酸调节pH值<3，加入乙酸乙酯和活性炭搅拌，过滤，滤饼用乙酸乙酯淋洗，合并滤液和淋洗液，旋蒸浓缩去除有机相，水相中加入乙酸乙酯萃取，萃取液合并旋蒸浓缩至大量晶体析出，过滤，干燥，得到17β-羟基-17α孕甾-4,6-二烯-3-酮-21-羧酸γ-内酯（4）。
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