CN116536279A - 一种基因工程菌及在制备去氢表雄酮上的应用 - Google Patents
一种基因工程菌及在制备去氢表雄酮上的应用 Download PDFInfo
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- CN116536279A CN116536279A CN202210103791.5A CN202210103791A CN116536279A CN 116536279 A CN116536279 A CN 116536279A CN 202210103791 A CN202210103791 A CN 202210103791A CN 116536279 A CN116536279 A CN 116536279A
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Abstract
本发明涉及去氢表雄酮的制备技术领域,公开了一种基因工程菌和使用该基因工程菌制备去氢表雄酮的方法。该基因工程菌为将含有SW基因片段的第一质粒、含有相连接的HY基因片段和GDH基因片段的第二质粒转入到宿主菌株中,并可表达SW基因、HY基因和GDH基因的重组菌,可同时表达酮还原酶(SW)、水解酶(HY)、葡萄糖脱氢酶(GDH),利用全细胞催化方法实现去氢表雄酮的高效合成。该方法在常温常压下通过一步反应便可获得去氢表雄酮,避免了中间水解产物的累积,同时利用体系自身实现NADH/NAD+的循环再生。该方法底物的底物浓度可达20~300 g/L,转化率为99%以上,产率95%以上,光学纯度大于99%,具有很好的应用开发前景。
Description
技术领域
本发明涉及去氢表雄酮的制备技术领域,具体涉及一种基因工程菌和使用该基因工程菌制备去氢表雄酮的方法。
背景技术
去氢表雄酮(dehydroepiandrosterone,DHEA)是哺乳动物中一种重要的内源性类固醇激素,用于治疗女性和男性的各种功能障碍,以及类固醇药物合成的中间体。目前合成DHEA的方法主要有化学法、发酵法以及化学-酶法。
传统的化学合成法多从4-雄烯-3-17-二酮(4-Androsten-4-ol-3,17-dione,4-AD)及其衍生物出发,经乙酰化、缩酮保护、还原、水解反应等步骤合成去氢表雄酮,不仅步骤繁琐、收率低,且环境污染严重。如中国专利CN107698643A的专利文献报道的化学法合成路线如下:此方法以4-AD为底物,通过乙酰化、缩酮、还原“一锅法”反应和水解反应制备去氢表雄酮,最终以88%左右的收率得到目标产物,产品中异构体占1~3%。精制收率为78%左右,纯度大于99%,可以看出为去除异构体的精制过程造成收率降低10%左右。另外,该工艺路线需用到大量的醋酐、二氯甲烷等有毒试剂,产生大量有毒废水,且使用到许多昂贵的催化剂如硼氢化镁、硼氢化钙等,工业化生产成本高。
直接利用微生物发酵转化制备去氢表雄酮的方法虽反应条件温和,对环境友好,但往往发酵反应周期长,底物投料量低且分离纯化困难,难以实现工业化生产。如中国专利CN110656146A的专利文献报道的发酵法的生物转化步骤如下:从植物甾醇出发,经3位羟基保护反应、分枝杆菌(Mycobacterium sp.)B-NRRL 3683生长细胞生物转化、水解、精制等步骤制备去氢表雄酮。该路线反应周期太长(10天左右)、底物投料量低(10g/L左右)且分离纯化步骤繁琐、收率低,生产成本高。
新兴的化学-酶法以4-AD为底物,经化学法异构生成5-AD,再利用酶将3位羰基不对称还原来两步法制备去氢表雄酮,此方法在制备5-AD时需用到大量的叔丁醇钾且杂质难除,酶法制备去氢表雄酮时还需添加昂贵的辅酶NAD和NADP,最终收率不高。如中国专利专利CN106086148A报道了化学-酶法合成法,路线如下:以4-AD为底物出发,依次经过化学方法的双键移位生成5-AD和生物方法的羰基还原制备去氢表雄酮,该方法在制备5-AD时反应条件苛刻,需氮气保护,且用到大量的叔丁醇和叔丁醇钾,5-AD的收率和纯度影响最终去氢表雄酮的纯度和收率。
因此开发合适的去氢表雄酮的制备方法,满足操作简洁、成本低并适合工业化生产的需求是去氢表雄酮制备技术发展的重要内容。
发明内容
针对现有去氢表雄酮的制备方法存在的不足,本发明的目的在于提供一种重组的基因工程菌,可同时表达特定基因,能够在在常温常压下通过一步反应便可获得去氢表雄酮。
本发明提供如下的技术方案:
一种酮还原酶SW,所述酮还原酶SW的氨基酸序列如SEQ NO.1所示。
该酮还原酶SW可以将5-雄烯二酮(5-AD)的3位的酮官能团还原成醇,得到去氢表雄酮,其人工氨基酸序列SEQ NO.1如下:
一种编码上述酮还原酶SW的SW基因片段,所述SW基因片段的核苷酸序列如SEQNO.4所示。其人工核苷酸序列SEQ NO.4如下:
一种含有上述SW基因片段的基因工程菌,所述基因工程菌为将含有SW基因片段的第一质粒、含有相连接的HY基因片段和GDH基因片段的第二质粒转入到宿主菌株中,并可表达SW基因、HY基因和GDH基因的重组菌株;
HY基因片段的核苷酸序列如SEQ NO.5所示;
GDH基因片段的核苷酸序列如SEQ NO.6所示。
上述SW基因片段可用于编码酮还原酶SW,HY基因片段可用于编码水解酶HY,GDH基因片段可用于编码葡萄糖脱氢酶GDH,使得重组的基因工程菌株可同时表达酮还原酶(SW)、水解酶(HY)、葡萄糖脱氢酶(GDH),利用全细胞催化方法实现去氢表雄酮的高效合成,其中各基因片段的核苷酸序列和对应所编码的酶的氨基酸序列如下所示。
水解酶HY的人工氨基酸序列SEQ NO.2如下所示:
编码水解酶HY的HY基因片段的人工核苷酸序列SEQ NO.5如下所示:
葡萄糖脱氢酶GDH的人工氨基酸序列SEQ NO.3如下所示:
编码葡萄糖脱氢酶GDH的人工核苷酸序列SEQ NO.6如下所示:
作为本发明的优选,所述第一质粒为pET28a-SW。
作为本发明的优选,所述第二质粒为pETDuet-HY-GDH。
作为本发明的优选,宿主菌株为大肠杆菌BL21(DE3)。
一种使用上述基因工程菌制备去氢表雄酮的方法,包括以下步骤:
将基因工程菌在培养基中扩大培养,结束后分离、收集湿菌体;
然后以雄烯二酮衍生物I为底物与菌体、烟酰胺腺嘌呤二核苷酸、葡萄糖和有机溶剂在水中混合,调节pH值范围为5.5~8,搅拌反应,结束后分离、纯化得到去氢表雄酮;
其中雄烯二酮衍生物I的结构式如下:
上述制备方法中发生的反应过程如下:
作为本发明的优选,基因工程菌的扩大培养过程如下:
(1-1)将基因工程菌接种至含50μg/ml卡那霉素、100μg/ml氨苄霉素的LB液体培养基中,37℃震荡培养8~10h,再以2%接种量(v/v)接种至含50μg/mL卡那霉素、100μg/mL氨苄霉素的种子培养基中,37℃,200rpm培养获得种子液;
(1-2)将种子液以体积浓度10%的接种量接种至装有发酵培养基的发酵罐中,37℃发酵培养14h后,将已灭菌的终浓度为0.6mM异丙基-β-D-硫代吡喃半乳糖苷添加到发酵罐中诱导培养;待培养至OD600达到70~100时放罐、离心分离收集湿菌体;
其中,所述种子培养基和发酵培养基的组成为:蛋白胨15g/L,酵母粉12g/L,NaCl10g/L,甘油15g/L,(NH4)2SO4 5g/L,KH2PO4 1.36g/L,K2HPO4·3H2O 2.28g/L,MgSO4·7H2O0.375g/L,溶剂为去离子水。
作为本发明的优选,
底物初始浓度为20~300g/L;
湿菌体的含水量75~80wt%,以湿重计添加量为30~150g/L;
烟酰胺腺嘌呤二核苷酸为0.3~0.5g/L;
葡萄糖的摩尔当量为底物当量的1.0~2.0倍;
反应温度为20~40℃。
作为本发明的优选,步骤(2)中的有机溶剂为甲苯、乙酸乙酯和叔丁醇中的一种或多种;
当有机溶剂为甲苯时,加入的甲苯的体积浓度为3~15%;
当有机溶剂为乙酸乙酯时,加入的乙酸乙酯的体积浓度为10%~30%;
当有机溶剂为叔丁醇时,加入的叔丁醇的体积浓度为10%~30%。
本发明的有益效果如下:
与现有技术相比,本发明的有益效果体现在:在基因工程菌中同时表达酮还原酶(SW)、水解酶(HY)、葡萄糖脱氢酶(GDH),利用全细胞催化方法实现去氢表雄酮的高效合成。该方法在常温常压下通过一步反应便可获得去氢表雄酮,避免了中间水解产物的累积,同时利用体系自身实现NADH/NAD+的循环再生。该方法的底物浓度可达20~300g/L,转化率为99%以上,产率95%以上,光学纯度大于99%,具有很好的应用开发前景。
附图说明
图1是实施例2的反应液的HPLC图谱。
图2是实施例4的反应液的HPLC图谱。
图3是实施例5的反应液的HPLC图谱。
具体实施方式
下面就本发明的具体实施方式作进一步说明。
如无特别说明,本发明中所采用的原料均可从市场上购得或是本领域常用的,如无特别说明,下述实施例中的方法均为本领域的常规方法。
一种酮还原酶SW,该酮还原酶SW的人工氨基酸序列如SEQ NO.1所示。
编码该酮还原酶SW的SW基因片段的人工核苷酸序列如SEQ NO.4所示。
一种基因工程菌,为将含有SW基因片段的第一质粒、含有相连接的HY基因片段和GDH基因片段的第二质粒转入到宿主菌株中,并可表达SW基因、HY基因和GDH基因的重组菌株;pETDuet-HY-GDH/pET28a-SW/BL21(DE3)。
该基因工程菌的制备过程如下:
(a)根据人工核苷酸序列SEQ NO.5基因合成HY基因片段,基因两端引入酶切位点NdeI和XhoI,连入pETDuet载体,获得质粒pETDuet-HY;
(b)根据人工核苷酸序列SEQ NO.6基因合成GDH基因片段,基因两端引入酶切位点BamHI和NotI,连入质粒pETDuet-HY,获得质粒pETDuet-HY-GDH;然后将质粒pETDuet-HY-GDH转入大肠杆菌BL21(DE3)获得基因工程菌株pETDuet-HY-GDH/BL21(DE3);
(c)根据人工核苷酸序列SEQ NO.4基因合成SW基因片段,基因两端引入酶切位点NdeI和XhoI,连入pET28a载体,获得质粒pET28a-SW;
(d)将质粒pET28a-SW转入大肠杆菌pETDuet-HY-GDH/BL21(DE3)获得双质粒基因工程菌株pETDuet-HY-GDH/pET28a-SW/BL21(DE3)。
实施例1基因工程菌的发酵罐扩大培养
将上述基因工程菌pETDuet-HY-GDH/pET28a-SW/BL21(DE3)接种至50μg/mL卡那霉素、100μg/mL氨苄霉素的LB培养基中,37℃,200rpm培养过夜,再以2%接种量(v/v)接种至含50μg/mL卡那霉素、100μg/mL氨苄霉素的种子培养基中,37℃,200rpm培养,在对数中期时以10%接种量(v/v)接种至含4L发酵培养基的7L发酵罐中,37℃培养14h,加入已灭菌的终浓度为0.6mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行诱导培养20h后放罐,利用蝶式离心机4000rpm离心收集菌体备用,湿菌体含水率为80wt%。
上述种子培养基和发酵培养基终浓度组成为:蛋白胨15g/L,酵母粉12g/L,NaCl10g/L,甘油15g/L,(NH4)2SO4 5g/L,KH2PO4 1.36g/L,K2HPO4 ·3H2O 2.28g/L,MgSO4·7H2O0.375g/L,溶剂为去离子水。
实施例2(制备去氢表雄酮,有机溶剂为甲苯)
反应体系(1L):将120g实施例1中收集的湿菌体细胞,247g葡萄糖,0.5g烟酰胺腺嘌呤二核苷酸(NAD)、100ml甲苯和640mL水以及底物雄性二酮衍生物I混合,底物浓度为300g/L,于30℃、200rpm条件下反应,反应过程中氨水自动调控pH值至6.5,反应8h结束,取样进行HPLC检测,结果如图1所示。从图1中可以看出底物转化率高,去氢表雄酮的产率高,纯度高,经计算,底物转化率为99%,ee值高达99%。反应结束后,旋蒸除去所有甲苯,离心,添加等体积的酸乙酯溶解产物,离心,取有机层,加水结晶即得到去氢表雄酮粗品。粗品用活性炭吸附除蛋白和重结晶来进一步提纯,真空干燥得到纯品约245g,收率93%,产物的HPLC纯度>99%,单杂小于0.5%。
实施例3(制备去氢表雄酮,有机溶剂为甲苯)
反应体系(100mL):将12g实施例1中收集的湿菌体细胞,6g葡萄糖,0.03g烟酰胺腺嘌呤二核苷酸(NAD)、10ml甲苯和64mL水以及底物雄性二酮衍生物I混合,底物浓度为60g/L,于30℃、200rpm条件下反应,反应过程中氨水自动调控pH值至6.5,反应8h,取样进行HPLC检测,转化率为99%,ee值高达99%;反应结束后,旋蒸除去所有乙酸乙酯,离心,添加等体积的丙酮溶解产物,离心,取有机层,加水结晶即得到去氢表雄酮粗品,粗品用活性炭吸附除蛋白和重结晶来进一步提纯、真空干燥得到纯品约4.8g,收率91%,HPLC纯度>99%,单杂小于0.5%。
实施例4(制备去氢表雄酮,有机溶剂为乙酸乙酯)
反应体系(100mL):将12g实施例1中收集的湿菌体细胞,6g葡萄糖,0.03g烟酰胺腺嘌呤二核苷酸(NAD)、20ml乙酸乙酯和64mL水以及底物雄性二酮衍生物I混合,底物浓度为20g/L,于30℃、200rpm条件下反应,反应过程中氨水自动调控pH值至6.5,反应8h,取样进行HPLC检测,结果如图2所示,其中近4.1min处的峰为杂质峰,底物转化率为99%,ee值高达99%;反应结束后,旋蒸除去所有乙酸乙酯,离心,添加等体积的丙酮溶解产物,离心,取有机层,加水结晶即得到去氢表雄酮粗品,粗品用活性炭吸附除蛋白和重结晶来进一步提纯、真空干燥得到纯品约1.53g,收率83%,HPLC纯度>99%,单杂小于0.5%。
实施例5(制备去氢表雄酮,有机溶剂为叔丁醇)
反应体系(100mL):将12g实施例1中收集的湿菌体细胞,6g葡萄糖,0.03g烟酰胺腺嘌呤二核苷酸(NAD)、20ml叔丁醇和64mL水以及底物雄性二酮衍生物I混合,底物浓度为20g/L,于30℃、200rpm条件下反应,反应过程中氨水自动调控pH值至6.5,反应20h,取样进行HPLC检测,结果如图3所示,其中近4.1min和13.8min处的峰为杂质峰,底物转化率为78%,ee值99%。
对比例1(制备去氢表雄酮,不添加有机溶剂)
反应体系(100ml):12g实施例1中收集的湿菌体细胞、6g葡萄糖、0.03g烟酰胺腺嘌呤二核苷酸(NAD)、100mL水,底物浓度为20g/L,于30℃、200rpm条件下反应,反应过程中用氨水自动调控pH值至6.5,反应20h,取样检测,转化率为8%,ee值99%。
从上述实验结果可以看出,采用甲苯和乙酸乙酯相对叔丁醇具有更高的底物转化率,且杂质含量低,其中甲苯在底物浓度相近时,较乙酸乙酯有更高的产物产率。而不添加上述有机溶剂时,底物转化率低。
序列表
<110> 杭州馨海酶源生物科技有限公司
<120> 一种基因工程菌及在制备去氢表雄酮上的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 258
<212> PRT
<213> 酮还原酶(SW)
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<213> 水解酶(HY)
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Ala Pro Lys Arg Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Val Gln Lys Lys Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Val Trp Leu Ala Ser Lys Glu Ser Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Lys Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
<210> 4
<211> 774
<212> DNA
<213> 编码基因(SW)
<400> 4
atgggtcgtc tggaaggtaa aaccgctatc gttaccggtg gtgctcaggg tatgggtgct 60
gctaccgttc gtgttatggt tgaagaaggt gctaaagttg ttatcgctga cctggctgaa 120
caggctggta aatctctggc tgctgaactg ggtgacgctg cttctttctg ccgtctggac 180
gtttctaacg aatctgactg gcagaaagtt ctggctcaca ccctggaagt tcacggtacc 240
gttaacgttc tggttaacaa cgctggtatc cagtacttcg ttggtgttga agacatcgaa 300
gctgaacgtg ttatgcgtct gttctctatc aacgttctgg gttctatgct gggtgttaaa 360
accgttgctc cgatcatgaa aaaagctggt gctggtgttg ttatcaacat ctcttctctg 420
gacggtttcc gtggtaccaa cggtatgtct ccgtacgttg cttctaaatg ggctgttcgt 480
ggtctgacca aagctcaggc tctggaactg ggtccggtta tccgtgttgt ttctgttcac 540
ccgggtggtg ttaacacccc gatgggtaac ccgaccggtg acaccggtga agctctgaac 600
gctccgtacg gtcgtgttcc gctgcgtcgt atcggtgaac cgatcgaagt tgctcgtgtt 660
accgctttca tggcttctga cgaagcttct tacgtttctg gttctgaact ggctgttgac 720
ggtggttgga ccgctggtca ctaccacgtt ggtctgccgg gtggtccgga agct 774
<210> 5
<211> 1029
<212> DNA
<213> 编码基因(HY)
<400> 5
atgaagctac tctctctgac cggtgtggct ggtgtgcttg cgacttgcgt tgcagccact 60
cctttggtga agcgtctacc ttccggttcg gaccctgcct tttcgcagcc caagtcggtg 120
ctcgatgcgg gtctgacctg ccagggtgct tcgccatcct cggtctccaa acccatcctt 180
ctcgtccccg gaaccggcac cacaggtcca cagtcgttcg actcgaactg gatccccctc 240
tcaacgcagt tgggttacac accctgctgg atctcacccc cgccgttcat gctcaacgac 300
acccaggtca acacggagta catggtcaac gccatcaccg cgctctacgc tggttcgggc 360
aacaacaagc ttcccgtgct tacctggtcc cagggtggtc tggttgcaca gtggggtctg 420
accttcttcc ccagtatcag gtccaaggtc gatcgactta tggcctttgc gcccgactac 480
aagggcaccg tcctcgccgg ccctctcgat gcactcgcgg ttagtgcacc ctccgtatgg 540
cagcaaacca ccggttcggc actcaccacc gcactccgaa acgcaggtgg tctgacccag 600
atcgtgccca ccaccaacct ctactcggcg accgacgaga tcgttcagcc tcaggtgtcc 660
aactcgccac tcgactcatc ctacctcttc aacggaaaga acgtccaggc acaggccgtg 720
tgtgggccgc tgttcgtcat cgaccatgca ggctcgctca cctcgcagtt ctcctacgtc 780
gtcggtcgat ccgccctgcg ctccaccacg ggccaggctc gtagtgcaga ctatggcatt 840
acggactgca accctcttcc cgccaatgat ctgactcccg agcaaaaggt cgccgcggct 900
gcgctcctgg cgccggcagc tgcagccatc gtggcgggtc caaagcagaa ctgcgagccc 960
gacctcatgc cctacgcccg cccctttgca gtaggcaaaa ggacctgctc cggcatcgtc 1020
accccctga 1029
<210> 6
<211> 786
<212> DNA
<213> 编码基因(GDH)
<400> 6
atgtacacgg atttaaaagg aaaagtcgtt gccattacgg gagcttcatc aggattagga 60
aaagcgatgg cgatccgctt cggacaggag caggcgaaag tcgtggttaa ctactatagt 120
aatgaaaaag acgcccaaac cgttaaagaa gagattcaaa aagcgggagg cgaagctgtc 180
atcgttcaag gagacgtcac aaaagaagaa gatgtcaaaa acatcgtgca gaccgcagtg 240
aaggaattcg gcacattgga tgtcatgatc aataacgccg gcatggaaaa tccggttcag 300
tcgcatgaaa tgccgcttaa agactggaac aaagttatca ataccaatct gaccggcgct 360
ttcttaggaa gccgcgaagc cataaaatat tacgtagaga atgatattca aggaaacgtc 420
attaacatgt caagcgtaca tgaaatgatt ccgtggccgc tgtttgtcca ctacgcggcg 480
agtaaaggcg gcatcaaatt gatgacggaa acgctggcgc ttgagtacgc gccgaagcgc 540
atccgtgtta acaatatcgg accgggcgcc atcaatacgc cgatcaacgc ggaaaaattt 600
gccgatcccg ttcagaaaaa agatgtggaa agcatgattc cgatgggata tatcggtgag 660
ccggaagaaa tcgcggctgt cgccgtctgg cttgcttcaa aggaatcaag ctatgtgacc 720
gggattacgc tgtttgctga cgggggaatg accaaatatc cgtctttcca ggcaggccgc 780
ggataa 786
Claims (10)
1.一种酮还原酶SW,其特征在于,所述酮还原酶SW的氨基酸序列如SEQ NO.1所示。
2.一种编码如权利要求1所述的酮还原酶SW的SW基因片段,其特征在于,所述SW基因片段的核苷酸序列如SEQ NO.4所示。
3.一种含有如权利要求2所述的SW基因片段的基因工程菌,其特征在于,所述基因工程菌为将含有SW基因片段的第一质粒、含有相连接的HY基因片段和GDH基因片段的第二质粒转入到宿主菌株中,并可表达SW基因、HY基因和GDH基因的重组菌株;
HY基因片段的核苷酸序列如SEQ NO.5所示;
GDH基因片段的核苷酸序列如SEQ NO.6所示。
4.根据权利要求3所示的基因工程菌,其特征在于,所述第一质粒为pET28a-SW。
5.根据权利要求3所述的基因工程菌,其特征在于,所述第二质粒为pETDuet-HY-GDH。
6.根据权利要求3至5任一所述的基因工程菌,其特征在于,宿主菌株为大肠杆菌BL21(DE3)。
7.一种使用如权利要求3至6任一所述的基因工程菌制备去氢表雄酮的方法,其特征在于,包括以下步骤:
将基因工程菌在培养基中扩大培养,结束后分离、收集湿菌体;
然后以雄烯二酮衍生物I为底物与菌体、烟酰胺腺嘌呤二核苷酸、葡萄糖和有机溶剂在水中混合,调节pH值范围为5.5~8,搅拌反应,结束后分离、纯化得到去氢表雄酮;
其中雄烯二酮衍生物I的结构式如下:
8.根据权利要求7所述的使用基因工程菌制备去氢表雄酮的方法,其特征在于,基因工程菌的扩大培养过程如下:
(1-1)将基因工程菌接种至含50μg/ml卡那霉素、100μg/ml氨苄霉素的LB液体培养基中,37℃震荡培养8~10h,再以2%接种量(v/v)接种至含50μg/mL卡那霉素、100μg/mL氨苄霉素的种子培养基中,37℃,200rpm培养获得种子液;
(1-2)将种子液以体积浓度10%的接种量接种至装有发酵培养基的发酵罐中,37℃发酵培养14h后,将已灭菌的终浓度为0.6mM异丙基-β-D-硫代吡喃半乳糖苷添加到发酵罐中诱导培养;待培养至OD600达到70~100时放罐、离心分离收集湿菌体;
所述种子培养基和发酵培养基的组成为:蛋白胨15g/L,酵母粉12g/L,NaCl 10g/L,甘油15g/L,(NH4)2SO4 5g/L,KH2PO4 1.36g/L,K2HPO4·3H2O 2.28g/L,MgSO4·7H2O 0.375g/L,溶剂为去离子水。
9.根据权利要求7所述的使用基因工程菌制备去氢表雄酮的方法,其特征在于,
底物初始浓度为20~300g/L;
湿菌体含水率为75~80wt%,湿菌体投放量以湿重计为30~150g/L;
烟酰胺腺嘌呤二核苷酸为0.3~0.5g/L;
葡萄糖的摩尔当量为底物当量的1.0~2.0倍;
反应温度为20~40℃。
10.根据权利要求7所述的使用基因工程菌制备去氢表雄酮的方法,其特征在于,步骤(2)中的有机溶剂为甲苯、乙酸乙酯和叔丁醇中的一种或多种;
当有机溶剂为甲苯时,加入的甲苯的体积浓度为3~15%;
当有机溶剂为乙酸乙酯时,加入的乙酸乙酯的体积浓度为10%~30%;
当有机溶剂为叔丁醇时,加入的叔丁醇的体积浓度为10%~30%。
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