CN109022473B - 一种酶法制备奥利司他中间体的方法 - Google Patents
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- CN109022473B CN109022473B CN201810917366.3A CN201810917366A CN109022473B CN 109022473 B CN109022473 B CN 109022473B CN 201810917366 A CN201810917366 A CN 201810917366A CN 109022473 B CN109022473 B CN 109022473B
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Abstract
本发明涉及医药技术领域,公开了一种酶法制备奥利司他中间体的方法,包括短链醇脱氢酶原始基因的合成、短链醇脱氢酶的表达、细胞破碎及酶催化等步骤,利用短链醇脱氢酶催化法制备高手性ee值(ee>99%)的奥利司他中间体(R)‑3‑羟基‑十四烷酸甲酯,以替代传统的化学合成方法,减少生产成本及对环境的污染。本发明提供的奥利司他中间体的合成方法,反应条件更温和,反应速度更快,反应收率和手性ee值高,且生产成本低,适合工业扩大化生产。
Description
技术领域
本发明涉及医药技术领域,尤其涉及一种酶法制备奥利司他中间体的方法。
背景技术
奥利司他,是目前全球唯一的OTC减肥药。奥利司他是长效和强效的特异性胃肠道脂肪酶抑制剂,它通过与胃和小肠腔内胃脂肪酶和胰脂肪酶的活性丝氨酸部位形成共价键使酶失活而发挥治疗作用,失活的酶不能将食物中的脂肪,主要是甘油三酯水解为可吸收的游离脂肪酸和单酰基甘油。未消化的甘油三酯不能被身体吸收,从而减少热量摄入,控制体重。该药无需通过全身吸收发挥药效。
奥利司他的结构式:
通过对奥利司他的结构分析可知,(R)-β-羟基十四烷酸酯是合成奥利司他的重要原料。因此,开发有效制备光学纯的(R)-β-羟基十四烷酸酯的方法具有重要的应用前景。
(R)-β-羟基十四烷酸酯生物催化的反应路线为:
R=CH3或C2H5
目前,(R)-3-羟基-十四烷酸甲酯的合成主要以化学合成为主,但该方法需要使用价格昂贵的手性配体和金属试剂,产品收率低,手性ee值不高,同时化学生产成本大且对环境不优化,不适于规模化生产。中国专利公开号CN101538285,公开日2009年9月23日,发明专利的名称为钌-手性双膦配体络合物及制备方法以及在β-羰基十四烷酸甲酯的催化氢化反应中的应用,该专利公开了一种钌-手性双膦配体络合催化剂以及其制备方法。利用该催化剂制备(R)-3-羟基-十四烷酸甲酯的手性ee值小于99%,催化剂成本较高,且催化反应需在强酸、高压条件下进行,对生产设备要求高且具有很大的腐蚀性。
发明内容
为了解决上述技术问题,本发明提供了一种酶法制备奥利司他中间体的方法。本发明利用短链醇脱氢酶催化法制备高手性ee值(ee>99%)的奥利司他中间体(R)-3-羟基-十四烷酸甲酯,减少生产成本及对环境的污染。
本发明的具体技术方案为:一种酶法制备奥利司他中间体的方法,包括以下步骤:
(1)短链醇脱氢酶原始基因的合成:按照大肠杆菌密码子分析用表对短链醇脱氢酶NA-ADH原始基因序列进行密码子优化,得到优化后的NA-ADH基因序列,对其进行目的基因的全合成,合成后的全基因两端分别带有NdeI和XhoI酶切位点并连接到pET26b(+)上,获得重组表达载体pET26b-NA-ADH,优化后的NA-ADH基因序列如SEQ ID NO.1所示,其编码的氨基酸序列如SEQ ID NO.2所示;
(2)短链醇脱氢酶的表达:将重组表达载体pET26b-NA-ADH通过热激转化法转到表达菌株BL21(DE3)中,挑取单菌落,将单菌落接入到含卡那霉素的LB培养液中培养,然后添加IPTG,诱导培养过夜;
(3)细胞破碎:将培养好的菌株离心,收集菌体,重悬菌体后对其进行超声破碎,所得液体即为含重组短链醇脱氢酶菌体破碎液;
(4)酶催化:将β-羰基十四烷酸甲酯加入反应器中,随后,将NAD+、含重组短链醇脱氢酶菌体破碎液、含重组葡萄糖脱氢酶菌体破碎液、葡萄糖、磷酸盐缓冲液的混合溶液加入反应器中,进行反应。
本发明首先合成出短链醇脱氢酶的原始基因,对其进行优化、重组,将重组的表达载体转移到菌株中进行表达,对表达菌株进行培养,然后破碎,得到含重组短链醇脱氢酶菌体破碎液。
在步骤(1)中,优化后的NA-ADH基因序列为:
ATGCCGCTTGAAATGACGATTGCTCTCAACAATGTGGTCGCCGTCGTCACCGGCGCGGCGGGAGGCATCGGCCGCGAACTGGTCAAGGCGATGAAGGCCGCCAACGCCATCGTCATCGCCACCGACATGGCGCCCTCGGCCGATGTCGAAGGCGCGGACCATTATCTCCAGCACGACGTGACGAGCGAGGCCGGCTGGAAGGCGGTCGCGGCACTGGCCCAGGAAAAGTACGGGCGCGTCGATGCGCTGGTGCACAACGCGGGCATCTCGATCGTCACGAAGTTCGAAGACACTCCGCTGTCCGATTTCCACCGCGTGAACACGGTCAACGTCGATTCCATCATCATCGGTACGCAGGTCCTGCTGCCGCTGCTCAAGGAAGGCGGCAAGGCGCGCGCAGGGGGCGCCTCGGTGGTCAACTTCTCCAGCGTCGCGGGTCTGCGCGGCGCGGCGTTCAATGCGGCCTATTGCACCAGCAAGGCGGCGGTGAAGATGCTCTCGAAGTGCCTCGGCGCGGAATTCGCGGCGCTCGGCTACAACATCCGCGTCAACTCCGTGCATCCGGGCGGCATCGATACCCCGATGCTCGGCTCGCTGATGGACAAGTACGTCGAACTCGGCGCTGCCCCCTCGCGCGAGGTGGCCCAGGCCGCGATGGAAATGCGCCACCCGATCGGTCGCATGGGTCGCCCTGCCGAAATGGGCGGCGGCGTGGTCTATCTCTGCTCCGACGCAGCAAGCTTCGTCACCTGCACGGAATTCGTGATGGACGGCGGCTTCAGCCAGGTC。
优化后的NA-ADH编码的氨基酸序列为:
MPLEMTIALNNVVAVVTGAAGGIGRELVKAMKAANAIVIATDMAPSADVEGADHYLQHDVTSEAGWKAVAALAQEKYGRVDALVHNAGISIVTKFEDTPLSDFHRVNTVNVDSIIIGTQVLLPLLKEGGKARAGGASVVNFSSVAGLRGAAFNAAYCTSKAAVKMLSKCLGAEFAALGYNIRVNSVHPGGIDTPMLGSLMDKYVELGAAPSREVAQAAMEMRHPIGRMGRPAEMGGGVVYLCSDAASFVTCTEFVMDGGFSQV。
经过优化重组的的表达载体在菌株中表达出的酶对催化制备奥利司他前驱体的反应具有相当高的催化活性和选择性,制备奥利司他前驱体的反应中底物的转化率高,产物的手性ee值大于99%。而现有技术中,制备奥利司他前驱体的反应中底物的转化率较低,一般为70~80%,产物的手性ee值最高为98%。虽然产物的手性ee值看似提升的不多,但从98%到大于99%的提升已经是十分巨大的提升,并且具有十分重要的意义。因为,药用领域对物质的纯度要求很高,制备的中间产物的纯度越高,理论上,目标产物制备过程中的副产物越少,越有利于进一步的提纯及进一步的反应。
作为优选,步骤(2)中,将所述单菌落接入到3~7mL含浓度为49~51μg/mL卡那霉素的LB培养液中,在36~38℃条件下振荡培养6~12h,得菌种液;每1mL菌种液加入到含浓度为49~51μg/mL卡那霉素的100mL TB培养基中,36~38℃下振荡培养至OD600至2.8~3.2,然后添加IPTG至IPTG的终浓度为0.05~0.15mM,20~30℃下诱导培养6~12h。
卡那霉素一种蛋白质生物合成抑制剂,被作为标记基因用于分子克隆中。本发明中卡那霉素作为表达载体的标记,在LB培养基和TB培养基里添加了卡那霉素,培养出来的菌为带有标记的目的菌。OD600指的是某种溶液在600nm波长处的吸光值。吸光值正比于溶液中的吸光物质的浓度。测量细菌培养液在600nm处的吸光值,得到的OD600的数值如果在0.6-0.8之间,表明细菌处于旺盛生长的对数生长期,OD600>3表明细菌已经饱和等。将菌种液在TB培养基中培养至饱和后再进行诱导。IPTG是一种作用极强的诱导剂,能够诱导菌体表现出与原来不同的性状,从而方便地挑选出基因重组体。
作为优选,步骤(2)中,所述LB培养液中含有以下成分的物质:每1L LB培养液中含有蛋白胨5~15g、酵母提取物2~8g、NaCl 5~15g,余量为去离子水。
作为优选,步骤(2)中,所述LB培养液中含有以下成分的物质:每1L LB培养液中含有蛋白胨10g、酵母提取物5g、NaCl 10g,余量为去离子水。
LB培养基中含有单菌落所需的营养物质。
作为优选,步骤(2)中,所述TB培养基中含有以下成分的物质:每1L TB培养液中含有蛋白胨10~15g、酵母提取物20~28g、甘油2~6mL、KH2PO4 2~2.7g、K2HPO4 12~13g,余量为去离子水。
作为优选,步骤(2)中,所述TB培养基中含有以下成分的物质:每1L TB培养液中含有蛋白胨12g、酵母提取物24g、甘油4mL、KH2PO4 2.31g、K2HPO4 12.54g,余量为去离子水。
TB培养基中含有菌种液扩大培养所需的营养物质和无机盐。
作为优选,所述培养基的制备方法为:将蛋白胨、酵母提取物、甘油加入900~950mL去离子水中,制成溶液A;将KH2PO4、K2HPO4溶于50~100mL去离子水中,制成溶液B;将溶液A和溶液B分别灭菌后冷却至20~60℃混合均匀。
作为优选,步骤(3)中,所述离心机的转速为8000~12000r/min,离心时间为8~12min;用浓度为0.05~0.15M、pH为6.8~7.2的磷酸盐缓冲液重悬藻体,菌体与磷酸缓冲溶液的固液比为1~3g/1mL。
作为优选,步骤(4)中,所述混合溶液中NAD+、含重组短链醇脱氢酶菌体破碎液、含重组葡萄糖脱氢酶菌体破碎液、葡萄糖、磷酸盐缓冲液的浓度分别为0.08~0.12g/L、10~50g/L、10~30g/L、100~200g/L、0.08~0.12M,底物β-羰基十四烷酸甲酯与混合溶液的固液比为50~100g/L,利用pH自动控制系统和1M Na2CO3溶液控制酶反应的pH为6.8~7.2,在30~35℃反应9~15h。
与现有技术对比,本发明的有益效果是:本发明提供的酶法制备奥利司他中间体的方法,对环境友好,制备出了高手性ee值(ee>99%)的奥利司他中间体(R)-3-羟基-十四烷酸甲酯。
具体实施方式
下面结合实施例对本发明作进一步的描述。在本发明中所涉及的装置、连接结构和方法,若无特指,均为本领域公知的装置、连接结构和方法。
一种酶法制备奥利司他中间体的方法,其中重组短链醇脱氢酶的制备包括以下步骤:(1)短链醇脱氢酶原始基因的合成:按照大肠杆菌密码子分析用表对短链醇脱氢酶NA-ADH原始基因序列进行密码子优化,得到优化后的NA-ADH基因序列,对其进行目的基因的全合成,合成后的全基因两端分别带有NdeI和XhoI酶切位点并连接到pET26b(+)上,获得重组表达载体pET26b-NA-ADH。
(2)短链醇脱氢酶的表达:将重组表达载体pET26b-NA-ADH通过热激转化法转到表达菌株BL21(DE3)中,挑取单菌落,将单菌落接入到5mL含浓度为50μg/mL卡那霉素的LB培养液中,在37℃条件下,振荡培养9h,得菌种液。每1mL菌种液加入到100mL含浓度为50μg/mL卡那霉素的TB培养基中,在37℃条件下振荡培养至OD600至3.0,然后添加IPTG至IPTG的终浓度为0.1mM,在25℃条件下诱导培养9h。
(3)细胞破碎:将培养好的菌株用离心机离心,离心机的转速为10000r/min,离心时间为10min,收集菌体,用浓度为0.1M、pH为7.0的磷酸盐缓冲液重悬菌体,菌体与磷酸缓冲溶液的固液比为2g/1mL,超声破碎后所得液体即为含重组短链醇脱氢酶菌体破碎液。
其中,LB培养液中含有以下成分的物质:每1L LB培养液中含有蛋白胨10g、酵母提取物5g、NaCl 10g,余量为去离子水。
TB培养基中含有以下成分的物质:每1L TB培养液中含有蛋白胨12g、酵母提取物24g、甘油4mL、KH2PO4 2.31g、K2HPO4 12.54g,余量为去离子水。其制备方法为:将蛋白胨、酵母提取物、甘油加入900mL去离子水中,制成溶液A;将KH2PO4、K2HPO4溶于100mL去离子水中,制成溶液B;将溶液A和溶液B分别灭菌后冷却至40℃混合均匀。
实施例1
将5g底物β-羰基十四烷酸甲酯加入到250mL反应器中,随后,将100mL含有0.1g/LNAD+、30g/L含重组短链醇脱氢酶菌体破碎液、10g/L含重组葡萄糖脱氢酶菌体破碎液、100g/L葡萄糖、0.1M磷酸盐缓冲液(pH 7.0)加入该反应器中,β-羰基十四烷酸甲酯与混合溶液的固液比为50g/L,利用pH自动控制系统和1M Na2CO3溶液控制酶反应的pH值为7.0左右。35℃反应12h。底物转化率大于98%,产物浓度为49.0g/L,产物手性ee值大于99%。
实施例2
将7.5g底物β-羰基十四烷酸甲酯加入到250mL反应器中,随后,将100mL含有0.1g/LNAD+、35g/L含重组短链醇脱氢酶菌体破碎液、10g/L含重组葡萄糖脱氢酶菌体破碎液、100g/L葡萄糖、0.1M磷酸盐缓冲液(pH 7.0)加入该反应器中,β-羰基十四烷酸甲酯与混合溶液的固液比为75g/L,利用pH自动控制系统和1M Na2CO3溶液控制酶反应的pH值为7.0左右。35℃反应12h。底物转化率大于96%,产物浓度为72.0g/L,产物手性ee值大于99%。
实施例3
将10g底物β-羰基十四烷酸甲酯加入到250mL反应器中,随后,将100mL含有0.1g/LNAD+、30g/L含重组短链醇脱氢酶菌体破碎液、10g/L含重组葡萄糖脱氢酶菌体破碎液、200g/L葡萄糖、0.1M磷酸盐缓冲液(pH 7.0)加入该反应器中,β-羰基十四烷酸甲酯与混合溶液的固液比为100g/L,利用pH自动控制系统和1M Na2CO3溶液控制酶反应的pH值为7.0左右。35℃反应12h。底物转化率大于78%,产物浓度为78.0g/L,产物手性ee值大于99%。
实施例4
将25g底物β-羰基十四烷酸甲酯加入到2L反应器中,随后,将500mL含有0.1g/LNAD+、30g/L含重组短链醇脱氢酶菌体破碎液、10g/L含重组葡萄糖脱氢酶菌体破碎液、100g/L葡萄糖、0.1M磷酸盐缓冲液(pH 7.0)加入该反应器中,β-羰基十四烷酸甲酯与混合溶液的固液比为50g/L,利用pH自动控制系统和1M Na2CO3溶液控制酶反应的pH值为7.0左右。35℃反应12h。底物转化率大于99%,产物浓度为49.5g/L,产物手性ee值大于99%。
实施例5
将50g底物β-羰基十四烷酸甲酯加入到2L反应器中,随后,将500mL含有0.1g/LNAD+、50g/L含重组短链醇脱氢酶菌体破碎液、30g/L含重组葡萄糖脱氢酶菌体破碎液、100g/L葡萄糖、0.1M磷酸盐缓冲液(pH 7.0)加入该反应器中,β-羰基十四烷酸甲酯与混合溶液的固液比为100g/L,利用pH自动控制系统和1M Na2CO3溶液控制酶反应的pH值为7.0左右。35℃反应12h。底物转化率大于85%,产物浓度为85g/L,产物手性ee值大于99%。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何限制,凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效结构变换,均仍属于本发明技术方案的保护范围。
序列表
<110> 浙江海洋大学
<120> 一种酶法制备奥利司他中间体的方法
<130> 2018
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 789
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 1
atgccgcttg aaatgacgat tgctctcaac aatgtggtcg ccgtcgtcac cggcgcggcg 60
ggaggcatcg gccgcgaact ggtcaaggcg atgaaggccg ccaacgccat cgtcatcgcc 120
accgacatgg cgccctcggc cgatgtcgaa ggcgcggacc attatctcca gcacgacgtg 180
acgagcgagg ccggctggaa ggcggtcgcg gcactggccc aggaaaagta cgggcgcgtc 240
gatgcgctgg tgcacaacgc gggcatctcg atcgtcacga agttcgaaga cactccgctg 300
tccgatttcc accgcgtgaa cacggtcaac gtcgattcca tcatcatcgg tacgcaggtc 360
ctgctgccgc tgctcaagga aggcggcaag gcgcgcgcag ggggcgcctc ggtggtcaac 420
ttctccagcg tcgcgggtct gcgcggcgcg gcgttcaatg cggcctattg caccagcaag 480
gcggcggtga agatgctctc gaagtgcctc ggcgcggaat tcgcggcgct cggctacaac 540
atccgcgtca actccgtgca tccgggcggc atcgataccc cgatgctcgg ctcgctgatg 600
gacaagtacg tcgaactcgg cgctgccccc tcgcgcgagg tggcccaggc cgcgatggaa 660
atgcgccacc cgatcggtcg catgggtcgc cctgccgaaa tgggcggcgg cgtggtctat 720
ctctgctccg acgcagcaag cttcgtcacc tgcacggaat tcgtgatgga cggcggcttc 780
agccaggtc 789
<210> 2
<211> 263
<212> PRT
<213> 菌株BL21 (DE3)
<400> 2
Met Pro Leu Glu Met Thr Ile Ala Leu Asn Asn Val Val Ala Val Val
1 5 10 15
Thr Gly Ala Ala Gly Gly Ile Gly Arg Glu Leu Val Lys Ala Met Lys
20 25 30
Ala Ala Asn Ala Ile Val Ile Ala Thr Asp Met Ala Pro Ser Ala Asp
35 40 45
Val Glu Gly Ala Asp His Tyr Leu Gln His Asp Val Thr Ser Glu Ala
50 55 60
Gly Trp Lys Ala Val Ala Ala Leu Ala Gln Glu Lys Tyr Gly Arg Val
65 70 75 80
Asp Ala Leu Val His Asn Ala Gly Ile Ser Ile Val Thr Lys Phe Glu
85 90 95
Asp Thr Pro Leu Ser Asp Phe His Arg Val Asn Thr Val Asn Val Asp
100 105 110
Ser Ile Ile Ile Gly Thr Gln Val Leu Leu Pro Leu Leu Lys Glu Gly
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Gly Lys Ala Arg Ala Gly Gly Ala Ser Val Val Asn Phe Ser Ser Val
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Ala Gly Leu Arg Gly Ala Ala Phe Asn Ala Ala Tyr Cys Thr Ser Lys
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Ala Ala Val Lys Met Leu Ser Lys Cys Leu Gly Ala Glu Phe Ala Ala
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Leu Gly Tyr Asn Ile Arg Val Asn Ser Val His Pro Gly Gly Ile Asp
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Thr Pro Met Leu Gly Ser Leu Met Asp Lys Tyr Val Glu Leu Gly Ala
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Ala Pro Ser Arg Glu Val Ala Gln Ala Ala Met Glu Met Arg His Pro
210 215 220
Ile Gly Arg Met Gly Arg Pro Ala Glu Met Gly Gly Gly Val Val Tyr
225 230 235 240
Leu Cys Ser Asp Ala Ala Ser Phe Val Thr Cys Thr Glu Phe Val Met
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Asp Gly Gly Phe Ser Gln Val
260
Claims (9)
1.一种酶法制备奥利司他中间体的方法,其特征在于包括以下步骤:
(1)短链醇脱氢酶原始基因的合成:按照大肠杆菌密码子分析用表对短链醇脱氢酶NA-ADH原始基因序列进行密码子优化,得到优化后的NA-ADH基因序列,对其进行目的基因的全合成,合成后的全基因两端分别带有NdeI和XhoI酶切位点并连接到pET26b(+)上,获得重组表达载体pET26b-NA-ADH,优化后的NA-ADH基因序列如SEQ ID NO.1所示,其编码的氨基酸序列如SEQ ID NO.2所示;
(2)短链醇脱氢酶的表达:将重组表达载体pET26b-NA-ADH通过热激转化法转到表达菌株BL21 (DE3)中,挑取单菌落,将单菌落接入到含卡那霉素的LB培养液中培养,然后添加IPTG,诱导培养过夜;
(3)细胞破碎:将培养好的菌株离心,收集菌体,重悬菌体后对其进行超声破碎,所得液体即为含重组短链醇脱氢酶菌体破碎液;
(4)酶催化:将β-羰基十四烷酸甲酯加入反应器中,随后,将NAD+、含重组短链醇脱氢酶菌体破碎液、含重组葡萄糖脱氢酶菌体破碎液、葡萄糖、磷酸盐缓冲液的混合溶液加入反应器中,进行反应,得到奥利司他中间体(R)-3-羟基-十四烷酸甲酯。
2.如权利要求1所述的一种酶法制备奥利司他中间体的方法,其特征在于:步骤(2)中,将所述单菌落接入到3~7mL含浓度为49~51μg/mL卡那霉素的LB培养液中,在36~38℃条件下振荡培养6~12h,得菌种液;每1mL菌种液加入到含浓度为49~51μg/mL卡那霉素的100mL TB培养基中,36~38℃下振荡培养至OD600至2.8~3.2,然后添加IPTG至IPTG的终浓度为0.05~0.15mM,20~30℃下诱导培养6~12h。
3.如权利要求1或2所述的一种酶法制备奥利司他中间体的方法,其特征在于:步骤(2)中,所述LB培养液中含有以下成分的物质:每1L LB培养液中含有蛋白胨5~15g、酵母提取物2~8g、NaCl 5~15g,余量为去离子水。
4.如权利要求3所述的一种酶法制备奥利司他中间体的方法,其特征在于:步骤(2)中,所述LB培养液中含有以下成分的物质:每1L LB培养液中含有蛋白胨10g、酵母提取物5g、NaCl 10g,余量为去离子水。
5.如权利要求2所述的一种酶法制备奥利司他中间体的方法,其特征在于:步骤(2)中,所述TB培养基中含有以下成分的物质:每1L TB培养基中含有蛋白胨10~15g、酵母提取物20~28g、甘油2~6mL、KH2PO4 2~2.7g、K2HPO4 12~13g,余量为去离子水。
6.如权利要求5所述的一种酶法制备奥利司他中间体的方法,其特征在于:步骤(2)中,所述TB培养基中含有以下成分的物质:每1L TB培养基中含有蛋白胨12g、酵母提取物24g、甘油4mL、KH2PO4 2.31g、K2HPO4 12.54g,余量为去离子水。
7.如权利要求6所述的一种酶法制备奥利司他中间体的方法,其特征在于:所述培养基的制备方法为:将蛋白胨、酵母提取物、甘油加入900~950mL去离子水中,制成溶液A;将KH2PO4、K2HPO4溶于50~100mL去离子水中,制成溶液B;将溶液A和溶液B分别灭菌后冷却至20~60℃混合均匀。
8.如权利要求1所述的一种酶法制备奥利司他中间体的方法,其特征在于:步骤(3)中,所述离心时的转速为8000~12000r/min,离心时间为8~12min;用浓度为0.05~0.15M、pH为6.8~7.2的磷酸盐缓冲液重悬菌体,菌体与磷酸盐缓冲溶液的固液比1~3g/1mL。
9.如权利要求1所述的一种酶法制备奥利司他中间体的方法,其特征在于:步骤(4)中,所述混合溶液中NAD+、含重组短链醇脱氢酶菌体破碎液、含重组葡萄糖脱氢酶菌体破碎液、葡萄糖、磷酸盐缓冲液的浓度分别为0.08~0.12g/L、10~50g/L、10~30g/L、100~200g/L、0.08~0.12M,β-羰基十四烷酸甲酯与混合溶液的固液比为50~100g/L,利用pH自动控制系统和1MNa2CO3溶液控制酶反应的pH为6.8~7.2,在30~35℃反应9~15h。
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