CN107022500B - 胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂的制备及其应用 - Google Patents
胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂的制备及其应用 Download PDFInfo
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Abstract
本发明属于生物工程领域，提供了一种胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂的制备方法：一个经过密码子优化的热带假丝酵母脂肪酶基因在巴斯德毕赤酵母GS115中进行胞内表达，经过初筛和复筛后得到最佳重组菌株3‑1；将重组菌株3‑1用5％的十六烷基三甲基溴化铵进行通透化，冷冻干燥后得到胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂。本发明还提供了该胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂在合成6‑O‑丁酰澳栗精胺中的应用方法：以四氢呋喃、澳栗精胺、丁酸乙烯酯和胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂为原料，在30℃、160r/min条件下进行合成反应。这两种方法简单高效，适于工业应用。
Description
技术领域
本发明属于生物工程领域，具体涉及经过密码子优化的热带假丝酵母脂肪酶基因在巴斯德毕赤酵母GS115中表达、优化表达条件、毕赤酵母全细胞催化剂的制备及其在合成澳栗精胺衍生物中的应用。
背景技术
脂肪酶(EC 3.1.1.3)属于α/β水解酶家族，它能催化水解、酯化、酯交换、酸解、醇解、氨解等多种反应，在食品、洗涤、制革、饲料、制药、能源等领域应用广泛。脂肪酶的来源主要有植物、动物和微生物，其中微生物脂肪酶具有催化活性多样性、稳定性好、高产量、可批量生产等优点，因此微生物脂肪酶的应用更为广泛。据估计，细菌28个属、放线菌4个属、酵母菌10个属、其他真菌23个属的微生物均可产脂肪酶。来源于洋葱伯克霍尔德菌、荧光假单胞菌、米黑根毛霉、疏棉状嗜热丝孢菌、皱褶假丝酵母和南极假丝酵母的脂肪酶是目前常见的商品脂肪酶。
全细胞生物催化是指利用完整的生物有机体(即全细胞、组织甚至个体)作为催化剂进行化学转化的过程，生物催化中常用的有机体主要是微生物，其本质是利用微生物细胞内的酶进行催化。目前，全细胞生物催化已成功应用于谷胱甘肽、L-甘油醛、(2R,5R)-己二醇、L -二羟苯丙氨酸、抗坏血酸-2-磷酸、烟酰胺、CDP-胆碱、羟脯氨酸等物质的制备。澳栗精胺是从栗豆树种子中提取的一种多羟基氢化中氮茚生物碱，1993年Hempel等人用X-射线衍射方法解析了澳栗精胺的结构，它具有抗癌、抗病毒等生理活性，并且比其他生物碱抑制α- 葡萄糖苷酶的活性更强。八十年代以来，各国科学家在澳栗精胺全合成、生理作用以及药效等各个方面进行大量的研究。然而澳栗精胺也会抑制肠道内的蔗糖酶，从而引起渗透性腹泻。但是，6-O-丁酰澳栗精胺能增加亲油性并且对肠道内的蔗糖酶抑制作用减小，因此对胃肠道毒性小。6-O-丁酰澳栗精胺的抗病毒活性比母体化合物高30倍，它无论在试管中还是在体内对许多病毒都显示出较强的抗病毒活性，这些病毒包括HIV-1、HSV-1、HSV-2和牛病毒性腹泻病毒。由于澳栗精胺上存在四个十分相近似的仲醇基而使得在6-位的选择性单酰化作用十分困难。P.S.Liu等人用选择性保护和去保护的方法合成了6-O-丁酰澳栗精胺。从澳栗精胺起始一共需要5步反应(Synthesis of potent anti-HIV agents:Estersof castanospermine, Tetrahedron Lett.,1990,31(20):2829-2832)。W.K.Anderson等人用一步合成方法制备6-O-丁酰澳栗精胺，他们用甲醇中二丁基锡氧化物处理澳栗精胺，然后将反应加热至回流后冷却，并用酰基氯和三乙胺处理，急骤层析后得到澳栗精胺的6-O-单酯的游离碱，产率在18％-44％范围内(A facile selective acylation ofcastanospermine,Tetrahedron Lett.,1990,31(2):169-170)。D.L. Delinck等人用枯草杆菌蛋白酶在吡啶中催化合成了一些澳栗精胺的衍生酯类物质 (Enzyme-catalyzedacylation of castanospermine and 1-deoxynojirimycin Tetrahedron Lett.,1990,31(22):3093-3096)。然而在以往的研究中，尚无利用全细胞生物催化合成6-O-丁酰澳栗精胺的报道。
发明内容
本发明的目的在于提供一种胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂的制备方法和一种利用胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂合成6-O-丁酰澳栗精胺的应用方法。
本发明实现目的技术路线如下：
一种胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂的制备方法，包括以下步骤：
(1)一个经过密码子优化的热带假丝酵母脂肪酶基因在巴斯德毕赤酵母GS115中进行胞内表达，经过初筛和复筛后得到最佳重组菌株3-1；
(2)将重组菌株3-1用5％的十六烷基三甲基溴化铵进行通透化，冷冻干燥后得到胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂。
该重组菌株3-1的培养基组成为酵母粉1％、蛋白胨2％、0.1mol/L磷酸钾缓冲液、无氨基酵母氮源1.34％、生物素4×10-5％、甲醇3％，且起始pH为7.0。
其中，热带假丝酵母脂肪酶基因的密码子优化方法为：根据巴斯德毕赤酵母密码子的偏好，利用Codon Optimization By Generalbiol软件进行密码子优化。
胞内表达的方法为：(1)经过密码子优化的热带假丝酵母脂肪酶基因与载体pPICZB连接，得到携带的重组载体。(2)将重组载体转化E.coli Top 10，筛选阳性克隆。(3)提取重组载体再转入毕赤酵母GS115，初筛和复筛后得到重组菌株3-1。(4)重组菌株3-1转接到50毫升发酵培养基(培养基组成为：酵母粉1％、蛋白胨2％、0.1mol/L磷酸钾缓冲液、无氨基酵母氮源1.34％、生物素4×10-5％、甲醇3％，起始pH＝7.0)培养，装瓶量为20％。在32℃、 180r/min培养96h，其中每24h补加0.5％的甲醇进行诱导，重组菌株3-1的脂肪酶得到最优表达。
透化处理的方法为：收集重组菌株3-1发酵液，离心弃上清液，取菌体沉淀。用十六烷基三甲基溴化铵溶液通透化，离心弃上清液。用水洗涤多次，冷冻干燥后得到毕赤酵母全细胞催化剂干粉。
重组菌株3-1在国家知识产权局指定的保藏单位保藏，保藏单位是中国典型培养物保藏中心，保藏单位地址是中国湖北省武汉市武汉大学，保藏日期为2017年2月27日，保藏编号为CCTCC NO:M 2017070，拉丁文命名为Pichia pastoris 3-1。
上述胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂在合成澳栗精胺-6-丁酯中的应用方法，包括以下步骤：
在10毫升具塞锥形瓶内加入4毫升四氢呋喃、4微摩尔澳栗精胺、8微摩尔丁酸乙烯酯和100毫克胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂，在30℃、160r/min条件下进行合成反应。12000r/min离心20min除去毕赤酵母全细胞催化剂,上清液用旋转蒸发仪浓缩，浓缩液再用半制备HPLC(Chromolith SemiPrep RP-18 endcapped 100-10mm HPLC柱) 纯化，使用H2O(0.1％HCOOH)/乙腈(0.1％HCOOH)(体积比为9：1)作为洗脱液，收集含有澳栗精胺-6-丁酯的洗脱液，旋转蒸发仪再次浓缩后得到澳栗精胺-6-丁酯。
上述合成6-O-丁酰澳栗精胺的方法简单高效，60分钟内6-O-丁酰澳栗精胺的产率达到 27.2％。
本发明的有益效果是：实现了胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂的制备；实现了利用胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞生物催化合成6-O-丁酰澳栗精胺，并在60分钟内澳栗精胺-6-丁酯的产率达到27.2％。
附图说明
图1是按照本发明的方法制备的胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂。
具体实施方式
下面将结合实施例来详细说明本发明所具有的有益效果，旨在帮助阅读者更好地理解本发明的实质，但不能对本发明的实施和保护范围构成任何限定。
实施例1
胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂的制备：
根据巴斯德毕赤酵母密码子的偏好，将热带假丝酵母脂肪酶基因进行密码子优化，然后在体外合成经过密码子优化的热带假丝酵母脂肪酶基因。该基因含有KpnⅠ和NotⅠ酶切位点。将该基因和pPICZB质粒分别用KpnⅠ和NotⅠ双酶切，然后将酶切产物连接，得到的重组质粒转化E.coli Top 10感受态细胞，筛选含有重组质粒的重组子。
从重组子中提取重组质粒，SalⅠ在37℃将重组质粒线性化2h，用电转法将重组质粒转入毕赤酵母GS11，电转条件为1500V、25μF、200Ω。电击后立刻加入1mL 1mol/L山梨醇，30℃静置30min，取100μL涂平板(1％酵母提取物，2％蛋白胨，2％葡萄糖，2％琼脂，25mg/ml博莱霉素)，用牙签挑平板上的菌落到含有三丁酸甘油酯的YPDS平板上(1％酵母提取物，2％蛋白胨，2％葡萄糖，2％琼脂，25mg/ml博莱霉素)，30℃培养一周后，选择水解圈大的菌株进行斜面保存并复筛。
将初筛得到的菌株分别转接到装有50mL BMMY培养基(1％酵母粉，2％蛋白胨，0.1mol/L 磷酸钾缓冲液pH＝6.0，YNB 1.34％，Biotin(4×10-5)％，甲醇3％)的250mL摇瓶中，30℃、 180r/min摇床培养，每24h补加0.5％的甲醇进行诱导培养，96h后取样，采用酵母蛋白提取试剂盒KGP6100提取脂肪酶，测定蛋白质浓度和脂肪酶活力，筛选得到比活力最高的菌株为菌株3-1。
重组菌株3-1转接到50毫升发酵培养基(培养基组成为：酵母粉1％、蛋白胨2％、0.1mol/L 磷酸钾缓冲液、无氨基酵母氮源1.34％、生物素4×10-5％、甲醇3％，起始pH＝7.0)培养，装瓶量为20％。在32℃、180r/min培养96h，其中每24h补加0.5％的甲醇进行诱导，重组菌株3-1的脂肪酶得到最优表达。
将重组菌株3-1发酵液50mL置于离心管中，于5000r/min转速下离心5-7分钟，弃上清液。用30mL 5％的十六烷基三甲基溴化铵溶液通透化5分钟，充分混匀。5000r/min转速下离心5分钟，弃上清液。加入30mL水，离心5分钟，弃上清液，重复3-4次，冷冻干燥后得到毕赤酵母全细胞催化剂(图1)。
实施例2
用实施例1制得的胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂催化澳栗精胺和丁酸乙烯酯合成6-O-丁酰澳栗精胺：
在10毫升具塞锥形瓶内加入4毫升四氢呋喃、4微摩尔澳栗精胺、8微摩尔丁酸乙烯酯和100毫克胞内表达热带假丝酵母脂肪酶的毕赤酵母全细胞催化剂，在30℃、160r/min条件下反应60分钟内6-O-丁酰澳栗精胺的产率达到27.2％。
密码子优化后的热带假丝酵母脂肪酶基因序列：
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ttgagcggcc tgtttttccc aatcgatgtt aagaactctt ggcaacttct agttagatcc 180
accgattcgt ttgggaatgc cacagcgatt gtggctacaa ttattgagcc attcaactca 240
gaccctagta aagtggtgtc ttatcaaaca tttgaggatt cggccaacat taattgttct 300
ccttcttacg gtatgcaatg gggagcctct atatctaccg ttgccactca gatcgatatg 360
tctttcatgg ttccgttgtt gaataacggc tactttcttg tctctccaga ttacgaggga 420
cctaaatcca cattcactgt tggacgtcaa agcggacacg gtactctcga ttcaattcgt 480
gctatcctcc aatcaggtaa ctttacgggg gtagatgagg acgcacaggt tgcaatgtgg 540
ggttactctg gaggctctct agcctcaggc tgggcagctg cacttcagcc acattatgcc 600
cctgagctag aagataacct gattggtgcc gcccttggtg gcttcgtcac caatatcacc 660
gccactgcag aagcaacaga tgggaaatta ctggcaggac tagtccctat cgcattgaac 720
ggtcttggta acgagtacaa cgacttccgt gaaatccttt acagcgaagt taaagaagga 780
ggaagggata aacttgccga cggcctaaac cactgcatga tacccgggat aattagattt 840
gctttttccc agttcttggc tggtaagaac cgcttgtttc caaatggtta cggtttattg 900
gacgatccaa ttgttaacag gaccattcaa gaaaataact tgatgtccgt gtccaaagaa 960
tatataccaa agatcccatt gtttgtatat catggaacat tggatgcagt agtgccaatt 1020
gttaatgtca agaagacata cgagaggtgg tgtgactggg gaatagagtc attcgaattc 1080
gctgaagacc tgctaaatgg tcatattagt gaaacattgg tcggtgctcc ggctgctctt 1140
acttggttgg aaagaagatt cgctggtttg gatcctgtta agggttgcca gcataccgct 1200
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Claims (1)
1.一株重组菌株3-1，其特征在于：保藏于中国典型培养物保藏中心，保藏单位地址是中国湖北省武汉市武汉大学，保藏日期为2017年2月27日，保藏编号为CCTCCNO:M2017070，拉丁文命名为Pichiapastoris 3-1。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101062954A (zh) * | 2006-05-16 | 2007-10-31 | 中国人民解放军军事医学科学院野战输血研究所 | 具有抗血管生成作用的融合蛋白及其编码基因与应用 |
| CN104388461A (zh) * | 2014-11-18 | 2015-03-04 | 昆明理工大学 | 一种以氯霉素为筛选标记的毕赤酵母表达载体及其应用 |
-
2017
- 2017-04-14 CN CN201710242688.8A patent/CN107022500B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101062954A (zh) * | 2006-05-16 | 2007-10-31 | 中国人民解放军军事医学科学院野战输血研究所 | 具有抗血管生成作用的融合蛋白及其编码基因与应用 |
| CN104388461A (zh) * | 2014-11-18 | 2015-03-04 | 昆明理工大学 | 一种以氯霉素为筛选标记的毕赤酵母表达载体及其应用 |
Non-Patent Citations (5)
| Title |
|---|
| Development of recombinant Escherichia coli whole-cell biocatalyst expressing a novel alkaline lipase-coding gene from Proteus sp. for biodiesel production;Bei Gao等;《Journal of Biotechnology》;20090115;第139卷(第2期);摘要 * |
| Enzyme-Catalyzed Regioselective Acylation of Castanospermine;Alexey L等;《AMERICAN CHEMICAL SOCIETY》;19900411;第112卷(第8期);摘要，第2853页左栏最后1段-右栏第1段 * |
| 张雨佳等.热带假丝酵母脂肪酶在毕赤酵母中的表达及其条件优化.《中国生物化学与分子生物学会2016年全国学术会议》.2016, * |
| 毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯;郑穗平等;《生物工程学报》;20091225;第25卷(第12期);第1934页右栏第3段 * |
| 热带假丝酵母脂肪酶在毕赤酵母中的表达及其条件优化;张雨佳等;《中国生物化学与分子生物学会2016年全国学术会议》;20161020;摘要 * |
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