CN114854807B - 一种生产海藻糖六磷酸的方法 - Google Patents
一种生产海藻糖六磷酸的方法 Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
本发明属于生产海藻糖六磷酸的技术领域。本发明提供了一种生产海藻糖六磷酸的方法，包括如下步骤：重组工程菌TY001和重组工程菌TY002分别接种至含有50μg/ml链霉素的2YT液体培养基中培养至OD为0.8；加入阿拉伯糖至终浓度为0.2mM，诱导培养后离心得菌体沉淀；配置催化反应体系；所述反应体系中含有浓度为4OD/ml的重组工程菌TY001、浓度为4OD/ml的重组工程菌TY002、浓度为100mM的蔗糖、浓度为100mM的葡萄糖六磷酸、浓度为100mM的尿苷二磷酸；催化反应体系在37℃和220rpm条件下催化反应6小时得海藻糖六磷酸。本发明的方法能催化生成海藻糖六磷酸，开拓了生化催化生成海藻糖六磷酸的新方法。
Description
技术领域
本发明属于生产海藻糖六磷酸的技术领域。
背景技术
本发明涉及一种生产海藻糖六磷酸的方法及其专用工程菌。海藻糖六磷酸，简称T6P,存在于微生物、植物体内，是海藻糖生物合成时的中间体，它可提高植物的抗旱性，延缓叶片衰老等作用。此外，在碳水化合物代谢中起着重要作用。
酶法合成海藻糖六磷酸主要是以尿苷二磷酸葡萄糖、葡萄糖六磷酸为底物，在海藻糖六磷酸合成酶作用下生成，在之前的实验中1小时可催化50mM的底物生成海藻糖六磷酸22mM，但是底物成本较高，限制了其工业化生产使用。
因此，构建一条安全、成本低、高效的海藻糖六磷酸合成路线，满足其工业化生产需求十分迫切。
发明内容
有鉴于此，本发明提供了一种生产海藻糖六磷酸的方法，包括如下步骤：重组工程菌TY001和重组工程菌TY002分别接种至含有50μg/ml链霉素的2YT液体培养基中培养至OD为0.8；加入阿拉伯糖至终浓度为0.2mM，诱导培养后离心得菌体沉淀；配置催化反应体系；所述反应体系中含有浓度为4OD/ml的重组工程菌TY001、浓度为4OD/ml的重组工程菌TY002、浓度为100mM的蔗糖、浓度为100mM的葡萄糖六磷酸、浓度为100mM的尿苷二磷酸；催化反应体系在37℃和220rpm条件下催化反应6小时得海藻糖六磷酸。
在本发明的具体实施例中，所述重组工程菌TY001的制备方法，包括如下步骤：将pBAD-hisB载体的XhoⅠ和SpeⅠ酶切位点间的片段替换为序列表中SEQ ID NO.2所示的DNA分子，得到重组表达载体pBAD-SS；将重组表达载体pBAD-SS导入大肠杆菌BW25113，得到重组工程菌TY001。
在本发明的具体实施例中，所述重组工程菌TY002的制备方法，包括如下步骤：将pBAD-hisB载体的XhoⅠ和SpeⅠ酶切位点间的片段替换为列表中SEQ ID NO.4所示序列的DNA分子，得到重组表达载体pBAD-TPS；将重组表达载体pBAD-TPS导入大肠杆菌BW25113，得到重组工程菌TY002。
在本发明的具体实施例中，所述培养的温度为37℃。
在本发明的具体实施例中，所述诱导培养的温度为30℃。
在本发明的具体实施例中，所述诱导培养的时间为16小时。
在本发明的具体实施例中，所述离心的温度为4℃。
在本发明的具体实施例中，所述离心的转数为8000rpm/min。
在本发明的具体实施例中，在配置催化反应体系前还包括将菌体菌体沉淀用50mM、pH6.8的磷酸缓冲液重悬，超声破碎20min。
本发明的方法能催化生成海藻糖六磷酸，开拓了生化催化生成海藻糖六磷酸的新方法。
附图说明
图1为海藻糖六磷酸标准品HPLC峰图。
图2为利用重组工程菌进行催化反应后的上清液HPLC峰图。
图3为利用重组工程菌生产尿苷二磷酸葡萄糖原理图。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
pBAD-hisB载体：Invitrogen公司，货号:854314DE。
大肠杆菌BW25113：武汉圣达威科技有限公司，货号：P1466。
2YT液体培养基：0.5％(质量百分含量)NaCl、1％(质量百分含量)酵母提取物、1.6％(质量百分含量)胰蛋白胨，蒸馏水定容。
海藻糖六磷酸标准品：北京佰凯生物科技有限公司，货号GC45076-10。
海藻糖六磷酸的结构如下所示：
实施例1
1.重组表达载体的构建
(1)、将pBAD-hisB载体的XhoⅠ和SpeⅠ酶切位点间的片段替换为序列表中SEQ IDNO.2所示的DNA分子，得到重组表达载体pBAD-SS(已经测序验证)。
序列表中SEQ ID NO.2的序列编码来源于拟南芥(Arabidopsis thaliana)的蔗糖合成酶。
序列表中SEQ ID NO.2所示的序列的DNA分子编码蛋白氨基酸序列如序列表中SEQID NO.1所示。
(2)、将pBAD-hisB载体的XhoⅠ和SpeⅠ酶切位点间的片段替换为列表中SEQ IDNO.4所示序列的DNA分子，得到重组表达载体pBAD-TPS(已经测序验证)。
序列表的序列4编码来源于大肠杆菌(Escherichia coli)的海藻糖磷酸合成酶。
序列表的序列4所示的DNA分子编码蛋白氨基酸序列如序列表中SEQ ID NO.3所示的蛋白质。
2.重组工程菌的制备
(1)、将制备的重组表达载体pBAD-SS导入大肠杆菌BW25113，得到重组工程菌TY001。
(2)、将制备的重组表达载体pBAD-TPS导入大肠杆菌BW25113，得到重组工程菌TY002。
3.利用重组工程菌生产海藻糖六磷酸
合成原理如图3所示。
(1)、将制备的重组工程菌TY001和重组工程菌TY002分别接种至含有50μg/ml链霉素的2YT液体培养基中，37℃、220rpm培养至OD为0.8，
向培养体系中加入阿拉伯糖(上海源叶生物科技有限公司,货号：S11032)，至阿拉伯糖在培养体系中的终浓度为0.2mM，30℃、220rpm诱导培养16小时后，收集培养体系，4℃、8000rpm/min离心10min，收集菌体沉淀。
(2)、将上述菌体沉淀(即细胞)用50mM、pH6.8的磷酸缓冲液重悬，超声(功率198W)破碎20min，向其中加入蔗糖、葡萄糖六磷酸、尿苷二磷酸和MgCl2配置催化反应体系；
反应体系中含有浓度为4OD/ml的TY001菌体(细胞)、浓度为4OD/ml的TY002菌体(细胞)、浓度为100mM的蔗糖、浓度为100mM的葡萄糖六磷酸(北京沃凯生物科技有限公司，货号A45380-1g)、浓度为100mM的尿苷二磷酸(北京翰隆达科技发展有限公司，货号：M1408-500mg)。
(3)、将上步的催化反应体系在37℃、220rpm催化反应6小时。
(4)、完成上步后，将催化反应体系4℃、12000rpm/min离心5min，取上清。
用0.22μM的滤膜过滤上清，收集滤液进行HPLC检测海藻糖六磷酸的产量。
海藻糖六磷酸标准品(北京佰凯生物科技有限公司，货号GC45076-10)的保留时间为24.750min(图1)。
利用重组工程菌TY001和TY992进行催化反应后的上清液检测结果如图2所示。
结果显示，上清液中也有保留时间为24.767min的峰值，表明转化液中生成了海藻糖六磷酸。上述重组工程菌可催化生成10mM海藻糖六磷酸。
序列表
<110> 中国科学院微生物研究所
广西科学院
<120> 一种生产海藻糖六磷酸的方法
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gctgatactc ttgctgattt cttcaccaag tgtaaggagg atccatctca ctgggatgag 2220
atctcaaaag gagggcttca gaggattgag gagaaataca cttggcaaat ctattcacag 2280
aggctcttga cattgactgg tgtgtatgga ttctggaagc atgtctcgaa ccttgaccgt 2340
cttgaggctc gccgttacct tgaaatgttc tatgcattga agtatcgccc attggctcag 2400
gctgttcctc ttgcacaaga tgattga 2427
<210> 3
<211> 474
<212> PRT
<213> 大肠杆菌(Escherichia coli)
<400> 3
Met Ser Arg Leu Val Val Val Ser Asn Arg Ile Ala Pro Pro Asp Glu
1 5 10 15
His Ala Ala Ser Ala Gly Gly Leu Ala Val Gly Ile Leu Gly Ala Leu
20 25 30
Lys Ala Ala Gly Gly Leu Trp Phe Gly Trp Ser Gly Glu Thr Gly Asn
35 40 45
Glu Asp Gln Pro Leu Lys Lys Val Lys Lys Gly Asn Ile Thr Trp Ala
50 55 60
Ser Phe Asn Leu Ser Glu Gln Asp Leu Asp Glu Tyr Tyr Asn Gln Phe
65 70 75 80
Ser Asn Ala Val Leu Trp Pro Ala Phe His Tyr Arg Leu Asp Leu Val
85 90 95
Gln Phe Gln Arg Pro Ala Trp Asp Gly Tyr Leu Arg Val Asn Ala Leu
100 105 110
Leu Ala Asp Lys Leu Leu Pro Leu Leu Gln Asp Asp Asp Ile Ile Trp
115 120 125
Ile His Asp Tyr His Leu Leu Pro Phe Ala His Glu Leu Arg Lys Arg
130 135 140
Gly Val Asn Asn Arg Ile Gly Phe Phe Leu His Ile Pro Phe Pro Thr
145 150 155 160
Pro Glu Ile Phe Asn Ala Leu Pro Thr Tyr Asp Thr Leu Leu Glu Gln
165 170 175
Leu Cys Asp Tyr Asp Leu Leu Gly Phe Gln Thr Glu Asn Asp Arg Leu
180 185 190
Ala Phe Leu Asp Cys Leu Ser Asn Leu Thr Arg Val Thr Thr Arg Ser
195 200 205
Ala Lys Ser His Thr Ala Trp Gly Lys Ala Phe Arg Thr Glu Val Tyr
210 215 220
Pro Ile Gly Ile Glu Pro Lys Glu Ile Ala Lys Gln Ala Ala Gly Pro
225 230 235 240
Leu Pro Pro Lys Leu Ala Gln Leu Lys Ala Glu Leu Lys Asn Val Gln
245 250 255
Asn Ile Phe Ser Val Glu Arg Leu Asp Tyr Ser Lys Gly Leu Pro Glu
260 265 270
Arg Phe Leu Ala Tyr Glu Ala Leu Leu Glu Lys Tyr Pro Gln His His
275 280 285
Gly Lys Ile Arg Tyr Thr Gln Ile Ala Pro Thr Ser Arg Gly Asp Val
290 295 300
Gln Ala Tyr Gln Asp Ile Arg His Gln Leu Glu Asn Glu Ala Gly Arg
305 310 315 320
Ile Asn Gly Lys Tyr Gly Gln Leu Gly Trp Thr Pro Leu Tyr Tyr Leu
325 330 335
Asn Gln His Phe Asp Arg Lys Leu Leu Met Lys Ile Phe Arg Tyr Ser
340 345 350
Asp Val Gly Leu Val Thr Pro Leu Arg Asp Gly Met Asn Leu Val Ala
355 360 365
Lys Glu Tyr Val Ala Ala Gln Asp Pro Ala Asn Pro Gly Val Leu Val
370 375 380
Leu Ser Gln Phe Ala Gly Ala Ala Asn Glu Leu Thr Ser Ala Leu Ile
385 390 395 400
Val Asn Pro Tyr Asp Arg Asp Glu Val Ala Ala Ala Leu Asp Arg Ala
405 410 415
Leu Thr Met Ser Leu Ala Glu Arg Ile Ser Arg His Ala Glu Met Leu
420 425 430
Asp Val Ile Val Lys Asn Asp Ile Asn His Trp Gln Glu Cys Phe Ile
435 440 445
Ser Asp Leu Lys Gln Ile Val Pro Arg Ser Ala Glu Ser Gln Gln Arg
450 455 460
Asp Lys Val Ala Thr Phe Pro Lys Leu Ala
465 470
<210> 4
<211> 1425
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 4
atgagtcgtt tagtcgtagt atctaaccgg attgcaccac cagacgagca cgccgccagt 60
gccggtggcc ttgccgttgg catactgggg gcactgaaag ccgcaggcgg actgtggttt 120
ggctggagtg gtgaaacagg gaatgaggat cagccgctaa aaaaggtgaa aaaaggtaac 180
attacgtggg cctcttttaa cctcagcgaa caggaccttg acgaatacta caaccaattc 240
tccaatgccg ttctctggcc cgcttttcat tatcggctcg atctggtgca atttcagcgt 300
cctgcctggg acggctatct acgcgtaaat gcgttgctgg cagataaatt actgccgctg 360
ttgcaagacg atgacattat ctggatccac gattatcacc tgttgccatt tgcgcatgaa 420
ttacgcaaac ggggagtgaa taatcgcatt ggtttctttc tgcatattcc tttcccgaca 480
ccggaaatct tcaacgcgct gccgacatat gacaccttgc ttgaacagct ttgtgattat 540
gatttgctgg gtttccagac agaaaacgat cgtctggcgt tcctggattg tctttctaac 600
ctgacccgcg tcacgacacg tagcgcaaaa agccatacag cctggggcaa agcatttcga 660
acagaagtct acccgatcgg cattgaaccg aaagaaatag ccaaacaggc tgccgggcca 720
ctgccgccaa aactggcgca acttaaagcg gaactgaaaa acgtacaaaa tatcttttct 780
gtcgaacggc tggattattc caaaggtttg ccagagcgtt ttctcgccta tgaagcgttg 840
ctggaaaaat atccgcagca tcatggtaaa attcgttata cccagattgc accaacgtcg 900
cgtggtgatg tgcaagccta tcaggatatt cgtcatcagc tcgaaaatga agctggacga 960
attaatggta aatacgggca attaggctgg acgccgcttt attatttgaa tcagcatttt 1020
gaccgtaaat tactgatgaa aatattccgc tactctgacg tgggcttagt gacgccactg 1080
cgtgacggga tgaacctggt agcaaaagag tatgttgctg ctcaggaccc agccaatccg 1140
ggcgttcttg ttctttcgca atttgcggga gcggcaaacg agttaacgtc ggcgttaatt 1200
gttaacccct acgatcgtga cgaagttgca gctgcgctgg atcgtgcatt gactatgtcg 1260
ctggcggaac gtatttcccg tcatgcagaa atgctggacg ttatcgtgaa aaacgatatt 1320
aaccactggc aggagtgctt cattagcgac ctaaagcaga tagttccgcg aagcgcggaa 1380
agccagcagc gcgataaagt tgctaccttt ccaaagcttg cgtag 1425
Claims (7)
1.生产海藻糖六磷酸的方法，其特征在于，包括如下步骤：
重组工程菌TY001和重组工程菌TY002分别接种至含有50μg/ml链霉素的2YT液体培养基中培养至OD为0.8；
加入阿拉伯糖至终浓度为0.2mM，诱导培养后离心得菌体沉淀；
配置催化反应体系；所述反应体系中含有浓度为4OD/ml的重组工程菌TY001、浓度为4OD/ml的重组工程菌TY002、浓度为100mM的蔗糖、浓度为100mM的葡萄糖六磷酸、浓度为100mM的尿苷二磷酸；
催化反应体系在37℃和220rpm条件下催化反应6小时得海藻糖六磷酸；
所述重组工程菌TY001的制备方法，包括如下步骤：将pBAD-hisB载体的XhoⅠ和SpeⅠ酶切位点间的片段替换为序列表中SEQ ID NO.2所示的DNA分子，得到重组表达载体pBAD-SS；将重组表达载体pBAD-SS导入大肠杆菌BW25113，得到重组工程菌TY001；
所述重组工程菌TY002的制备方法，包括如下步骤：将pBAD-hisB载体的XhoⅠ和SpeⅠ酶切位点间的片段替换为列表中SEQ ID NO.4所示序列的DNA分子，得到重组表达载体pBAD-TPS；将重组表达载体pBAD-TPS导入大肠杆菌BW25113，得到重组工程菌TY002。
2.依据权利要求1所述生产海藻糖六磷酸的方法，其特征在于，所述培养的温度为37℃。
3.依据权利要求1所述生产海藻糖六磷酸的方法，其特征在于，所述诱导培养的温度为30℃。
4.依据权利要求1所述生产海藻糖六磷酸的方法，其特征在于，所述诱导培养的时间为16小时。
5.依据权利要求1所述生产海藻糖六磷酸的方法，其特征在于，所述离心的温度为4℃。
6.依据权利要求1所述生产海藻糖六磷酸的方法，其特征在于，所述离心的转数为8000rpm/min。
7.依据权利要求1所述生产海藻糖六磷酸的方法，其特征在于，在配置催化反应体系前还包括将菌体沉淀用50mM、pH6.8的磷酸缓冲液重悬，超声破碎20min。
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