CN109988799B - 一种甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷中的应用 - Google Patents
一种甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷中的应用 Download PDFInfo
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- CN109988799B CN109988799B CN201910066879.2A CN201910066879A CN109988799B CN 109988799 B CN109988799 B CN 109988799B CN 201910066879 A CN201910066879 A CN 201910066879A CN 109988799 B CN109988799 B CN 109988799B
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Abstract
本发明公开了一种甘油‑2‑α‑葡萄糖基化酶在制备2‑α‑甘油葡萄糖苷中的应用,所述甘油‑2‑α‑葡萄糖基化酶氨基酸序列为SEQ ID NO.2所示;本发明所述生产2‑α‑GG的重组大肠杆菌,即重组大肠杆菌(Escherichia coli)IEF‑bpmsp208能够在胞内高效合成蔗糖磷酸化酶,以甘油和蔗糖为底物,高效催化甘油的糖基化反应,反应18‑24小时,可得到大于10%的2‑α‑GG溶液,且对底物蔗糖的转化率大于92%,2‑α‑GG的产物浓度高,转化率高,有利于2‑α‑GG分离纯化。
Description
(一)技术领域
本发明涉及一种含甘油-2-α-葡萄糖基化酶在生产2-α-甘油葡萄糖苷(2-O-α-D-glucosylglycerol,以下简称2-α-GG)中的应用。
(二)背景技术
2-α-甘油葡萄糖苷(2-α-GG)是一类由甘油分子与葡萄糖分子以糖苷键结合的物质,结构式如图1所示,其通过糖苷键将一个D-吡喃葡萄糖和甘油2号位的羟基偶联,是自然界多种植物、藻类和细菌对抗干旱和高盐环境的天然保护剂。葡萄糖分子的构型结合甘油分子的位置分为多种,目前已经鉴定出六种,但作为天然渗透压抵抗分子的是2-α-GG。它是微生物在胁迫条件下合成的一种渗透保护物质,同时还是一种大分子稳定剂,能够在高温或冷冻干燥条件下稳定蛋白质或酶等生物大分子结构,也可用于蛋白质药物等的长期保存;由于具有低吸水性、高保湿性的特点,用于提高皮肤保湿效果,是一种良好的化妆品添加剂,抗氧化和抗衰老等功效。更有研究发现2-α-GG还具有治疗过敏性呼吸系统疾病等多种人体保健功效。
2-α-GG在化妆品、医药等行业具有广泛的应用前景,但局限于2-α-GG的生产技术,高纯度的产品价格高居不下。目前的生产方法包括化学法合成、酶法合成和植物或藻类中直接提取。其中化学法缺乏立体和区域专一性,产物多样,得率低;许多酶法转化的产物亦含有多个糖基化位点的产物,难以得到单一产品2-α-GG;直接提取的方法,其得率太低,成本高,难以产业化。目前,2-α-GG的生产技术侧重于筛选专一性更好的酶法转化和微生物代谢工程直接发酵生产。
(三)发明内容
本发明目的是提供一种甘油-2-α-葡萄糖基化酶在制备2-α-GG中应用,实现了用酶法高效、专一性的生产2-α-GG。
本发明采用的技术方案是:
本发明提供一种甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷(2-α-GG)中的应用,所述甘油-2-α-葡萄糖基化酶氨基酸序列为SEQ ID NO.2所示,编码基因核苷酸序列为SEQ ID NO.1所示。所述甘油-2-α-葡萄糖基化酶来源于Leuconostocpseudomesenteroides。
本发明所述甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷(2-α-GG)中的应用方法为:将含甘油-2-α-葡萄糖基化酶基因的重组大肠杆菌经发酵培养获得的发酵液或发酵离心的湿菌体用缓冲液悬浮的菌悬液为催化剂,以甘油为底物,以蔗糖为辅助底物,在30-45℃的条件下进行反应,获得含2-α-甘油葡萄糖苷的反应液,将反应液分离纯化,获得2-α-甘油葡萄糖苷。所述发酵液或菌悬液中湿菌体含量为5-100g/L(优选30-50g/L),甘油的终浓度为100-150g/L(优选120-140g/L),蔗糖终浓度为300-400g/L(优选330-350g/L)。
进一步,所述反应pH为6.0-8.0,反应时间为24h。
进一步,所述缓冲液为pH7.0、2.5mM磷酸缓冲液。
本发明所述重组大肠杆菌构建方法:将SEQ ID NO.1所示甘油-2-α-葡萄糖基化酶基因(lpmsp)克隆到pET28a质粒上,构建pET28a-lpmsp重组表达质粒,并转化到大肠杆菌E.coli BL21(DE3)中,得到重组大肠杆菌E.coli BL21(DE3)(pET28a-lpmsp)。
本发明所述催化剂可以为重组大肠杆菌E.coli BL21(DE3)(pET28a-lpmsp)细胞发酵液或固体菌剂或细胞匀浆后的粗酶液,所述固体菌剂是向所述发酵菌剂中添加吸附剂制成,所述吸附剂为轻质碳酸钙或草炭;所述的细胞匀浆后的粗酶液是将发酵液用生产用自来水稀释2-10倍后,进行高压匀浆后获得的粗酶液。
本发明所述湿菌体摇瓶制备方法:(1)将含甘油-2-α-葡萄糖基化酶基因的重组大肠杆菌接种在含50μg/ml卡那霉素的种子培养基中,30-37℃、180-250rpm(优选37℃、220rpm)培养至对数生长中期,获得种子液;所述种子培养基终浓度组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO40.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH7.0;
(2)发酵培养:将种子液以体积浓度5%的接种量接种到含卡那霉素50mg/L的发酵培养基中,在30-37℃培养4-6h(优选37℃、4h);加入终浓度为18-22g/L(优选20g/L)的α-乳糖,在22-25℃继续发酵12-18h(优选25℃、12h),取发酵液离心,收集湿菌体细胞;所述发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.3g/L,溶剂为去离子水,pH6.8-7.0。
本发明所述湿菌体发酵罐制备方法:(1)将含甘油-2-α-葡萄糖基化酶基因的重组大肠杆菌接种在含50mg/L卡那霉素的种子培养基中,30-37℃、180-250rpm培养至对数生长中期,获得种子液;所述种子培养基终浓度组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH6.8-7.0;
(2)发酵培养:将新鲜培养的种子液按照体积浓度5%的接种量,接种到1.5L的含质量浓度0.05%消泡剂和50mg/L卡那霉素的发酵培养基中,37℃培养4h;加入终浓度为20g/L的α-乳糖,控制发酵温度25℃,溶解氧DO控制大于20%,用25%的氨水控制发酵pH6.8,继续发酵12h,得到用于生产2-α-GG的重组大肠杆菌发酵液,湿菌体含量为30g/L;所述发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.3g/L,溶剂为去离子水,pH6.8-7.0。
与现有报道技术相比,包括植物提取法、化学合成法、微生物直接发酵法、α-糖苷酶生物催化法以及其他微生物来源的蔗糖磷酸化酶生物催化法(Tan XM,Luo Q,LuXF.Appl Microbiol Biot 2016,100:6131-6139),本发明有益效果主要体现在:①采用生物法一步生物酶转化法生产单一2-α-GG产物,具有高度区域和立体专一糖基化的特点;②蔗糖磷酸化酶的催化活性高,18-24h内催化底物甘油100-150g/L的转化率达到92%以上;③该催化反应生成的2-α-GG含量高,其反应液中的浓度在10%以上。
本发明所述生产2-α-GG的重组大肠杆菌,即重组大肠杆菌(Escherichia coli)IEF-bpmsp208能够在胞内高效合成蔗糖磷酸化酶,以甘油和蔗糖为底物,高效催化甘油的糖基化反应,反应18-24小时,可得到大于10%的2-α-GG溶液,且对底物蔗糖的转化率大于92%,2-α-GG的产物浓度高,转化率高,有利于2-α-GG分离纯化。
(四)附图说明
图1为2-α-GG结构式。
图2为pET28a-lpmsp载体的结构示意图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
本发明实施例中如无特殊说明所用方法均为常规方法,所用试剂均可从商业途径获得。
LB培养基:酵母粉5.0g/L、蛋白胨10.0g/L、NaCl 10.0g/L,溶剂为去离子水,pH值6.5-7.0。
种子培养基:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO43.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH6.8-7.0。
发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.3g/L,溶剂为去离子水,pH6.8-7.0。
实施例1、生产2-α-GG的催化菌剂的制备
一、含甘油-2-α-葡萄糖基化酶的大肠杆菌Escherichia coli-lpmsp的构建
采用细菌基因组DNA提取试剂盒提取对数生长中期的假肠膜明串珠菌L.pseudomesenteroides的基因组DNA(NCBI登入号为MK370897),以此为模板,用以下引物进行PCR扩增:
lpmSP-F:
GCCTGGTGCCGCGCGGCAGCCATATGGAAATTCAAAACAAAGCAATG;
lpmSP-R:
GTCGACGGAGCTCGAATTCGGATCCTTAGTTCTGAGTCAAATCATC。
采用南京诺唯赞生物科技有限公司(Vazyme Biotech Co.,Ltd)的高效保真酶Phanta Max Super-Fidelity DNA Polymerase进行PCR扩增,其PCR扩增程序为:95℃3min;95℃15s、58℃15s、72℃1.5min,30个循环;72℃5min。
将所得的PCR产物采用PCR产物回收试剂盒纯化,采用南京诺唯赞生物科技有限公司的一步克隆试剂盒One Step Cloning Kit,克隆到pET28a+质粒的Nde I和BamH I之间。pET28a+质粒用TaKaRa公司的Nde I和BamH I进行双酶切,37℃静置4h后,用DNA胶回收试剂盒纯化酶切后的pET28a+质粒。将纯化的PCR产物和酶切纯化的pET28a+质粒进行连接反应(根据试剂盒说明书操作),转化E.coli BL21(DE3)的高效感受态细胞中,在含终浓度50mg/L卡那霉素的LB平板上筛选。菌落PCR验证阳性克隆子,并提取质粒进行测序分析,获得含有甘油-2-α-葡萄糖基化酶基因(lpmsp,核苷酸序列为SEQ ID NO.1所示,编码的甘油-2-α-葡萄糖基化酶氨基酸序列为SEQ ID NO.2所示)的阳性克隆子。阳性克隆子含有的重组表达载体命名为pET28a-lpmsp(图2)。
含重组质粒pET28a-lpmsp的阳性克隆子E.coli BL21(DE3)(pET28a-lpmsp)记为大肠杆菌(Escherichia coli)-lpmsp。
二、生产2-α-GG的发酵液菌剂的制备
大肠杆菌-lpmsp在含有50μg/ml卡那霉素的种子培养基中,37℃、220rpm培养至对数生长中期,获得种子液。
将新鲜培养的种子液以体积浓度5%的接种量接种到含卡那霉素50mg/L的发酵培养基中,37℃培养4h后;加入终浓度为20g/L的α-乳糖,控制发酵温度25℃,继续发酵12h,获得含有湿菌体30g/L的发酵液,用作生产2-α-GG的催化菌剂。
将新鲜发酵液,用生产用水稀释3倍,使湿菌体的含量为10g/L,采用高压细胞匀浆仪破碎细胞后,得到粗酶液,需尽快用于催化反应,避免长时间保存。
实施例2菌剂在生产2-α-GG中的应用
一、催化剂的活性检测
将实施例1方法制备的湿菌体细胞1g重新悬浮于20mL的pH7.0、2.5mM磷酸缓冲液中;加入终浓度132g/L的甘油和342g/L的蔗糖,在30℃、220rpm条件下水浴摇床催化24h,反应液用于HPLC分析,测得残留的底物蔗糖浓度为18.81g/L,形成的产物2-α-GG浓度为43.6g/L,底物蔗糖转化率为94.5%。
液相色谱检测条件。样品前处理:100μL反应液,加入到900μL的0.01mol/L稀盐酸中;10000×g离心5min,用0.22μm滤膜过滤,将滤液加入液相样品瓶中;色谱柱:Sugar-Ca柱,300×7.8mm;柱温:80℃,检测器温度:55℃;流动相:纯水;流速:0.5mL·min-1;检测器:示差测器;进样量:10μL。底物蔗糖的出峰时间一般为10-11min,甘油的出峰时间一般为18-19min,产物2-α-GG的出峰时间为12-13min。
二、2L发酵罐中的催化剂制备及其在1L反应体系中催化反应的应用
(1)菌种活化
大肠杆菌-lpmsp在含有50μg/ml卡那霉素的种子培养基中,37℃、220rpm培养至对数生长中期,获得种子液。
(2)2L发酵罐中的催化剂制备
将新鲜培养的种子液按照体积浓度5%的接种量,接种到1.5L的含质量浓度0.05%消泡剂和50mg/L卡那霉素的发酵培养基中,37℃培养4h;加入终浓度为20g/L的α-乳糖,控制发酵温度25℃,溶解氧DO控制大于20%,用25%的氨水控制发酵pH6.8,继续发酵12h,得到用于生产2-α-GG的大肠杆菌-lpmsp发酵液,湿菌体含量为30g/L。
(3)1L发酵液中的转化
取步骤(2)制备的1L大肠杆菌-lpmsp发酵液(湿菌体含量为30g/L),用1mol/LNaOH调节pH7.0后,加入342g蔗糖和138g甘油,放在30℃水浴锅上,安装全自动机械搅拌器,100rpm进行催化反应,反应进行24h。
(4)2-α-GG产物浓度液相色谱检测(方法同一)
经过24h转化反应,测得残留的底物为蔗糖为27.6g/L,形成的产物甘油葡萄糖101g/L,底物蔗糖转化率为92.9%。其产量和转化率高于文献报道的α-糖苷酶和化学合成等方法(Tan XM,Luo Q,Lu XF.Appl Microbiol Biot 2016,100:6131-6139)。
序列表
<110> 浙江工业大学
<120> 一种甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1473
<212> DNA
<213> 未知(Unknown)
<400> 1
atggaaattc aaaacaaagc aatgttgatc acttatgctg attcgttggg caaaaactta 60
aaagatgttc atcaagtctt gaaagaagat attggagatg cgattggtgg ggttcacttg 120
ttgccattct tcccttcaac aggtgatcgt ggctttgcac cagccgatta tactcgtgtt 180
gatgccgcat ttggtgattg gaaagatgtc gaagcattgg gtgaagaata ctatttgatg 240
tttgacttca tgattaacca tatttctcgt gaatcagtga tgtatcagga ttttaaaaag 300
aaccatgacg attcaaaata taaagatttc tttatccgtt gggaaaagtt ctgggcaaag 360
gccggcgaaa accgtccaac acaagccgat gttgacttaa tttacaagcg taaagataag 420
gcaccaacgc aagaaatcac ttttgatgat ggcacaacag aaaacttgtg gaatactttt 480
ggtgaagaac aaattgacat tgatgttaat tcagccattg ccaaggaatt tattaagaca 540
acccttgaag acatggtaaa acatggtgct aacttgattc gtttggatgc ctttgcgtat 600
gcagttaaaa aagttgacac aaatgacttc ttcgttgagc cagaaatctg ggacactttg 660
aatgaagtac gtgaaatttt gacaccatta aaggctgaaa ttttaccaga aattcatgaa 720
cattactcaa tccctaaaaa gatcaatgat catggttact tcacctatga ctttgcatta 780
ccaatgacaa cgctttacac attgtattca ggtaagacaa atcaattggc aaagtggttg 840
aaaatgtcat caatgaagca attcacaaca ttggacacgc atgatggtat tggtgttgtc 900
gatgcccgtg atattctaac tgatgatgaa attgattacg cttctgagca actttacaag 960
gttggcgcta atgttaaaaa gacatattca tcagcttcat acaacaacct tgatatctac 1020
caaattaact caacttatta ttcagcattg ggaaatgatg atgcagcata cttgttgagt 1080
cgtgtcttcc aagtctttgc gcctggaatt ccacaaattt attacgttgg tttgttggca 1140
ggtgaaaacg atatcgcgct tttggagtca actaaagaag gtcgtaatat taaccgtcat 1200
tactatacgc gtgaagaagt taagtcagaa gttaagcgac cagttgttgc taacttattg 1260
aagctattgt catggcgtaa tgaaagccca gcattcgatt tggctggttc aatcacagtt 1320
gacacgccaa ctgatacaac gattgtggtg acacgtcaag atgaaaatgg tcaaaacaaa 1380
gcggtattaa cagctgatgc ggcgaacaag acttttgaaa tcgttgaaaa tggtcaaaca 1440
gttatgagca gtgatgattt gactcagaac taa 1473
<210> 2
<211> 490
<212> PRT
<213> 未知(Unknown)
<400> 2
Met Glu Ile Gln Asn Lys Ala Met Leu Ile Thr Tyr Ala Asp Ser Leu
1 5 10 15
Gly Lys Asn Leu Lys Asp Val His Gln Val Leu Lys Glu Asp Ile Gly
20 25 30
Asp Ala Ile Gly Gly Val His Leu Leu Pro Phe Phe Pro Ser Thr Gly
35 40 45
Asp Arg Gly Phe Ala Pro Ala Asp Tyr Thr Arg Val Asp Ala Ala Phe
50 55 60
Gly Asp Trp Lys Asp Val Glu Ala Leu Gly Glu Glu Tyr Tyr Leu Met
65 70 75 80
Phe Asp Phe Met Ile Asn His Ile Ser Arg Glu Ser Val Met Tyr Gln
85 90 95
Asp Phe Lys Lys Asn His Asp Asp Ser Lys Tyr Lys Asp Phe Phe Ile
100 105 110
Arg Trp Glu Lys Phe Trp Ala Lys Ala Gly Glu Asn Arg Pro Thr Gln
115 120 125
Ala Asp Val Asp Leu Ile Tyr Lys Arg Lys Asp Lys Ala Pro Thr Gln
130 135 140
Glu Ile Thr Phe Asp Asp Gly Thr Thr Glu Asn Leu Trp Asn Thr Phe
145 150 155 160
Gly Glu Glu Gln Ile Asp Ile Asp Val Asn Ser Ala Ile Ala Lys Glu
165 170 175
Phe Ile Lys Thr Thr Leu Glu Asp Met Val Lys His Gly Ala Asn Leu
180 185 190
Ile Arg Leu Asp Ala Phe Ala Tyr Ala Val Lys Lys Val Asp Thr Asn
195 200 205
Asp Phe Phe Val Glu Pro Glu Ile Trp Asp Thr Leu Asn Glu Val Arg
210 215 220
Glu Ile Leu Thr Pro Leu Lys Ala Glu Ile Leu Pro Glu Ile His Glu
225 230 235 240
His Tyr Ser Ile Pro Lys Lys Ile Asn Asp His Gly Tyr Phe Thr Tyr
245 250 255
Asp Phe Ala Leu Pro Met Thr Thr Leu Tyr Thr Leu Tyr Ser Gly Lys
260 265 270
Thr Asn Gln Leu Ala Lys Trp Leu Lys Met Ser Ser Met Lys Gln Phe
275 280 285
Thr Thr Leu Asp Thr His Asp Gly Ile Gly Val Val Asp Ala Arg Asp
290 295 300
Ile Leu Thr Asp Asp Glu Ile Asp Tyr Ala Ser Glu Gln Leu Tyr Lys
305 310 315 320
Val Gly Ala Asn Val Lys Lys Thr Tyr Ser Ser Ala Ser Tyr Asn Asn
325 330 335
Leu Asp Ile Tyr Gln Ile Asn Ser Thr Tyr Tyr Ser Ala Leu Gly Asn
340 345 350
Asp Asp Ala Ala Tyr Leu Leu Ser Arg Val Phe Gln Val Phe Ala Pro
355 360 365
Gly Ile Pro Gln Ile Tyr Tyr Val Gly Leu Leu Ala Gly Glu Asn Asp
370 375 380
Ile Ala Leu Leu Glu Ser Thr Lys Glu Gly Arg Asn Ile Asn Arg His
385 390 395 400
Tyr Tyr Thr Arg Glu Glu Val Lys Ser Glu Val Lys Arg Pro Val Val
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Ala Asn Leu Leu Lys Leu Leu Ser Trp Arg Asn Glu Ser Pro Ala Phe
420 425 430
Asp Leu Ala Gly Ser Ile Thr Val Asp Thr Pro Thr Asp Thr Thr Ile
435 440 445
Val Val Thr Arg Gln Asp Glu Asn Gly Gln Asn Lys Ala Val Leu Thr
450 455 460
Ala Asp Ala Ala Asn Lys Thr Phe Glu Ile Val Glu Asn Gly Gln Thr
465 470 475 480
Val Met Ser Ser Asp Asp Leu Thr Gln Asn
485 490
Claims (8)
1.一种甘油-2-α-葡萄糖基化酶在制备2-α-甘油葡萄糖苷中的应用,其特征在于所述甘油-2-α-葡萄糖基化酶氨基酸序列为SEQ ID NO.2所示。
2.如权利要求1所述的应用,其特征在于所述甘油-2-α-葡萄糖基化酶编码基因的核苷酸序列为SEQ ID NO.1所示。
3.如权利要求1所述的应用,其特征在于所述应用的方法为:将含甘油-2-α-葡萄糖基化酶基因的重组大肠杆菌经发酵培养获得的发酵液或发酵离心的湿菌体用缓冲液悬浮的菌悬液为催化剂,以甘油为底物,以蔗糖为辅助底物,在30-45℃的条件下进行反应,获得含2-α-甘油葡萄糖苷的反应液,将反应液分离纯化,获得2-α-甘油葡萄糖苷。
4.如权利要求3所述的应用,其特征在于所述发酵液或菌悬液中湿菌体含量为5-100g/L,甘油的终浓度为100-150g/L,蔗糖终浓度为300-400g/L。
5.如权利要求3所述的应用,其特征在于反应pH为6.0-8.0,反应时间为24h。
6.如权利要求3所述的应用,其特征在于缓冲液为pH7.0、2.5mM磷酸缓冲液。
7.如权利要求3所述的应用,其特征在于所述发酵液的摇瓶制备方法:(1)将含甘油-2-α-葡萄糖基化酶基因的重组大肠杆菌接种在含50mg/L卡那霉素的种子培养基中,30-37℃、180-250rpm培养至对数生长中期,获得种子液;所述种子培养基终浓度组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH6.8-7.0;
(2)发酵培养:将种子液以体积浓度5%的接种量接种到含卡那霉素50mg/L的发酵培养基中,在30-37℃培养4-6h;加入终浓度为18-22g/L的α-乳糖,在22-25℃继续发酵12-18h,取发酵液离心,收集湿菌体细胞;所述发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.3g/L,溶剂为去离子水,pH6.8-7.0。
8.如权利要求3所述的应用,其特征在于所述发酵液的发酵罐制备方法:
(1)将含甘油-2-α-葡萄糖基化酶基因的重组大肠杆菌接种在含50mg/L卡那霉素的种子培养基中,30-37℃、180-250rpm培养至对数生长中期,获得种子液;所述种子培养基终浓度组成:酵母粉5g/L、蛋白胨10g/L、NaHPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.49g/L,溶剂为去离子水,pH6.8-7.0;
(2)发酵培养:将新鲜培养的种子液按照体积浓度5%的接种量,接种到含质量浓度0.05%消泡剂和50mg/L卡那霉素的发酵培养基中,37℃培养4h;加入终浓度为20g/L的α-乳糖,在发酵温度25℃、溶解氧DO大于20%、pH6.8条件下发酵培养,获得发酵液;所述发酵培养基质量终浓度组成:酵母粉12g/L、蛋白胨15g/L、甘油10g/L、Na2HPO4·12H2O 8.9g/L、KH2PO4 3.4g/L、NH4Cl 2.67g/L、Na2SO4 0.71g/L、MgSO4·7H2O 0.3g/L,溶剂为去离子水,pH6.8-7.0。
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