CN114058610B - 一种高活性的蔗糖异构酶及其应用 - Google Patents
一种高活性的蔗糖异构酶及其应用 Download PDFInfo
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- CN114058610B CN114058610B CN202111442626.4A CN202111442626A CN114058610B CN 114058610 B CN114058610 B CN 114058610B CN 202111442626 A CN202111442626 A CN 202111442626A CN 114058610 B CN114058610 B CN 114058610B
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Abstract
本发明涉及酶催化技术领域，尤其是一种高活性的蔗糖异构酶及其应用，该蔗糖异构酶氨基酸序列如SEQ ID NO：1所示。使用时，取蔗糖母液，加入PB、纯水，加入该蔗糖异构酶的粗酶液，30℃反应。其中，酶的最高底物浓度≤750g/L，底物：粗酶液投比≤200：1。本发明的高活性蔗糖异构酶比野生型蔗糖异构酶具有更高的底物浓度，更快的反应速度，投酶量低，可有效缩减成本；转化率高，大于90％。
Description
技术领域
本发明涉及酶催化技术领域，具体领域为一种高活性的蔗糖异构酶。
背景技术
蔗糖是一种常用的甜味剂。一旦摄取，蔗糖降解为葡萄糖和果糖，从而升高血糖水平。过量摄入蔗糖很容易导致肥胖，并与健康问题有关，例如心脏病和2型糖尿病。随着生活水平和健康意识的提高，人们开始关注可以替代蔗糖的、更健康的功能性甜味剂。
随着生活水平的提高和健康意识的发展，人们更加注重饮食。在甜味剂方法，人们更喜欢低热量和保健功能产品，如D-塔格糖、阿洛酮糖、异麦芽酮糖和低聚果糖等。作为蔗糖的替代品，异麦芽酮糖具有许多健康优势，例如，延长能量释放、减慢消化、更高的稳定性、降低胰岛素水平、防止蛀牙和血糖指数。因此，异麦芽酮糖被认为是的肠胃外治疗糖尿病营养物。另外，异麦芽酮糖作为一种还原性功能二糖，是目前唯一没有用量限定的甜味剂。
异麦芽酮糖，俗称帕拉金糖，由d-葡萄糖和d-果糖通过α-1,6-糖苷键连接而成，不同于蔗糖中的α-1,2-糖苷键。异麦芽酮糖具有与蔗糖相似的物理和感官特性。然而，与蔗糖相比，它有几个优点：由于异麦芽酮糖的甜度低于蔗糖(约为甜度的50％)，是一种低热量的非致龋营养糖；此外，异麦芽酮糖被酶缓慢水解并以葡萄糖和果糖的形式被吸收到机体中，从而降低胰岛素水平和血糖指数。因此，异麦芽酮糖有潜力成为肥胖症和糖尿病的蔗糖替代品。
异麦芽酮糖的合成通常涉及化学或酶促方法。化学法总是伴随着某些副产物的形成，颜色呈褐色，增加了产品的分离纯化难度；此外，这些困难增加了生产成本和能源消耗。因此，利用生物转化技术以蔗糖为底物合成异麦芽酮糖成为研究热点。此外，作为高效、绿色的生物催化剂，酶具有更温和的反应条件、更容易加工和更高的产品收率等优势，使得酶法在各个行业中越来越重要。
在工业上，异麦芽酮糖主要由蔗糖经细菌发酵制得。这些异麦芽酮糖生产菌株可以合成蔗糖异构酶，该酶负责将蔗糖异构化为异麦芽酮糖和其他副产物，如海藻糖、果糖和葡萄糖。但蔗糖底物浓度有限，使得发酵过程中异麦芽酮糖产量降低。
目前，蔗糖异构酶已有多种微生物来源得到披露，但根据文献报道，用蔗糖异构酶制备异麦芽酮糖存在以下问题：
(1)随反应时间延长，杂糖比例增多，如葡萄糖、海藻酮糖等总计占比在10％-20％不等。极端的情况下，曾经有过海藻酮糖占比高达50％的结果出现。
(2)高浓度底物的反应体系可以有效增加时空生产效率，减小能耗，并且有文献报道在800g/L蔗糖的高渗透压下微生物无法生长，故可以在开放式设备中生产。
因此，开发一种高酶活的蔗糖异构酶或突变体，使得反应时间缩短，进而在一定程度上减小杂糖占比，具有很大的实用价值。
发明内容
本发明的目的在于提供一种高活性的蔗糖异构酶及其应用。
为实现上述目的，本发明提供如下技术方案：
一种高活性的蔗糖异构酶，其氨基酸序列如SEQ ID NO：1所示。
本发明的蔗糖异构酶的底物浓度和反应速度均高于野生型蔗糖异构酶的底物浓度和反应速度。其转化率大于90％。
本发明高活性的蔗糖异构酶可用于催化生产异麦芽酮糖中，使用方法为：取蔗糖母液，加入PB、纯水，加入所述蔗糖异构酶的粗酶液，30℃反应。
经过大量实验验证，该蔗糖异构酶的最高底物浓度≤750g/L；底物：粗酶液投比≤200：1。
其中，所述粗酶液的制备方法包括以下步骤：
(1)通过引物拼接的方法，将SEQ ID NO：1所示蛋白的对应编码多核苷酸序列进行组装，并克隆到原核表达载体，以实现在大肠杆菌中的高表达；
(2)摇瓶发酵
挑取含有表达载体的大肠杆菌单菌落接种于10mL高压灭菌后的培养基A中，30℃，250rpm过夜培养；
次日取1L三角瓶，按1：100的接种比实施例接入到100mL高压灭菌后的培养基B中，于30℃中培养至菌体OD 5-6，立刻将三角瓶置于25℃摇床中，250rpm培养1小时；加IPTG至终浓度0.1mM，并于25℃，250rpm继续培养16小时；
培养结束后，将培养液于4℃，12000g下离心20分钟收集湿菌体；然后将菌体沉淀用蒸馏水清洗两次，收集菌体，-70℃保存；同时取2克菌体加入6mL纯水超声破碎后进行SDS-PAGE检测，并将粗酶液-20℃保存；
(3)分批补料发酵
分批补料发酵在计算机控制的生物反应器中进行，初级接种菌种制备200mL培养物，OD2.0时接入；在整个发酵过程中，温度保持在37℃，发酵过程中溶解氧浓度由搅拌速率和通气供应级联控制在30％自动控制，而培养基的pH值由50％正磷酸和30％氨水维持在7.0；
发酵过程中，当出现大幅的溶氧回升时，开始补料，补料溶液含有9％w/v蛋白胨、9％w/v酵母提取物、14％w/v甘油；当OD600为35.0时，用0.2mM IPTG诱导16小时；取2克菌体加入6mL纯水超声破碎后进行SDS-PAGE检测，并将粗酶液-20℃保存。
进一步的，步骤(2)中所述培养基A为：胰蛋白胨10g/L，酵母提取物5g/L，磷酸氢二钠3.55g/L，磷酸二氢钾3.4g/L，氯化铵2.68g/L，硫酸钠0.71g/L，七水硫酸镁0.493g/L，六水氯化铁0.027g/L，甘油5g/L，葡萄糖0.8g/L，添加卡那霉素至50mg/L。
所述培养基B为：胰蛋白胨10g/L，酵母提取物5g/L，磷酸氢二钠3.55g/L，磷酸二氢钾3.4g/L，氯化铵2.68g/L，硫酸钠0.71g/L，七水硫酸镁0.493g/L，六水氯化铁0.027g/L，甘油5g/L，葡萄糖0.3g/L，添加卡那霉素至50mg/L。
进一步的，步骤(3)中所用的培养基为：酵母抽提物24g/L，蛋白胨12g/L，葡萄糖0.4％，2.31g/L磷酸二氢酶和12.54g/L磷酸氢二钾，pH 7.0。
与现有技术相比，本发明的有益效果是：
本发明的高活性蔗糖异构酶比野生型蔗糖异构酶具有更高的底物浓度，更快的反应速度，投酶量低，可有效缩减成本；转化率高，大于90％；尤其适用于工业大规模生产异麦芽酮糖，可取得较好的社会效益和经济价值。
附图说明
图1为实施例2的TLC检测图谱；从左至右依次为蔗糖标品，异麦芽糖标品，反应样品；
图2为20g/L异麦芽酮糖标品检测图谱；其中，出峰时间为8.8min；
图3为实施例4的检测结果，8.8min为目标产物。
图4为实施例8的检测结果，8.8min为目标产物，8.5min为残余底物峰。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
以下实施例中涉及到的检测条件如下：
(1)液相检测条件
流动相：乙腈：水＝70：30
检测器：示差检测器
流速：1mL/min
柱温：40℃
示差检测器池温度：40℃
使用250mm*4.6um氨基柱。
(2)TLC检测条件
展开剂：将正丁醇：丙酮：水按4:3:1混合再加一滴乙酸，跑2次(跑一次吹干再跑一次)。
显色剂：将4％苯胺-4％二苯胺-85％磷酸按体积比5:5:1混合使用，显色温度85℃，显色时间10分钟。
实施例1粗酶液的制备
(1)通过引物拼接的方法，将SEQ ID NO：1所示蛋白的对应编码多核苷酸序列进行组装，并克隆到原核表达载体，以实现在大肠杆菌中的高表达，并命名为SP1。具体操作如下：
将引物SP1-1～SP1-48(如SEQ ID NO：3-50所示)加双蒸水溶解后，加到如下的反应体系中，使得各引物的终浓度为30nM，首尾引物的终浓度为0.6μM。
| 2mM dNTP mix(2mM each dNTP) | 5μl |
| 10×Pfu buffer | 5μl |
| Pfu DNA polymerase(10U/μl) | 0.5μl |
| ddH2O | 使得反应体系总体积至50μl |
将配制好的PCR反应体系置于博日XP cycler基因扩增仪中，按下列程序进行扩增：98℃30s，55℃45s，72℃120s，35x。将PCR得到的DNA片段进行切胶纯化克隆并测序。
同样的，将对照野生型蔗糖异构酶蛋白SEQ ID NO：2的对应编码多核苷酸序列进行组装，并克隆到原核表达载体，命名为SP2。所用到的引物SP2-1～SP2-48如SEQ ID NO：51-98所示。
(2)摇瓶发酵
挑取含有表达载体的大肠杆菌单菌落接种于10mL高压灭菌后的培养基中：胰蛋白胨10g/L，酵母提取物5g/L，磷酸氢二钠3.55g/L，磷酸二氢钾3.4g/L，氯化铵2.68g/L，硫酸钠0.71g/L，七水硫酸镁0.493g/L，六水氯化铁0.027g/L，甘油5g/L，葡萄糖0.8g/L，添加卡那霉素至50mg/L。30℃，250rpm过夜培养。次日取1L三角瓶，按1：100的接种比实施例接入到100mL高压灭菌后的培养基中：胰蛋白胨10g/L，酵母提取物5g/L，磷酸氢二钠3.55g/L，磷酸二氢钾3.4g/L，氯化铵2.68g/L，硫酸钠0.71g/L，七水硫酸镁0.493g/L，六水氯化铁0.027g/L，甘油5g/L，葡萄糖0.3g/L，添加卡那霉素至50mg/L。于30℃中培养至菌体OD 5-6，立刻将三角瓶置于25℃摇床中，250rpm培养1小时。加IPTG至终浓度0.1mM，并于25℃，250rpm继续培养16小时。培养结束后，将培养液于4℃，12000g下离心20分钟收集湿菌体。然后将菌体沉淀用蒸馏水清洗两次，收集菌体，-70℃保存。同时取2克菌体加入6mL纯水超声破碎后进行SDS-PAGE检测，并将粗酶液-20℃保存。
(3)分批补料发酵：
分批补料发酵在计算机控制的生物反应器(上海国强)中进行，反应器容量为15L，工作体积为8L，所用到的培养基为酵母抽提物24g/L，蛋白胨12g/L，葡萄糖0.4％，2.31g/L磷酸二氢酶和12.54g/L磷酸氢二钾，pH 7.0。初级接种菌种制备200mL培养物，OD2.0时接入。在整个发酵过程中，温度保持在37℃，发酵过程中溶解氧浓度由搅拌速率(rpm)和通气供应级联控制在30％自动控制，而培养基的pH值由50％(v/v)正磷酸和30％(v/v)氨水维持在7.0。发酵过程中，当出现大幅的溶氧回升时，开始补料。补料溶液含有9％w/v蛋白胨、9％w/v酵母提取物、14％w/v甘油。当OD600约为35.0(湿重约为60g/L)时，用0.2mM IPTG诱导16小时。取2克菌体加入6mL纯水超声破碎后进行SDS-PAGE检测，并将粗酶液-20℃保存。
实施例2 650g/L底物浓度催化反应
先配制蔗糖母液(800g/L)，取800g蔗糖，加水定容至1L加热助溶，待用。
取1.62mL蔗糖母液(800gL)，加入1M PH6.0的PB 0.1mL，加入0.14mL纯水，加入粗酶液SP1 0.14mL，30℃反应。6小时点板检测无底物剩余。TLC结果见图1。
实施例3 700g/L底物浓度催化反应
取1.75mL蔗糖母液(800g/L)，加入1M pH6.0的PB缓冲液0.1mL，加入粗酶液SP1用量为56μL，补水至2mL，30℃摇床反应。6小时点板检测无底物剩余。
实施例4 700g/L底物浓度催化反应
取1.75mL蔗糖母液(800g/L)，加入1M pH5.8的PB缓冲液0.1mL，加入粗酶液SP1用量为28μL，补水至2mL。6小时点板检测无底物剩余。
即底物：粗酶液的投比为50:1时，6小时可完全转化底物。且目的产物转化率大于90％，杂糖占比小于10％。液相检测结果见图3。
实施例5 700g/L底物浓度催化反应
取1.75mL蔗糖母液(800g/L)，加入1M pH5.8的PB缓冲液0.1mL，加入粗酶液SP1用量为14μL，补水至2mL。6小时点板检测仍有底物剩余，表明此酶的极限投比在100:1以内。
实施例6 750g/L底物浓度催化反应
用50mM pH6.0的PB缓冲液配制蔗糖825g/L，取1.82mL使用，加入水0.12mL以及粗液液SP1 56μL，此时总体系为2mL。6小时点板检测有底物剩余。
实施例7 800g/L底物浓度催化反应
由于蔗糖在高于800g/L的浓度以上易析晶，无法预先配制母液，故使用前用50mMpH5.8的PB缓冲液配制蔗糖825g/L，加热助溶后，取2mL加入SP1 56μL(此时底物浓度为800g/L)，无需再加入水，30℃摇床反应。反应6小时，24小时点板显示，底物均未反应完。由此可知，800g/L近饱和状态下底物：粗酶液为30:1时无法有效完成催化，表明此酶的最高底物浓度要控制在750g/L以内。
实施例8 700g/L底物对照反应
取1.75mL蔗糖母液(800g/L)，加入1M pH5.8的PB缓冲液0.1mL，加入SEQ ID NO：2的对照粗酶液SP2为28μL，补水至2mL。6小时点板检测仍有底物剩余。液相检测结果见图4。
通过上述实验结果的对比可以看出，本发明的蔗糖异构酶比野生型蔗糖异构酶具有更高的底物浓度；比野生型蛋白在高浓度底物时反应速度更快，而且投酶量低，可有效缩减成本；转化率高，大于90％。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 南京诺云生物科技有限公司
<120> 一种高活性的蔗糖异构酶及其应用
<160> 98
<170> SIPOSequenceListing 1.0
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<210> 2
<211> 576
<212> PRT
<213> 野生型蔗糖异构酶(Artificial Sequence)
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Met Phe Gly Thr Gln Gln Pro Leu Leu Asn Glu Lys Ser Ile Glu Gln
1 5 10 15
Ser Lys Thr Ile Pro Lys Trp Trp Lys Glu Ala Val Phe Tyr Gln Val
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Tyr Pro Arg Ser Phe Lys Asp Thr Asn Gly Asp Gly Ile Gly Asp Ile
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Asn Gly Ile Ile Glu Lys Leu Asp Tyr Leu Lys Ala Leu Gly Ile Asp
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Ala Ile Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn Gly
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Tyr Asp Ile Arg Asp Tyr Arg Lys Ile Met Lys Glu Tyr Gly Thr Met
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Glu Asp Phe Asp Arg Leu Ile Ser Glu Met Lys Lys Arg Asn Met Arg
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Leu Met Ile Asp Val Val Ile Asn His Thr Ser Asp Gln Asn Glu Trp
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Phe Val Lys Ser Lys Ser Ser Lys Asp Asn Pro Tyr Arg Gly Tyr Tyr
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Phe Trp Lys Asp Ala Lys Glu Gly Gln Ala Pro Asn Asn Tyr Pro Ser
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Phe Phe Gly Gly Ser Ala Trp Gln Lys Asp Glu Lys Thr Asn Gln Tyr
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Tyr Leu His Tyr Phe Ala Lys Gln Gln Pro Asp Leu Asn Trp Asp Asn
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Pro Lys Val Arg Gln Asp Leu Tyr Thr Met Leu Arg Phe Trp Leu Asp
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Lys Gly Val Ser Gly Leu Arg Phe Asp Thr Val Ala Thr Tyr Ser Lys
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Ile Pro Asp Phe Pro Asn Leu Thr Gln Gln Gln Leu Lys Asn Phe Ala
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Ala Glu Tyr Thr Lys Gly Pro Asn Ile His Arg Tyr Val Asn Glu Met
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Asn Lys Glu Val Leu Ser His Tyr Asp Ile Ala Thr Ala Gly Glu Ile
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Phe Gly Asp Asp Arg Pro Gln Trp Arg Glu Pro Ser Ala Lys Ala Leu
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Ser Glu Leu Gly Met Thr Asn Tyr Pro Phe Lys Ala Ile Asp Glu Phe
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Asp Asp Ile Glu Val Lys Gly Phe Trp His Asp Tyr Val Glu Thr Gly
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<210> 3
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tgtttaactt taagaaggag atatacatat gaaatacctg ctgccg 46
<210> 4
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gctgggcagc gaggagcagc agaccagcag cagcggtcgg cagcaggtat ttcatatgta 60
<210> 5
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctcctcgctg cccagccggc gatggccatg ttcggtaccc agcagccgct gctgaacgaa 60
<210> 6
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctttccacca tttcgggatg gttttagact gttcgataga tttttcgttc agcagcggct 60
<210> 7
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccatcccgaa atggtggaaa gaagctgttt tctaccaggt ttacccgcgt tctttcaaag 60
<210> 8
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gataccgttg atgtcaccga taccgtcacc gttggtgtct ttgaaagaac gcgggtaaac 60
<210> 9
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atcggtgaca tcaacggtat catcgaaaaa ctggactacc tgaaagctct gggtatcgac 60
<210> 10
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggtgttcgga gagtcgtagt gcgggttgat ccagatagcg tcgataccca gagctttcag 60
<210> 11
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
actacgactc tccgaacacc gacaacggtt acgacatccg tgactaccgt aaaatcatga 60
<210> 12
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atcagacggt cgaagtcttc catggtaccg tattctttca tgattttacg gtagtcacgg 60
<210> 13
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggaagacttc gaccgtctga tctctgaaat gaaaaaacgt aacatgcgtc tgatgatcga 60
<210> 14
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
accattcgtt ctggtcagag gtgtggttga taacaacgtc gatcatcaga cgcatgttac 60
<210> 15
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ctctgaccag aacgaatggt tcgttcagtc taaatcttct aaagacaacc cgtaccgtgg 60
<210> 16
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ttcggagcct gaccttcttt agcgtctttc cagaagtagt aaccacggta cgggttgtct 60
<210> 17
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
taaagaaggt caggctccga acaactaccc gtctttcttc ggtggttctg cttggcagaa 60
<210> 18
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tagcgaagta gtgcaggtag tactggttgg ttttttcgtc tttctgccaa gcagaaccac 60
<210> 19
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
actacctgca ctacttcgct aaacagcagc cggacctgaa ctgggacaac ccgaaagttc 60
<210> 20
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
ttgtccagcc agaaacgcag catggtgtac aggtcctgac gaactttcgg gttgtcccag 60
<210> 21
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tgcgtttctg gctggacaaa ggtgtttctg gtctgcgttt cgacaccgtt gctacctact 60
<210> 22
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
agctgctgct gggtcaggtt cgggaagtcc gggattttag agtaggtagc aacggtgtcg 60
<210> 23
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ctgacccagc agcagctgaa aaacttcgct gctgaataca ccaaaggtcc gaacatccac 60
<210> 24
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
agtgagacag aacttctttg ttcatttcgt taacgtaacg gtggatgttc ggacctttgg 60
<210> 25
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
atgaacaaag aagttctgtc tcactacgac atcgctaccg ctggtgaaat cttcggtgtt 60
<210> 26
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
acgacgacgg tcgaagaatt tgatagactg gtccagcgga acaccgaaga tttcaccagc 60
<210> 27
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
attcttcgac cgtcgtcgtg acgaactgaa catcgctttc accttcgacc tgatccgtct 60
<210> 28
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tccagtcttt acgacgccaa cgctggtcag agtcacggtc cagacggatc aggtcgaagg 60
<210> 29
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tggcgtcgta aagactggaa actgtctcag ttccgtcaga tcatcgacaa cgttgaccgt 60
<210> 30
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ttgtccagga agaaagcgtt ccaaccgtat tcaccagcgg tacggtcaac gttgtcgatg 60
<210> 31
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gaacgctttc ttcctggaca accacgacaa cccgcgtgct gtttctcact tcggtgacga 60
<210> 32
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
gccagagctt tagcagacgg ttcacgccac tgcggacggt cgtcaccgaa gtgagaaaca 60
<210> 33
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
cgtctgctaa agctctggct accctgaccc tgacccagcg tgctaccccg ttcatctacc 60
<210> 34
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gctttgaacg ggtagttggt catacccagt tcagaaccct ggtagatgaa cggggtagca 60
<210> 35
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gaccaactac ccgttcaaag ctatcgacga attcgacgac atcgaagtta aaggtttctg 60
<210> 36
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
tcagctttaa ctttaccggt ttcaacgtag tcgtgccaga aacctttaac ttcgatgtcg 60
<210> 37
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
tgaaaccggt aaagttaaag ctgacgaatt cctgcagaac gttcgtctga cctctcgtga 60
<210> 38
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gtttttagaa ccgtcccact ggaacggggt acgagagttg tcacgagagg tcagacgaac 60
<210> 39
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ccagtgggac ggttctaaaa acgctggttt cacctctggt aaaccgtggt tcaaagttaa 60
<210> 40
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
aacctgagaa acagcgttga tttcctggta gttcgggtta actttgaacc acggtttacc 60
<210> 41
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
atcaacgctg tttctcaggt tacccagccg gactctgttt tcaactacta ccgtcagctg 60
<210> 42
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ccgtaggtca gagccgggat gtcgtgacgg atgttgatca gctgacggta gtagttgaaa 60
<210> 43
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
ccggctctga cctacggtac ctacaccgac ctggacccgg ctaacgactc tgtttacgct 60
<210> 44
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
aacaaccagg tatttttcag cacccagaga acgggtgtaa gcgtaaacag agtcgttagc 60
<210> 45
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gtgctgaaaa atacctggtt gttgttaact tcaaagaaca gatgatgcgt tacaaactgc 60
<210> 46
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
agagtcgatg ataacttttt cgatagacag gttgtccggc agtttgtaac gcatcatctg 60
<210> 47
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
ctatcgaaaa agttatcatc gactctaact ctaaaaacgt tgttaaaaaa aacgactctc 60
<210> 48
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
gtagatacca gactgccacg gtttcagttc cagcagagag tcgttttttt taacaacgtt 60
<210> 49
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
cgtggcagtc tggtatctac aaactgaacc agtaactcga gcaccaccac caccaccact 60
<210> 50
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
cggatctcag tggtggtggt ggtgg 25
<210> 51
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
tgtttaactt taagaaggag atatacatat gaaatacctg ctgccg 46
<210> 52
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
gctgggcagc gaggagcagc agaccagcag cagcggtcgg cagcaggtat ttcatatgta 60
<210> 53
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
ctcctcgctg cccagccggc gatggccatg ttcggtaccc agcagccgct gctgaacgaa 60
<210> 54
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
ctttccacca tttcgggatg gttttagact gttcgataga tttttcgttc agcagcggct 60
<210> 55
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
ccatcccgaa atggtggaaa gaagctgttt tctaccaggt ttacccgcgt tctttcaaag 60
<210> 56
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
gataccgttg atgtcaccga taccgtcacc gttggtgtct ttgaaagaac gcgggtaaac 60
<210> 57
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
atcggtgaca tcaacggtat catcgaaaaa ctggactacc tgaaagctct gggtatcgac 60
<210> 58
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
ggtgttcgga gagtcgtagt gcgggttgat ccagatagcg tcgataccca gagctttcag 60
<210> 59
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
actacgactc tccgaacacc gacaacggtt acgacatccg tgactaccgt aaaatcatga 60
<210> 60
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
atcagacggt cgaagtcttc catggtaccg tattctttca tgattttacg gtagtcacgg 60
<210> 61
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
ggaagacttc gaccgtctga tctctgaaat gaaaaaacgt aacatgcgtc tgatgatcga 60
<210> 62
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
accattcgtt ctggtcagag gtgtggttga taacaacgtc gatcatcaga cgcatgttac 60
<210> 63
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
ctctgaccag aacgaatggt tcgttaaatc taaatcttct aaagacaacc cgtaccgtgg 60
<210> 64
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
ttcggagcct gaccttcttt agcgtctttc cagaagtagt aaccacggta cgggttgtct 60
<210> 65
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
taaagaaggt caggctccga acaactaccc gtctttcttc ggtggttctg cttggcagaa 60
<210> 66
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
tagcgaagta gtgcaggtag tactggttgg ttttttcgtc tttctgccaa gcagaaccac 60
<210> 67
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
actacctgca ctacttcgct aaacagcagc cggacctgaa ctgggacaac ccgaaagttc 60
<210> 68
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
ttgtccagcc agaaacgcag catggtgtac aggtcctgac gaactttcgg gttgtcccag 60
<210> 69
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
tgcgtttctg gctggacaaa ggtgtttctg gtctgcgttt cgacaccgtt gctacctact 60
<210> 70
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
agctgctgct gggtcaggtt cgggaagtcc gggattttag agtaggtagc aacggtgtcg 60
<210> 71
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
ctgacccagc agcagctgaa aaacttcgct gctgaataca ccaaaggtcc gaacatccac 60
<210> 72
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
agtgagacag aacttctttg ttcatttcgt taacgtaacg gtggatgttc ggacctttgg 60
<210> 73
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
atgaacaaag aagttctgtc tcactacgac atcgctaccg ctggtgaaat cttcggtgtt 60
<210> 74
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
acgacgacgg tcgaagaatt tgatagactg gtccagcgga acaccgaaga tttcaccagc 60
<210> 75
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
attcttcgac cgtcgtcgtg acgaactgaa catcgctttc accttcgacc tgatccgtct 60
<210> 76
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
tccagtcttt acgacgccaa cgctggtcag agtcacggtc cagacggatc aggtcgaagg 60
<210> 77
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
tggcgtcgta aagactggaa actgtctcag ttccgtcaga tcatcgacaa cgttgaccgt 60
<210> 78
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
ttgtccagga agaaagcgtt ccaaccgtat tcaccagcgg tacggtcaac gttgtcgatg 60
<210> 79
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
gaacgctttc ttcctggaca accacgacaa cccgcgtgct gtttctcact tcggtgacga 60
<210> 80
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
gccagagctt tagcagacgg ttcacgccac tgcggacggt cgtcaccgaa gtgagaaaca 60
<210> 81
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
cgtctgctaa agctctggct accctgaccc tgacccagcg tgctaccccg ttcatctacc 60
<210> 82
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
gctttgaacg ggtagttggt catacccagt tcagaaccct ggtagatgaa cggggtagca 60
<210> 83
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
gaccaactac ccgttcaaag ctatcgacga attcgacgac atcgaagtta aaggtttctg 60
<210> 84
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
tcagctttaa ctttaccggt ttcaacgtag tcgtgccaga aacctttaac ttcgatgtcg 60
<210> 85
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
tgaaaccggt aaagttaaag ctgacgaatt cctgcagaac gttcgtctga cctctcgtga 60
<210> 86
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
gtttttagaa ccgtcccact ggaacggggt acgagagttg tcacgagagg tcagacgaac 60
<210> 87
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
ccagtgggac ggttctaaaa acgctggttt cacctctggt aaaccgtggt tcaaagttaa 60
<210> 88
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
aacctgagaa acagcgttga tttcctggta gttcgggtta actttgaacc acggtttacc 60
<210> 89
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
atcaacgctg tttctcaggt tacccagccg gactctgttt tcaactacta ccgtcagctg 60
<210> 90
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
ccgtaggtca gagccgggat gtcgtgacgg attttgatca gctgacggta gtagttgaaa 60
<210> 91
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
ccggctctga cctacggtac ctacaccgac ctggacccgg ctaacgactc tgtttacgct 60
<210> 92
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
aacaaccagg tatttttcag cacccagaga acgggtgtaa gcgtaaacag agtcgttagc 60
<210> 93
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
gtgctgaaaa atacctggtt gttgttaact tcaaagaaca gatgatgcgt tacaaactgc 60
<210> 94
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
agagtcgatg ataacttttt cgatagacag gttgtccggc agtttgtaac gcatcatctg 60
<210> 95
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
ctatcgaaaa agttatcatc gactctaact ctaaaaacgt tgttaaaaaa aacgactctc 60
<210> 96
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
gtaaacacca gactgccacg gtttcagttc cagcagagag tcgttttttt taacaacgtt 60
<210> 97
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
cgtggcagtc tggtgtttac aaactgaacc agtaactcga gcaccaccac caccaccact 60
<210> 98
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
cggatctcag tggtggtggt ggtgg 25
Claims (4)
1.一种高活性的蔗糖异构酶，其特征在于：其氨基酸序列如SEQ ID NO：1所示。
2.权利要求1所述高活性的蔗糖异构酶在催化生产异麦芽酮糖中的应用。
3.根据权利要求2所述高活性的蔗糖异构酶在催化生产异麦芽酮糖中的应用，其特征在于：取蔗糖母液，加入PB缓冲液、纯水，加入权利要求1所述蔗糖异构酶的粗酶液，30℃反应；所述蔗糖母液浓度≤750g/L，蔗糖母液：粗酶液投比≤200：1，所述投比为蔗糖母液中蔗糖质量与粗酶液体积之比；
所述粗酶液的制备方法包括以下步骤：
(1)通过引物拼接的方法，将SEQ ID NO：1所示蛋白的对应编码多核苷酸序列进行组装，并克隆到原核表达载体，以实现在大肠杆菌中的高表达；
(2)摇瓶发酵
挑取含有表达载体的大肠杆菌单菌落接种于10mL高压灭菌后的培养基A中，30℃，250rpm过夜培养；所述培养基A为：胰蛋白胨10g/L，酵母提取物5g/L，磷酸氢二钠3.55g/L，磷酸二氢钾3.4g/L，氯化铵2.68g/L，硫酸钠0.71g/L，七水硫酸镁0.493g/L，六水氯化铁0.027g/L，甘油5g/L，葡萄糖0.8g/L，添加卡那霉素至50mg/L；
次日取1L三角瓶，按1：100的接种比例接入到100mL高压灭菌后的培养基B中，于30℃中培养至菌体OD 5-6，立刻将三角瓶置于25℃摇床中，250rpm培养1小时；加IPTG至终浓度0.1mM，并于25℃，250rpm继续培养16小时；所述培养基B为：胰蛋白胨10g/L，酵母提取物5g/L，磷酸氢二钠3.55g/L，磷酸二氢钾3.4g/L，氯化铵2.68g/L，硫酸钠0.71g/L，七水硫酸镁0.493g/L，六水氯化铁0.027g/L，甘油5g/L，葡萄糖0.3g/L，添加卡那霉素至50mg/L；
培养结束后，将培养液于4℃，12000g下离心20分钟收集湿菌体；然后将菌体沉淀用蒸馏水清洗两次，收集菌体，-70℃保存；同时取2克菌体加入6mL纯水超声破碎后进行SDS-PAGE检测，并将粗酶液-20℃保存；
(3)分批补料发酵
分批补料发酵在计算机控制的生物反应器中进行，初级接种菌种制备200mL培养物，OD2.0时接入；在整个发酵过程中，温度保持在37℃，发酵过程中溶解氧浓度由搅拌速率和通气供应级联控制在30％自动控制，而培养基的pH值由50％正磷酸和30％氨水维持在7.0；
发酵过程中，当出现溶氧回升时，开始补料，补料溶液含有9％w/v蛋白胨、9％w/v酵母提取物、14％w/v甘油；当OD600为35.0时，用0.2mM IPTG诱导16小时；取2克菌体加入6mL纯水超声破碎后进行SDS-PAGE检测，并将粗酶液-20℃保存。
4.根据权利要求3所述高活性的蔗糖异构酶在催化生产异麦芽酮糖中的应用，其特征在于：粗酶液制备方法的步骤(3)中所用的培养基为：酵母抽提物24g/L，蛋白胨12g/L，葡萄糖0.4％，2.31g/L磷酸二氢酶和12.54g/L磷酸氢二钾，pH 7.0。
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Denomination of invention: A highly active sucrose isomerase and its application Granted publication date: 20230811 Pledgee: Bank of China Limited Nanjing Chengdong Branch Pledgor: NANJING NUOYUN BIOTECHNOLOGY Co.,Ltd. Registration number: Y2024980013829 |
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