CN112831486A - 一种内切木聚糖苷酶及其制备低聚木糖的应用 - Google Patents
一种内切木聚糖苷酶及其制备低聚木糖的应用 Download PDFInfo
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- CN112831486A CN112831486A CN202011314412.4A CN202011314412A CN112831486A CN 112831486 A CN112831486 A CN 112831486A CN 202011314412 A CN202011314412 A CN 202011314412A CN 112831486 A CN112831486 A CN 112831486A
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- podoxyn11
- oligosaccharide
- xylanase
- xylo
- endoxylanase
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Abstract
一种内切木聚糖苷酶及其制备低聚木糖的应用，涉及食品和饲料添加剂领域，氨基酸序列如SEQ ID NO.2所示。本发明所述内切木聚糖酶podoxyn11在中温条件下稳定性良好。本发明所述内切木聚糖水解木聚糖类半纤维素的产物中低木糖得率高，且主要成分为木二糖和木三糖。
Description
技术领域
本发明属于基因工程技术，具体涉及食品和饲料添加剂领域，尤其涉及一种内切木聚糖苷酶及其制备低聚木糖的应用。
背景技术
肠道微生物菌群的分布可能会直接影响到生命体的健康，双歧杆菌、乳杆菌等肠道益生菌的大量增殖，能够产生多种有机酸，抑制有害菌的生长，同时这些益生菌能够改善便秘，治疗慢性腹泻，针对肝脏保护，改善高血压、动脉硬化等症状，且在降低血清胆固醇等方面具有显著的效果。
低聚木糖（Xylooligosaccharides, XOS），又称木寡糖。一般，低聚木糖指地是由2~10个木糖以 β-1,4-糖苷键连接形成低聚合度糖的总称，是新型的益生元和功能性低聚糖之一。其生理功能和理化性质方面均优于其它种类的低聚糖（水苏糖、棉籽糖、异麦芽酮糖、乳酮糖、低聚果糖、低聚半乳糖、低聚异麦芽糖等），尤其是对双歧杆菌增殖能力显著，也是目前使用效价最高的功能性低聚糖。同时它还具有良好的理化特性，如优良的热稳定性、酸稳定性和贮藏稳定性，甜度适中，感官特性适于与食品配伍。此外，低聚木糖主要是以富含木聚糖的木质纤维类为原料进行制备，而生物质来源的木质纤维素在自然界中广泛分布，同时我国农林剩余物资源也极为丰富。由此可见，低木聚糖被认为是一种具有开发和应用潜力的食品添加剂和饲料添加剂。
目前，有诸多专利文件报道水热法和酸水解法在制备低聚木糖方面的诸多应用，如专利文件（申请号201611114439 .2，公开号CN 106755615 A）中提出了一种水热法与稀乙酸水解法联合降解木质纤维素生物质的方法，其特征在于(1)将木质纤维素生物质干燥并粉碎后，以水为反应溶剂，进行水热法预处理，反应结束后迅速冷却，至固液分离，收集固体；(2)以100mmol/L以下 浓度的乙酸水溶液为反应溶剂，将步骤(1)中获得的固体进行水热法水解反应，反应结束后 迅速冷却，固液分离，收集水解液；(3)对收集的水解液进行精制得到低聚木糖产品。专利文件(申请号201310422674.6，103468834B)提出了一种中性亚硫酸盐处理植物纤维制备低聚木糖的方法，它包括如下步骤：1)将富含半纤维素的农林废弃物和生物质残渣切段或粉粹备料；2)将步骤1)得到的物料加入中性亚硫酸盐水溶液中，在pH5.0-7.0、140-170℃条件下充分混合反应10-60分钟；3)步骤2)反应结束后固液分离去除固体残渣，液体经超滤纯化收集透过液，再经纳滤除盐和多余二氧化硫即得到木糖含量较低、主要富含聚合度在2-6的低聚木糖水溶液。但上述水热及酸水解法制备低聚糖的过程中，木聚糖过度降解生成单糖是目前仍需解决的问题。酶法制备因其具有较高选择性，被认为是具有应用潜力的一种定向制备低聚木糖的方法，它是将富含木聚糖的木质纤维材料经过热处理或者酸碱预处理后再用木聚糖酶水解，获得聚合度2-6之间的低聚木糖。
发明内容
解决的技术问题：为解决低聚木糖制备过程中的过度水解解生成木糖问题，本发明提供了一种内切木聚糖苷酶及其制备低聚木糖的应用，其水解产物主要为木二糖和木三糖，且几乎不产生木糖。
技术方案：一种内切木聚糖酶podoxyn11，氨基酸序列如SEQ ID NO.2所示。
编码所述内切木聚糖酶podoxyn11的核苷酸，序列如SEQ ID NO.1所示。
制备所述内切木聚糖酶podoxyn11的方法，步骤为，为获得所述的内切木聚糖酶podoxyn11的DNA片段，将该DNA片段插入表达载体获得重组质粒，将重组质粒转化宿主菌，以及其诱导表达条件和后续目的蛋白的纯化。
含有所述的编码内切木聚糖酶podoxyn11核苷酸的重组质粒。
所述内切木聚糖酶podoxyn11在制备低聚木糖中的应用。
一种通过所述内切木聚糖酶podoxyn11转化农林生物质废弃物来源的半纤维素制备低聚木糖的方法，以杨木木聚糖为例，所述内切木聚糖酶podoxyn11在pH 4-6.5，温度45℃-55℃，酶解杨木木聚糖。pH优选为5.5，温度优选为45℃。
有益效果：（1）本发明所述内切木聚糖酶podoxyn11在中温条件下稳定性良好。（2）本发明所述内切木聚糖水解木聚糖类半纤维素的产物中低木糖得率高，且主要成分为木二糖和木三糖。
附图说明
图1为实施例2纯化的内切木聚糖酶的纯度鉴定结果图；其中泳道M为蛋白Marker(购自Thermo scientific公司，货号2661)，泳道1为本专利所述内切木聚糖酶podoxyn11的纯酶蛋白。
图2为实施例3本发明所述述内切木聚糖酶podoxyn11的酶学性质测定结果图，其中a为最适反应pH的测定结果图，横坐标为pH，纵坐标为相对酶活力，单位%；b为最适反应温度的测定结果图，横坐标为温度，单位摄氏度（℃），纵坐标为相对酶活力，单位%；c为酶蛋白pH稳定性的测定图；d为酶蛋白温度稳定性的测定图。
图3所示为实施例4本发明所述内切木聚糖酶podoxyn11对杨木木聚糖酶水解产物中低聚糖含量和种类的分析结果图，其中a：测定反应产物中还原糖及低聚木糖的含量图，b：测定产物中低聚木糖的含量图。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改和替换，均属于本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
为了进一步理解本发明，下面结合实施例对本发明进行详细阐述，其中，如无特殊说明，实施例中涉及的各种反应试剂均可以通过商业渠道购买得到；如无特殊说明，实施例中涉及的具体操作参见《分子克隆实验指南 第三版》。
实施例1 本发明所述内切木聚糖酶podoxyn11的获得及重组质粒PTRC-Xyn的构建
按照已知的拟杆菌全基因组序列筛选得到本专利所述编码内切木聚糖酶podoXyn11的基因片段，由于该种拟杆菌密码子的偏好性与表达宿主大肠杆菌BL21(DE3)具有一定差异，且可能对目的蛋白的成功表达产生影响，因此我们根据宿主菌的密码子偏好性对目的蛋白的编码基因进行密码子优化，同时优化该编码内切木聚糖酶podoXyn11的mRNA结构，提高其稳定性，优化后的编码内切木聚糖酶podoXyn11基因片段由南京金斯瑞生物科技有限公司合成，基因片段序列如SEQ ID NO:2所示。
优化后的片段被亚克隆至表达载体pTrc99A,即得到极耐热内切木聚糖酶podoXyn11的重组表达质粒PTRC-Xyn。
实施例2本发明所述内切木聚糖酶podoXyn11的制备
将重组质粒PTRC-Xyn转化大肠杆菌JM109(DE3) 宿主菌（购自Novagen公司），在含有卡那霉素（50 μg/mL）的LB平板（LB培养基：胰蛋白胨 10 g/L，酵母提取物 5 g/L，NaCl 5g/L，琼脂 15 g/L）上经过37℃培养过夜，挑转化子到200 mL的LB培养基中（50 μg/mL 卡那霉素）37℃，200 rpm振荡培养至OD600为0.6-1.2时，加入终浓度为1-2.5 mM 异丙基β-D-硫代吡喃半乳糖苷（IPTG）诱导剂，24-28℃下培养6 h，用高速冷冻离心机将培养液在4℃下，以13,000 rpm离心15 min，收集菌体。
由于重组质粒PTRC-Xyn中含有His-tag标签，通过His•Bind Purification Kit（购自Novagen公司）进行纯化，得到纯化的重组酶。具体操作过程：
A.样品的处理
（1）将洗涤过的菌体，用1×Binding Buffer 8mL重悬，超声波破壁。
（2）破壁后，13,000 g离心30 min，取上清即为样品。
（3）由于该种蛋白具有较好的热稳定性，因此通过热处理(70℃下保温15min后)可将大部分杂蛋白变性形成不溶性沉淀，13,000 g离心30 min，取上清即为热处理后的样品。
B.处理柱子
（1）取1 mL填料装柱。
（2）用3mL的无菌水洗柱子。
（3）用5mL的1×Charge Buffer洗柱子。
（4）用3mL的1×Binding Buffer洗柱子。
C.上样
（1）将样品加入柱子，控制流速约每分钟6滴。
（2）用3 mL 1×Binding Buffer洗柱子，除去未结合的蛋白质。
（3）用4 mL含有20 mM咪唑的洗脱液洗柱子，除去杂蛋白。
（4）用80 mmol/L咪唑的洗脱液洗柱子，将目的蛋白洗脱下来。
（5）用4 mL 1×Strip Buffer洗柱子。
通过此过程得到纯化的内切木聚糖酶，通过SDS-PAGE电泳后染色鉴定内切木聚糖酶podoXyn11的纯度，结果如图1所示。
由图1结果可见，目的蛋白通过HisTag标签纯化后，其洗脱液中内切木聚糖酶podoXyn11纯度较高，在24 kDa处有单一的条带，达到电泳纯级别。
实施例3 本发明所述内切木聚糖酶podoXyn11的定性测定
1、酶活的测定方法
酶活力测定方法参照文献报道，反应体系为200 μL，其中10 μL 500 mmol/L柠檬酸-柠檬酸三钠冲溶液，170 μL 1.5 %榉木木聚糖溶液，置于酶最适反应温度下孵育10min后，向反应体系中加入稀释至适宜浓度的酶液20 μL混匀后，孵育20 min。向离心管中加入300 μL DNS试剂终止反应，沸水浴10min 后冰浴至室温，在550 nm下读取吸光度值。酶活力单位(U)定义为：在测定条件下，每分钟产生1 μmol还原糖所需要的酶量为1个酶活力单位。
2、最适反应pH的测定
在不同的pH（3.0-7.5，25 mmol/L磷酸盐缓冲液）条件下，45℃下分别测定酶活，以上述pH条件中测得酶活力的最高值为100%，计算相对酶活力值，结果如图2：a所示。
由图2：a 结果可知，本发明所述内切木聚糖酶的最适反应pH为5.5，且在pH5-7之间仍能显示出较高的酶活力，所以该种酶反应适宜pH范围较宽。
3、最适反应温度的测定
在60-100℃范围内，每隔5℃，分别测定酶活。缓冲为100 mmol/L磷酸盐缓冲液，pH6.0，以上述温度条件中测得酶活力的最高值为100%，计算相对酶活力值，结果如图2：b所示。
由图2：b结果可知，本发明所述内切木聚糖酶的最适反应温度为95℃，且该种酶在80-100℃之间仍具有较高的酶活力。
4、酶蛋白pH稳定性的测定
在pH4.0-7.5范围内（25 mmol/L磷酸盐缓冲液），在35℃下保温5 h后分别测定残余酶活力，以上述pH条件中测得酶活力的最高值为100%，计算相对酶活力值，结果如图2：c所示。
由图2：c结果可见，本发明所述内切木聚糖酶在其最适合反应pH条件下稳定性良好，且在中性条件下显示出具有更好的稳定性。
5、酶蛋白温度稳定性的测定
取适量酶也在不同温度条件下(45℃，40℃，35℃)进行孵育，定时取样，分别取样酶 液样品中的残余酶活力，以未孵育酶液的酶活力值为100％，计算相对酶活力值，结果如图2： d所示。
由图2：d结果可知，本发明所述内切木聚糖酶在最适合反应温度下保温3h仍具有较好 的稳定性。
实施例4本发明所述内切木聚糖酶水解杨木木聚糖制备低聚木糖的应用
酶水解体系为30 mL，称取木聚糖60 mg置于50 mL锥形瓶中，以浓度为20g/L的木聚糖为底物，加入0.5 mol/L柠檬酸-柠檬酸三钠缓冲溶液 (pH值 5.5) 2 mL，添加适量重组木聚糖酶液及适量蒸馏水，充分混匀，置于45 ℃下进行水解反应。在反应体系内添加木聚糖酶至不同终浓度（10 U/g，15 U/g，20 U/g，30 U/g，35 U/g，40 U/g，45 U/g），测定反应产物中还原糖及低聚木糖的含量，结果如图3：a所示，添加不同浓度木聚糖酶均可有效水解杨木木聚糖，优选浓度为35U/g。在反应体系中添加木聚糖酶至终浓度35 U/g，进行酶水解并定时取样，测定产物中低聚木糖的含量，结果如图3：b所示，在反应14 h后，反应体系的酶解率可达79.50%，产物中低聚木聚糖的主要成分是木二糖（10.56 g/L）和木三糖（4.09 g/L），几乎不产生木糖。
序列表
<110> 中国林业科学研究院林产化学工业研究所
<120> 一种内切木聚糖苷酶及其制备低聚木糖的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 577
<212> DNA
<213> 人工序列(Artificial Sequence)
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aggaacagac tattggcaaa attggaccga tggtggcggg acagtaaatg ctactaatgg 60
atctggtggg aattacagtg ttacatggtc aaatactggt aactttgttg ttggtaaagg 120
ttggaatact ggatccccat ctagaacaat aaactacaat gcaggcgtct gggcaccatc 180
cggcaatggg tatttaactc tttatgggtg gacgagaaac tccctcatag aatattacgt 240
cgttgatagt tggggcactt atagacctac tggaacctat aaaggtaccg tgactagtga 300
tggaggcaca tatgatatat atacgactac gagaacgaac gccccttcta ttgatggtac 360
ggcaaccttc acacagtttt ggagtgtaag acaatccaag cgagcaacgg gaagcaatgt 420
cgcgatcact tttgccaacc acgttaatgc ttggaaaagt tacggaatga atctgggcag 480
cagttggtct taccaggtat tagcgacaga aggatatcaa agtagtggga gctcaaacgt 540
aacagtatgg caccaccacc accaccacca ccactaa 577
<210> 2
<211> 190
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Thr Asp Tyr Trp Gln Asn Trp Thr Asp Gly Gly Gly Thr Val Asn
1 5 10 15
Ala Thr Asn Gly Ser Gly Gly Asn Tyr Ser Val Thr Trp Ser Asn Thr
20 25 30
Gly Asn Phe Val Val Gly Lys Gly Trp Asn Thr Gly Ser Pro Ser Arg
35 40 45
Thr Ile Asn Tyr Asn Ala Gly Val Trp Ala Pro Ser Gly Asn Gly Tyr
50 55 60
Leu Thr Leu Tyr Gly Trp Thr Arg Asn Ser Leu Ile Glu Tyr Tyr Val
65 70 75 80
Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr
85 90 95
Val Thr Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Thr
100 105 110
Asn Ala Pro Ser Ile Asp Gly Thr Ala Thr Phe Thr Gln Phe Trp Ser
115 120 125
Val Arg Gln Ser Lys Arg Ala Thr Gly Ser Asn Val Ala Ile Thr Phe
130 135 140
Ala Asn His Val Asn Ala Trp Lys Ser Tyr Gly Met Asn Leu Gly Ser
145 150 155 160
Ser Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Gln Ser Ser Gly
165 170 175
Ser Ser Asn Val Thr Val Trp His His His His His His His
180 185 190
Claims (5)
1.一种内切木聚糖酶podoxyn11，其特征在于，氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述内切木聚糖酶podoxyn11的核苷酸，其特征在于，序列如SEQ IDNO.1所示。
3.制备权利要求1所述内切木聚糖酶podoxyn11的方法，其特征在于步骤为，为获得所述的内切木聚糖酶podoxyn11的DNA片段，将该DNA片段插入表达载体获得重组质粒，将重组质粒转化宿主菌，以及其诱导表达条件和后续目的蛋白的纯化。
4.含有权利要求2所述的编码内切木聚糖酶podoxyn11核苷酸的重组质粒。
5.权利要求1所述内切木聚糖酶podoxyn11在制备低聚木糖中的应用。
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