CN116262925A - 一种β-1,3-葡聚糖酶及其制备方法和应用 - Google Patents
一种β-1,3-葡聚糖酶及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种β‑1,3‑葡聚糖酶及其制备方法和应用,属于生物技术领域。本发明利用基因工程技术手段,从纤维化纤维微细菌中获得内切β‑1,3‑葡聚糖酶CcGluE‑CDMΔH1的基因,将内切β‑1,3‑葡聚糖酶CcGluE‑CDMΔH1的基因克隆到大肠杆菌表达载体上,获得可异源表达该酶的大肠杆菌重组菌株,用该菌株异源表达制备的内切β‑1,3‑葡聚糖酶,在不同的pH下产生不同聚合度分布的葡寡糖,可广泛应用于农业、食品、饲料添加、医药及葡寡糖的制备等领域。
Description
技术领域
本发明属于生物技术领域,具体涉及一种内切β-1,3-葡聚糖酶及其制备方法和应用;本发明提供了该内切β-1,3-葡聚糖酶的重组质粒和重组基因工程菌株及其在多糖降解和寡糖制备方面的应用;本发明提供的内切β-1,3-葡聚糖酶可广泛应用于农业、食品、饲料添加、医药及寡糖制备等领域。
背景技术
β-葡聚糖是自然界中常见的多糖,广泛分布于动物,植物,真菌和细菌。常见的β-1,3葡聚糖有酵母多糖(zymosan)、昆布多糖(laminarin)、可徳兰多糖(curdlan)、香菇多糖(lentinan)及裂褶多糖(Schizophyllan)等。昆布多糖和可徳兰多糖是其中比较常见的两类。可德兰是β-1,3连接而成的线性单糖,不溶于水,是良好的食品添加剂和胶凝剂。昆布多糖是β-1,3及少量β-1,6支链连接而成的,是海藻的储能物质,含有β-1,6支链提高了β-葡聚糖的溶解性。
近年来β-1,3葡聚糖的生物活性广受关注,截至2021年,在Scopus(http://www.scopus.com)上发表的文章标题、摘要和关键词中包含“β-葡聚糖”的文章数量有11834,研究表明β-葡聚糖的聚合度,分支程度等特性会影响糖与其受体(特别是dectin-1)相互作用的方式。Adams等人研究了分子量及分支程度对葡聚糖结合Dectin-1的影响,发现在分子量相同时,含1,6支链会显著提升其与受体的亲和力。在分子量方面,Elizabeth等人发现庚糖是人葡聚糖模式识别受体的最小识别单元,加入戊糖己糖不会对其结合产生竞争作用。Li等人验证了高聚合度可德兰寡糖诱发植物免疫能力强于低聚合度,即使DP改变1,也会对活性产生显著影响。
综上所述,高DP的含分支的β-1,3-葡寡糖具有更好的生物活性。目前获得β-1,3-葡寡糖的方式主要有2种,化学法和酶法。目前商品化的葡寡糖主要是化学法降解,之后利用HPAEC-PAD分离,高温高压,强酸强碱,对设备的要求很高,对环境的伤害也不小,产率不高,步骤复杂,因此单价非常昂贵,通常以mg为单位进行销售。以1,3葡二糖为例,其市场价高达2065.5元/50mg,折合每毫克41.31元,高DP的寡糖因其产量更低,活性更强,纯化效率更低,在市场上难觅踪迹,八糖及以上DP的产品屈指可数,国产纤维八糖甚至达到5088元/5mg。酶法制备β-1,3-葡寡糖是相对温和和环保的方法,然而商品化的酶具有降解产物DP低,对支链底物活性低的不足。
发明内容
为解决背景技术中所述的技术问题,本发明的目的是提供了一种β-1,3-葡聚糖酶及其制备方法和应用。
本发明目的是通过以下方式实现:
本发明的第一个目的是提供一种β-1,3-葡聚糖酶CcGluE-CDMΔH1的编码基因,所述的编码基因具有如下特征之一:
1)序列表中SEQ ID NO.1所示的脱氧核糖核酸(DNA)序列;
2)编码序列表中SEQ ID NO.2的氨基酸序列的脱氧核糖核酸(DNA)序列;
3)与SEQ ID NO.1限定的脱氧核糖核酸(DNA)序列的同源性达到80%以上,且能编码具有内切β-1,3-葡聚糖酶活性的蛋白质的脱氧核糖核酸(DNA)序列;
4)对序列表中SEQ ID NO.1的脱氧核糖核酸(DNA)序列进行一个或两个以上核苷酸取代、缺失或添加,得到的能够编码具有内切β-1,3-葡聚糖酶活性的蛋白质的脱氧核糖核酸(DNA)序列。
本发明第二个目的是提供一种β-1,3-葡聚糖酶GluE-CDMΔH1,所述的β-1,3-葡聚糖酶GluE-CDMΔH1具有如下特征之一:
1)序列表中的SEQ ID NO.2所示的氨基酸序列;
2)将序列表中的SEQ ID NO.2所示的氨基酸序列进行一个或两个以上氨基酸取代、缺失或添加而形成具有内切β-1,3-葡聚糖酶活性的氨基酸序列。
本发明第三个目的是提供上述的β-1,3-葡聚糖酶GluE-CDMΔH1的制备方法,将编码基因GluE-CDMΔH1克隆入重组表达载体,导入宿主细胞,获得重组表达的内切β-1,3-葡聚糖酶。
进一步地,所述的重组表达载体选自:大肠杆菌表达载体、酵母表达载体、枯草杆菌表达载体、乳酸菌表达载体、链霉菌表达载体、噬菌体载体、丝状真菌表达载体、植物表达载体、昆虫表达载体或哺乳动物细胞表达载体。
进一步地,所述的宿主细胞为重组菌和转基因细胞系,包括大肠杆菌宿主细胞、酵母菌宿主细胞、枯草杆菌宿主细胞、乳酸菌宿主细胞、放线菌宿主细胞、丝状真菌宿主细胞、昆虫细胞或哺乳动物细胞。
进一步地,所述宿主细胞选自Escherichia coli BL21、Escherichia coliJM109、Escherichia coli DH5α、Saccharomyces cerevisiae、Pichia pastoris、Kluyveromyces lactis、Bacillus subtilis R25、Bacillus subtilis9920、Lactic acidbacteria COCC101、Streptomyces spp.、Trichoderma viride、Trichoderma reesei、Aspergillus niger、Aspergillus nidulans、Bombyxmori、Antharaea eucalypti、中国仓鼠卵巢细胞CHO、幼小仓鼠肾脏细胞BHK或中国仓鼠肺细胞CHL。
本发明还提供上述β-1,3-葡聚糖酶CcGluE-CDMΔH1在多糖降解和寡糖制备中的应用。
进一步地,所述的多糖包括可德兰多糖和昆布多糖。
进一步地,通过改变反应体系的pH来调控产物聚合度,生产不同活性的寡糖产品。
进一步地,所述的寡糖包括二糖、三糖、四糖、五糖和六糖。
本发明相对于现有技术具有的有益效果如下:
1.目前研究报道的多数β-1,3-葡聚糖酶具有水解昆布多糖的活力,但对可德兰多糖的水解能力较弱,而本发明中提供的内切β-1,3-葡聚糖酶CcGluE-CDMΔH1对可德兰多糖的水解能力明显优于对昆布多糖的水解活性,同时解决了现有β-1,3-葡寡糖生产成本高的难题,具有重要的实用价值,可应用于大规模的工业化生产。
2.本发明所提供的内切β-1,3-葡聚糖酶CcGluE-CDMΔH1在降解可德兰多糖时,在不同pH下,产生的产物聚合度分布不同,但与野生酶相比,其产物聚合度均有所提高。
3.本发明的内切β-1,3-葡聚糖酶CcGluE-CDMΔH1可广泛应用农业、食品、饲料添加、医药及葡寡糖的制备等领域。
附图说明
为了更清楚地说明本发明实施例,下面将对实施例涉及的附图进行简单地介绍。
图1:内切β-1,3-葡聚糖酶基因CcGluE-CDMΔH1琼脂糖凝胶电泳检测。
图2:内切β-1,3-葡聚糖酶CcGluE-CDMΔH1(A)及gluE(B)表达及纯化的SDS-PAGE图;A图各泳道加入的样品分别是:泳道6-E.coli BL21(DE3)/pET28a-GluE-CDMΔH1诱导菌体上清;泳道5-纯化时流穿;泳道4-GluE-CDMΔH1 20mM咪唑洗脱,泳道3-GluE-CDMΔH140mM咪唑洗脱,泳道2-GluE-CDMΔH1 200mM咪唑洗脱,即纯化蛋白,泳道1-预染蛋白分子量标准;B图各泳道加入的样品分别是:泳道1-E.coli BL21(DE3)/pET28a-gluE未诱导菌体沉淀;泳道2-E.coli BL21(DE3)/pET28a-gluE诱导后菌体沉淀;泳道3-预染蛋白分子量标准;泳道4-gluE纯化蛋白。
图3:pH值对内切β-1,3-葡聚糖酶CcGluE-CDMΔH1的影响曲线。
图4:温度对内切β-1,3-葡聚糖酶CcGluE-CDMΔH1的影响曲线。
图5:不同pH条件下CcGluE-CDMΔH1降解Curdlan产物区别。
图6:CcGluE-CDMΔH1降解昆布六糖产物图。
图7:不同pH下的CcGluE-CDMΔH1的Tm。
具体实施方式
下面结合实施例对本发明进行详细的说明,但本发明的实施方式不限于此,显而易见地,下面描述中的实施例仅是本发明的部分实施例,对于本领域技术人员来讲,在不付出创造性劳动性的前提下,获得其他的类似的实施例均落入本发明的保护范围。
实施例1内切β-1,3-葡聚糖酶全长基因克隆
本发明的内切β-1,3-葡聚糖酶CcGluE-CDMΔH1的基因序列是通过PCR技术从纤维化纤维微细菌菌株(Cellulosimicrobium cellulans)中扩增出的内切β-1,3-葡聚糖酶gluE编码基因(命名为gluE),再用RF克隆将其去掉碳水化合物结合域和Ser144-Ser147改造而成的。该基因编码区长726bp,属于多糖裂解酶16家族。
以pET28a-CcGluE质粒为模板,获得的ccgluE的π-helix后部分基因为模板,将246位氨基酸突变为终止密码子,以F:GTCTACGACAACGGCTCGGGCTCGTCG TAACCGGGGAACC和R:GGGCAGGCCGGTGCCGGGGTTCCCCGGTTACG ACGAGCCC为引物进行RF克隆,PCR反应体系为:10×PCR,Buffer 5μL,25mM MgSO4 3μL,dNTP 5μL,10μmol/L的正向引物和反向引物各1.5μL,模板DNA 1μL,KOD 1μL,H2O 32μL。
PCR反应条件:①变性温度98℃反应2min,循环1次;②变性温度98℃反应10s,退火温度55℃反应30s,延伸温度72℃反应35s,循环30次;③延伸温度72℃反应2min,循环1次。
通过琼脂糖凝胶电泳检测产物。将获得的上述PCR产物与获得的ccgluE的π-helix前部分基因作为模板,以F:GGGAATTC CATATG GCGCCGGGCGACCTC与R:GCGC CTCGAGTCAGAGGGTCCACTG作为引物进行PCR。
PCR反应体系(50μL):模板DNA 2μL(上述PCR产物各1μL),Phusion mix 25μL,10μmol/L的正向引物和反向引物各2.5μL,H2O 18μL。
PCR反应条件:①变性温度98℃反应2min,循环1次;②变性温度98℃反应30s,退火温度55℃反应30s,延伸温度72℃反应60s,循环30次;③延伸温度72℃反应2min,循环1个。
PCR扩增产物即为全长目的基因,通过琼脂糖凝胶电泳检测,DNA胶回收试剂盒进行胶回收。胶回收后用NdeI和XhoI 37℃双酶切消化6h,与同样酶切后的pET28a通过T4 DNA连接酶室温连接12h,转入E.coli TOP 10感受态细胞,在含kan抗性的平板上37℃生长过夜,用通用引物5’-TAATACGACTCACTATAGG和3’-GCTAGTTATTGCTCAGCGG进行菌落PCR验证后,提质粒送华大基因(北京)测序,命名测序正确的质粒为pET28a-CcGluECDMΔH1。
实施例2CcGluECDMΔH1及gluE基因在大肠杆菌中的重组表达及纯化
将pET28a-gluE和pET28a-CcGluECDMΔH1分别转化E.coli BL21(DE3),对其进行诱导表达及纯化。用聚丙烯酰胺凝胶电泳检测内切β-1,3-葡聚糖酶CcGluECDMΔH1的表达及纯化情况,结果如图2所示,纯化后的内切β-1,3-葡聚糖酶CcGluECDMΔH1在电泳胶上呈单一条带,且位置与预测的分子量相吻合。
改造前gluE表达量较少,为2mg/L培养基左右,经过改造后,表达量大大提高,可达100mg/L培养基,增长近50倍。
实施例3内切β-1,3-葡聚糖酶CcGluECDMΔH1的酶学性质分析
(1)内切β-1,3-葡聚糖酶CcGluECDMΔH1活力的测定
用缓冲液(40mM pH 6醋酸/醋酸钠缓冲液)配制0.5%可德兰/昆布多糖底物溶液,取4μg酶与1000μL底物混合。60℃反应5min,取128μL加入96μL DNS终止反应,煮沸10min,最后加1376μL的ddH2O,于540nm处测定OD值,用葡萄糖生成量表示酶活力,对照组加灭活的酶液,其余操作与实验组相同。酶活(U)单位定义:每分钟释放1μmol的葡萄糖需要的酶量被定义为一个酶活单位。
(2)pH对重组酶CcGluECDMΔH1的影响
在60℃的条件下,分别以0.5%,40mM柠檬酸/柠檬酸钠pH3-5,40mM Na2HPO3/NaH2PO3 pH6-8的可德兰多糖/昆布多糖为底物,按照标准检测方法测定其活性,根据酶在不同pH下的相对活性绘制曲线,确定酶的最适反应pH。以灭活的酶作为对照,以活性最高的值为100%,测定在各个反应pH下酶的相对活性。结果如图3所示,CcGluECDMΔH1的最适反应pH为6-7,随着pH的升高或降低,CcGluECDMΔH1的活性均降低。
(3)温度对重组酶CcGluECDMΔH1的影响
在pH6的条件下,以0.5%可德兰多糖为底物,0.2ml 40mM Na2HPO3/NaH2PO3 pH60.5%Curdlan缓冲液反应5min(30min,60min,120min),煮沸10min,反应温度:20,30,40,50,60,70℃下按照标准方法测定重组酶的活性,根据酶在不同温度下的相对活性绘制曲线。以灭活的酶为对照,以反应最高酶活力为100%计算相对酶活。结果如图4所示,CcGluECDMΔH1的最适反应温度为60℃,说明该重组酶是一种中等嗜热的葡聚糖酶。
实施例4pH对CcGluECDMΔH1降解产物的影响
在60℃的条件下,分别以0.5%,40mM柠檬酸/柠檬酸钠pH3-5,40mM Na2HPO3/NaH2PO3 pH6-8的可德兰多糖为底物,按照标准检测方法测定其活性。将降解产物煮沸后,于离心机12000rpm离心5min,经0.22μm滤膜过滤之后,10μL的样品注射到PA-100分析柱,1mL/min的流速、30℃条件下,进行产物检测。梯度洗脱程序:流动相A为100mmol/L NaOH,流动相B为100mmol/L NaOH和500mmol/L CH3COONa,洗脱梯度如下:1-30min,A/B,100/0(v/v)至40/60(v/v);30-35min,A/B,40/60(v/v)至0/100(v/v);35-50min,A/B,100/0(v/v)。电极的脉冲电位和持续时间如下:E1=+200mV(t1=300ms);E2=+700mV(t2=100ms);E3=-900mV(t3=100ms),数据由Coul Array软件分析。
结果如图5所示,在pH6时,产物的聚合度较高,六糖含量超过20%,二糖三糖为主要产物。而在pH8时,二糖超过50%,六糖仅有10%不到,pH影响产物聚合度的分布。
实施例5酶CcGluECDMΔH1的降解产物分析
将1ml 0.5%昆布多糖与纯化的CcGluECDMΔH1酶液(4μg)的比例混合后,在60℃条件下反应0,5,10min,通过Sevage法除蛋白后,对其产物进行液相检测。如图6所示,CcGluECDMΔH1对可德兰多糖的降解可以生成多种聚合度的寡糖,主要产物为三糖和四糖,其中四糖最多。
实施例6不同pH下的CcGluE-CDMΔH1的Tm研究
将纯化的不同pH下的CcGluE-CDMΔH1稀释为0.1mg/ml,分别置换为40mM柠檬酸/柠檬酸钠pH4-5,40mM Na2HPO3/NaH2PO3 pH6-8,进行DSC测量。温度范围20-80℃,升温速率1℃/min。发现在pH4-6时,CcGluE-CDMΔH1的Tm变化了16.3℃,说明不同pH时CcGluE-CDMΔH1的构象发生改变。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 中国科学院大连化学物理研究所
<120> 一种β-1,3-葡聚糖酶及其制备方法和应用
<130> 20211207
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 723
<212> DNA
<213> 人工序列
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atggcgccgg gcgacctcct gtggtccgac gagttcgacg gcgcggcggg ctcggcgccg 60
aacccggccg tctggaacca cgagaccggc gcgcacgggt ggggcaacgc cgagctccag 120
aactacacgg cctcgcgcgc caactccgcg ctcgacggcc agggcaacct cgtcatcacc 180
gcgcgtcgcg agggcgacgg gtcgtacacg tcggcccgca tgacgaccca gggcaagtac 240
cagccgcagt acgggcgcat cgaggcgcgc atccagatcc cgcgcggcca ggggatctgg 300
ccggcgttct ggatgctcgg cgggagcttc cccgggacgc cgtggccgtc gtcgggcgag 360
atcgacatca tggagaacgt cgggttcgag ccgcaccgcg tgcacggcac ggtgcacggc 420
ccggggtact ccggcggctc cggcatcacg ggcatgtacc agcacccgca gggctggtcg 480
ttcgcggaca cgttccacac gttcgcggtc gactggaagc cgggggagat cacgtggttc 540
gtcgacggcc agcagttcca ccgcgtcacg cgcgcgagcg tcggcgcgaa cgcctgggtg 600
ttcgaccagc cgttcttcct catcctcaac gtcgcggtcg gcgggcagtg gcccggctac 660
cccgacggca cgacccagct cccgcagcag atgaaggtcg actacgtgcg cgtctacgac 720
aac 723
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<212> PRT
<213> 人工序列
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Met Ala Pro Gly Asp Leu Leu Trp Ser Asp Glu Phe Asp Gly Ala Ala
1 5 10 15
Gly Ser Ala Pro Asn Pro Ala Val Trp Asn His Glu Thr Gly Ala His
20 25 30
Gly Trp Gly Asn Ala Glu Leu Gln Asn Tyr Thr Ala Ser Arg Ala Asn
35 40 45
Ser Ala Leu Asp Gly Gln Gly Asn Leu Val Ile Thr Ala Arg Arg Glu
50 55 60
Gly Asp Gly Ser Tyr Thr Ser Ala Arg Met Thr Thr Gln Gly Lys Tyr
65 70 75 80
Gln Pro Gln Tyr Gly Arg Ile Glu Ala Arg Ile Gln Ile Pro Arg Gly
85 90 95
Gln Gly Ile Trp Pro Ala Phe Trp Met Leu Gly Gly Ser Phe Pro Gly
100 105 110
Thr Pro Trp Pro Ser Ser Gly Glu Ile Asp Ile Met Glu Asn Val Gly
115 120 125
Phe Glu Pro His Arg Val His Gly Thr Val His Gly Pro Gly Tyr Gly
130 135 140
Ile Thr Gly Met Tyr Gln His Pro Gln Gly Trp Ser Phe Ala Asp Thr
145 150 155 160
Phe His Thr Phe Ala Val Asp Trp Lys Pro Gly Glu Ile Thr Trp Phe
165 170 175
Val Asp Gly Gln Gln Phe His Arg Val Thr Arg Ala Ser Val Gly Ala
180 185 190
Asn Ala Trp Val Phe Asp Gln Pro Phe Phe Leu Ile Leu Asn Val Ala
195 200 205
Val Gly Gly Gln Trp Pro Gly Tyr Pro Asp Gly Thr Thr Gln Leu Pro
210 215 220
Gln Gln Met Lys Val Asp Tyr Val Arg Val Tyr Asp Asn Gly Ser Gly
225 230 235 240
Ser Ser
Claims (10)
1.一种β-1,3-葡聚糖酶CcGluE-CDMΔH1的编码基因,其特征在于,所述的编码基因具有如下特征之一:
1)序列表中SEQ ID NO.1所示的脱氧核糖核酸(DNA)序列;
2)编码序列表中SEQ ID NO.2的氨基酸序列的脱氧核糖核酸(DNA)序列;
3)与序列表中SEQ ID NO.1限定的脱氧核糖核酸(DNA)序列的同源性达到80%以上,且能编码具有内切β-1,3-葡聚糖酶活性的蛋白质的脱氧核糖核酸(DNA)序列;
4)对序列表中SEQ ID NO.1的脱氧核糖核酸(DNA)序列进行一个或两个以上核苷酸取代、缺失或添加,得到的能够编码具有内切β-1,3-葡聚糖酶活性的蛋白质的脱氧核糖核酸(DNA)序列。
2.权利要求1所述的编码基因编码的β-1,3-葡聚糖酶GluE-CDMΔH1,其特征在于,所述的β-1,3-葡聚糖酶GluE-CDMΔH1具有如下特征之一:
1)序列表中的SEQ ID NO.2所示的氨基酸序列;
2)将序列表中的SEQ ID NO.2所示的氨基酸序列进行一个或两个以上氨基酸取代、缺失或添加而形成具有内切β-1,3-葡聚糖酶活性的氨基酸序列。
3.权利要求2所述的β-1,3-葡聚糖酶GluE-CDMΔH1的制备方法,其特征在于,将权利要求1所述的β-1,3-葡聚糖酶CcGluE-CDMΔH1的编码基因克隆入重组表达载体,导入宿主细胞,获得重组表达的β-1,3-葡聚糖酶。
4.根据权利要求3所述的制备方法,其特征在于,所述的重组表达载体选自:大肠杆菌表达载体、酵母表达载体、枯草杆菌表达载体、乳酸菌表达载体、链霉菌表达载体、噬菌体载体、丝状真菌表达载体、植物表达载体、昆虫表达载体或哺乳动物细胞表达载体。
5.根据权利要求3所述的制备方法,其特征在于,所述的宿主细胞为重组菌和转基因细胞系,包括大肠杆菌宿主细胞、酵母菌宿主细胞、枯草杆菌宿主细胞、乳酸菌宿主细胞、放线菌宿主细胞、丝状真菌宿主细胞、昆虫细胞或哺乳动物细胞。
6.根据权利要求5所述的制备方法,其特征在于,所述宿主细胞选自EscherichiacoliBL21、Escherichia coliJM109、Escherichia coliDH5α、Saccharomyces cerevisiae、Pichia pastoris、Kluyveromyces lactis、Bacillus subtilis R25、Bacillussubtilis9920、Lactic acid bacteria COCC101、Streptomyces spp.、Trichodermaviride、Trichoderma reesei、Aspergillus niger、Aspergillus nidulans、Bombyxmori、Antharaea eucalypti、中国仓鼠卵巢细胞CHO、幼小仓鼠肾脏细胞BHK或中国仓鼠肺细胞CHL。
7.权利要求2所述的β-1,3-葡聚糖酶CcGluE-CDMΔH1在多糖降解和寡糖制备中的应用。
8.根据权利要求7所述的应用,其特征在于,所述的多糖包括可德兰多糖和昆布多糖。
9.根据权利要求7所述的应用,其特征在于,通过改变反应体系的pH来调控产物聚合度,生产不同活性的寡糖产品。
10.根据权利要求7所述的应用,其特征在于,所述的寡糖包括二糖、三糖、四糖、五糖和六糖。
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