CN110819610A - 一种极耐热甘露聚糖酶及其制备方法与应用 - Google Patents
一种极耐热甘露聚糖酶及其制备方法与应用 Download PDFInfo
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- CN110819610A CN110819610A CN201910983725.XA CN201910983725A CN110819610A CN 110819610 A CN110819610 A CN 110819610A CN 201910983725 A CN201910983725 A CN 201910983725A CN 110819610 A CN110819610 A CN 110819610A
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Abstract
本发明属于生物技术领域,具体涉及一种极耐热甘露聚糖酶及其制备方法与应用。本发明将来源于超嗜热神袍菌Thermotoga sp. RQ2基因组中的糖苷水解酶基因在大肠杆菌中进行了异源表达,得到了一种重组菌,重组菌在一定的培养条件下培养收集菌体,分离纯化后得到极耐热甘露聚糖酶,制备的重组酶成功地以可溶蛋白形式表达。初步分析表明本发明提供的极耐热甘露聚糖酶对金属离子Co2+敏感,具有极好的酶耐热性,具有较高的热稳定性及动力学性能;在纺织、食品、饲料以及造纸等领域具有一定的生产应用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及一种极耐热甘露聚糖酶及其制备方法与应用。
背景技术
甘露聚糖酶,即β-甘露聚糖酶为一种多功能的生物质降解剂,在纺织、食品、饲料、造纸和木质纤维素生物质转化等工业领域具有广泛的应用。耐热酶因其具有较高的催化效率、较高的热稳定性、较高的有机溶剂耐受性、降低染菌风险等特性在工业应用过程中具有显著优势。但是现有甘露聚糖酶的异源表达水平较低或形成大量无活性包涵体,特别是耐热甘露聚糖酶广泛存在表达量低、热稳定性较差、最适pH范围较窄等问题,严重制约其工业化应用。
超嗜热神袍菌属(Thermotoga spp.)是一类超嗜热细菌,与其他细菌和古细菌相比,编码着最多数量的糖苷水解酶。到目前为止,超嗜热神袍菌属中仍然有大量糖苷水解酶的生理生化特性未得到全面解析。在国内外的报道中, Thermotoga sp. RQ2基因组中GenBank登录号为TRQ2_1073的糖苷水解酶基因被定义为内切葡聚糖酶,即纤维素酶。该酶与Dictyoglomus turgidum DSM 6724(WP_012583041.1)和Dictyoglomus thermophilumH-6-12(WP_012547750.1)相似性仅为59%,进行的氨基酸序列同源性分析(blastp)揭示没有发现与其他菌属的甘露聚糖酶与之具有同源性。基于此,能否可以直接从超嗜热菌中获得一种新的纤维素生物质降解酶,具有非常高的研究价值。
发明内容
有鉴于此,本发明的目的在于提供一种极耐热甘露聚糖酶及其制备方法与应用。
为实现上述发明目的,本发明采取的技术方案为:
在一些实施方案中,本发明提供了一种极耐热甘露聚糖酶,其氨基酸序列如SEQ IDNO.1所示,由于密码子的简并性,存在多种能够编码本发明所述的极耐热甘露聚糖酶的核苷酸序列,在一些实施方案中,本发明也提供了编码氨基酸序列如SEQ ID NO.1所示的极耐热甘露聚糖酶的DNA分子,其核苷酸序列如SEQ ID NO.2所示。
所述的极耐热甘露聚糖酶的适宜温度为75-90ºC,适宜pH为5.0~6.0。
所述的极耐热甘露聚糖酶的最适温度为85ºC,最适pH为5.5。
本发明还提供了所述的极耐热甘露聚糖酶的制备方法,具体为获得本发明所述的极耐热甘露聚糖酶的DNA分子,将该DNA分子与表达载体结合获得重组质粒,将重组质粒转化表达宿主菌诱导表达,分离纯化后获得。
在一些实施方案中,本发明还提供了所述的极耐热甘露聚糖酶的制备方法,具体包括如下步骤:
(1)以提取的超嗜热神袍菌Thermotoga sp. RQ2基因组DNA为模板,通过核苷酸序列如SEQ ID NO.3所示的上游引物和核苷酸序列如SEQ ID NO.4所示的下游引物扩增得如SEQID NO.2所示的核苷酸序列,将得到的基因序列与表达载体连接得到重组质粒,将重组质粒转化到宿主菌得到基因工程菌;
(2)将步骤(1)得到的基因工程菌预培养后接种于含有抗生素的LB液体培养基中30℃培养至OD600达到1-2,然后将培养基转移至42℃继续培养6-8h,离心收集菌体;
(3)将步骤(2)得到的菌体破碎后热处理,经镍离子柱柱纯化后得到极耐热甘露聚糖酶。
在一些实施方案中,步骤(1)中所述的宿主菌为大肠杆菌BL21,所述的表达载体为pHsh质粒(含热激启动子)。
步骤(2)中所述的接种为1%的接种量,所述的抗生素为氨苄青霉素。
在一些实施方案中,步骤(3)中所述破碎为将收集的菌体用25mM邻苯二甲酸氢钾-咪唑缓冲溶液(PI缓冲液)重新悬浮细胞进行超声破碎,75ºC热处理20min,离心收集上清液得到粗酶液。
在一些实施方案中,步骤(3)中所述纯化为将粗酶液用0.22μm水系滤膜过滤后通过镍柱进行纯化,有活性的洗脱峰用透析袋(25mM的PI缓冲,pH6.8)进行脱盐,得到电泳纯的蛋白。
本发明还提供了一种包含如SEQ ID NO.2所示的核苷酸序列的重组质粒,所述的重组质粒其核苷酸序列如SEQ ID NO.5所示。
本发明还提供了一种极耐热甘露聚糖酶在纺织、食品、饲料或造纸领域的应用。
与现有技术相比,本发明的有益效果是:
本发明将来源于超嗜热神袍菌Thermotoga sp. RQ2基因组中GenBank登录号为TRQ2_1073的糖苷水解酶基因在大肠杆菌中进行了异源表达,得到了一种重组菌,重组菌在一定的培养条件下培养收集菌体,分离纯化后得到极耐热甘露聚糖酶,制备的重组酶成功地以可溶蛋白形式表达。本发明提供的极耐热甘露聚糖酶的适宜温度为75-90ºC,适宜pH为5.0~6.0。最适温度为85℃,最适pH为5.5。初步性质分析表明提供的极耐热甘露聚糖酶对金属离子钴离子(Co2+)敏感,具有极好的酶耐热性,具有较高的热稳定性及动力学性能;在纺织、食品、饲料、造纸和木质纤维素生物质等领域具有一定的生产应用价值。
附图说明
图1是重组酶在不同温度下的相对酶活力对照图;
图2是重组酶在不同pH下的相对酶活力对照图;
图3是重组酶在不同温度下的稳定性对照图;
图4是重组酶在不同pH下的稳定性对照图;
图5是重组酶在不同Co2+浓度时的酶活力对照图;
图6 是实施例7中Lineweaver-Burk方程测定试验组和对照组的双倒数图,图中A是对照组,B是试验组。
具体实施方式
以下实施例中进一步定义本发明,根据以上的描述和这些实施例,本领域技术人员可以确定本发明的基本特征,并且在不偏离本发明精神和范围的情况下,可以对本发明做出各种修改和改变,以使其使用各种用途和条件。下述实施例中所使用的实验原料如无特殊说明,均可通过商业途径得到。除特殊注明外,本发明所采用的均为该领域现有技术。
对羟基苯甲酸酰肼(PAHBAH)和还原性碳水化合物在碱性溶液中会发生显著的显色反应,本发明根据上述反应测定甘露聚糖酶的活性,包括以下步骤:以0.5%的槐豆胶(LBG)为底物,反应体系为200μL,将100μL LBG底物加入到1.5mL离心管中,加入95μL邻苯二甲酸氢钾-咪唑缓冲溶液(PI缓冲液,25mM,pH6.0),之后添加5μL适当稀释的酶液,在一定温度的水浴中反应5分钟,之后立即将离心管放在冰上冷却。往离心管中添加600μL反应终止溶液(0.5M NaOH溶液与PAHBAH溶液的体积比为4:1)终止反应。将离心管放在沸水浴中煮沸约10分钟,之后取出离心管离心并放在冰上冷却。取100μL溶液到酶标板上,使用酶标仪测定其在410nm处的吸光度,并从甘露糖标准曲线上查出相应的还原糖含量并折算成酶活单位。在上述反应条件下,每分钟催化反应生成1μmol甘露糖所需的酶量,定义为一个酶活力单位,用U表示。每次反应设置3个平行组和1个对照组实验。
实施例1:基因工程菌的构建及重组酶的表达和纯化
(1)基因工程菌大肠杆菌BL21/ pHsh-Thman5的构建
依据NCBI公布的TRQ2_1073基因序列,通过软件SnapGene获得克隆该基因的引物序列,上游引物TmanF的核苷酸序列如SEQ ID No.3所示,下游引物TmanR的核苷酸序列如SEQ IDNo.4所示;提取超嗜热神袍菌Thermotoga sp. RQ2的基因组,以此为模板,通过引物TmanF和TmanR扩增甘露聚糖酶的基因核苷酸序列如SEQ ID NO.2所示,将得到的基因序列与pHsh质粒(仙奕生物科技(南京)有限公司)连接,导入大肠杆菌BL21得到基因工程菌BL21/pHsh-Thman5;
(2)基因工程菌的发酵方法
将基因工程菌BL21/ pHsh-Thman5于30℃LB液体培养基中预培养过夜,以1%的接种量接种于含有100µg/mL氨苄青霉素的LB液体培养基中30ºC、200r/min培养至OD600达到1-2,将培养基转移至42ºC、200r/min继续培养6-8h,离心收集菌体;
(3)极耐热甘露聚糖酶的纯化
将收集得到的菌体用25mM PI缓冲液重新悬浮进行超声破碎,75ºC热处理20min,离心收集上清液得到粗酶液,粗酶液通过GE公司的1mL预装镍柱进行纯化,首先用镍柱平衡缓冲液进行平衡,然后上样,流速为1mL/min,上样结束后用平衡缓冲溶液继续冲洗未结合的杂蛋白,然后用含有500mM的咪唑的洗脱缓冲液洗脱目的蛋白,将穿透峰和洗脱峰收集,将SDS-PAGE检验的洗脱峰(25mM PI缓冲液,pH6.8)通过透析袋透析四次,用于进一步测定蛋白量和酶活。
实施例2:极耐热甘露聚糖酶最适温度的测定
采用25mM PI缓冲溶液(pH6.8),在不同温度(60-90ºC)下,每隔5ºC分别测定实施例1中得到的重组酶的酶活。以最高酶活为100%计算相对酶活,图1是重组酶在不同温度下的相对酶活力对照图;由图1可知,在75-90ºC之间酶活均比较高,达到70%以上,85ºC为最适温度,此时酶活最高。可见,实施例1中制备的重组酶属于超嗜热甘露聚糖酶。
实施例3:极耐热甘露聚糖酶最适pH的测定
采用25mM PI缓冲溶液,在85ºC,pH4-7.5范围内分别测定实施例1中得到的重组酶的酶活变化情况。以最高酶活为100%,计算相对酶活。图2是重组酶在不同pH下的相对酶活力对照图;如图2所示,重组酶的最适反应pH为5.5,当pH为5.0~6.0时均有较高的酶活力。
实施例4:极耐热甘露聚糖酶温度稳定性的评价
将实施例1中得到的重组酶分别置于85ºC、90ºC和95ºC下处理5h,每小时取样并在最适温度及pH环境中检测残余酶活,以处理0h的酶活为100%计算。图3是重组酶在不同温度下的稳定性对照图;由图3可见,制备的重组酶在85ºC和90ºC条件下的半衰期分别为5h和4h,属于极耐热酶。
实施例5:极耐热甘露聚糖酶pH稳定性的评价
重组酶在不同pH条件(pH3.0-8.0)下于85ºC保温1h测得残余酶活,分别以不同pH条件下,不保温处理的酶的酶活为100%,计算相对酶活。图4是重组酶在不同pH下的稳定性对照图;由图4可见,重组酶在pH5-8条件下比较稳定,且在pH5.5条件下,保温1h后仍残留90%以上酶活。
实施例6:不同Co2+浓度对极耐热甘露聚糖酶活性的影响
分别配制不同浓度的Co2+(0-2.5mM),在温度为85ºC,pH为5.5的条件下考察Co2+浓度对重组酶活性的影响,以未添加金属离子的酶活为100%计算。图5是重组酶在不同Co2+浓度时的酶活力对照图;由图5可见,Co2+浓度对极耐热甘露聚糖酶的活性具有正影响。Co2+的浓度为0.6~2.5mM时能有效激活重组酶,当Co2+的浓度为1.0mM时酶蛋白比活力为416U/mg,而未添加Co2+时,酶蛋白的比活力为176U/mg,当Co2+的浓度为1.5mM时重组酶的酶活力提高到270%。
实施例7:极耐热甘露聚糖酶的动力学参数测定
以添加1mM Co2+的重组酶为试验组,未添加Co2+的重组酶为对照组测试Co2+对极耐热甘露聚糖酶的动力学影响。在温度为85ºC,pH为5.5的条件下分别检测添加不同浓度(0-15mg/mL)0.5%槐豆胶(LBG)底物的初始酶活,根据Lineweaver-Burk双倒数作图。以底物1/[S]为X轴,1/[V]为Y轴作图拟合出线性方程,计算不同底物的Km和Vmax值。图6是Lineweaver-Burk方程测定试验组和对照组的双倒数图,图中A是对照组,B是试验组。如表1所示,添加了1mMCo2+时极耐热甘露聚糖酶具有更高的催化效率。
表1.重组极耐热甘露聚糖酶的动力学分析
重组酶 | K<sub>m</sub>(mg/mL) | V<sub>max</sub>(U/mg) |
对照组 | 8.3 | 833 |
试验组 | 8.0 | 1000 |
以上显示和描述了本发明的基本原理、主要特征以及本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
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cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 900
ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atggggggat 960
cattgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1020
gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1080
actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1140
aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1200
cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1260
tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagactgat 1320
cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 1380
tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 1440
ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 1500
ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 1560
cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 1620
aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 1680
agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 1740
tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 1800
ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 1860
cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 1920
atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 1980
ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggg tatctttata 2040
gtcctgtcgg ggtttcgcac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 2100
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 2160
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 2220
ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 2280
tgagagagga agcggaagag cccccttgaa tgtgggggaa acatccccat gatccaatga 2340
cctgttaacc gtcgacaaga aggagatata ccctatgaat aacaccattc caagatggcg 2400
tggcttcaac cttctggaag ccttttccat taaaagtacc ggaaatttta aagaggaaga 2460
ttttttgtgg atggctcagt gggactttaa ttttgttaga atccctatgt gtcatcttct 2520
ctggtcagac tggggcaact catttattat cagagaagat ttttttgaga aaatcgatcg 2580
tgtaattttc tggggagaga aatatggaat acatatatgt atttctctcc acagggcacc 2640
tggctattct gttaacaagg aagtagaaga gaaaaccaat ctgtggaaag atgaaacagc 2700
tcaagaagcg ttcattcatc actggtcttt tatcgcacgt cgttacaaag gaatttcttc 2760
cacacacctg agttttaact taataaatga gcctccattt cctgatccac aaatcatgag 2820
tgttgaagat cacaactctc ttatcaagag aactattaca gaaattcgaa aaatagatcc 2880
cgaaagatta attatgatag atggattagg ctatgggaat attccagtgg atgatttaac 2940
aattgagaat acagtgcaat catgcagagg gtacattccc ttcagtgtta ctcattacaa 3000
agcggaatgg gtggatagta aggactttcc tgctcctgag tggccaaatg gatggcattt 3060
tggggaatac tggaacagag aaaagttatt ggaacattac ttaacgtgga taaaactcag 3120
acaaaaagga atagaagtat tctgtggaga aatgggagct tacaacaaaa cacctcacga 3180
tgtggtttta aaatggcttg aagatctttt agaaattttt aaaactttga acatagggtt 3240
tgccttatgg aattttagag ggccttttgg tattttagat tcagaaagga aagacgttga 3300
atacgaagaa tggtatggac ataaactgga taggaaaatg ttggaactat tgagaaaata 3360
tcatcatcat catcatcatt aga 3383
序列表
<110> 江苏大学
<120> 一种极耐热甘露聚糖酶及其制备方法与应用
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 335
<212> PRT
<213> 极热嗜热菌(Thermotoga hypogea)
<400> 1
Met Asn Asn Thr Ile Pro Arg Trp Arg Gly Phe Asn Leu Leu Glu Ala
1 5 10 15
Phe Ser Ile Lys Ser Thr Gly Asn Phe Lys Glu Glu Asp Phe Leu Trp
20 25 30
Met Ala Gln Trp Asp Phe Asn Phe Val Arg Ile Pro Met Cys His Leu
35 40 45
Leu Trp Ser Asp Trp Gly Asn Ser Phe Ile Ile Arg Glu Asp Phe Phe
50 55 60
Glu Lys Ile Asp Arg Val Ile Phe Trp Gly Glu Lys Tyr Gly Ile His
65 70 75 80
Ile Cys Ile Ser Leu His Arg Ala Pro Gly Tyr Ser Val Asn Lys Glu
85 90 95
Val Glu Glu Lys Thr Asn Leu Trp Lys Asp Glu Thr Ala Gln Glu Ala
100 105 110
Phe Ile His His Trp Ser Phe Ile Ala Arg Arg Tyr Lys Gly Ile Ser
115 120 125
Ser Thr His Leu Ser Phe Asn Leu Ile Asn Glu Pro Pro Phe Pro Asp
130 135 140
Pro Gln Ile Met Ser Val Glu Asp His Asn Ser Leu Ile Lys Arg Thr
145 150 155 160
Ile Thr Glu Ile Arg Lys Ile Asp Pro Glu Arg Leu Ile Met Ile Asp
165 170 175
Gly Leu Gly Tyr Gly Asn Ile Pro Val Asp Asp Leu Thr Ile Glu Asn
180 185 190
Thr Val Gln Ser Cys Arg Gly Tyr Ile Pro Phe Ser Val Thr His Tyr
195 200 205
Lys Ala Glu Trp Val Asp Ser Lys Asp Phe Pro Ala Pro Glu Trp Pro
210 215 220
Asn Gly Trp His Phe Gly Glu Tyr Trp Asn Arg Glu Lys Leu Leu Glu
225 230 235 240
His Tyr Leu Thr Trp Ile Lys Leu Arg Gln Lys Gly Ile Glu Val Phe
245 250 255
Cys Gly Glu Met Gly Ala Tyr Asn Lys Thr Pro His Asp Val Val Leu
260 265 270
Lys Trp Leu Glu Asp Leu Leu Glu Ile Phe Lys Thr Leu Asn Ile Gly
275 280 285
Phe Ala Leu Trp Asn Phe Arg Gly Pro Phe Gly Ile Leu Asp Ser Glu
290 295 300
Arg Lys Asp Val Glu Tyr Glu Glu Trp Tyr Gly His Lys Leu Asp Arg
305 310 315 320
Lys Met Leu Glu Leu Leu Arg Lys Tyr His His His His His His
325 330 335
<210> 2
<211> 1008
<212> DNA
<213> 极热嗜热菌(Thermotoga hypogea)
<400> 2
atgaataaca ccattccaag atggcgtggc ttcaaccttc tggaagcctt ttccattaaa 60
agtaccggaa attttaaaga ggaagatttt ttgtggatgg ctcagtggga ctttaatttt 120
gttagaatcc ctatgtgtca tcttctctgg tcagactggg gcaactcatt tattatcaga 180
gaagattttt ttgagaaaat cgatcgtgta attttctggg gagagaaata tggaatacat 240
atatgtattt ctctccacag ggcacctggc tattctgtta acaaggaagt agaagagaaa 300
accaatctgt ggaaagatga aacagctcaa gaagcgttca ttcatcactg gtcttttatc 360
gcacgtcgtt acaaaggaat ttcttccaca cacctgagtt ttaacttaat aaatgagcct 420
ccatttcctg atccacaaat catgagtgtt gaagatcaca actctcttat caagagaact 480
attacagaaa ttcgaaaaat agatcccgaa agattaatta tgatagatgg attaggctat 540
gggaatattc cagtggatga tttaacaatt gagaatacag tgcaatcatg cagagggtac 600
attcccttca gtgttactca ttacaaagcg gaatgggtgg atagtaagga ctttcctgct 660
cctgagtggc caaatggatg gcattttggg gaatactgga acagagaaaa gttattggaa 720
cattacttaa cgtggataaa actcagacaa aaaggaatag aagtattctg tggagaaatg 780
ggagcttaca acaaaacacc tcacgatgtg gttttaaaat ggcttgaaga tcttttagaa 840
atttttaaaa ctttgaacat agggtttgcc ttatggaatt ttagagggcc ttttggtatt 900
ttagattcag aaaggaaaga cgttgaatac gaagaatggt atggacataa actggatagg 960
aaaatgttgg aactattgag aaaatatcat catcatcatc atcattag 1008
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaataaca ccattccaag 20
<210> 4
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctaatgatga tgatgatgat gatattttct caatagttcc 40
<210> 5
<211> 3383
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caccaccacc accactaata agcttgaagg ccgcttccga aaggaagcgg cttttttgcc 60
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 120
tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 180
ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 240
tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 300
agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 360
cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 420
tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 480
gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 540
cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 600
atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 660
agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 720
gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 780
ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 840
cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 900
ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atggggggat 960
cattgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1020
gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1080
actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1140
aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1200
cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1260
tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagactgat 1320
cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 1380
tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 1440
ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 1500
ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 1560
cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 1620
aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 1680
agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 1740
tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 1800
ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 1860
cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 1920
atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 1980
ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggg tatctttata 2040
gtcctgtcgg ggtttcgcac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 2100
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 2160
ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta 2220
ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag 2280
tgagagagga agcggaagag cccccttgaa tgtgggggaa acatccccat gatccaatga 2340
cctgttaacc gtcgacaaga aggagatata ccctatgaat aacaccattc caagatggcg 2400
tggcttcaac cttctggaag ccttttccat taaaagtacc ggaaatttta aagaggaaga 2460
ttttttgtgg atggctcagt gggactttaa ttttgttaga atccctatgt gtcatcttct 2520
ctggtcagac tggggcaact catttattat cagagaagat ttttttgaga aaatcgatcg 2580
tgtaattttc tggggagaga aatatggaat acatatatgt atttctctcc acagggcacc 2640
tggctattct gttaacaagg aagtagaaga gaaaaccaat ctgtggaaag atgaaacagc 2700
tcaagaagcg ttcattcatc actggtcttt tatcgcacgt cgttacaaag gaatttcttc 2760
cacacacctg agttttaact taataaatga gcctccattt cctgatccac aaatcatgag 2820
tgttgaagat cacaactctc ttatcaagag aactattaca gaaattcgaa aaatagatcc 2880
cgaaagatta attatgatag atggattagg ctatgggaat attccagtgg atgatttaac 2940
aattgagaat acagtgcaat catgcagagg gtacattccc ttcagtgtta ctcattacaa 3000
agcggaatgg gtggatagta aggactttcc tgctcctgag tggccaaatg gatggcattt 3060
tggggaatac tggaacagag aaaagttatt ggaacattac ttaacgtgga taaaactcag 3120
acaaaaagga atagaagtat tctgtggaga aatgggagct tacaacaaaa cacctcacga 3180
tgtggtttta aaatggcttg aagatctttt agaaattttt aaaactttga acatagggtt 3240
tgccttatgg aattttagag ggccttttgg tattttagat tcagaaagga aagacgttga 3300
atacgaagaa tggtatggac ataaactgga taggaaaatg ttggaactat tgagaaaata 3360
tcatcatcat catcatcatt aga 3383
Claims (10)
1.一种极耐热甘露聚糖酶,其氨基酸序列如SEQ ID NO.1所示。
2.编码SEQ ID NO.1所示氨基酸序列的DNA分子,其特征在于,其核苷酸序列如SEQ IDNO.2所示。
3.根据权利要求1所述的极耐热甘露聚糖酶,其特征在于,所述极耐热甘露聚糖酶的适宜温度为75-90ºC,适宜pH为5.0~6.0。
4.根据权利要求3所述的极耐热甘露聚糖酶,其特征在于,所述极耐热甘露聚糖酶的最适温度为85ºC,最适pH为5.5。
5.权利要求1所述极耐热甘露聚糖酶的制备方法,其特征在于,包括以下步骤:
(1)以提取的超嗜热神袍菌Thermotoga sp. RQ2基因组DNA为模板,通过核苷酸序列如SEQ ID NO.3所示的上游引物和核苷酸序列如SEQ ID NO.4所示的下游引物扩增得如SEQID NO.2所示的核苷酸序列,将得到的基因序列与表达载体连接得到重组质粒,将重组质粒转化到宿主菌得到基因工程菌;
(2)将步骤(1)得到的基因工程菌预培养后接种于含有抗生素的LB液体培养基中30℃培养至OD600达到1-2,然后将培养基转移至42℃继续培养6-8h,离心收集菌体;
(3)将步骤(2)得到的菌体破碎后热处理,经镍离子柱柱纯化后得到极耐热甘露聚糖酶。
6.根据权利要求5所述的制备方法,其特征在于,步骤(1)中所述的宿主菌为大肠杆菌BL21,所述的表达载体为pHsh质粒;步骤(2)中所述的接种为1%的接种量,所述的抗生素为氨苄青霉素。
7.根据权利要求5所述的制备方法,其特征在于,步骤(3)中所述破碎为将收集的菌体用25mM邻苯二甲酸氢钾-咪唑缓冲溶液重新悬浮细胞进行超声破碎,75ºC热处理20min,离心收集上清液得到粗酶液。
8.根据权利要求5所述的制备方法,其特征在于,步骤(3)中所述纯化为将粗酶液用0.22μm水系滤膜过滤后通过镍柱进行纯化,有活性的洗脱峰用透析袋进行脱盐,得到电泳纯的蛋白。
9.一种重组质粒,其特征在于,包含权利要求2所述的核苷酸序列,所述的重组质粒其核苷酸序列如SEQ ID NO.5所示。
10.权利要求1所述的耐热甘露聚糖酶应用在纺织、食品、饲料或造纸领域。
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