CN112795556A - 一种β-N-乙酰氨基葡萄糖苷酶159及其克隆表达与应用 - Google Patents
一种β-N-乙酰氨基葡萄糖苷酶159及其克隆表达与应用 Download PDFInfo
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Abstract
本发明公开了一种β‑N‑乙酰氨基葡萄糖苷酶159及其克隆表达与应用。通过基因工程手段将β‑N‑乙酰氨基葡萄糖苷酶159的基因构建在质粒pET‑28a(+)上,并转入E.coli BL21(DE3)中表达,该重组酶分子量大小为92kDa,通过对比分析,β‑N‑乙酰氨基葡萄糖苷酶159属于糖苷水解酶GH20家族。对该重组酶进行酶学性质研究表明,纯酶的比活力为16.67 U/mg,其最适温度为40℃,最适pH为9.0,在pH高于7的条件下能保持高于80%的酶活,与其他几丁质酶协同作用时,β‑N‑乙酰氨基葡萄糖苷酶159表现出较高的pH稳定性和协同效应,为高效制备N‑乙酰氨基葡萄糖提供了理论基础。
Description
技术领域
本发明属于基因工程领域,具体涉及一种β-N-乙酰氨基葡萄糖苷酶159及其克隆表达与应用。
背景技术
几丁质又称甲壳素、壳多糖,是由N-乙酰氨基葡萄糖通过β-1,4-糖苷键连接的聚合物,是地球上除纤维素外含量最高的多聚糖。其分布十分广泛,主要存在于虾蟹壳、昆虫的外骨骼和真菌细胞壁中,其水解产物N-乙酰氨基葡萄糖可以保护软骨组织及骨关节。几丁质酶可以防治植物的真菌类病害,在食品、医药、农业以及化妆品等行业具有广泛应用。
以几丁质为底物制备N-乙酰氨基葡萄糖(GlcNAc)的方法主要可分为是化学法和酶法,目前工业上多采用化学法,此法存在反应过程控制较难,伴有副产物产生,且产物的生物活性差等缺陷。而且对环境污染严重,三废处理成本高。因此,寻找绿色高效的GlcNAc的制备方法具有重要意义。
几丁质酶(Chitinase, EC3.2.1.14)是一种糖苷水解酶,能专一水解几丁质生产GlcNAc及几丁寡糖的糖苷水解酶,按切割糖苷键的方式的不同可分为外切几丁质酶(EC3.2.1.20),内切几丁质酶(EC 3.2.1.14)及β-N-乙酰氨基葡萄糖苷酶(NAGases,EC3.2.1.52),外切几丁质酶从几丁质糖链的非还原末端将几丁质降解为(GlcNAc)2,内切几丁质酶与外切几丁质酶有所不同,其可随机作用于几丁质长链的任何一个糖苷键,并将其水解成几丁寡糖;而N-乙酰氨基葡萄糖苷酶主要水解几丁寡糖为单糖GlcNAc,因其反应条件温和、控制性强、产物生物活性好且绿色环保,这种生物酶法处理几丁质将成为现代几丁质产业发展方向,具有巨大的发展前景。
目前,GlcNAc已广泛用于生产唾液酸、生物乙醇和单细胞蛋白质;在医药领域可以作为肠胃炎、骨关节炎的药物,还可应用于癌症诊断(Chen J.K.,Shen C.R.and LiuC.L.N-Acetylglucosamine:Production andApplications.MarineDrugs,2010,8:2493-2516)。因此,N-乙酰氨基葡萄糖的绿色生物生产具有重要的意义和应用价值。近年来,酶法制备N-乙酰氨基葡萄糖因其高效无污染而被广泛关注。如专利号2015107793579利用几丁质酶与几丁质的亲和吸附可高效生产N-乙酰氨基葡萄糖。Suresh等(Suresh P.V.andKumarP.K.A.Enhanced degradation of a-chitin materials prepared fromshrimpprocessing byproduct and production of N-acetyl-D-glucosamine bythermoactivechitinases from soil mesophilic fungi.Biodegradation,2012,23:597-607)利用几丁质酶和β-N-乙酰氨基葡萄糖苷酶水解几丁质粉末生产N-乙酰氨基葡萄糖,得率为23.7%。因此,挖掘具有更高酶活的新型β-N-乙酰氨基葡萄糖苷酶对于几丁寡糖的高效炼制具有重要意义。
发明内容
针对现有技术的不足,本发明的目的在于提供一种β-N-乙酰氨基葡萄糖苷酶159及其克隆表达与应用,所述β-N-乙酰氨基葡萄糖苷酶159的基因来源于菌种Chitinibacter sp.GC72,重组β-N-乙酰氨基葡萄糖苷酶159酶活力较高;该酶的最适温度为40℃,在温度低于40℃的条件下159较稳定,残余酶活力保持在70%以上;最适pH 9.0,在pH 7.0-9.0下都展现出了较高的稳定性,残余酶活力均保持在80%以上;
以pNP-GlcNAc为底物研究,该酶的V max及K m分别为20.02μmol/(min•mg)和0.055mmol/L;该重组酶分别与来自Chitinolyticbacter meiyuanensis、SYBC-H1、Chitinibactersp.GC72、变形斑沙雷氏菌的几丁质酶同时加入到胶体几丁质中,可以显著提高胶体几丁质降解效率。
一种β-N-乙酰氨基葡萄糖苷酶159,其氨基酸序列如SEQ ID No.2所示。
SEQ ID No.2
MNKPAGAHLKLTWANKTNAHNGDNFLAALTLTNLSDQPLPASGWAIYFNTCRKIKPETVSGNVLVTHVNGDFWSLEPKAEFGALQPGETRTFDYEGLFWVISETDAPLGFYIVYDVGTPNAQMEVIGDPEIAEFATPAQRNRNAADQVALSNAAQTYADNAALTLLPADSLSKITPTPLSYTAAAGEFVINKNTYIVHSADLANEAALLQASLHDVSGKTLQRAAITPAGAATIKLQISAVNVSDTLAPNEAYQLDVTPQGIVITGASAAGVCNGIQSLRQLLPVAAFLNPQAELAVGACKVVDAPRFAYRGMHLDVGRNFTSKETILRLLECMSLYKLNQFHFHLTDDEGWRLEIPSLPELTEIGSLRGYTADETDNLVPCFGSGGDVAGKAGSGYYSKADFIEILKYATARHIEVVPEIDVPGHARAAIKAMNVRYTRLMAEGREAEAKQYLLCDFNDASVYESVQLWHDNVICIGQESCYTFIETVLHDVKAMFEEAGAPFTTMHTGGDEVPHGAWEQSPVCQAFMKEQGMTQIVELQNYFLARYRDLLKKYGLVFGGWEEIALVKKEVNGVHTHGPNPEFVNANFRPYVWNNVWGWGQEDFAYQLANAGYKTVLSNVTNLYFDLAYAKDPLEPGYYWGGFIETRGAFEFVPLDIFTTATVNLFGHALDKDNIASKVRLTPEGTKNIIGIQGQLWAENIRNRGRLEYLAMPRTIALAERAWAKDPAWTSIADAAARQSKIDADWNEFANRLGQRELPRLDGFPRSEDFGGYGYRIPLPGVKVEAGQLYANTSAPGLAIRYTTDGSDPTPASALYTGPVAATATMKVAAFSSNNRR
一种核苷酸,编码上述β-N-乙酰氨基葡萄糖苷酶159的核苷酸序列如SEQ ID No.1所示。
SEQ ID No.1:
ATGAACAAGCCAGCAGGTGCGCATTTAAAATTAACATGGGCCAATAAAACCAACGCTCATAACGGCGATAACTTTCTCGCTGCTTTGACGCTGACCAATCTATCCGATCAGCCCCTGCCAGCATCAGGCTGGGCCATCTATTTCAATACCTGTCGCAAAATCAAGCCTGAAACCGTATCGGGCAATGTGCTGGTCACGCACGTCAATGGCGACTTCTGGAGCCTTGAGCCCAAAGCTGAATTCGGCGCATTGCAGCCGGGTGAAACCCGCACTTTCGATTACGAAGGCCTGTTCTGGGTGATTAGCGAAACCGACGCGCCGCTGGGTTTCTATATTGTTTACGACGTGGGCACGCCGAATGCGCAGATGGAAGTGATTGGCGACCCGGAAATTGCCGAGTTTGCCACGCCAGCGCAGCGCAATCGCAATGCTGCCGATCAGGTCGCGCTGAGCAATGCTGCACAAACCTATGCCGATAATGCCGCTCTGACTCTGTTGCCAGCCGATAGCCTGAGCAAAATCACGCCAACCCCGCTGAGCTACACTGCCGCAGCGGGCGAGTTTGTAATCAATAAAAATACCTATATCGTGCATTCGGCTGATCTGGCCAATGAAGCGGCTTTGCTGCAAGCCAGCCTGCATGATGTGAGCGGCAAAACATTGCAACGCGCGGCGATTACGCCAGCGGGCGCGGCAACGATCAAACTGCAAATTAGCGCGGTGAACGTTAGCGATACTTTGGCGCCTAACGAGGCTTACCAGCTCGACGTAACGCCACAAGGCATCGTAATTACCGGCGCGAGCGCTGCGGGCGTGTGCAACGGCATTCAAAGTTTACGTCAATTATTGCCAGTGGCGGCCTTCCTGAACCCGCAAGCAGAACTCGCCGTTGGCGCGTGTAAAGTTGTGGATGCGCCACGTTTTGCCTACCGCGGCATGCACCTCGATGTGGGCCGTAACTTCACCAGCAAAGAAACGATTCTGCGCCTGCTTGAATGCATGTCGCTATACAAGCTGAACCAGTTCCACTTCCACCTCACCGACGACGAAGGCTGGCGCTTGGAAATTCCTAGCCTGCCTGAACTAACCGAAATCGGTAGCTTGCGCGGTTACACGGCGGACGAAACCGACAATCTGGTGCCATGCTTTGGCTCGGGCGGCGACGTGGCGGGTAAGGCGGGTAGTGGTTATTACAGCAAAGCTGATTTCATCGAAATCTTGAAATACGCCACCGCGCGCCATATCGAAGTCGTGCCAGAAATCGACGTGCCAGGCCATGCGCGTGCCGCGATCAAGGCGATGAATGTGCGCTACACGCGTTTGATGGCCGAAGGCCGCGAGGCTGAAGCCAAGCAATACCTGTTGTGCGACTTTAACGACGCATCGGTGTATGAGTCCGTGCAGCTCTGGCACGACAATGTAATCTGTATTGGTCAGGAATCCTGCTACACCTTTATCGAAACCGTGCTACACGATGTGAAAGCCATGTTTGAAGAAGCGGGCGCGCCGTTTACGACGATGCATACCGGCGGCGATGAAGTACCGCACGGTGCGTGGGAACAATCGCCGGTTTGCCAAGCCTTTATGAAAGAGCAGGGCATGACGCAAATCGTCGAGCTGCAAAACTACTTCCTCGCCCGCTACCGCGATCTGCTGAAAAAATACGGCTTGGTGTTTGGCGGCTGGGAAGAAATTGCACTGGTGAAAAAAGAAGTCAACGGTGTACATACCCACGGCCCGAATCCAGAATTCGTCAACGCCAACTTCCGCCCGTACGTGTGGAATAACGTATGGGGCTGGGGTCAGGAAGACTTTGCTTACCAGTTGGCTAACGCCGGCTATAAAACGGTCTTGTCGAACGTGACGAATCTGTATTTTGACTTGGCCTACGCGAAAGACCCACTGGAGCCGGGTTACTACTGGGGCGGCTTTATCGAAACGCGTGGTGCGTTTGAGTTTGTACCACTCGATATTTTCACGACGGCGACGGTTAATTTGTTTGGCCATGCGTTGGATAAAGACAATATCGCCAGCAAAGTACGCCTGAC
本发明中通过比较氨基酸序列同源性,本发明的β-N-乙酰氨基葡萄糖苷酶159属于糖苷水解酶GH20家族。
一种重组质粒,含有编码β-N-乙酰氨基葡萄糖苷酶159的核苷酸序列。
一种重组菌株,表达上述重组质粒的菌株。
上述重组菌株的构建方法,包括以下步骤:
步骤1,PCR扩增SEQ ID NO:1所示的序列,所用扩增引物如下:
159-F-Primer:5’-ATTCTAGCT ATGAACAAGCCAGCAGGTG-3’,
159-R-Primer:5’-ATTCCGCTCCGCCAGCAAAGTACGCCTGAC-3’;
上述PCR扩增的反应条件为:95℃预变性5 min;94℃ 30sec、55℃ 30sec、72℃40sec,30个循环;最后在72℃下延伸10min;
步骤2,构建重组质粒pET-28a(+)-159
将步骤1扩增的序列和pET-28a(+)质粒均用NdeI和XhoI限制性内切酶进行双酶切,酶切产物进行回收纯化,再用T4DNA连接酶连接纯化的酶切产物,得到重组质粒pET-28a(+)-159;
步骤3,将得到的重组质粒pET-28a(+)-159导入大肠杆菌BL21(DE3),得到含有重组质粒pET-28a(+)-159的大肠杆菌BL21(DE3),命名为重组菌;
步骤4,挑选重组菌的单克隆,摇床过夜培养后,将种子液以体积分数1%-5%接种量接入含有100μg/L卡那霉素LB培养基,37℃下培养菌OD600为0.4-0.6时,加入诱导剂在18℃诱导14-16h后收集发酵液,再在4℃进行6000-8000rpm的速度离心,收集菌株;
步骤5,向菌株中加入10m L50mM PBS缓冲液重悬细胞,超声破碎后,离心收集上清后冷冻备用。
上述β-N-乙酰氨基葡萄糖苷酶159在降解胶体几丁质上的应用。
有益效果:
与现有技术相比,本发明一种β-N-乙酰氨基葡萄糖苷酶159及其克隆表达与应用的优势在于:
1 、本发明获得了β-N-乙酰氨基葡萄糖苷酶基因的核苷酸序列,该序列首次从Chitinibacter sp.GC72中克隆,且成本低廉,分子水平操作简单,易于克隆表达,重复性较好,在工业中具有潜在应用价值;
2、本发明构建了β-N-乙酰氨基葡萄糖苷酶159原核表达载体,成功获得分子量约为92kD的酶蛋白,蛋白表达效果好,酶活高,且制备过程简单,为β-N-乙酰氨基葡萄糖苷酶159的性能研究提供了简便方法;
3、本发明中β-N-乙酰氨基葡萄糖苷酶159在25-40℃时热稳定性良好,酶解最适宜的反应温度为40℃;在pH 9.0酶活达到最高,在pH7.0-9.0,酶均有活性,从结果表明β-N-乙酰氨基葡萄糖苷酶159的稳定性较高,酶的反应条件温和,适应pH范围广,具有工业化潜在价值;
4、本发明中β-N-乙酰氨基葡萄糖苷酶159对pNP-GlcNAc具有较高的降解活性,在工业化生产N-乙酰氨基葡萄糖中具有良好的生产应用前景。
附图说明
图1为本发明β-N-乙酰氨基葡萄糖苷酶159的SDS-PAGE检测结果;
图2为本发明中温度对β-N-乙酰氨基葡萄糖苷酶159活性的影响,其中(a)为最适温度,(b)为温度稳定性;
图3为本发明中pH对β-N-乙酰氨基葡萄糖苷酶159活性的影响;其中,(a)为最适pH;(b)为pH稳定性;
图4为β-N-乙酰氨基葡萄糖苷酶159与其他几丁质酶的协同效应。
具体实施方案
下面通过实施例对本发明做进一步描述,但不用于限制本发明的保护范围。实施例中的实验方法,如无特殊说明,均为常规方法。
下述实例中所用到的材料、试剂等,如无特殊说明,均可从商业途径获得。
实施例1 菌株来源
本发明所用菌株Chitinibacter sp.GC72是从南京市池塘淤泥中分离得到的。现保藏于中国典型培养物保藏中心(保藏号:CCTCC M 2014113)
实施例2
克隆β-N-乙酰氨基葡萄糖苷酶159,包括以下步骤:
步骤1,β-N-乙酰氨基葡萄糖苷酶的基因,其核苷酸序列如SEN ID NO:1所示;
步骤2,设计PCR扩增引物
159-F-Primer:5’-ATTCTAGCT ATGAACAAGCCAGCAGGTG-3’;
159-R-Primer:5’-ATTCCGCTCCGCCAGCAAAGTACGCCTGAC-3’,扩增出该基因的全长;
步骤3,上述PCR扩增的反应条件为:95℃预变性5 min;94℃ 30sec、55℃ 30sec、72℃ 40sec,30个循环;最后在72℃下延伸10min;
步骤4,用限制性内切酶NdeI和XhoI双酶切PCR扩增产物,回收酶切产物;
步骤5,用限制性内切酶NdeI和XhoI双酶切载体pET-28a(+),回收载体骨架(约5400 bp);
步骤6,对上述步骤4的酶切产物和步骤5的载体骨架用T4连接酶进行连接,得到重组质粒pET-28a(+)-159。
实施例3
β-N-乙酰氨基葡萄糖苷酶159基因的克隆表达,包括以下步骤:
第一步,将重组质粒pET-28a(+)-159导入大肠杆菌BL21(DE3),得到含有重组质粒pET-28a(+)-159的大肠杆菌,命名为重组菌;
第二步,挑选重组菌(表达含有His标签pET-28a(+)-159)的单克隆,接至含100μg/mL的卡那霉素5 mL LB培养基在37℃、200rmp的振荡摇床中过夜培养;
第三步,将上述菌液以1:100的体积比接种至100 mL LB含100μg/mL卡那霉素的液体培养基,37℃、200 rpm/min的振荡培养至OD600达到0.6-0.8;
第四步,然后加入IPTG(诱导剂)至浓度为1mmol/L,20℃、200rmp振荡培养20h;
第五步,收集上述菌液到离心管中,4℃,6000rpm离心10min,弃上清,收集沉淀;
第六步,向细菌沉淀中加入10 mL50mM PBS缓冲液重悬菌体;
第七步,冰上超声破碎细菌,超声功率为300W,每次超声处理2s,间隔3s,共超声处理10 min;
第八步,4℃,6000 rpm/min离心10 min,收集细菌裂解液上清,并置于冰上;
第九步,收集的上清液用AKTA蛋白纯化系统和该公司的HIS-TAG柱重组蛋白进行蛋白纯化,纯化后的重组蛋白溶液进行SDS-PAGE,获得92kDa的蛋白条带(结果如图1所示)。
实施例4
β-N-乙酰氨基葡萄糖苷酶159活性的鉴定
蛋白含量测定:以牛血清白蛋白(BSA)为标准品绘制标准曲线,采用考马斯亮蓝法测定蛋白含量。取20μL稀释100倍的蛋白溶液,加入700μL Bradford 溶液,迅速混匀。室温反应 3 min 后,以加入等量蒸馏水为空白对照,使用分光光度计检测 595nm 波长下的吸光值。
β-N-乙酰氨基葡萄糖苷酶的酶活测定:取100μL 2mM pNP-GlcNAG为底物,加入50μL 200 mM磷酸盐缓冲液(pH 6.0),混匀,65℃预热1 min,然后加入50μL稀释100倍的酶液,反应10min后,加入200μL、0.5M NaOH溶液终止反应。待冷却后,以等量蒸馏水为空白对照,使用分光光度计检测410nm波长下的吸光值。
酶活力单位(U)定义:在上述条件下,每分钟生成1μmoL对硝基苯酚所需的 β-N-乙酰氨基葡萄糖苷酶酶量为1个酶活力单位。酶比活力为每毫克蛋白质所具有的酶活力单位数,计算公式:比活力=酶活力/蛋白含量。
实施例5
β-N-乙酰氨基葡萄糖苷酶159酶学性质的研究
一、酶活测定
具体见实施例4。
二、温度对β-N-乙酰氨基葡萄糖苷酶159的影响
1、最适反应温度
取 100μL 20mM pNP-GlcNAG为底物,加入50μL 200 mM磷酸盐缓冲液(pH 6.0),混匀,然后加入50μL稀释100倍的酶液,在30℃、35℃、40℃、45℃、50℃、55℃、 60℃、65℃、70℃、80℃温度梯度范围内反应10min后,加入200μL 0.5M NaOH溶液终止反应。待冷却后,以等量蒸馏水为空白对照,使用分光光度计检测410 nm 波长下的吸光值,确定不同温度下β-N-乙酰氨基葡萄糖苷酶159的酶活。根据酶在不同温度下的相对活性绘制曲线(以酶活力的最高点为100%),确定β-N-乙酰氨基葡萄糖苷酶159的最适反应温度。结果如图2(a)所示,β-N-乙酰氨基葡萄糖苷酶159的最适的反应温度为40℃。
2、β-N-乙酰氨基葡萄糖苷酶159的热稳定性
将相同体积和浓度的酶液分别置于不同温度(30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、80℃) 下孵育30h,冰水浴10min冷却然后检测残余酶活,并和未处理的酶活进行比较,计算相对活性。结果如图2(b)所示,在温度低于40℃的条件下β-N-乙酰氨基葡萄糖苷酶159较稳定,残余酶活力保持在70%以上。研究发现大部分β-N-乙酰氨基葡萄糖苷酶在不高于40℃的条件下稳定性较高;在45-60℃时其热稳定性都较差,β-N-乙酰氨基葡萄糖苷酶159相较于其他来源如苜蓿链霉菌的β-N-乙酰氨基葡萄糖苷酶热稳定性相近。
三、pH对β-N-乙酰氨基葡萄糖苷酶159的影响
1、最适pH
取100μL 20mM pNP-GlcNAG为底物,加入50μL不同pH缓冲液(柠檬酸盐缓冲液的pH:4.0-6.5,磷酸盐缓冲液的pH:5.5-8.0,Tris-HCl的pH 7.5-9.0,甘氨酸-NaOH的pH 9.0-10.5),混匀,然后加入50μL稀释100倍的酶液,以pNP-GlcNAG为底物,在40℃下反应10min后,加入200μL、0.5M NaOH溶液终止反应,根据酶在不同的pH值下的相对活性绘制曲线,待冷却后,以等量蒸馏水为空白对照,使用分光光度计检测410nm 波长下的吸光值,确定酶的最适反应pH 值。结果如图3(a)所示,β-N-乙酰氨基葡萄糖苷酶159的最适pH是9.0。
2、β-N-乙酰氨基葡萄糖苷酶159的pH值稳定性
将相同体积和浓度的酶液分别置于不同pH的缓冲液(柠檬酸盐缓冲液的pH:4.0-6.5,磷酸盐缓冲液的pH:5.5-8.0,Tris-HCl的pH:7.5-9.0,甘氨酸-NaOH的pH:9.0-10.5)中,于40℃下放置30min,然后在最适反应温度下检测残余酶活,并和未处理的酶活进行比较,计算相对活性。结果如图3(b)所示,β-N-乙酰氨基葡萄糖苷酶159在弱碱性条件(7.0-9.0)下都展现出了较高的稳定性,残余酶活力均保持在80%以上。β-N-乙酰氨基葡萄糖苷酶159具有较适pH以及良好的pH值稳定性,可以适应弱碱性的生产条件。
四、β-N-乙酰氨基葡萄糖苷酶159动力学参数的测定
用50 mM、pH 6.0磷酸缓冲液配制10个不同浓度(0.015、0.03、0.045、0.06、0.075、0.09、0.105、0.12、0.135、0.15mM)的pNP-β-N-乙酰氨基葡萄糖苷作为酶水解的底物,然后加入的50μL稀释100倍的β-N-乙酰氨基葡萄糖苷酶在40℃下反应 5min,通过sigmaplot软件计算出Vmax、Km值和标准偏差,β-N-乙酰氨基葡萄糖苷酶159的Vmax及Km分别为20.02μmol/(min·mg)和0.055 mmol/L。
五、协同效应的测定
取50μL稀释100倍的酶液与200μL来自Chitinolyticbacter meiyuanensisSYBC-H1、Chitinibactersp.GC72、变形斑沙雷氏菌的几丁质酶,加入到500μL胶体几丁质中,用纯水补齐至1mL,另外一组中,只将200μL其他几丁质酶加入到500μL胶体几丁质中,并用纯水补齐至1mL。37℃下反应30min,沸水浴5 min后中止反应,冷却至室温后加入1mL DNS试剂,沸水浴5min,离心后取上清,在波长 540 nm下测吸光度。以灭活的等量酶液作空白对照加入1mL的DNS溶液煮沸5min,在540nm波长下测取吸光值。结果如图4所示,与没有加入β-N-乙酰氨基葡萄糖苷酶159(对照组)和空白对照相比,加入β-N-乙酰氨基葡萄糖苷酶159的实验组的胶体几丁质降解效率均有显著提高。
序列表
<110> 南京工业大学
<120> 一种β-N-乙酰氨基葡萄糖苷酶159及其克隆表达与应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 838
<212> PRT
<213> 氨基酸序列(Amino acid sequence)
<400> 1
Met Asn Lys Pro Ala Gly Ala His Leu Lys Leu Thr Trp Ala Asn Lys
1 5 10 15
Thr Asn Ala His Asn Gly Asp Asn Phe Leu Ala Ala Leu Thr Leu Thr
20 25 30
Asn Leu Ser Asp Gln Pro Leu Pro Ala Ser Gly Trp Ala Ile Tyr Phe
35 40 45
Asn Thr Cys Arg Lys Ile Lys Pro Glu Thr Val Ser Gly Asn Val Leu
50 55 60
Val Thr His Val Asn Gly Asp Phe Trp Ser Leu Glu Pro Lys Ala Glu
65 70 75 80
Phe Gly Ala Leu Gln Pro Gly Glu Thr Arg Thr Phe Asp Tyr Glu Gly
85 90 95
Leu Phe Trp Val Ile Ser Glu Thr Asp Ala Pro Leu Gly Phe Tyr Ile
100 105 110
Val Tyr Asp Val Gly Thr Pro Asn Ala Gln Met Glu Val Ile Gly Asp
115 120 125
Pro Glu Ile Ala Glu Phe Ala Thr Pro Ala Gln Arg Asn Arg Asn Ala
130 135 140
Ala Asp Gln Val Ala Leu Ser Asn Ala Ala Gln Thr Tyr Ala Asp Asn
145 150 155 160
Ala Ala Leu Thr Leu Leu Pro Ala Asp Ser Leu Ser Lys Ile Thr Pro
165 170 175
Thr Pro Leu Ser Tyr Thr Ala Ala Ala Gly Glu Phe Val Ile Asn Lys
180 185 190
Asn Thr Tyr Ile Val His Ser Ala Asp Leu Ala Asn Glu Ala Ala Leu
195 200 205
Leu Gln Ala Ser Leu His Asp Val Ser Gly Lys Thr Leu Gln Arg Ala
210 215 220
Ala Ile Thr Pro Ala Gly Ala Ala Thr Ile Lys Leu Gln Ile Ser Ala
225 230 235 240
Val Asn Val Ser Asp Thr Leu Ala Pro Asn Glu Ala Tyr Gln Leu Asp
245 250 255
Val Thr Pro Gln Gly Ile Val Ile Thr Gly Ala Ser Ala Ala Gly Val
260 265 270
Cys Asn Gly Ile Gln Ser Leu Arg Gln Leu Leu Pro Val Ala Ala Phe
275 280 285
Leu Asn Pro Gln Ala Glu Leu Ala Val Gly Ala Cys Lys Val Val Asp
290 295 300
Ala Pro Arg Phe Ala Tyr Arg Gly Met His Leu Asp Val Gly Arg Asn
305 310 315 320
Phe Thr Ser Lys Glu Thr Ile Leu Arg Leu Leu Glu Cys Met Ser Leu
325 330 335
Tyr Lys Leu Asn Gln Phe His Phe His Leu Thr Asp Asp Glu Gly Trp
340 345 350
Arg Leu Glu Ile Pro Ser Leu Pro Glu Leu Thr Glu Ile Gly Ser Leu
355 360 365
Arg Gly Tyr Thr Ala Asp Glu Thr Asp Asn Leu Val Pro Cys Phe Gly
370 375 380
Ser Gly Gly Asp Val Ala Gly Lys Ala Gly Ser Gly Tyr Tyr Ser Lys
385 390 395 400
Ala Asp Phe Ile Glu Ile Leu Lys Tyr Ala Thr Ala Arg His Ile Glu
405 410 415
Val Val Pro Glu Ile Asp Val Pro Gly His Ala Arg Ala Ala Ile Lys
420 425 430
Ala Met Asn Val Arg Tyr Thr Arg Leu Met Ala Glu Gly Arg Glu Ala
435 440 445
Glu Ala Lys Gln Tyr Leu Leu Cys Asp Phe Asn Asp Ala Ser Val Tyr
450 455 460
Glu Ser Val Gln Leu Trp His Asp Asn Val Ile Cys Ile Gly Gln Glu
465 470 475 480
Ser Cys Tyr Thr Phe Ile Glu Thr Val Leu His Asp Val Lys Ala Met
485 490 495
Phe Glu Glu Ala Gly Ala Pro Phe Thr Thr Met His Thr Gly Gly Asp
500 505 510
Glu Val Pro His Gly Ala Trp Glu Gln Ser Pro Val Cys Gln Ala Phe
515 520 525
Met Lys Glu Gln Gly Met Thr Gln Ile Val Glu Leu Gln Asn Tyr Phe
530 535 540
Leu Ala Arg Tyr Arg Asp Leu Leu Lys Lys Tyr Gly Leu Val Phe Gly
545 550 555 560
Gly Trp Glu Glu Ile Ala Leu Val Lys Lys Glu Val Asn Gly Val His
565 570 575
Thr His Gly Pro Asn Pro Glu Phe Val Asn Ala Asn Phe Arg Pro Tyr
580 585 590
Val Trp Asn Asn Val Trp Gly Trp Gly Gln Glu Asp Phe Ala Tyr Gln
595 600 605
Leu Ala Asn Ala Gly Tyr Lys Thr Val Leu Ser Asn Val Thr Asn Leu
610 615 620
Tyr Phe Asp Leu Ala Tyr Ala Lys Asp Pro Leu Glu Pro Gly Tyr Tyr
625 630 635 640
Trp Gly Gly Phe Ile Glu Thr Arg Gly Ala Phe Glu Phe Val Pro Leu
645 650 655
Asp Ile Phe Thr Thr Ala Thr Val Asn Leu Phe Gly His Ala Leu Asp
660 665 670
Lys Asp Asn Ile Ala Ser Lys Val Arg Leu Thr Pro Glu Gly Thr Lys
675 680 685
Asn Ile Ile Gly Ile Gln Gly Gln Leu Trp Ala Glu Asn Ile Arg Asn
690 695 700
Arg Gly Arg Leu Glu Tyr Leu Ala Met Pro Arg Thr Ile Ala Leu Ala
705 710 715 720
Glu Arg Ala Trp Ala Lys Asp Pro Ala Trp Thr Ser Ile Ala Asp Ala
725 730 735
Ala Ala Arg Gln Ser Lys Ile Asp Ala Asp Trp Asn Glu Phe Ala Asn
740 745 750
Arg Leu Gly Gln Arg Glu Leu Pro Arg Leu Asp Gly Phe Pro Arg Ser
755 760 765
Glu Asp Phe Gly Gly Tyr Gly Tyr Arg Ile Pro Leu Pro Gly Val Lys
770 775 780
Val Glu Ala Gly Gln Leu Tyr Ala Asn Thr Ser Ala Pro Gly Leu Ala
785 790 795 800
Ile Arg Tyr Thr Thr Asp Gly Ser Asp Pro Thr Pro Ala Ser Ala Leu
805 810 815
Tyr Thr Gly Pro Val Ala Ala Thr Ala Thr Met Lys Val Ala Ala Phe
820 825 830
Ser Ser Asn Asn Arg Arg
835
<210> 2
<211> 2048
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaacaagc cagcaggtgc gcatttaaaa ttaacatggg ccaataaaac caacgctcat 60
aacggcgata actttctcgc tgctttgacg ctgaccaatc tatccgatca gcccctgcca 120
gcatcaggct gggccatcta tttcaatacc tgtcgcaaaa tcaagcctga aaccgtatcg 180
ggcaatgtgc tggtcacgca cgtcaatggc gacttctgga gccttgagcc caaagctgaa 240
ttcggcgcat tgcagccggg tgaaacccgc actttcgatt acgaaggcct gttctgggtg 300
attagcgaaa ccgacgcgcc gctgggtttc tatattgttt acgacgtggg cacgccgaat 360
gcgcagatgg aagtgattgg cgacccggaa attgccgagt ttgccacgcc agcgcagcgc 420
aatcgcaatg ctgccgatca ggtcgcgctg agcaatgctg cacaaaccta tgccgataat 480
gccgctctga ctctgttgcc agccgatagc ctgagcaaaa tcacgccaac cccgctgagc 540
tacactgccg cagcgggcga gtttgtaatc aataaaaata cctatatcgt gcattcggct 600
gatctggcca atgaagcggc tttgctgcaa gccagcctgc atgatgtgag cggcaaaaca 660
ttgcaacgcg cggcgattac gccagcgggc gcggcaacga tcaaactgca aattagcgcg 720
gtgaacgtta gcgatacttt ggcgcctaac gaggcttacc agctcgacgt aacgccacaa 780
ggcatcgtaa ttaccggcgc gagcgctgcg ggcgtgtgca acggcattca aagtttacgt 840
caattattgc cagtggcggc cttcctgaac ccgcaagcag aactcgccgt tggcgcgtgt 900
aaagttgtgg atgcgccacg ttttgcctac cgcggcatgc acctcgatgt gggccgtaac 960
ttcaccagca aagaaacgat tctgcgcctg cttgaatgca tgtcgctata caagctgaac 1020
cagttccact tccacctcac cgacgacgaa ggctggcgct tggaaattcc tagcctgcct 1080
gaactaaccg aaatcggtag cttgcgcggt tacacggcgg acgaaaccga caatctggtg 1140
ccatgctttg gctcgggcgg cgacgtggcg ggtaaggcgg gtagtggtta ttacagcaaa 1200
gctgatttca tcgaaatctt gaaatacgcc accgcgcgcc atatcgaagt cgtgccagaa 1260
atcgacgtgc caggccatgc gcgtgccgcg atcaaggcga tgaatgtgcg ctacacgcgt 1320
ttgatggccg aaggccgcga ggctgaagcc aagcaatacc tgttgtgcga ctttaacgac 1380
gcatcggtgt atgagtccgt gcagctctgg cacgacaatg taatctgtat tggtcaggaa 1440
tcctgctaca cctttatcga aaccgtgcta cacgatgtga aagccatgtt tgaagaagcg 1500
ggcgcgccgt ttacgacgat gcataccggc ggcgatgaag taccgcacgg tgcgtgggaa 1560
caatcgccgg tttgccaagc ctttatgaaa gagcagggca tgacgcaaat cgtcgagctg 1620
caaaactact tcctcgcccg ctaccgcgat ctgctgaaaa aatacggctt ggtgtttggc 1680
ggctgggaag aaattgcact ggtgaaaaaa gaagtcaacg gtgtacatac ccacggcccg 1740
aatccagaat tcgtcaacgc caacttccgc ccgtacgtgt ggaataacgt atggggctgg 1800
ggtcaggaag actttgctta ccagttggct aacgccggct ataaaacggt cttgtcgaac 1860
gtgacgaatc tgtattttga cttggcctac gcgaaagacc cactggagcc gggttactac 1920
tggggcggct ttatcgaaac gcgtggtgcg tttgagtttg taccactcga tattttcacg 1980
acggcgacgg ttaatttgtt tggccatgcg ttggataaag acaatatcgc cagcaaagta 2040
cgcctgac 2048
<210> 3
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
attctagcta tgaacaagcc agcaggtg 28
<210> 4
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
attccgctcc gccagcaaag tacgcctgac 30
Claims (6)
1.一种β-N-乙酰氨基葡萄糖苷酶159,其特征在于,所述β-N-乙酰氨基葡萄糖苷酶159的氨基酸序列如SEQ ID No.2所示。
2.一种核苷酸,其特征在于,编码权利要求1所述的β-N-乙酰氨基葡萄糖苷酶159的核苷酸序列如SEQ ID No.1所示。
3.一种重组质粒,其特征在于,含有权利要求2所述的编码β-N-乙酰氨基葡萄糖苷酶159的核苷酸序列。
4.一种重组菌株,其特征在于,表达权利要求3所述的重组质粒的菌株。
5.基于权利要求4所述的重组菌株的构建方法,包括以下步骤:
步骤1,PCR扩增SEQ ID NO:1所示的序列
扩增引物为159-F-Primer:5’-ATTCTAGCT ATGAACAAGCCAGCAGGTG-3’;
159-R-Primer:5’-ATTCCGCTCCGCCAGCAAAGTACGCCTGAC-3’;
上述PCR扩增的反应条件为:95℃预变性5 min;94℃ 30sec、55℃ 30sec、72℃ 40sec,30个循环;最后在72℃下延伸10min;
步骤2,构建重组质粒pET-28a(+)-159
将步骤1扩增的序列和pET-28a(+)质粒均用NdeI和XhoI限制性内切酶进行双酶切,酶切产物进行回收纯化,再用T4DNA连接酶连接纯化的酶切产物,得到重组质粒pET-28a(+)-159;
步骤3,将得到的重组质粒pET-28a(+)-159导入大肠杆菌BL21(DE3),得到含有重组质粒pET-28a(+)-159的大肠杆菌BL21(DE3),命名为重组菌;
步骤4,挑选重组菌的单克隆,摇床过夜培养后,将种子液以体积分数1%-5%接种量接入含有100μg/L卡那霉素LB培养基,37℃下培养菌OD600为0.4-0.6时,加入诱导剂在18℃诱导14-16h后收集发酵液,再在4℃进行6000-8000g离心,收集菌株;
步骤5,向沉淀中加入10mL 50mM PBS缓冲液重悬细胞,超声破碎后,离心收集上清后冷冻备用。
6.基于权利要求1所述的β-N-乙酰氨基葡萄糖苷酶159在降解胶体几丁质上的应用。
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CN114230621A (zh) * | 2021-12-28 | 2022-03-25 | 中国海洋大学 | N-乙酰氨基葡萄糖基甘油酯及其制备方法 |
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CN113528553A (zh) * | 2021-07-07 | 2021-10-22 | 扬州日兴生物科技股份有限公司 | 一种密码子优化的n-乙酰氨基葡萄糖转移酶基因及其应用 |
CN113528553B (zh) * | 2021-07-07 | 2023-09-12 | 扬州日兴生物科技股份有限公司 | 一种密码子优化的n-乙酰氨基葡萄糖转移酶基因及其应用 |
CN114230621A (zh) * | 2021-12-28 | 2022-03-25 | 中国海洋大学 | N-乙酰氨基葡萄糖基甘油酯及其制备方法 |
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