CN116426504A - 一种嗜酸、嗜盐、嗜热、耐受离子液纤维素酶及其应用 - Google Patents
一种嗜酸、嗜盐、嗜热、耐受离子液纤维素酶及其应用 Download PDFInfo
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Abstract
本发明公开了一种嗜酸、嗜盐、嗜热、耐受离子液纤维素酶及其应用。包括提供了其氨基酸、核苷酸序列及其应用。最适反应pH为4.0,最适反应温度为50~55℃,最适盐浓度为10%NaCl溶液,最适离子液浓度为25%。在pH3.0~4.6缓冲体系中,保持80%以上相对活性;pH2.0~6.0下孵育24h后,仍保持100%以上相对活性;在40~60℃均保持60%以上相对活性,80℃下孵育1h后,仍保持40%以上相对活性。5%~35%浓度NaCl溶液中,保持160%以上相对活性;水解产物中葡萄糖含量均提高50%以上。可应用于食品、饲料、纺织以及工业生产中,提高纤维素的降解率。
Description
技术领域
本发明涉及基因工程技术领域,更具体的说是涉及一种嗜酸、嗜盐、嗜热、耐受离子液纤维素酶及其应用。
背景技术
纤维素是地球上一种可再生有机碳,是植物生物量中最丰富的组成部分,占植物干重的35~50%。纤维素的水解需要多种纤维素酶的协同作用,可分为三大类:内切葡聚糖酶(EC 3.2.1.4),外切葡聚糖酶(EC 3.2.1.91)和β-葡糖苷酶(EC 3.2.1.21)。这些酶共同作用,将纤维素分解成更小的低聚糖,葡萄糖作为最终产物。纤维素酶在纺织、纸浆漂白、生物乙醇生产以及人类和动物食品制备方面具有潜在的用途。
纤维素酶在自然界中广泛存于细菌、古菌、真菌、和动物体内。目前,用于生产的纤维素酶主要来自于真菌,如木霉属(Trichoderma)、曲霉属(Aspergillus)和青霉属(Penicillium)。但它们都存在一定的劣势,如高温、高盐和残留离子液环境,而在食品加工、饲料处理、离子液预处理木质纤维素、盐碱环境或海洋来源木质纤维素的降解等对于嗜酸、嗜盐、嗜热、耐受离子液纤维素酶具有非常大的潜在需求,故,需要进一步挖掘获得新的嗜酸、嗜盐、嗜热、耐受离子液纤维素酶。
因此,提供一种纤维素酶,具有嗜酸、嗜盐、嗜热、耐受离子液等特点,是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了一种嗜酸、嗜盐、嗜热、耐受离子液纤维素酶及其应用。
为了实现上述目的,本发明采用如下技术方案:
一种嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶,其氨基酸序列如SEQ ID NO.2所示。
本发明还提供了一种编码权利要求1所述嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因,基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了包含上述编码嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因的重组载体。
优选的:重组载体的制备方法为将嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因插入到质粒pET-28a上的EcoR Ⅰ和Hind Ⅲ限制性酶切位点之间得到重组质粒。
本发明还提供了包含上述编码嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因或上述重组载体的重组菌株。
优选的:重组菌株为:用重组质粒转化大肠杆菌即得。
本发明还提供了述上嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶在食品、饲料、纺织以及工业生产中的应用。
优选的:纤维素酶提高酸性、高盐、高温和离子液残留条件下纤维素的降解率;酸性pH2.0~6.0;高盐:5%~35%浓度NaCl溶液;高温:40~60℃;离子液:1-乙基-3-甲基咪唑乙酸盐。
本发明还提供了上述基因、上述的重组载体或上述重组菌株在产业化生产嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶中的应用。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一种嗜酸、嗜盐、嗜热、耐受离子液纤维素酶及其应用,取得的技术效果为本发明提供了纤维素酶基因(B4-Cel9),携带该基因的重组质粒以及重组菌株,该纤维素酶基因可以编码出一种纤维素酶,该酶最适反应温度为50~55℃,最适反应pH为4.0,最适盐浓度为10%NaCl溶液,最适离子液(1-乙基-3-甲基咪唑乙酸盐)浓度为25%。可应用于食品、饲料、纺织以及工业生产中,提高酸性、高盐、高温和离子液残留条件下纤维素的降解率,解决纤维素资源利用率低的问题。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1附图为本发明提供的纤维素酶基因PCR扩增电泳图,其中,泳道第1条为Marker,片段由上到下依次是2000bp、1000bp、750bp、500bp、200bp、100bp,泳道第2条为扩增目的基因条带。
图2附图为本发明提供的纤维素酶基因SDS-PAGE电泳图,泳道1为Marker,片段由上到下依次是180KD、130KD、100KD、70KD、55KD、40KD、35KD、25KD、15KD、10KD,其中泳道2为IPTG诱导的E.coli BL21/pET-28a-B4-Cel9总蛋白、泳道3为B4-Cel9纯化重组酶。
图3附图为本发明提供的pH对纤维素酶B4-Cel9活性的影响示意图。
图4附图为本发明提供的不同pH缓冲液对纤维素酶B4-Cel9稳定性的影响示意图。
图5附图为本发明提供的温度对纤维素酶B4-Cel9活性的影响示意图。
图6附图为本发明提供的不同温度对纤维素酶B4-Cel9稳定性的影响示意图。
图7附图为本发明提供的不同浓度NaCl对纤维素酶B4-Cel9活性的影响示意图。
图8附图为本发明提供的不同浓度离子液(IL)对纤维素酶B4-Cel9活性的影响示意图。
图9附图为本发明提供的不同浓度离子液(IL)对纤维素酶B4-Cel9稳定性的影响示意图。
图10附图为本发明提供的纤维素酶B4-Cel9对离子液处理后桦木木屑(A)和玉米芯(B)水解后葡萄糖产量的影响示意图。
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。
本发明实施例公开了一种嗜酸、嗜盐、嗜热、耐受离子液纤维素酶及其应用。
实施例中所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。例如:
基因:本实施例在对新疆自治区克拉玛依盐碱地(45.56104°N,85.20898°E)采集的沙土样品进行富集培养和宏基因组测序后,通过生物信息学分析并筛选出纤维素酶的功能基因,并对该功能基因进行PCR扩增、鉴定、克隆表达、纤维素酶的纯化和酶学性质,克隆表达载体pET-28a(+)。
酶类及其他生化试剂:限制性内切酶(EcoR Ⅰ和Hind Ⅲ)购自Thermo Scientific公司,pEASY-Uni无缝克隆和组装试剂盒购自北京全式金生物技术有限公司,其它都为生化试剂均可从国内普通生化试剂公司购买得到。
培养基:
富集培养基(g/L):MgSO4 0.5,NaCl 200,KNO3 1,K2HPO4 0.5,滤纸2,pH 8.0。
LB培养基(g/L):酵母提取物5,胰蛋白10,NaCl 10,pH7.4;使用抗生素为卡那霉素(Kanamycin),终浓度为50mg/L,固体分离培养基添加2%的琼脂。
实施例1
沙土样品富集培养和总DNA的提取
将新疆自治区克拉玛依盐碱地沙土作为实验样品,称取0.5g样品加入100mL富集培养基中,置于28℃培养箱静置培养30天。然后,室温10000×g离心20分钟,收取富集培养物进行总DNA的提取,使用土壤DNA提取试剂盒(品牌MOBIO,美国,货号:12888-50),参照商家提供的使用说明书进行操作。后送测序公司进行宏基因组测序和注释,通过数据分析筛选出纤维素酶的功能基因,获得该纤维素酶功能基因B4-Cel9的DNA序列(核苷酸序列:ATGACAAGACGTCACGCAAGCTCGCTCGCCGCTGCGCTGCTGGCCGCCCTGGCCGTGGTGGCAGGGCTGCTGGTCACGGCCACTCCCGCCCAGGCGGACGAGGAGATCTGCGAGCGCTACGGCACCACCGTCATCCAGGGCAGCTACGTGGTGCAGAACAACCGGTGGGGCACCGATGCCACCCAGTGCATCAATGTCACCGACAATGGATTCGAGGTCACCCGCGCGGACGGCGGGGTGCCGACCAATGGGGCGCCCAAGTCCTATCCGTCGCTGTACGTGGGCTGCCATTACACCAACTGCTCGCCGGGCACCAACCTTCCGCTGCGGGTGGACTCCATCGCCTCCGCGCCGAGCAGCATCTCCTACGGATACACCGATGGTTCCGTGTACAACGCCGCCTATGACATCTGGCTCGATCCGACGCCCAAAACGGACGGGGTGAGCCAGACCGAGATCATGATCTGGTTCAACCGCGTCGGCCCCGTCCAGCCCATCGGTTCGCCGGTCGGCACCGCCACGGTCGGCGGCCGCAGCTGGGAGGTCTGGACCGGCAGCAACGGGATGAACGACGTGATCTCCTTCCTGGCGCCGTCCGCGATCAGCAGCTGGAGCTTCGACACCAAGGACTTCATCGACGAGGCCGTCAGTCACGGCCTGGCCGCGCCGGACTGGTACCTGACCAGCATCCAGGCCGGTTTCGAGCCCTGGGAGAACGGCACCGGGCTGTCGGTGAACTCCTTCTCCGCGTCCGTCAACGAGGGCCAGGACCCCTCCGACCCGTCCGACCCCACGGACCCGAGCGATCCGGCGGACCCCTCCGACCCGTCGGACCCGTCGGACCCGACCGATCCCGGCACCGGCGACTGCCAGGTCGAGTACGGCGTCAACGCCTGGAACACCGGCTTCACGGCTGAGGTGAGCGTCACCAACTCCGGGACGGCGGCCGTCAACGGCTGGGAGCTGGCCTTCACGCTGCCGGACGGGCAGTCGGTCGGCCAGGCGTGGAACGCCGAGATCGACGGCACCAGCGGGCCGGTGACGGCGCGCAACGTCGGCCACAACGCGCAGATCGCCCCGGGCGGGACCGTCTCGTTCGGCTTCCAGGGCAGCCACTCCGGGTCGGCCGGCGCCCCGTCGGCGTTCACCCTGAACGGGGTCCCCTGCGGCTAG,如SEQ ID NO.1所示;氨基酸序列:MTRRHASSLAAALLAALAVVAGLLVTATPAQADEEICERYGTTVIQGSYVVQNNRWGTDATQCINVTDNGFEVTRADGGVPTNGAPKSYPSLYVGCHYTNCSPGTNLPLRVDSIASAPSSISYGYTDGSVYNAAYDIWLDPTPKTDGVSQTEIMIWFNRVGPVQPIGSPVGTATVGGRSWEVWTGSNGMNDVISFLAPSAISSWSFDTKDFIDEAVSHGLAAPDWYLTSIQAGFEPWENGTGLSVNSFSASVNEGQDPSDPSDPTDPSDPADPSDPSDPSDPTDPGTGDCQVEYGVNAWNTGFTAEVSVTNSGTAAVNGWELAFTLPDGQSVGQAWNAEIDGTSGPVTARNVGHNAQIAPGGTVSFGFQGSHSGSAGAPSAFTLNGVPCG,如SEQID NO.2所示)。
实施例2
以上述总DNA为模板,通过引物(B4-Cel9-F:CAAATGGGTCGCGGATCCGAAATGACAAGACGTCACGCAAGCTCG和B4-Cel9-R:GTGCTCGAGTGCGGCCGCAAGCTAGCCGCAGGGGACCCCGTTCAG)对其功能基因进行PCR扩增,划线序列代表与pET28a(+)载体的重叠序列片段,扩增使用高保真酶金牌Mix(北京擎科生物科技有限公司),扩增条件为:预变性94℃,3min,变性98℃,15s,退火58℃,30s,延伸72℃,50s,32个循环,最后延伸72℃,5min。PCR产物经1.0%琼脂糖凝胶电泳鉴定,120V,30min,紫外灯下观察。见图1,得到一段约1200bp片段,与理论大小1173bp接近,确定其为目的基因,胶回收。
使用限制性内切酶(EcoRⅠ和HindⅢ)对载体pET-28a(+)按说明书进行双酶切,并对酶切产物进行胶回收,通过pEASY-Uni无缝克隆和组装试剂盒(北京擎科生物科技有限公司)将酶切后的载体和含重叠序列的PCR产物按说明书进行无缝拼接,反应条件为50℃孵育15min,连接产物通过Ca2+化学转化法导入到大肠杆菌菌株E.coli DH5α感受态中,通过菌落PCR对阳性克隆子进行筛选,最后通过擎科生物科技有限责任公司测序进一步验证,获得重组载体pET-28a-B4-Cel9。
实施例3
重组蛋白的诱导表达
将实施例2构建好的重组载体pET-28a-B4-Cel9,转化E.coli BL21(DE3),获得重组菌株E.coli BL21(DE3)-B4-Cel9。从平板上挑取单菌落分接种于含50μg/mL卡那霉素抗性的LB液体培养基中37℃,180rpm,进行过夜培养,以1%的接种量接入含50μg/mL卡那霉素的LB液体培养基中培养,37℃,180rpm,培养约2h至OD600≈0.6,加入IPTG,至终浓度为0.2mM,转至25℃,180rpm,继续培养6h;然后4℃,10000×g离心20min,收获菌体,保存至-80℃冰箱,备用。
实施例4
重组蛋白的分离纯化
将菌体用含10mM咪唑的PBS溶液(pH 7.6)在离心管中进行重新悬浮,置于冰水混合物中超声波破碎30min,之后使用高速冷冻离心机离心,条件为4℃、10000×g,离心处理20min,收集上清液。使用Ni-NTA层析柱进行分离纯化,步骤为:使用5倍柱体积的平衡液(10mM咪唑的PBS溶液,pH7.6)对Ni-NTA层析柱进行平衡,将上述上清液加入Ni-NTA层析柱中进行上样,重复过柱一次,过柱后使用10倍柱体积的平衡液进行洗涤,最后使用3倍柱体积的洗脱液(含250mM咪唑的PBS溶液,pH7.6)对目的蛋白进行洗脱,用1.5mL离心管分管收集蛋白液,每管收集1.0mL。收集后的蛋白液用Bradford法测量蛋白质浓度,并进行SDS-PAGE电泳分析,目的蛋白成功得到纯化,纯化结果如图2所示。其中泳道2为IPTG诱导的E.coli BL21/pET-28a-B4-Cel9总蛋白、泳道3为B4-Cel9纯化重组酶。纯化重组酶B4-Cel9在40~55KD处有明显条带,与重组蛋白理论预测的分子量(44.03kD)一致。
实施例5
纤维素酶的酶学性质分析
(1)纤维素酶活力测定方法:运用3,5-二硝基水杨酸(DNS)测定法测定还原糖释放量。使用羧甲基纤维素钠作为底物测定纤维素酶活性。取10μL酶液加入到90μL含有1%(w/v)羧甲基纤维素钠的缓冲液中,在最适温度下反应30min,加入150μLDNS试剂终止反应。沸水浴10min后,取150μL反应混合液加入在96孔培养板中,使用酶标仪在540nm处测定其吸收值。一个单位(U)被定义为每分钟从纤维素底物中释放1μmol葡萄糖当量还原糖所需的酶量。每组实验设置3个平行。
(2)pH对纤维素酶B4-Cel9活性和稳定性的影响
经纯化的实施例4表达的纤维素酶在50℃条件下,于不同的pH缓冲液中进行酶促反应以测定其最适pH。所用缓冲液为pH4.0~7.6柠檬酸一磷酸氢二钠系列缓冲液(2.0、2.6、3.0、3.6、4.0、4.6、5.0、5.6、6.0、6.6、7.0、7.6),pH8.0~9.0的甘氨酸-盐酸缓冲液(8.0、8.6、9.0),纯化的纤维素酶在不同pH的缓冲体系,50℃下测定的pH适性结果(图3)表明:该纤维素酶的最适pH为4.0,该酶在pH3.0~4.6缓冲体系中,保持80%以上相对活性。
用pH为(2.0~9.0)的缓冲液,每1pH为一个梯度,分别将纯酶液与pH缓冲液按1:4比例混匀,将上述酶液在4℃下分别放置24h后再测定pH的稳定性;阳性对照使用pH7.6的PBS溶液相同比例稀释(1:4),直接用于活性测定,其余条件不变,以阳性对照的酶活力作为100%。测定结果如图4所示,结果表明:pH2.0~6.0的范围内,经过24h的孵育处理,保持100%以上相对活性。
(3)温度对纤维素酶B4-Cel9活性和稳定性的影响
纯化的纤维素酶在pH4.0条件下,测定不同温度(25~70℃)下的酶活性,分析实验结果(图5)表明:最适温度为50-55℃,在40~60℃均保持60%以上相对活性。
为测定温度对该纤维素酶的影响,将酶液分别放置于不同温度(25~80℃)孵育1小时后,在pH4,50℃条件下测定残留的纤维素酶活性。结果(图6)表明:该酶在25~60℃下孵育1h后,保持60%以上相对活性;在80℃下孵育1h后,仍保持40%以上相对活性。
(4)不同浓度NaCl对纤维素酶B4-Cel9活性的影响。
为测定盐浓度对该纤维素酶的影响,在pH4.0,50℃条件下,测定不同NaCl浓度(0、5%、10%、15%、20%、25%、30%、35%)下,纤维素酶活力。结果(图7)表明:该酶最适反应盐浓度为15%,高浓度NaCl溶液对该酶具有激活作用,5%~35%浓度NaCl溶液中,该酶保持110%以上相对活性;在15%浓度NaCl溶液中,该酶保持140%以上相对活性。
(5)不同浓度离子液对纤维素酶B4-Cel9活性及其稳定性的影响
为测定离子液对该纤维素酶的影响,在pH4.0,50℃条件下,测定不同浓度(0、5%、10%、15%、20%、25%、30%、、35%、40%、45%、50%)离子液(1-乙基-3-甲基咪唑乙酸盐)下,纤维素酶活力。结果(图8)表明:该酶最适反应离子液浓度为25%,高浓度离子液溶液对该酶具有激活作用,在5~40%浓度离子液中,该酶保持110%以上相对活性;在25%浓度离子液中,该酶保持150%以上相对活性;在50%浓度离子液中,该酶仍保持60%以上相对活性。
为测定离子液对该纤维素酶稳定性的影响,将酶液置于不同浓度(0、10%、20%、30%、40%、50%)离子液下,孵育12h和24h后,在pH4.0,50℃条件下,测定纤维素酶活力。结果(图9)表明:在5%~50%浓度离子液中,孵育12h和24h后,该酶仍保持100%以上相对活性。
(6)纤维素酶B4-Cel9对离子液处理木质纤维素水解的影响
为测定纤维素酶B4-Cel9对离子液处理木质纤维素水解的影响,将该酶加入Trichoderma reesei来源纤维素酶系(生工生物工程<上海>股份有限公司,货号A002610),协同水解离子液(1-乙基-3-甲基咪唑乙酸盐)处理后的桦木木屑和玉米芯,并使用葡萄糖氧化酶-过氧化物酶检测试剂盒检测水解产物中葡萄糖含量。
离子液(1-乙基-3-甲基咪唑乙酸盐)处理后的桦木木屑和玉米芯方法:称取2g桦木屑(20目)和2g玉米芯粉末(80目)分别与20mL离子液混匀,然后置于100℃烘箱中热处理24h,至桦木木屑和玉米芯成胶状,然后加入20mL纯水,过滤出离子液处理后的桦木木屑和玉米芯。直接将离子液处理后的桦木木屑和玉米芯加入到100mL的pH5.0缓冲液中,混匀后取1mL加入Trichoderma reesei来源纤维素酶系至浓度为0.1mg/mL,加入重组纤维素酶B4-Cel9至浓度为0.02mg/mL,于pH5.0,50℃条件下,反应0~48h,每隔6h取样测定葡萄糖含量。以未加入纤维素酶B4-Cel9的水解体系为对照进行比较,每组实验设置3个平行。结果(图10)表明:加入纤维素酶B4-Cel9的桦木木屑和玉米芯水解体系,产物中葡萄糖含量均比对照组高;在离子液处理后的桦木木屑和玉米芯水解体系中,加入纤维素酶B4-Cel9协同水解48h后,产物中葡萄糖含量均提高50%以上。
以上特性表明,此酶可以适用于酸性、高盐、高温和含离子液环境中,在食品、饲料、纺织以及工业行业生产等行业具有广泛的应用前景。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (9)
1.一种嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶,其特征在于,其氨基酸序列如SEQ IDNO.2所示。
2.一种编码权利要求1所述嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO.1所示。
3.包含权利要求2所述编码嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因的重组载体。
4.根据权利要求3所述的重组载体,其特征在于,所述重组载体的制备方法为将嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因插入到质粒pET-28a上的EcoRⅠ和HindⅢ限制性酶切位点之间得到重组质粒。
5.包含权利要求2所述编码嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶的基因或权利要求3或4所述重组载体的重组菌株。
6.根据权利要求5所述的重组菌株,其特征在于,所述重组菌株为:用重组质粒转化大肠杆菌即得。
7.权利要求1所述嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶在食品、饲料、纺织以及工业生产中的应用。
8.根据权利要求7所述的应用,其特征在于,所述纤维素酶提高酸性、高盐、高温和离子液残留条件下纤维素的降解率;所述酸性pH2.0~6.0;所述高盐:5%~35%浓度NaCl溶液;所述高温:40~60℃;所述离子液:1-乙基-3-甲基咪唑乙酸盐。
9.权利要求2所述基因、权利要求3或4所述的重组载体或权利要求5或6所述重组菌株在产业化生产嗜酸、嗜盐、嗜热、耐受离子液的纤维素酶中的应用。
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