CN104560846A - 一种双活性木聚糖降解酶工程菌及其应用 - Google Patents
一种双活性木聚糖降解酶工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种双活性木聚糖降解酶工程菌,以及该菌在制备低聚木糖中的应用。一种双活性木聚糖降解酶工程菌是将木聚糖酶和α-葡萄糖醛酸酶基因分别置于不同表达盒下,通过优化翻译起始区使其高效表达而构建得到共表达载体pET-28a-xynB-aguA,并转化大肠杆菌宿主细胞后获得的重组菌。利用该菌生产双活性木聚糖降解酶,不仅能提高酶水解木聚糖的活性,而且可简化酶制备和水解操作步骤,降低生产成本低;相对单一木聚糖酶,利用所得到的双活性酶水解木聚糖制备低聚木糖,具有转化效率高,木二糖产量和纯度高,分离纯化容易、化学污染少等优点。
Description
技术领域
本发明涉及应用微生物技术领域,具体涉及一种双活性木聚糖降解酶工程菌,以及该菌在制备低聚木糖中的应用。
技术背景
低聚木糖又称木寡糖,由2~7个木糖以β-1,4-糖苷键连接而成,以木二糖,木三糖为主。各项研究表明,低聚木糖,尤其是木二糖,具有显著的双歧杆菌增殖能力,不被消化性,无龋齿性以及促进人体对钙的吸收等特性,因此,低聚木糖作为一种新型功能性低聚糖成为食品工业研究开发的热点之一。
目前低聚木糖生产方法包括:酸水解法、热水抽提、霉菌深层发酵和酶法等,木聚糖酶水解法是低聚木糖生产的主要方法,即将玉米芯用氢氧化钠溶液浸提,然后用酸中和、过滤,滤液经过处理后直接添加木聚糖酶来制备低聚糖。但是,在玉米芯,甘蔗渣和秸秆等农林废弃物中约94%的半纤维素是***糖葡萄糖醛酸木聚糖,这类木聚糖是一条以β-1,4糖苷键相联的木聚糖主链,上面带有1,3-***糖或1,2-4-O-甲基葡萄糖醛酸构成的侧枝,尤其是4-O-甲基葡萄糖醛酸侧枝,无论是草类,软木还是硬木来源的木聚糖,其均中含有,这些侧链的存在妨碍了木聚糖酶对木聚糖底物糖苷键的水解[9-10],研究表明,木聚糖酶和α-葡萄糖醛酸酶具有协同水解作用,不仅能加速低聚糖的产生,而且能使酶解产物中带有葡萄糖醛酸侧枝的大片段得到有效水解,提高低聚木糖的产量和纯度。但是2种酶的大量制备势必带来能耗和成本的增加,因此,构建一种双活性木聚糖降解酶工程菌,使其既产α-葡萄糖醛酸酶又产木聚糖酶,不仅提高酶水解活性,而且简化酶制备和水解操作步骤,成为获得更高效降解秸秆木聚糖和价格低廉的生物催化剂的途径。目前尚未见报道。
发明内容
本发明的目的是提供一种双活性木聚糖降解酶工程菌,以及该菌在制备低聚木糖中的应用。
本发明的一种双活性木聚糖降解酶工程菌,是将木聚糖酶和α-葡萄糖醛酸酶基因分别置于不同表达盒下,通过优化翻译起始区使其高效表达而构建得到共表达载体pET-28a-xynB-aguA,并转化大肠杆菌宿主细胞后获得的重组菌。
其中所述的共表达载体pET-28a-xynB-aguA是将木聚糖酶基因及其表达元件和翻译起始区序列SEQ:ID NO.1或3和α-葡萄糖醛酸酶基因及其表达元件和翻译起始区序列SEQ:IDNO.2或4,分别在XbaⅠ/XhoⅠ,StuI/XhoI位点***pET-28a而获得。
本发明还公开了所述的双活性木聚糖降解酶工程菌在用木聚糖制备低聚木糖中的应用。
上述应用,具体是利用所述的双活性木聚糖降解酶工程菌产生的双活性木聚糖降解酶的粗酶液水解木聚糖底物制备低聚木糖。
所述的木聚糖底物,是从农林废弃物、食品加工的下脚料中提取的木聚糖中任何一种,或农林废弃物汽瀑液。
双活性木聚糖降解酶水解木聚糖底物的反应条件:温度65-90℃,pH5-8。
本发明利用基因重组技术将极端嗜热袍菌属(Thermotiga)的木聚糖酶(XynB)和α-葡萄糖醛酸酶(AguA)基因连接到大肠杆菌(E.coli)表达载体pET-28a(+)上,且分别置于不同表达盒下,并通过利用优化翻译起始区使木聚糖酶和α-葡萄糖醛酸酶高效生产的引物,其序列如SEQ:ID NO.5、ID NO.6、ID NO.7和ID NO.8所示,构建得到表达载体pET-28a-xynB-aguA,转化大肠杆菌E.coli JM109(DE3)后获得重组工程菌E.coli JM109(DE3)/pET-28a-xynB-aguA;该菌在含有30-50μg/mL卡那霉素抗性的LB培养基中30℃温度培养至对数生长期,经异丙基硫代半乳糖苷(IPTG)或乳糖和37-42℃诱导培养可获得高效表达的双活性木聚糖降解酶;进一步利用所得的双活性木聚糖降解酶对木聚糖的降解表现出比单一木聚酶更高效率,且所得酶解液中木二糖的含量和纯度更高。
本发明通过因重组技术将性能优良的极端嗜热袍菌属(Thermotiga)的木聚糖酶(XynB)和α-葡萄糖醛酸酶(AguA)基因分别置于不同表达盒下并通过优化翻译起始区二级结构进行独立共表达获得双活性木聚糖降解酶,不仅能提高酶水解木聚糖的活性,而且可简化酶制备和水解操作步骤,降低生产成本低;所得到的双活性木聚糖降解酶对木聚糖降解获得木二糖含量和纯度更高的低聚木糖,这不仅排除了供体菌共生的其他一系列酶系的干扰,而且相对单一木聚糖酶,具有转化效率高,反应副产物少,产物分离和纯化容易等优点。
附图说明
图1重组工程菌E.coli JM109(DE3)/pET-28a-xynB-aguA在不同诱导时间和诱导剂浓度下的粗酶液SDS-PAGE图谱
M:蛋白分子量标样;1:空载对照;2:优化前对照;3:诱导培养6h的细胞上清液;4:诱导培养8h的细胞上清液;5:诱导10h的细胞上清液,6:50mM IPTG下诱导的的细胞上清液;7:100mM IPTG下诱导的细胞上清液;8:800mM IPTG下诱导的细胞上清液。
图2重组工程菌E.coli JM109(DE3)/pET-28a-xynB-aguA在不同诱导温度下的粗酶液SDS-PAGE图谱和XynB/AguA比例分析
M:蛋白分子量标样;1,7,10:空载对照;2,3:在37℃诱导条件的全细胞液和细胞上清液;4:空载对照;5,6:在39℃诱导条件的全细胞液和细胞上清液;8,9:在41℃诱导条件的全细胞液和细胞上清液;11,12:在42℃诱导条件的全细胞液和细胞上清液
图3双活性木聚糖降解酶和单一木聚糖酶水解桦木木聚糖的还原糖释放量
图4双活性木聚糖降解酶和单一木聚糖酶水解桦木木聚糖的TLC图谱1:木二糖标样;2,11:木糖标样;3-6:单一木聚糖酶水解桦木木聚糖水解桦木木聚糖1、2、4、8小时的酶解液;7-10:双活性木聚糖降解酶水解桦木木聚糖1、2、4、8小时的酶解液。
具体实施方式
以下是本该发明的具体实施例,但本发明的内容并不仅局限于此。
实施例1:
根据海栖热袍菌Thermotiga maritima的木聚糖酶B(XynB)基因(TM0070)序列设计引物,其序列具体是:
5’-TGCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACAATGAGT
CAGAATGTAAGCTTGAGAGAACTCGCGGAAAAGCTGAACATCTATATTGG-3’(SEQ IDNO.5)和5’-CGGCTCGAG AGGCCTCAAAAAACCCCTCAAGA
CCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTATTTTCTTTCTTCTATCTTTTTCTCCAG-3’(SEQ ID NO.6),以海栖热袍菌基因组为模板进行PCR扩增,用Pyrobest DNA酶对模板进行扩增。PCR扩增参数均设为:先在95℃预变性5min,加Pyrobest DNA聚合酶1μl;然后94℃变性40s,54℃退火40s,72℃延伸1min20s,循环35次后,72℃保温10min。PCR产物核酸胶验证正确后,割胶回收PCR产物,序列如SEQ ID NO.3,并用XbaⅠ和XhoⅠ分别单酶切并与用XbaⅠ和XhoⅠ双酶切表达质粒pET-28a(Novagen公司)连接构建重组质粒pET-28a-xynB。
实施例2:根据海栖热袍菌Thermotiga maritima的α-葡萄糖醛酸酶(AguA)基因(TM0055)序列设计引物,其序列具体是:5’-AAAAGGCCTTGACCCCCTTGAATGTGGGGGGAAACATCCCCATGATCCAATGACCTGTTAACCGTCGACAAGAAGGAGATATACAATGGACTACCGTATGTGCTGGCTGGAGTACCGTGGTCTGCCGGCTGATGTCGCCGGAA-3’(SEQ ID NO.7)和5’-CCGCTCGAGCGGATATATCTTTCTTCCCTT-3’(SEQ ID NO.8)扩增带表达元件的α-葡萄糖醛酸酶基因片段SEQ ID NO.4,然后用StuI和XhoI双酶切并与用StuI和XhoI双酶切的重组质粒pET-28a-xynB连接,获得重组表达质粒pET-28a-xynB-aguA。
实施例3:通过生物技术公司(上海生工公司)分别合成木聚糖酶基因及其表达元件和翻译起始区序列SEQ:ID NO.1和α-葡萄糖醛酸酶基因及其表达元件和翻译起始区序列SEQ:ID NO.2,然后用XbaⅠ和XhoⅠ双酶切SEQ:ID NO.1序列并与用XbaⅠ和XhoⅠ双酶切的质粒pET-28a连接(Novagen公司)构建出重组质粒pET-28a-xynB,进一步用StuI和XhoI双酶切后再与用StuI和XhoI双酶切的SEQ:ID NO.2序列连接,获得重组表达质粒pET-28a-xynB-aguA。
实施例4:
将重组质粒pET-28a-xynB-aguA转化宿主,在含有30-50μg/mL卡那霉素抗性的LB培养基中30℃温度培养至对数生长期,经异丙基硫代半乳糖苷(IPTG)或乳糖在37-42℃下诱导培养6-10h,然后将菌液在0-10℃、转速4000-6000rpm条件下离心收集沉淀物并用水洗涤3次,再用0.02-0.5M pH6.0-8.0的缓冲液重悬,置冰水浴中超声破碎或用高压破碎,破碎后的菌液在60-70℃温度条件下热处理10-20min,在0-4℃、10000-13000rpm条件下离心10-30min,取细胞上清液即得双活性木聚糖降解酶的粗酶液;不同诱导时间和诱导剂浓度下的双活性木聚糖降解酶产量经SDS-PAGE电泳检测分析,结果(见图1)表明XynB和AguA的产量比对照组显著提高;不同诱导温度的双活性木聚糖降解酶粗酶液的木聚糖酶和葡萄糖醛酸酶活性比例(XynB/AguA)的分析结果见图2,显示出随着诱导温度增加其XynB/AguA比例随之增加。
实施例5:粗酶液与桦木木聚糖(Sigma)混合,在pH6.2和温度80℃下振荡反应60min后,酶解产物采用3,5-二硝基水杨酸(DNS)法检测还原糖释放量的结果(见图3)显示双活性木聚糖降解酶对木聚糖的降解比单一木聚酶具有更高的水解效率;进一步经TLC法检测酶解产物的结果(见图4)发现,与单一木聚酶相比,双活性木聚糖降解酶对木聚糖的降解所制得酶解液中木二糖的含量和纯度更高,进一步将富含木二糖更高更纯度低聚木糖酶解液经真空冷冻干燥即得槲皮素产品。
实施例6:与实施例4基本相同,不同之处在于所用的水解底物是从甘蔗渣中用碱抽提到的木聚糖,水解反应条件为:pH7.0,温度75℃下,保温时间70min。
实施例7:与实施例4基本相同,不同之处在于所用的水解底物是从玉米芯中用碱抽提到的木聚糖,水解反应条件为:pH5.0,温度65℃下,保温时间2小时。
实施例8:与实施例4基本相同,不同之处在于所用的水解底物是稻草汽瀑液,水解反应条件为:pH5.8,温度70℃下,保温时间90min。
Claims (6)
1.一种双活性木聚糖降解酶工程菌,其特征在于所述双活性木聚糖降解酶工程菌是将木聚糖酶和α-葡萄糖醛酸酶基因分别置于不同表达盒下,通过优化翻译起始区使其高效表达而构建得到共表达载体pET-28a-xynB-aguA,并转化大肠杆菌宿主细胞后获得的重组菌。
2.根据权利要求1所述的双活性木聚糖降解酶工程菌,其特征在于:其中所述的共表达载体pET-28a-xynB-aguA是将木聚糖酶基因及其表达元件和翻译起始区序列SEQ:ID NO.1或ID NO.3和α-葡萄糖醛酸酶基因及其表达元件和翻译起始区序列SEQ:ID NO.2或IDNO.4,分别在XbaⅠ/XhoⅠ,StuI/XhoI位点***pET-28a而获得。
3.权利要求1所述的双活性木聚糖降解酶工程菌在用木聚糖制备低聚木糖中的应用。
4.根据权利要求3述的应用,其特征在于利用所述的双活性木聚糖降解酶工程菌产生的双活性木聚糖降解酶的粗酶液水解木聚糖底物制备低聚木糖。
5.根据权利要求4所述的应用,其特征在于所述的木聚糖底物,是从农林废弃物、食品加工的下脚料中提取的木聚糖中任何一种,或农林废弃物汽瀑液。
6.根据权利要求4所述的应用,其特征在于双活性木聚糖降解酶水解木聚糖底物的反应条件:温度65-90℃,pH5-8。
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