CN104946614A - 一种重组木聚糖酶的制备及其应用于木薯渣降解的方法 - Google Patents
一种重组木聚糖酶的制备及其应用于木薯渣降解的方法 Download PDFInfo
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Abstract
本发明属于生物工程技术领域,涉及一种重组木聚糖酶的制备以及应用该重组木聚糖酶降解木薯渣的方法。本发明将Clostridium clariflavum DSM19732的木聚糖酶基因与质粒pET-28a连接,转化大肠杆菌BL21,诱导表达,细胞破碎得到重组木聚糖酶,将该酶处理木薯渣,降低了木薯渣中纤维素和半纤维素的含量。用该方法制备的重组木聚糖酶,具有良好的酶学性质,处理木薯渣后可提高木薯渣中木质纤维素的降解效率。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种重组木聚糖酶的制备,以及利用该重组木聚糖酶降解木薯渣的方法。
背景技术
木聚糖酶是指能够专一降解木聚糖为低聚木糖和木糖的一类酶的总称,由于木聚糖是植物半纤维的主要成分,在自然界中广泛存在,无论是作为工业生产原料或加工副产物中的木聚糖,其有效降解都具有十分重要的意义。因此从六十年代起,人们就已经关注木聚糖酶的研究,涉及领域包括食品、饲料、造纸、能源工业等方面。
目前,木聚糖酶产生菌包括真菌、细菌及基因工程菌,不同来源的木聚糖酶其酶学性质和底物的专一性有所不同,因此针对不同需要,选择相应的木聚糖酶才能充分发挥酶解作用。
木薯是世界三大薯类作物之一,作为工业原料,其加工后产生大量的残渣。木薯渣的累积不仅造成资源的浪费,而且会对环境造成污染。木薯渣中含有大量的木质纤维素资源,如能将其有效利用,则可变费为利,但目前对木薯渣中半纤维素的降解尚无高效的方法。Clostridium clariflavum DSM19732能够以木薯渣为底物,在55℃下生长良好,推测该菌应具有相应的半纤维素降解酶系,而针对其所产木聚糖酶尚无文献报道。
因此,开发Clostridium clariflavum DSM19732木聚糖酶,并将其用于木薯渣中木质纤维素的降解,具有一定的经济和社会效益。
发明内容
本发明的一个目的在于获得重组Clostridium clariflavum DSM19732木聚糖酶的制备方法,开发新型木聚糖酶。
本发明的另一个目的是应用上述重组木聚糖酶处理木薯渣,提高木薯渣中木质纤维素的降解效率,解决木薯渣中木质纤维素资源利用率低的问题。
为了实现上述目的,本发明采用了以下技术方案:
1)目的木聚糖酶基因的获得:用细菌基因组DNA提取试剂盒提取含有Clostridium clariflavum DSM19732的基因组,得到基因组DNA溶液;
2)重组表达载体的构建:根据数据库中报道的Clostridium clariflavumDSM19732木聚糖酶基因序列设计带有限制性内切酶BamH I和Not I的引物,以步骤1)所得基因组DNA溶液为模板,进行PCR扩增,并对扩增产物进行纯化,然后用限制性内切酶BamH Ⅰ和Not Ⅰ双酶切已纯化的PCR产物和质粒pET-28a,用T4连接酶16℃过夜连接,得到重组表达载体;
3)重组木聚糖酶的诱导表达:将步骤2)中所得到的重组表达载体转化到大肠杆菌BL21感受态细胞中,培养细胞,诱导重组木聚糖酶的表达;
4)诱导重组木聚糖酶的表达条件:菌体生长OD600值为0.6-1.6;诱导剂IPTG浓度为0.05-1mmol/L;诱导温度为16-37℃;诱导时间为4-8h;
5)重组木聚糖酶液制备:收获步骤4)的菌体,8000-10000rpm离心15-20min,沉淀用20mmol/L、pH 6.0-7.5Tris-HCl缓冲液重悬,混匀后超声破碎10-15min,之后8000-10000rpm离心15-20min,取上清液即为木聚糖酶液;
6)重组木聚糖酶应用于木薯渣降解:在20目的木薯渣中加入质量体积比浓度为1%-3%的20mmol/L、pH 6.0-7.5Tris-HCl缓冲液,加入100-400U步骤5)的酶液及100-5000U纤维素酶,55-60℃恒温48-96h,测量木薯渣中纤维素、半纤维素含量以及木薯渣的失重率。
以上技术方案中所有基本分子生物学操作均参照“分子克隆实验指南”(第三版,科学出版社,2002年)。
1个酶活单位(U)定义为在给定条件下,每分钟降解1%的木聚糖溶液生成1μmol还原糖需要的酶量。
所得重组木聚糖酶具有以下特性:
1)最适反应温度
在40-80℃表现出催化活力,最适反应温度为60℃;
2)最适反应pH
在pH4-8范围内表现出催化活力,最适pH为6.0;
3)热稳定性
在40-60℃表现出很好的热稳定性,60℃保温30min,活力仍保持在90%以上;
4)pH稳定性
在pH 5-8具有良好的稳定性,pH8,4℃保持60min,仍保持90%的酶活力。
木薯渣的处理效果:与仅添加纤维素酶相比,同时添加重组木聚糖酶与纤维素酶,木薯渣失重率提高5%-10%;木薯渣中纤维素含量降低2-5%;半纤维素含量降低2-7%。
本发明采用的重组木聚糖酶的制备方法,较从Clostridium clariflavumDSM19732中直接获取木聚糖酶,重组大肠杆菌细胞易于培养,产酶周期短。
与仅添加纤维素酶处理木薯渣的工艺相比,添加本发明制备的木聚糖酶,可降低木薯渣中纤维素和半纤维素含量。
实施例1
重组木聚糖酶的制备,具体如下:
1)目的木聚糖酶基因的获得:用细菌基因组DNA提取试剂盒提取含有Clostridium clariflavum DSM19732的基因组,得到基因组DNA溶液;
2)重组表达载体的构建:根据数据库中报道的Clostridium clariflavumDSM19732木聚糖酶基因序列设计带有限制性内切酶BamH I和Not I的引物以步骤1)所得基因组DNA溶液为模板,进行PCR扩增,并对扩增产物进行纯化,然后用限制性内切酶BamH Ⅰ和Not Ⅰ双酶切已纯化的PCR产物和质粒pET-28a,用T4连接酶16℃过夜连接,得到重组表达载体;
3)重组木聚糖酶的获得:将步骤2)中所得到的重组表达载体转化到大肠杆菌BL21感受态细胞中进行培养,当OD600为1.25时,添加终浓度为0.1mmol/L的诱导剂IPTG,23℃诱导6h,8000rpm离心15min,收集菌体,并用20mmol/L、pH 7.0Tris-HCl缓冲液重悬,混匀后超声破碎10min,之后8000rpm离心15min,取上清液,即得到15.2U/mg蛋白的木聚糖酶液。
实施例2
利用本发明得到的重组木聚糖酶处理木薯渣,具体如下:
在20目的木薯渣中加入质量体积比浓度为1%的20mmol/L、pH 7.0Tris-HCl缓冲液,加入270U的重组木聚糖酶液及4000U纤维素酶,55℃恒温96h,较仅添加纤维素酶处理效果相比,木薯渣的失重率增加了9%,纤维素含量下降了2.5%,半纤维素含量下降了4%。
Claims (2)
1.一种重组木聚糖酶的制备方法,其特征在于,包括以下步骤:
步骤一、用细菌基因组DNA提取试剂盒提取含有Clostridium clariflavumDSM19732的基因组,得到基因组DNA溶液;
步骤二、根据数据库中报道的Clostridium clariflavum DSM19732木聚糖酶基因序列设计引物,以步骤一所得基因组DNA溶液为模板,进行PCR扩增,得到木聚糖酶基因,并与质粒pET-28a连接,得到重组表达载体;
步骤三、将步骤二中所得到的重组表达载体转化到大肠杆菌BL21感受态细胞中,培养细胞,诱导重组木聚糖酶的表达;
步骤四、收集步骤三中的菌体细胞,用pH 6.0-7.5的Tris-HCl缓冲液重新悬浮,超声破碎细胞并离心,取上清即为木聚糖酶液。
2.一种如权利要求1所述的重组木聚糖酶应用于木薯渣降解的方法,其特征在于,在20目的木薯渣中加入质量体积比浓度为1%-3%pH6.0-7.5的Tris-HCl缓冲液,加入100-400U重组木聚糖酶及100-5000U纤维素酶,55-60℃恒温48-96h。
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CN106834408B (zh) * | 2017-03-20 | 2019-10-25 | 江西农业大学 | 一种猪粪和稻秆协同高效厌氧消化的方法 |
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