CN113621596A - 一种化学修饰的木聚糖酶及其制备方法和应用 - Google Patents
一种化学修饰的木聚糖酶及其制备方法和应用 Download PDFInfo
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- CN113621596A CN113621596A CN202110992324.8A CN202110992324A CN113621596A CN 113621596 A CN113621596 A CN 113621596A CN 202110992324 A CN202110992324 A CN 202110992324A CN 113621596 A CN113621596 A CN 113621596A
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- Prior art keywords
- xylanase
- dextran
- chemically modified
- enzyme
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Abstract
本发明公开了一种化学修饰的木聚糖酶及其制备方法和应用,属于酶工程领域。利用NaIO4的氧化特点,将右旋糖酐上的羟基氧化为醛基后对木聚糖酶进行化学修饰。修饰后的酶拥有右旋糖酐结构,可以与木质纤维素中纤维素结合,达到一种固定化作用,从而使酶活力提高了237%,同时拥有更高的最大反应速率Vmax,该方法克服了木聚糖酶在木质纤维素中反应速率低的缺点,提高了木聚糖酶的催化活性,具有广阔的工业应用前景。
Description
技术领域
本发明属于酶工程领域,具体涉及一种化学修饰的木聚糖酶及其制备方法和应用。
背景技术
木聚糖是植物半纤维素的主要成分,是除纤维素之外自然界最为丰富的多糖。木聚糖主链是由β-D-毗喃木糖残基通过β-1,4-糖苷键连接而成的多聚物。植物中的木聚糖位于木质素和纤维素之间,木聚糖和木质纤维的其它成分之间存在着不同程度的相互作用。木聚糖主要以共价键与木质素相连接,以非共价键与纤维素作用。木质纤维素降解的难点之一就是由于其复杂的结构导致降解困难,因此通过对木聚糖酶进行化学修饰,使其与右旋糖酐相连,使其能够与纤维素结合,达到一种固定化的作用,从而提高亲和性,促进木质纤维素的降解。虽然酶的化学修饰可以改变酶的某些性质,提高其使用效率,但在修饰过程中往往会破坏酶分子的结构,降低了酶的活性,所以需要对酶的化学修饰条件进行优化,包括修饰时间、温度和pH,以降低酶活性的损失。
发明内容
本发明提供一种化学修饰的木聚糖酶及其制备方法和应用,本发明合理设计并优化了修饰方法,克服了现有技术的不足,用该方法化学修饰的木聚糖酶对纤维素具有较高的亲和性,提高了木质纤维素的降解效果。
本发明的第一个目的是提供一种化学修饰的木聚糖酶,所述木聚糖酶的氨基酸序列如SEQ ID NO:1所示,编码基因如SEQ ID NO:2所示,化学修饰剂为右旋糖酐。
进一步的,所述化学修饰剂为含有醛基的右旋糖酐,与木聚糖酶通过氨基酸的氨基进行偶联。
进一步的,所述含有醛基的右旋糖酐采用NaIO4氧化右旋糖酐的邻羟基获得。
进一步的,所述含有醛基的右旋糖酐采用150kDa的右旋糖酐。步骤如下:将木聚糖酶编码基因片段和表达载体pET28a(+)相连接,并将连接产物转化至大肠杆菌BL21,获得重组菌株;培养重组菌株,诱导表达;发酵液破碎离心后盐析纯化,获得所述木聚糖酶的酶液。
本发明的第二目的是提供化学修饰的木聚糖酶的制备方法,包括如下步骤:
(1)木聚糖酶的制备
将木聚糖酶编码基因与表达载体pEt28a(+)相连接,并将连接产物转化至大肠杆菌BL21(DE3)细胞,获得重组菌株;培养重组菌株进行发酵表达,发酵液破碎离心后,将上清液进行硫酸铵沉淀、离子交换柱纯化,获得木聚糖酶液。
(2)含醛基右旋糖酐的制备
避光下将2.4g NaIO4溶于10mL纯水制得NaIO4溶液,将其倒入10mL浓度为2g/mL的右旋糖酐溶液中,室温下避光搅拌8h。反应完成后加入5mL的乙二醇反应2h后,反应液在8000-14000的透析袋中,过夜透析。
(3)木聚糖酶化学修饰物的制备
本发明的第三目的是提供上述化学修饰的木聚糖酶的应用,用于木质纤维素快速降解。
本发明的有益技术效果是:与未修饰的木聚糖酶相比,修饰后的木聚糖酶和纤维素具有了一定的结合力,达到一种固定化作用,从而使其在木质纤维素的分解过程中具有更高的反应速率,同时其米氏方程的Km下降,表明与木聚糖的亲和力也得到增加。
附图说明
图1不同时间下修饰反应后的酶活检测
图2不同pH下修饰反应后的酶活检测
图3不同温度下修饰反应后的酶活检测
图4修饰酶与未修饰酶的紫外吸收图谱。
图5修饰酶与未修饰酶的米氏方程
图6修饰酶与未修饰酶和木质纤维素结合电镜图
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完整的描述,显然,所描述的实施例仅是本发明一部分实施例。基于本发明的实施例,本领域的普通技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例,都属于本发明的保护范围。
实验试剂和材料
1.菌株及载体:大肠杆菌E.coli BL21(DE3)和表达载体pET28a由南京工业大学提供,该材料也可通过商业途径购买。
2.酶类及其他生化试剂:DNA聚合酶及dNTP购自南京诺唯赞生物科技股份有限公司,其他都为国产试剂(均可从普通生化试剂公司买到)
3.培养基:LB培养基:Peptone 10g/L,Yeast extract 5g/L,NaCl 10g/L,蒸馏水溶解。固体培养基在此基础上加2%(w/v)琼脂。
发酵培养基:
说明:以下实施例中未作具体说明的分子生物学实验方法,均参照《分子克隆实验指南》(第三版)一书中所列具体方法实行,或按照试剂盒和产品说明书进行。
实施例1所述木聚糖酶的表达和酶活检测
以来源于黑曲霉XylB为模板,设计含有EcoRI和SalI的突变片段,通过一步克隆将其连接到pET28a(+)上得到目的质粒。将质粒化转至大肠杆菌BL21(DE3)中,转化液涂布于LB平板中37℃培养12h,长出的单菌落为表达菌株。挑取单菌落接种至5mL LB培养基中37℃200rpm下培养12h,再进一步接种到200mL LB培养基中扩大培养,当菌液OD600长至0.6~0.8时,加入0.1mM IPTG诱导培养12h。
XylB-F上游引物为:5’-ATGGGTCGCGGATCCGAATTCATGTTTAAATTTA AAAAAAATTTC-3’
XylB-R上游引物为:5’-TGCGGCCGCAAGCTTGTCGACCCATACAGTCAC GTTAGAGCTA-3’
酶活检测:0.2mL酶液与pH为7.0的1mL 1%木聚糖溶液在55℃下反应15min后,取0.1mL反应液向其中加入0.3mLDNS和0.3mL水,沸水浴反应10min,定容至4mL,测定OD540通过标准曲线计算生成还原糖含量。
酶活定义:在一定条件下,将每分钟分解木聚糖而产生1μmol还原糖所需的酶量定义为一个活力单位。
实施例2所述木聚糖酶的发酵生产和纯化
取50μL保种菌液接种至5mL LB培养基中37℃200rpm下培养12h,再进一步接种到200mL发酵培养基中扩大培养,当菌液OD600长至0.6~0.8时,加入0.1mM IPTG诱导培养12h。发酵结束后,对发酵液进行超声破碎,破碎后离心取上清,向其中加入同体积饱和度100%的硫酸铵溶液,4℃下搅拌2h后,再静止6h,离心得到沉淀,加入pH 7.0的PBS溶解沉淀保存。实施例3所述木聚糖酶化学修饰的方法及条件优化
右旋糖酐的氧化:将2g分子量为150kDa的右旋糖酐和2.4gNaIO4分别溶于10mL水中,避光下将NaIO4倒入右旋糖酐溶液中,30℃下避光搅拌反应6h,再加入5mL乙二醇继续反应2h。反应完成后过夜透析得到含醛基的右旋糖酐溶液即修饰剂。
最适修饰时间测定。取1mL修饰剂用pH7.5的PBS定容至8mL,再加入0.2g木聚糖,最后加入2mL酶液,30℃下搅拌反应不同时间(4h、8h、12h、16h、20h、24h),再加入0.1g氰基硼氢化钠继续反应1h,反应完成后,在4℃下,透析4h即得到木聚糖酶修饰物。
最适修饰pH测定。取1mL修饰剂用不同pH(5.5、6.0、6.5、7.0、7.5、8.0)的PBS定容至8mL,再加入0.2g木聚糖,最后加入2mL酶液,30℃下搅拌反应8h,再加入0.1g氰基硼氢化钠继续反应1h,反应完成后,在4℃下,透析4h即得到木聚糖酶修饰物。
最适修饰温度测定。取1mL修饰剂用pH7.5的PBS定容至8mL,再加入0.2g木聚糖,最后加入2mL酶液,在不同温度(4℃、20℃、25℃、30℃、35℃)下搅拌反应8h,再加入0.1g氰基硼氢化钠继续反应1h,反应完成后,在4℃下,透析4h即得到木聚糖酶修饰物。
对比不同修饰条件后的相对酶活可以看出,修饰时间控制在8h为宜,时间过短修饰效果不明显,过长则大量损失酶活。pH应控制在中性偏碱性,在此条件下酶分子处于非质子化状态,易于反应,同时温度需保持在室温,以便反应进行。
木聚糖酶最佳修饰方法:取1mL修饰剂用pH7.5的PBS定容至8mL,再加入0.2g木聚糖,最后加入2mL酶液,30℃下搅拌反应8h,再加入0.1g氰基硼氢化钠继续反应1h,反应完成后,在4℃下,透析4h即得到木聚糖酶修饰物。
实施例4木聚糖酶化学修饰物的降解木质纤维素的米氏方程
分别配制不同浓度的经过稀硫酸预处理过的木质纤维素溶液,在55℃下,反应5min,检测还原糖生成量,计算米氏方程。具体实验结果如下:
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Tyr Gly Trp Thr Arg Ser Pro Leu Ile Glu Tyr Tyr Val Val Asp Ser
100 105 110
Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly Thr Val Lys Ser
115 120 125
Asp Gly Gly Thr Tyr Asp Ile Tyr Thr Thr Thr Arg Tyr Asn Ala Pro
130 135 140
Ser Ile Asp Gly Asp Arg Thr Thr Phe Thr Gln Tyr Trp Ser Val Arg
145 150 155 160
Gln Thr Lys Arg Pro Thr Gly Ser Asn Ala Thr Ile Thr Phe Ser Asn
165 170 175
His Val Asn Ala Trp Lys Ser His Gly Met Asn Leu Gly Ser Asn Trp
180 185 190
Ala His Gln Val Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser
195 200 205
Asn Val Thr Val Trp
210
<210> 2
<211> 639
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgtttaaat ttaaaaaaaa tttcctggta ggtctgagcg cagcgctgat gtccatctct 60
ctgttctccg caaccgcgag cgcggcgtcc actgattact ggcagaactg gaccgatggt 120
ggtggcattg taaacgcagt gaacggctct ggcggcaact actctgttaa ctggtccaac 180
accggcaact tcgtggttgg taaaggttgg accaccggta gcccgttccg tactatcaac 240
tacaacgctg gcgtatgggc acctaacggc aatggttatc tgaccctgta tggttggacc 300
cgttctccgc tgatcgagta ctatgttgtg gatagctggg gcacctatcg tccgaccggt 360
acttacaagg gcaccgtgaa aagcgatggc ggcacctacg acatctatac cactacccgt 420
tacaatgcgc cgtccatcga cggtgatcgt accaccttca cccagtactg gtccgttcgt 480
cagactaaac gcccgactgg ctctaacgcg accatcacct tttctaacca tgtcaacgcg 540
tggaaatccc acggcatgaa cctgggtagc aactgggcgc accaggttat ggcgaccgaa 600
ggctaccagt cttctggtag ctctaacgtg actgtatgg 639
Claims (6)
1.一种化学修饰的木聚糖酶,其特征在于:所述木聚糖酶的氨基酸序列如SEQ ID NO:1所示,编码基因如SEQ ID NO:2所示,化学修饰剂为右旋糖酐。
2.根据权利要求1所述的化学修饰的木聚糖酶,其特征在于:所述化学修饰剂为含有醛基的右旋糖酐。
3.根据权利要求1所述的化学修饰的木聚糖酶,其特征在于:化学修饰剂与木聚糖酶通过氨基酸的氨基进行偶联。
4.根据权利要求2所述的化学修饰的木聚糖酶,其特征在于:所述含有醛基的右旋糖酐采用NaIO4氧化右旋糖酐的邻羟基获得。
5.根据权利要求2所述的化学修饰的木聚糖酶,其特征在于:所述含有醛基的右旋糖酐采用150kDa的右旋糖酐。
6.权利要求1所述的化学修饰的木聚糖酶的制备方法,其特征在于包括如下步骤:
(1)木聚糖酶的制备
将木聚糖酶编码基因与表达载体pEt28a(+)相连接,并将连接产物转化至大肠杆菌BL21(DE3) 细胞,获得重组菌株;培养重组菌株进行发酵表达,发酵液破碎离心后,将上清液进行硫酸铵沉淀、离子交换柱纯化,获得木聚糖酶液;
(2)含醛基右旋糖酐的制备
避光下将2.4g NaIO4溶于10mL纯水制得NaIO4溶液,将其倒入10mL浓度为2g/mL的右旋糖酐溶液中,室温下避光搅拌8h,
反应完成后加入5mL的乙二醇反应2h后,反应液在8000-14000的透析袋中,过夜透析;
(3)木聚糖酶化学修饰物的制备
取1mL修饰剂与2mL木聚糖酶酶液,用pH 7.5的PBS溶液定容到10mL,加入0.1g木聚糖,35℃pH 7.0下反应6h后,加入0.1g氰基硼氢化钠,继续反应1h后,4℃下透析4h,得到木聚糖酶化学修饰物。
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