CN105349511B - 木聚糖酶和编码该酶的基因及其在废纸脱墨中的应用 - Google Patents
木聚糖酶和编码该酶的基因及其在废纸脱墨中的应用 Download PDFInfo
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- CN105349511B CN105349511B CN201510944820.0A CN201510944820A CN105349511B CN 105349511 B CN105349511 B CN 105349511B CN 201510944820 A CN201510944820 A CN 201510944820A CN 105349511 B CN105349511 B CN 105349511B
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- xylanase
- deinking
- zytase
- gene
- waste paper
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- C12N9/2477—Hemicellulases not provided in a preceding group
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Abstract
本发明公开了一种木聚糖酶和编码该酶的基因及其在废纸脱墨中的应用。木聚糖酶，其氨基酸序列如SEQ ID NO. 1 所示。编码所述的木聚糖酶的基因，核苷酸序列如SEQ ID NO.2所示。一种重组质粒，它包含所述的木聚糖酶基因。一种重组菌，它含有所述的重组质粒。所述木聚糖酶在废纸脱墨中的应用。将待脱墨的废纸放入含有0.6‑1.2U/mg木聚糖酶的缓冲溶液中，在50‑70℃，pH6.0‑10.0条件下进行脱墨反应。本发明提供的木聚糖酶基因利用密码子优化技术实现在大肠杆菌中的高表达；表达的木聚糖酶无纤维素酶活性，具有耐高温、耐高碱性质；该木聚糖酶可应用于废纸脱墨，且具有比商业酶更高的效率，表现出良好的工业应用价值。
Description
技术领域
本发明涉及基因工程和微生物技术领域，具体涉及一种木聚糖酶和编码该酶的基因及其在废纸脱墨中的应用。
背景技术
半纤维素是仅次于纤维素的第二丰富的可再生资源，其高效应用对于人类解决当前资源危机、能源危机、环境污染等问题具有极其重要的意义。半纤维素结构与组成十分复杂，包括木聚糖、甘露聚糖、阿拉伯聚糖、阿拉伯半乳聚糖和葡聚糖等多种组分。其中木聚糖是细胞中最具代表性的半纤维素，它可以作为再生资源被人们利用。木聚糖是一种复杂的多聚五碳糖，主要由β-1,4-D-木糖苷键连接起来，并带有多种取代基，它的完全降解需要多种水解酶的协同作用。其中最主要的糖苷水解酶是内切-β-1,4-木聚糖酶，它可以降解木聚糖主链的β-1,4-糖苷键。目前木聚糖酶在造纸、食品、饲料、生物转化等行业得到了日益广泛的应用。在造纸工业中，木聚糖酶可以应用于生物制浆，纸浆漂白，废纸脱墨处理等，尤其在废纸脱墨中，与传统的化学脱墨法相比，利用木聚糖酶进行脱墨，不需要大量的化学药品，因而对环境的影响较小，脱墨废水的BOD和COD也较低，而且酶法脱墨的效果与化学法相近，但纤维的物理性能则优于化学法脱墨，因此具有很好的发展前景。
在用木聚糖酶脱墨之前， 纸浆处于大约高温 (55-70℃)及高碱性pH (pH9-11)下。高温和高碱性状态对木聚糖酶制剂提出了严格的要求。许多可商购的野生型木聚糖酶的缺点是这些酶呈现酸性的最佳pH及大约55℃的最佳温度，因而会大大影响脱墨的效果。因此获得新型的具有耐高温及高碱性的木聚糖酶的研究具有重要的价值。
Planomicrobium glaciei CHR43是一种极端嗜冷菌，由于其独特的生存能力，我们推测来自于这种细菌的木聚糖酶很可能具有耐高/低温和高pH适应性。目前关于来源于Planomicrobium glaciei CHR43的木聚糖酶基因及其表达与应用还没有相关的报道。
发明内容
本发明的目的是弥补现有木聚糖酶温度和pH不能满足工业需求的不足，提供一种木聚糖酶和编码该酶的基因及其在废纸脱墨中的应用。
为了实现上述目的，本发明采用的技术方案为：
一种木聚糖酶，其氨基酸序列如SEQ ID NO. 1 所示。
编码所述的木聚糖酶的基因。优选核苷酸序列如SEQ ID NO.2所示。
一种重组质粒，它包含所述的木聚糖酶基因。
一种重组菌，它含有所述的重组质粒。
木聚糖酶的制备方法，在营养培养基中培养所述的重组菌，收集具有木聚糖酶活性的多肽。
所述木聚糖酶在废纸脱墨中的应用。将待脱墨的废纸放入含有0.6-1.2U/mg木聚糖酶的缓冲溶液中，在50-70℃，pH6.0-10.0条件下进行脱墨反应。
进一步优选为，将待脱墨的废纸放入含有1 U/mg木聚糖酶的缓冲溶液中，在70℃，pH7.0条件下进行脱墨反应。
应当理解，本领域技术人员可根据本发明公开的氨基酸序列，在不影响其活性的前提下，取代、缺失和/或增加一个或几个氨基酸，得到所述蛋白的突变序列。因此，本发明的木聚糖酶还包括由SEQ ID No.1所示的氨基酸序列中经取代、缺失或添加一个或几个氨基酸且具有同等活性的由SEQ ID No.1所示的蛋白质衍生的蛋白质。例如通过在末端添加标签序列，如His-tag 或Strep-tag 而衍生的蛋白质。
优选的，木聚糖酶的衍生蛋白的氨基酸序列与SEQ ID No.1所示的氨基酸序列的同源性可为70％以上，优选80％以上，更优选90％以上。
本发明还提供的含有所述基因或基因簇的载体、细胞系及宿主菌均属于本发明的保护范围。
本领域技术人员能够理解，作为遗传密码简并性的结果，许多不同的多核苷酸能够编码相同的多肽。另外，应当理解，本领域技术人员能够使用常规的技术进行核苷酸取代，所述取代不会影响本发明中所使用的多核苷酸编码的多肽序列。另外，还可以使用本领域中的已知的方法对多核苷酸进行修饰，以增强本发明多核苷酸在体内的活性或者存活期。
本发明中，可选用本领域己知的各种载体，如质粒，粘粒，噬菌体及反转录病毒等。
重组表达载体可以用本领域熟知的方法导入宿主细胞中，这些方法包括：氯化钙热激法，电转化法，PEG介导法，基因枪法等。
木聚糖酶基因的获得、重组质粒和重组工程菌的构建：
本发明中，木聚糖酶基因是通过如下方法获得的：利用美国国家生物技术信息中心(NCBI)的数据库，分析、筛选、发现Planomicrobium glaciei CHR43中有一段序列与Bacillus sp. Strain NG-27中的木聚糖酶(Genbank号：AAB70918)序列具有较高同源性，很有可能是编码木聚糖酶的基因序列。对该序列进行筛选、优化（优化包括部分位点的突变、插入等），得到具有SEQ ID NO.2的核苷酸序列。基因由金斯瑞公司按照SEQ ID NO.2 核苷酸序列进行人工合成，在序列两端分别添加BamH I和Xho I酶切位点，连接至pETDuet-1质粒载体上的BamH I和Xho I酶切位点之间，得到重组质粒pETDuet-1-xyn2。
转化大肠杆菌DH5α感受态细胞，筛选并通过测序鉴定阳性重组质粒，提取重组质粒，转化大肠杆菌 BL21（DE3）感受态细胞，获得含有重组质粒的重组工程菌。
上述木聚糖酶的蛋白纯化和酶学活性测定。
将获得的重组工程菌转接培养，IPTG 诱导酶蛋白表达。通过超声波破碎细胞，高速离心和 Ni-NTA sepharose4B 亲和层析分离纯化；然后通过 SDS-PAGE 电泳鉴定蛋白分子大小；最后浓缩，测定蛋白浓度，分析蛋白的酶学性质。本发明提供的木聚糖酶在pH6.0-10.0范围内保持较高活性，最适pH为 7.0；在50-70℃保持较高活性，适宜反应温度是 70℃。
有益效果：
本发明从极端嗜冷菌Planomicrobium glaciei CHR43中获得一种类似木聚糖酶基因，利用密码子优化技术进行筛选、优化得到本发明的木聚糖酶基因，并通过表达获得相关酶蛋白。在大肠杆菌中的高表达，表达的木聚糖酶无纤维素酶活性，具有耐高温、耐高碱性质；该木聚糖酶可应用于废纸脱墨，且具有比商业酶更高的效率，表现出良好的工业应用价值。
本发明木聚糖酶的最适pH是7.0，最适宜反应温度是 70℃，并且有较好的耐高温性和高碱性。对废纸具有很好的脱墨效率，和购于上海源叶生物科技有限公司的商业酶（型号：MFCD00132594）（157%）相比，达到了267%，且高于目前已报道的来自于Bacillus sp.CKBx1D的木聚糖酶（200%） （Maity et al. Applied Biochemistry and Biotechnology.2012, 167: 1208-1219.）。
附图说明
图1木聚糖酶表达并纯化后的 SDS-PAGE 分析；其中 ：1为木聚糖酶表达纯化后的条带；M为蛋白分子量标准。
图2pH对木聚糖酶活性的影响及其pH稳定性。
图3 温度对木聚糖酶活性的影响。
图4 木聚糖酶的热稳定性。
图5 酶法脱墨后的废纸表面形态，其中：A为对照组；B为本发明木聚糖酶组；C为商业酶组。
具体实施方式
本发明的实施将采用本领域技术人员的能力范围之内的化学、分子生物学等领域的传统技术。另外，除非另有说明，在本文中，核酸以5’至3’的方向从左向右书写，氨基酸序列则以氨基端到羧基端的方向从左向右书写。下列实施例中未注明具体条件的实验方法，通常按照常规条件操作，例如《分子克隆实验指南》，或者制造厂商所建议的条件。
试验材料和试剂
1、 菌株：大肠杆菌菌株Escherichia coli DH5α、Escherichia coli BL21（DE3）购于Novagen 公司。
2、生化试剂 ：木聚糖购自 Sigma 公司 ； 其它都为国产试剂 （均可从普通生化试剂公司购买得到） 。
3、 培养基 ：LB 培养基 ： Peptone 10g， Yeast extract 5g， NaCl 10g， 加蒸馏水至 1000ml， pH 自然（约为 7） 。固体培养基在此基础上加 2.0%（w/v） 琼脂。
实施例1：木聚糖酶基因的获得、重组质粒和重组工程菌的构建：
木聚糖酶基因的获得：
利用美国国家生物技术信息中心(NCBI)的数据库，分析、筛选、发现Planomicrobium glaciei CHR43中有一段序列与Bacillus sp. Strain NG-27中的木聚糖酶(Genbank号：AAB70918)序列具有较高同源性，很有可能是编码木聚糖酶的基因序列。对该序列进行筛选、优化（优化包括部分位点的突变、插入等），得到具有SEQ ID NO.2的核苷酸序列。木聚糖酶基因由金斯瑞公司按照SEQ ID NO.2 核苷酸序列进行人工合成，在序列两端分别添加BamH I和Xho I酶切位点，连接至pETDuet-1质粒载体上的BamH I和Xho I酶切位点之间，得到重组质粒pETDuet-1-xyn2。
将合成的含有木聚糖酶基因的重组质粒pETDuet-1-xyn2导入到 100μl DH5α感受态细胞中，冰浴 30min，42℃热激 60s，再立即冰浴3min，加入 1ml LB 液体培养基，37℃，震荡培养 1h。低速离心菌液，弃去部分上清，将剩余菌体重悬，涂布于含有 100μg/ml 氨苄青霉素的 LB 平板上，于 37℃培养箱中培养过夜。挑取单菌落进行菌液 PCR 鉴定，测序确定阳性克隆，用质粒小提试剂盒提取该阳性克隆质粒。
重组质粒转化E.coli BL21（DE3）获得重组工程菌：
将重组质粒转化大肠杆菌 BL21（DE3）感受态细胞中，于含有 100μg/ml 氨苄青霉素的LB平板上37℃培养过夜。挑取单菌克隆于含有 100μg/ml 氨苄青霉素的 LB 液体培养基中培养，用 20% 的甘油保存菌种，获得以 E.coli BL21（DE3）为宿主菌构建的木聚糖酶xyn2重组菌株。
实施例 2 木聚糖酶xyn2的表达和纯化
表达：
重组菌先分别接种于 10ml 含 100μg/ml 氨苄青霉素的 LB 液体培养基中，37℃、220rpm 振荡培养过夜。然后以1%的量转接到500ml 含 100μg/ml 氨苄青霉素的LB液体培养基，37℃、220rpm 振荡培养至 OD600 达到0.6左右，加入 IPTG 至终浓度1mM，37℃、220rpm 振荡培养 6 小时。离心收集菌体。
纯化：
向菌体加入适量 pH7.0 PBS buffer （137mmol/L NaCl ，2.7mmol/L KCl ，4.3mmol/L Na2HPO4 ，1.4mmol/L KH2PO4 ，用HCl调节溶液的pH值至7.0）重悬，超声破碎重组菌，高速离心，收集上清。将上清于65℃温育半小时，可沉淀大量非耐热的宿主菌蛋白，再高速离心收集上清。将收集上清过Ni-NTA亲和层析柱，先用6个柱体积的banding buffer（20mM Tris-HCl pH7.5，300mMNaCl，40mM 咪唑）洗柱，再用少量Elution Buffer（50mMTris-HCl pH7.5，300mMNaCl，500mM 咪唑）洗脱，SDS-PAGE 电泳检测洗脱液，即发现得到高纯度的目的蛋白。此结果如图 1 所示。然后将收集的蛋白液加入到超滤管，离心浓缩得到纯化的蛋白液。
实施例3 木聚糖酶的酶学性质测定
使用DNS法测定木聚糖酶活性：2mL 反应体系中加入200μL 酶，底物浓度为0.9%，在温度50℃，pH4.8的条件下反应10min，加入2mL DNS终止反应后与沸水浴10min。冷却至室温后加水至15mLl后于540nm 波长检测其吸收值，对照标准曲线得到还原糖含量，酶活力单位（U）定义为每分钟释放 1mM 还原糖的酶量。依照此方法测定木聚糖酶在pH值 3.0 到12.0范围内的活性。结果如图2所示，从图中可以看出木聚糖酶最适pH为 7.0，在pH6.0-10.0范围内保持较高活性。还测定木聚糖酶在30℃到90℃范围内的活性，木聚糖酶适宜反应温度是 70℃，且在50-70℃保持较高活性。
实施例4重组木聚糖酶xyn2应用于废纸脱墨：
将一张100%表面积被油墨覆盖的来自于惠普打印机的废纸裁剪成3x3的小纸片，将纸片置于5ml分别含有1 U/mg本发明木聚糖酶、商业酶的pH7.0的PBS缓冲中，于70℃、pH7.0下反应12小时，设置不加酶的反应为对照组，反应结束后用紫外分光光度计测定反应液在596nm处的吸光值，同时用显微镜观察纸张表面形态（图5）。结果显示木聚糖酶的脱墨效率为267%，高于购自上海源叶生物科技有限公司的商业酶（型号：MFCD00132594）（157%），且高于目前已报道的来自于Bacillus sp. CKBx1D的木聚糖酶（200%） （Maity et al.Applied Biochemistry and Biotechnology. 2012, 167: 1208-1219.）
<110> 南京工业大学
<120> 木聚糖酶和编码该酶的基因及其在废纸脱墨中的应用
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<213> 人工序列
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tgtcaagctt tagccgaacg ttataaagat tctttttcca ttggtgcagc gttggagcct 60
gaacatcttg agggccgctc agctgacgtt gtcaagttcc actttaattc gatcgtagcc 120
gaaaacgcaa tgaaagcgcc caatatacag ccacaagagg gagtgttcaa ctttgaagag 180
actgatcgaa ttgttcagtt cgctaaggaa aatcaactcg agctacggtt tcataccctg 240
gtctggcacg ccgaaatccc ggagtggttc acattagaca aaaacgggca ggaaatgatg 300
gaggaaacgg atcctttgaa gagagaggaa aataaacaac ttctcctaaa gaggctggag 360
gaccatatac gtgtaattgc aagtcgctac cgagaagata tcaaatattg ggacgtggtt 420
aacgaggtca tagatgaaaa tgcgcggaac agcaagaaat taagagagtc tttttggtac 480
gaattgactg gtaccgacta tattaagaca gctttccttg ccacgaggaa atacgcaggc 540
gaggatgcga agctctttat caatgaattc aacactgaga tggaacccaa acgttcctat 600
ctactggagt tagtaaagga attgatggct gacggagtgc caatagatgg gattggtcac 660
cagtcacatt acaccatcgg ctggccgcct cttcaagaca tagaggaatc gattctccta 720
tttagtagct tcggactgga taatcagatc acagagttag acgtttctat atattcctca 780
gaacccgaga aaatttacgg gtcggatgaa gagatcccag aagagttgct tcaagaacag 840
gccaagtatt acggtgacct ctttgagcta tatgaagagc tggatgacca aataagtagc 900
attacgttct ggggcttagc agataaccgc tcttggttga attttcgagc gcaggacctt 960
tccaacggaa atgggaaaga tgctccgttc gtctttgact catactataa cgtaaagcct 1020
gccttctggg caatcgtgaa actcgaa 1047
Claims (8)
1.一种木聚糖酶，其氨基酸序列如SEQ ID NO. 1 所示，所述木聚糖酶在温度50~70℃、pH 6.0~10.0范围内保持较高活性，有较好的耐高温性和高碱性，对废纸具有很好的脱墨效率。
2.编码权利要求1所述的木聚糖酶的基因，其核苷酸序列如SEQ ID NO.2所示。
3.一种重组质粒，其特征在于，它包含权利要求2所述的木聚糖酶基因。
4.一种重组菌，其特征在于，它含有权利要求3所述的重组质粒。
5.木聚糖酶的制备方法，其特征在于，在营养培养基中培养权利要求4所述的重组菌，收集具有木聚糖酶活性的多肽。
6.权利要求1所述木聚糖酶在废纸脱墨中的应用。
7.根据权利要求6所述的应用，其特征在于，将待脱墨的废纸放入含有0.6-1.2U/mg木聚糖酶的缓冲溶液中，在50-70℃，pH6.0-10.0条件下进行脱墨反应。
8.根据权利要求7所述的应用，其特征在于，将待脱墨的废纸放入含有1 U/mg木聚糖酶的缓冲溶液中，在70℃，pH7.0条件下进行脱墨反应。
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