CN114574453A - 一种宏基因组来源的耐热耐酸性漆酶及其编码基因 - Google Patents

一种宏基因组来源的耐热耐酸性漆酶及其编码基因 Download PDF

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CN114574453A
CN114574453A CN202210165040.6A CN202210165040A CN114574453A CN 114574453 A CN114574453 A CN 114574453A CN 202210165040 A CN202210165040 A CN 202210165040A CN 114574453 A CN114574453 A CN 114574453A
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laccase
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张士成
刘怡
罗刚
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Abstract

本发明提供一种从宏基因组中筛选的具有耐热性、耐酸性的漆酶及其编码基因，利用基因重组技术，将漆酶的编码基因异源表达，表征其物理化学性质。本发明所发现的漆酶具有耐热和耐酸的优越特性，在高温、酸碱、有机溶剂等工业条件下具有很大的应用潜力。

Description

一种宏基因组来源的耐热耐酸性漆酶及其编码基因
技术领域
本发明属于基因工程和酶工程技术领域，具体涉及一种从宏基因组来源的耐热耐酸性漆酶及其编码基因。
背景技术
漆酶（Laccase，EC.1.10.3.2)是一类多铜氧化酶，催化底物范围较广泛，可包括酚类和非酚类物质。漆酶因其绿色催化过程及其唯一绿色副产物“水”而得到研究者们的重点关注。
木质纤维素类生物质废弃物是第二代生产生物燃料的原料，在地球上广泛存在且可再生，其主要成分之一的木质素占木质纤维素类生物质原料干重的15-30%，且含有其中40%左右的能量，是地球上产量最大且可再生的芳香类化合物。漆酶其对木质素具有降解效力已被证实，其催化断裂木质素结构中的C-C键与C-O键，木质素低聚物和芳香性单体产品的生成，同时不破坏纤维素与半纤维素的结构，有利于糖或生物乙醇类生物燃料产量的提高，提高木质纤维素的整体转化水平。
高通量测序和宏基因组学技术能获取大量微生物的功能基因，为挖掘新的性能优异的生物酶而创造了可能性。但从宏基因组学中筛选出来目标酶的基因，还需利用实际的酶催化反应来确认其真实活性和各种物理化学性质，因为目前的技术发展还不能建立基因序列与功能之间的有效性关系。基因重组技术可以实现将一段目的基因插入到质粒中形成含有目的基因的重组质粒，再将重组质粒导入微生物载体如大肠杆菌进行诱导表达，最后分离纯化出目标酶液，再经过特定底物的催化实验鉴定目的酶的活性。
而相比于常温下进行催化反应的酶，耐热性酶更能适应高温（55~90℃）、酸/碱及有机溶剂的工业反应条件，为绿色的工业化应用提供更多的可能，具有更大的工业价值。
发明内容
本发明的目的在于提供一种宏基因组来源的耐热耐酸性漆酶及其编码基因。经过对该基因表达蛋白的化学测试发现该酶有较强的耐热（70℃）和耐酸（pH4.71）性能，能在木质素的生物降解过程及工业化应用中具有很大的潜力。
本发明提出的一种宏基因组来源的耐热耐酸性漆酶，所述漆酶为AA1_Bin30_Lac，漆酶AA1_Bin30_Lac是具有如下（a1）或（a2）特征的蛋白质：
（a1）其氨基酸序列与SEQ ID NO.1所示的氨基酸序列一致；
（a2）将（a1）经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的突变体，该突变体具有与SEQ ID NO.1所示的蛋白序列至少90%以上的同源性及至少90%以上的漆酶活性；
其中：SEQ ID NO.1由658个氨基酸残基组成。
本发明提出的一种宏基因组来源的耐热耐酸性漆酶的编码基因，所述漆酶AA1_Bin30_Lac的编码基因从宏基因组中筛选获得；
其中，所述基因具有（b1）-（b3）中任一所述核苷酸序列：
（b1）SEQ ID NO.2所示的核苷酸序列；
（b2）与（b1）互补的核苷酸序列；
（b3）与（b1）或（b2）互补所示的核苷酸序列具有≥90%一致性且编码相同功能蛋白质的核苷酸序列。
本发明提出的一种核酸构建体，其包含与一种或多种调控序列可操作性地连接的上述编码基因，所述调控序列在合适的表达宿主中指导所述漆酶的产生。
本发明提出的一种重组表达载体，其包含上述的核酸构建体。所述的载体为原核表达载体如pET系列载体，酵母表达载体或昆虫病毒表达载体BV。
进一步优选为BV系列载体，如Baculovirus / Insect Cells expressionsystem。
本发明提出的提供一种宿主，其由上述重组表达载体经转化或转染原核生物或真核生物宿主得到。
所述宿主，其为细菌、酵母或动物细胞；优选为昆虫细胞，进一步优选为草地贪夜蛾细胞。
本发明提出的一种宏基因组来源的耐热耐酸性漆酶的制备方法，具体步骤如下：
（1）在有助于产生漆酶的条件下培养上述宿主，其中所述宿主细胞包含SEQ IDNO.2所述核苷酸；
（2）回收上述漆酶。
本发明的有益效果在于：本发明提供的一种耐热耐热耐酸性漆酶具有发展为木质素降解工业酶的巨大潜力，其最适的高温反应条件不仅能提高木质素降解反应速率，还能推动反应平衡右移，提高反应效率，增加反应物产量。
附图说明
图1为实施例1得到的漆酶AA1_Bin30_Lac的电泳图。
图2为 ABTS标准底物下AA1_Bin30_Lac最适pH。
具体实施方式
下面通过实施例进一步说明本发明。
实施例1： 漆酶AA1_Bin30_Lac基因序列的确定
利用dbCAN2对比工具，将宏基因组与CAZyme数据库进行比对，筛选比对到AA1family的基因序列，再经过蛋白质一级结构模拟预测（ExPASy-ProtParam）、二级结构模拟预测（SOPMA）、三维结构模板预测（SWISS-MODEL）筛选出结构稳定性最强的三个漆酶基因，通过异源表达和活性测试，确定漆酶AA1_Bin30_Lac的热稳定性最强，由此确定漆酶AA1_Bin30_Lac的基因序列。
实施例2：漆酶AA1_Bin30_Lac的克隆、异源表达及分离纯化
首先化学合成SEQ ID NO.2的核苷酸序列；
将该段SEQ ID NO.2核苷酸序列亚克隆到昆虫细胞的表达载体(pFastBac1)中；
将上述所获得的pFastBac1转化到大肠杆菌菌株DH10Bac中以产生重组杆粒，用标准方法纯化重组杆粒；
将上述重组杆粒转染到草地贪夜蛾(Spodoptera frugiperda, Sf)细胞中产生P1代病毒悬液；
收集P1代获得的细胞和培养基样本，并进行分析，以检测目标蛋白的表达；
用P1代病毒株感染Sf细胞，扩增出重组病毒；
获得的P2病毒株用于进行表达测试；
用不同数量的P2代培养液感染草地贪夜蛾细胞和粉纹夜蛾细胞，以确定最佳的M.O.I.和培养时间，用SDS-PAGE分析目标蛋白表达；
最后按照说明书的标准方法进行镍柱亲和层析，分离纯化出目标蛋白；
层析后收集的样品用SDS-PAGE检测纯度，证明已经获得漆酶AA1_Bin30_Lac的电泳纯酶（图1所示）。
实施例3：漆酶AA1_Bin30_Lac活性及稳定性分析
选择标准底物ABTS来测定漆酶AA1_Bin30_Lac的活性；
建立反应体系1 mL，含Tris-HCl溶液600μL, CuSO4300μL, ABTS 50μL，酶液50μL；
设置温度范围(60℃、65℃、70℃、75℃、80℃)、pH范围(5.14、4.87、4.81、 4.70)和CuSO4浓度(150 mM、200 mM、250 mM、300 mM)，考察这些因素对漆酶活性的影响程度；
在60℃下反应5min，然后于420nm下测定吸光度值的变化，定义每分钟氧化1μmol·L-1ABTS所需的酶量为1个漆酶活力单位；
漆酶AA1_Bin30_Lac的在60℃，pH4.87条件下的最适CuSO4浓度为200mM（72.5±25.37 U)，（图2所示）。
序列表
<110> 复旦大学
<120> 一种宏基因组来源的耐热耐酸性漆酶及其编码基因
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序列表
<110> 复旦大学
<120> 一种宏基因组来源的耐热耐酸性漆酶及其编码基因
<130> 11
<160> 2
<170> SIPOSequenceListing 1.0
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<213> 植物()
<400> 1
atgaaccgta tcatccgtaa cgtgatcctg tacttcctga tcttcctggt ggtcatcggt 60
atcatcagcg tgttcactgg ccagaacaac caggccgagg agctgaagag ctcccagttc 120
atgcaggccc tggaaaacaa cgaaatcaag gagatggaaa tgcgtccttc caacggcgtg 180
atgcgcatcg tgggtaccct gagcaccgag gaccagacct tcgtgactaa catccctgac 240
tccagcggta tcatcgagga ggtgtacaac actgcctccg accagggtat cctgaaggtg 300
gacgaagaag aacagcctaa cggcttcctg gccttcctga ctaccatgat ccctttcgtg 360
atcatcttca tcctgttctt cttcctgctg aaccaggccc agggtggcgg tagccgcgtg 420
atgaacttcg gtaaaagcaa ggccaagatg tactccgaag acaagaagaa ggtgcgcttc 480
cgtgacgtcg ccggtgctga cgaggaaaag caggaactgg tggaagtcgt ggacttcctg 540
aaggaccctc gcaagttcac cgccatcggc gccaagatcc ctaagggtgt cctgctggtg 600
ggcccccccg gaactggtaa aactctgctg gcccgtgccg tggccggcga agctggtgtt 660
cccttcttct ccatcagcgg cagcgacttc gtggagatgt tcgtcggcgt cggtgcctcc 720
cgtgtccgtg acctgttcga gaacgccaag aagaacgccc cctgcatcat cttcattgac 780
gaaatcgacg ctgtgggtcg ccagcgcggc gctggtgttg gtggaggtca cgacgaacgc 840
gagcagaccc tgaaccagct gctggtcgag atggacggct tcggcgaaaa cgagggcatc 900
atcatcatcg ctgctactaa ccgtcccgac atcctggacc ctgccctgct gcgccctggc 960
agattcgacc gtcagatcac cgtcgaccgc cctgacctga agggtcgtga agaggtcctg 1020
aaggtgcacg ctcgtaacaa gcccctgagc gacgacgtca gcctgaagac catcgctatg 1080
cgtacccctg gcttcagcgg cgccgacctg gagaacctgc tgaacgaagc cgccctggtg 1140
gccgctcgta ctgacaagca gaagatcgac atggaggacg tcgacgaagc tatcgaccgt 1200
gtgatcgctg gccccgctaa gaagagccgc gtcatcagcg aaaaggagcg taacatcgtg 1260
gcctaccacg aatccggcca caccgtgatc ggcatggtcc tggacgacgc cgacatggtg 1320
cacaaggtga ctatcgtgcc tcgtggccag gccggtggtt acgctgtcat gctgcctcgt 1380
gaggaccgtt acttcatgac caagcccgaa ctgctggaca agatcactgg tctgctgggt 1440
ggccgtgtgg ctgaggaggt cattttcggt gaggtgagca ccggcgccca caacgacttc 1500
cagcgcgcca ccagcatcgc tcgcaagatg gtcatggaat acggtatgtc cgacaagatc 1560
ggccctctgc agttcggcaa cacttccggc caggtcttcc tgggtcgcga catgcagagc 1620
gaacagaact acagcgacgc tatcgcttac gagatcgacc aggaaatcca gcagttcatc 1680
cagagctgct acgaccgtgc caagcagatc ctgaccgaaa accgcgagaa gctggaactg 1740
atcgctaaga ccctgctgga aatcgagact ctggacgccc gtcagatcaa gagcctgttc 1800
gagaagggta tcctgcccga acccgaggtc gtcagcgtgc aggacggcga gaaggccagc 1860
actgacagca aggacgagac caaggtcgag gagaaggagt ccgacgtgaa ggtgaacatc 1920
cagagcaagg tggacgacgt ggaaacccag cagcctgaca ctgacaagaa ggaaggcagc 1980
caccaccacc accatcac 1998
<210> 2
<211> 658
<212> PRT
<213> 植物()
<400> 2
Met Asn Arg Ile Ile Arg Asn Val Ile Leu Tyr Phe Leu Ile Phe Leu
1 5 10 15
Val Val Ile Gly Ile Ile Ser Val Phe Thr Gly Gln Asn Asn Gln Ala
20 25 30
Glu Glu Leu Lys Ser Ser Gln Phe Met Gln Ala Leu Glu Asn Asn Glu
35 40 45
Ile Lys Glu Met Glu Met Arg Pro Ser Asn Gly Val Met Arg Ile Val
50 55 60
Gly Thr Leu Ser Thr Glu Asp Gln Thr Phe Val Thr Asn Ile Pro Asp
65 70 75 80
Ser Ser Gly Ile Ile Glu Glu Val Tyr Asn Thr Ala Ser Asp Gln Gly
85 90 95
Ile Leu Lys Val Asp Glu Glu Glu Gln Pro Asn Gly Phe Leu Ala Phe
100 105 110
Leu Thr Thr Met Ile Pro Phe Val Ile Ile Phe Ile Leu Phe Phe Phe
115 120 125
Leu Leu Asn Gln Ala Gln Gly Gly Gly Ser Arg Val Met Asn Phe Gly
130 135 140
Lys Ser Lys Ala Lys Met Tyr Ser Glu Asp Lys Lys Lys Val Arg Phe
145 150 155 160
Arg Asp Val Ala Gly Ala Asp Glu Glu Lys Gln Glu Leu Val Glu Val
165 170 175
Val Asp Phe Leu Lys Asp Pro Arg Lys Phe Thr Ala Ile Gly Ala Lys
180 185 190
Ile Pro Lys Gly Val Leu Leu Val Gly Pro Pro Gly Thr Gly Lys Thr
195 200 205
Leu Leu Ala Arg Ala Val Ala Gly Glu Ala Gly Val Pro Phe Phe Ser
210 215 220
Ile Ser Gly Ser Asp Phe Val Glu Met Phe Val Gly Val Gly Ala Ser
225 230 235 240
Arg Val Arg Asp Leu Phe Glu Asn Ala Lys Lys Asn Ala Pro Cys Ile
245 250 255
Ile Phe Ile Asp Glu Ile Asp Ala Val Gly Arg Gln Arg Gly Ala Gly
260 265 270
Val Gly Gly Gly His Asp Glu Arg Glu Gln Thr Leu Asn Gln Leu Leu
275 280 285
Val Glu Met Asp Gly Phe Gly Glu Asn Glu Gly Ile Ile Ile Ile Ala
290 295 300
Ala Thr Asn Arg Pro Asp Ile Leu Asp Pro Ala Leu Leu Arg Pro Gly
305 310 315 320
Arg Phe Asp Arg Gln Ile Thr Val Asp Arg Pro Asp Leu Lys Gly Arg
325 330 335
Glu Glu Val Leu Lys Val His Ala Arg Asn Lys Pro Leu Ser Asp Asp
340 345 350
Val Ser Leu Lys Thr Ile Ala Met Arg Thr Pro Gly Phe Ser Gly Ala
355 360 365
Asp Leu Glu Asn Leu Leu Asn Glu Ala Ala Leu Val Ala Ala Arg Thr
370 375 380
Asp Lys Gln Lys Ile Asp Met Glu Asp Val Asp Glu Ala Ile Asp Arg
385 390 395 400
Val Ile Ala Gly Pro Ala Lys Lys Ser Arg Val Ile Ser Glu Lys Glu
405 410 415
Arg Asn Ile Val Ala Tyr His Glu Ser Gly His Thr Val Ile Gly Met
420 425 430
Val Leu Asp Asp Ala Asp Met Val His Lys Val Thr Ile Val Pro Arg
435 440 445
Gly Gln Ala Gly Gly Tyr Ala Val Met Leu Pro Arg Glu Asp Arg Tyr
450 455 460
Phe Met Thr Lys Pro Glu Leu Leu Asp Lys Ile Thr Gly Leu Leu Gly
465 470 475 480
Gly Arg Val Ala Glu Glu Val Ile Phe Gly Glu Val Ser Thr Gly Ala
485 490 495
His Asn Asp Phe Gln Arg Ala Thr Ser Ile Ala Arg Lys Met Val Met
500 505 510
Glu Tyr Gly Met Ser Asp Lys Ile Gly Pro Leu Gln Phe Gly Asn Thr
515 520 525
Ser Gly Gln Val Phe Leu Gly Arg Asp Met Gln Ser Glu Gln Asn Tyr
530 535 540
Ser Asp Ala Ile Ala Tyr Glu Ile Asp Gln Glu Ile Gln Gln Phe Ile
545 550 555 560
Gln Ser Cys Tyr Asp Arg Ala Lys Gln Ile Leu Thr Glu Asn Arg Glu
565 570 575
Lys Leu Glu Leu Ile Ala Lys Thr Leu Leu Glu Ile Glu Thr Leu Asp
580 585 590
Ala Arg Gln Ile Lys Ser Leu Phe Glu Lys Gly Ile Leu Pro Glu Pro
595 600 605
Glu Val Val Ser Val Gln Asp Gly Glu Lys Ala Ser Thr Asp Ser Lys
610 615 620
Asp Glu Thr Lys Val Glu Glu Lys Glu Ser Asp Val Lys Val Asn Ile
625 630 635 640
Gln Ser Lys Val Asp Asp Val Glu Thr Gln Gln Pro Asp Thr Asp Lys
645 650 655
Lys Glu

Claims (9)

1.一种宏基因组来源的耐热耐酸性漆酶，其特征在于：所述漆酶为AA1_Bin30_Lac，漆酶AA1_Bin30_Lac是具有如下（a1）或（a2）特征的蛋白质：
（a1）其氨基酸序列与SEQ ID NO.1所示的氨基酸序列一致；
（a2）将（a1）经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的突变体，该突变体具有与SEQ ID NO.1所示的蛋白序列至少90%以上的同源性及至少90%以上的漆酶活性；
其中：SEQ ID NO.1由658个氨基酸残基组成。
2.一种如权利要求1所述的宏基因组来源的耐热耐酸性漆酶的编码基因，其特征在于：所述漆酶AA1_Bin30_Lac的编码基因从宏基因组中筛选获得；
其中，所述基因具有（b1）-（b3）中任一所述核苷酸序列：
（b1）SEQ ID NO.2所示的核苷酸序列；
（b2）与（b1）互补的核苷酸序列；
（b3）与（b1）或（b2）互补所示的核苷酸序列具有≥90%一致性且编码相同功能蛋白质的核苷酸序列。
3.一种核酸构建体，其特征在于：包含与一种或多种调控序列可操作性地连接的上述编码基因，所述调控序列在合适的表达宿主中指导所述漆酶的产生。
4.一种重组表达载体，其特征在于：包含上述的核酸构建体。
5.所述的载体为原核表达载体如pET系列载体，酵母表达载体或昆虫病毒表达载体BV。
6.根据权利要求4所述的重组表达载体，其特征在于： BV系列载体为Baculovirus /Insect Cells expression system。
7.一种如权利要求5所述的重组表达载体得到的宿主，其特征在于：由所述重组表达载体经转化或转染原核生物或真核生物宿主得到。
8.根据权利要求6所述的宿主，其特征在于：所述宿主为细菌、酵母或动物细胞。
9.根据权利要求1所述的一种宏基因组来源的耐热耐酸性漆酶的制备方法，其特征在于：具体步骤如下：
（1）在有助于产生漆酶的条件下培养上述宿主，其中所述宿主细胞包含SEQ ID NO.2所述核苷酸；
（2）回收上述漆酶。
CN202210165040.6A 2022-02-23 2022-02-23 一种宏基因组来源的耐热耐酸性漆酶及其编码基因 Pending CN114574453A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114958788A (zh) * 2022-06-24 2022-08-30 武汉轻工大学 一种耐高温漆酶及基因与菌株和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430181A (zh) * 2021-08-09 2021-09-24 云南师范大学 一种来源亚洲象肠道宏基因组的细菌漆酶及其基因

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113430181A (zh) * 2021-08-09 2021-09-24 云南师范大学 一种来源亚洲象肠道宏基因组的细菌漆酶及其基因

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114958788A (zh) * 2022-06-24 2022-08-30 武汉轻工大学 一种耐高温漆酶及基因与菌株和应用
CN114958788B (zh) * 2022-06-24 2023-04-25 武汉轻工大学 一种耐高温漆酶及基因与菌株和应用

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