CN111004789A - 一种耐硫酸铵的木糖苷酶突变体v322dh328dt329e - Google Patents

一种耐硫酸铵的木糖苷酶突变体v322dh328dt329e Download PDF

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CN111004789A
CN111004789A CN201911269838.XA CN201911269838A CN111004789A CN 111004789 A CN111004789 A CN 111004789A CN 201911269838 A CN201911269838 A CN 201911269838A CN 111004789 A CN111004789 A CN 111004789A
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周峻沛
黄遵锡
张蕊
李娜
韩楠玉
唐湘华
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Abstract

本发明涉及基因工程及蛋白质改造技术领域，公开了一种耐硫酸铵的木糖苷酶突变体V322DH328DT329E，突变体V322DH328DT329E的氨基酸序列如SEQ ID NO.1所示。经15.0～30.0％(w/v)的KCl、Na2SO4和(NH4)2SO4处理60min后，V322DH328DT329E的活性分别为63～85％、60～81％和99～107％。本发明的耐硫酸铵的木糖苷酶突变体V322DH328DT329E可应用于农业、制革、污水处理等行业。

Description

一种耐硫酸铵的木糖苷酶突变体V322DH328DT329E
技术领域
本发明属于基因工程技术领域，涉及蛋白质改造技术，具体为一种耐硫酸铵的木糖苷酶突变体V322DH328DT329E。
背景技术
木聚糖是一种半纤维素，广泛存在于植物的细胞壁中，在高等植物和农业废料中约占干重的20％。内切木聚糖酶(endo-1,4-β-D-xyla nase，EC 3.2.1.8)作用于木聚糖的主链骨架，可随机地切割木聚糖从而生成低聚木糖，木糖苷酶(β-D-xylosidase，EC3.2.1.37)可将低聚木糖水解为木糖(Collins et al.FEMS Microbiology Reviews,2005,29:3～23.)，木糖可作为微生物等生物利用的碳源，或作为原料生产乙醇、乳酸、木糖醇等。
除了木聚糖外，植物的糖蛋白中也含有木糖，其可被木糖苷酶降解(Leszczuk etal.Plant Physiology and Biochemistry,2019,139:681～690.)；此外，广泛存在于动物体内的蛋白聚糖也含有木糖，其也可被木糖苷酶降解(Takagaki et al.The JournalofBiological Chemistry,1990,265:854～860.)。
盐广泛存在于在自然界和各种生产实践中，包括农业、制革、污水、洗涤、食品、造纸等。例如，氯化钾和硫酸铵是在农业种植中应用相对广泛的一种化肥；在皮革软化过程中，需要添加硫酸钠，在此过程中加入木聚糖酶，可达到促进皮纤维松散，提高成品革的柔软度、手感和物理机械性能的效果(专利：ZL201710574969.3)。不耐盐的木糖苷酶将无法和化肥同时施用，不利于降解农业废料中的低聚木糖，从而导致木聚糖的循环利用降低，进一步导致土壤肥力降低。由于高盐浓度下的盐析作用，大部分的酶在高盐浓度下不具有良好的催化活性。因此，为了使酶在高盐环境生物技术领域具有更好的应用性，需要提高酶在盐中的稳定性。
发明内容
针对上述技术问题，本发明的目的旨在提供耐硫酸铵的木糖苷酶突变体V322DH328DT329E，V322DH328DT329E可应用于农业、制革、污水处理等行业。
为了达到上述技术目的，本发明具体通过以下技术方案实现：
本发明通过蛋白质改造技术，设计了耐硫酸铵的木糖苷酶突变体V322DH328DT329E，所述的突变体V322DH328DT329E的氨基酸序列如SEQ ID NO.1所示，与GenBank记录的木糖苷酶序列AQM74402(SEQ ID NO.3)相比，V322DH328DT329E的第322位、328位和329位氨基酸分别为天冬氨酸、天冬氨酸和谷氨酸，而AQM74402的第322位、328位和329位氨基酸分别为缬氨酸、组氨酸和苏氨酸。
所述的突变体V322DH328DT329E在不同盐中的稳定性不同：V322DH328DT329E经3.0～30.0％(w/v)的KCl处理60min后，活性剩余63～93％；该突变体经3.0～30.0％(w/v)的Na2SO4处理60min后，活性剩余60～96％；该突变体经3.0～10.0％(w/v)的(NH4)2SO4处理60min后，活性剩余56～86％，经15.0～30.0％(w/v)的(NH4)2SO4处理60min后，活性为99～107％。
本发明提供了所述的耐硫酸铵的木糖苷酶突变体V322DH328DT329E的编码基因v322dh328dt329e，其核苷酸序列如SEQ ID NO.2所示。
本发明的另一目的在于提供包含木糖苷酶突变体V322DH328DT329E编码基因的重组载体。
本发明的另一目的在于提供包含木糖苷酶突变体V322DH328DT329E编码基因的重组菌。
另外，本发明所述的木糖苷酶突变体V322DH328DT329E在农业、制革和污水处理的应用也在本发明的保护范围内。
本发明所述的耐硫酸铵的木糖苷酶突变体V322DH328DT329E的制备方法，具体包括以下步骤：
1)合成突变体V322DH328DT329E的编码基因v322dh328dt329e(SEQ ID NO.2)；
2)将(1)中合成的序列和表达载体pEasy-E1相连接，即可获得包含v322dh328dt329e的表达载体；
3)将连接产物转化大肠杆菌BL21(DE3)，获得表达突变体V322DH328DT329E的重组菌株；
4)培养重组菌株，诱导木糖苷酶突变体V322DH328DT329E表达；
5)回收并纯化所表达的木糖苷酶突变体V322DH328DT329E。
本发明的有益效果为：
与野生酶HJ14GH43和突变酶V322D相比，突变酶V322DH328DT329E在高浓度(NH4)2SO4中的稳定性得到了增强。经15.0～30.0％(w/v)的(NH4)2SO4处理60min后，野生酶HJ14GH43的活性从111％降到38％，突变酶V322D的活性从104％降到60％，而V322DH328DT329E的活性几乎没有下降，最低也可达99％。本发明的耐硫酸铵的木糖苷酶突变体V322DH328DT329E可应用于农业、制革、污水处理等行业。
附图说明
图1是野生酶HJ14GH43和突变酶V322DH328DT329E的SDS-PAGE分析，其中，M：蛋白质Marker；W：HJ14GH43；Mut：V322DH328DT329E；
图2是纯化的野生酶HJ14GH43和突变酶V322DH328DT329E在KCl中的稳定性；
图3是纯化的野生酶HJ14GH43和突变酶V322DH328DT329E在Na2SO4中的稳定性；
图4是纯化的野生酶HJ14GH43和突变酶V322DH328DT329E在(NH4)2SO4中的稳定性。
具体实施方式
下面将结合本发明具体的实施例，对本发明技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明以下实施例中的实验材料和试剂：
1、菌株及载体：大肠杆菌Escherichia coli BL21(DE3)和表达载体pEasy-E1购自北京全式金生物技术有限公司。
2、酶类及其它生化试剂：pNP(p-nitrophenol)和pNPX(p-nitrophenyl-β-d-xylopyranoside)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：Peptone10 g，Yeast extract5g，NaCl10 g，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。
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)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1表达载体的构建和转化
1)根据GenBank记录的木糖苷酶核苷酸序列KY391885(SEQ ID NO.4)，合成野生木糖苷酶HJ14GH43的编码基因hJ14GH43；另合成突变酶V322D(SEQ ID NO.5)的编码基因v322d(SEQ ID NO.6)及突变酶V322DH328DT329E的编码基因v322dh328dt329e(SEQ IDNO.2)；
2)将(1)中合成的序列分别和表达载体pEasy-E1相连接，即可分别获得包含hJ14GH43、v322d和v322dh328dt329e的表达载体；
3)将连接产物分别转化大肠杆菌BL21(DE3)，获得分别表达野生酶HJ14GH43、突变酶V322D及V322DH328DT329E的重组菌株。
实施例2野生酶HJ14GH43和突变酶V322D及V322DH328DT329E的制备
将含hJ14GH43、v322d和v322dh328dt329e的重组菌株以0.1％的接种量分别接种于LB(含100μg mL-1Amp)培养液中，37℃快速振荡16h。
然后将此活化的菌液以1％接种量接种到新鲜的LB(含100μg mL-1Amp)培养液中，快速振荡培养约2～3h(OD600达到0.6-1.0)后，加入终浓度0.1mM的IPTG进行诱导，于20℃继续振荡培养约20h。12000rpm离心5min，收集菌体。用适量的pH7.0 Tris～HCl缓冲液悬浮菌体后，于低温水浴下超声波破碎菌体。以上胞内浓缩的粗酶液经12,000rpm离心10min后，吸取上清并用Nickel-NTAAgarose和0～500mM的咪唑分别亲和和洗脱目的蛋白。
SDS-PAGE结果(图1)表明，野生酶HJ14GH43和突变酶V322D及V322DH328DT329E在大肠杆菌中都得到了表达，经纯化后，产物均为单一条带。
实施例3纯化的野生酶HJ14GH43和突变酶V322D及V322DH328DT329E的性质测定
采用pNP法测定纯化的野生酶HJ14GH43和突变酶V322D及V322DH328DT329E的活性：将pNPX溶于缓冲液中，使其终浓度为2mM；反应体系含50μL适量酶液，450μL的2mM底物；底物在反应温度下预热5min后，加入酶液再反应适当时间，然后加2mL1M Na2CO3终止反应，冷却至室温后在405nm波长下测定释放出的pNP；1个酶活单位(U)定义为每分钟分解底物产生1μmol pNP所需的酶量。
1)纯化的野生酶HJ14GH43和突变酶V322D及V322DH328DT329E在KCl中的稳定性
将纯化的酶液置于3.0～30.0％(w/v)KCl水溶液中，在20℃下处理60min，然后在pH7.0及20℃下进行酶促反应，以未处理的酶液作为对照。以pNPX为底物，反应10min，测定纯化的HJ14GH43和突变酶V322D及V322DH328DT329E的酶学性质。
结果表明：在高浓度KCl中，突变酶V322D及V322DH328DT329E的稳定性比较相似，均优于野生酶HJ14GH43的稳定性，经3.0～30.0％(w/v)的KCl处理60min后，野生酶HJ14GH43的活性从114％降至28％，突变酶V322D的活性为68～130％，突变酶V322DH328DT329E的活性为63～93％(图2)。
2)纯化的野生酶HJ14GH43和突变酶V322D及V322DH328DT329E在Na2SO4中的稳定性
将纯化的酶液置于3.0～30.0％(w/v)Na2SO4水溶液中，在20℃下处理60min，然后在pH7.0及20℃下进行酶促反应，以未处理的酶液作为对照。以pNPX为底物，反应10min，测定纯化的HJ14GH43和突变酶V322D及V322DH328DT329E的酶学性质。
结果表明：在高浓度Na2SO4中，突变酶V322D及V322DH328DT329E的稳定性比较相似，均优于野生酶HJ14GH43的稳定性，经3.0～30.0％(w/v)的Na2SO4处理60min后，野生酶HJ14GH43的活性剩余47～86％，突变酶V322D的活性为60～105％，突变酶V322DH328DT329E的活性为60～96％(图3)。
3)纯化的野生酶HJ14GH43和突变酶V322D及V322DH328DT329E在(NH4)2SO4中的稳定性
将纯化的酶液置于3.0～30.0％(w/v)(NH4)2SO4水溶液中，在20℃下处理60min，然后在pH7.0及20℃下进行酶促反应，以未处理的酶液作为对照。以pNPX为底物，反应10min，测定纯化的HJ14GH43和突变酶V322D及V322DH328DT329E的酶学性质。
结果表明：野生酶HJ14GH43和突变酶V322DH328DT329E在(N H4)2SO4中的稳定性不同，经3.0～10.0％(w/v)的(NH4)2SO4处理60min后，野生酶HJ14GH43和突变酶V322D保持稳定，而突变酶V322DH328DT329E的酶活剩余56～86％，但是经15.0～30.0％(w/v)的(NH4)2SO4处理60min后，野生酶HJ14GH43的活性从111％降到38％，突变酶V322D的活性从104％降到60％，而V322DH328DT329E的活性几乎没有下降，最低也可达99％(图4)。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
序列表
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Ile Gly Ser Leu Thr Glu Lys Pro Gln His Leu Arg Leu Phe Gly Arg
355 360 365
Glu Ser Leu Thr Ser Lys Phe Thr Gln Ala Phe Val Ala Arg Arg Trp
370 375 380
Gln Ser Phe Tyr Phe Glu Ala Glu Thr Ala Val Ser Phe Phe Pro Glu
385 390 395 400
Asn Phe Gln Gln Ala Ala Gly Leu Val Asn Tyr Tyr Asn Thr Glu Asn
405 410 415
Trp Thr Ala Leu Gln Val Thr Tyr Asp Glu Glu Leu Gly Arg Thr Leu
420 425 430
Glu Leu Ser Val Cys Gln Asn Leu Ala Phe Ser Gln Pro Leu Thr His
435 440 445
Lys Ile Ile Ile Pro Asp Glu Val Thr Tyr Val Tyr Leu Lys Val Thr
450 455 460
Val Arg Lys Glu Thr Tyr Lys Tyr Ser Tyr Ser Phe Asp Gln Lys Glu
465 470 475 480
Trp Lys Glu Ile Asp Val Pro Phe Glu Ser Ile His Leu Ser Asp Asp
485 490 495
Phe Ile Arg Gly Gly Gly Phe Phe Thr Gly Ala Phe Val Gly Met Gln
500 505 510
Cys Gln Asp Thr Ser Gly Glu Arg Leu Pro Ala Asp Phe His Tyr Phe
515 520 525
Arg Tyr Glu Glu Thr Asp Glu
530 535
<210> 2
<211> 1608
<212> DNA
<213> 突变体(v322dh328dt329e)
<400> 2
atgaagatta ccaatccagt gctcaaaggg tttaatcctg atccaagtat ttgccgtgta 60
ggagaagatt attatatggc cgtctctaca tttgaatggt ttccaggggt gcaaatttat 120
cattcaaagg atctcgtcca ttggcgtctt gctgcgcgtc cattgcaaaa aacgtcgcag 180
ctggatatga aggggaatcc tgactctggc ggggtatggg cgccgtgctt aagctatgct 240
gatgggcagt tttggcttat ttattcagat atcaaagtag tggatggccc atttaaagac 300
ggtcataatt atttggtcac ggcaagcgag gtggacggcg attggagtga accgatcctg 360
ctcaacagct ctggctttga tccatcttta ttccatgatc acagcgggaa gaaatacgtc 420
ttaaatatgc tgtgggatca tagggaaaag catcattcgt ttgcaggtat tgccttgcag 480
gaatatagtg tggctgaaaa gaagctcatc ggtcaaagga aggtcatttt taaaggcaca 540
ccgattaaac tgacagaagc gccgcatctg tatcatatcg gtgactacta ctatttatta 600
acggcagaag gaggtacccg gtatgagcat gcagcaacga tcgcccggtc ctcgcatatt 660
gaagggcctt atgaggttca tcctgataac ccgattgtaa gtgccttcca tgtgcctgaa 720
catccgcttc aaaaatgcgg gcatgcttca atcgttcaaa cgcatacaaa tgaatggtat 780
ctcgctcatc tcactggccg cccgattcaa tccagcaagg aatcgatttt tcaacagaga 840
gggtggtgcc ctttaggaag agaaacagcg atccaaaagc ttgaatggaa ggatggatgg 900
ccttatgttg taggcggaaa agaggggacg ctagaggttg aagcgccaaa gatcgaagaa 960
aaggattttg caccaaccta tgatgaagtc gatgaattta aagaatcaac tctaaataga 1020
cactttcaaa cattaagaat tccgtttacc gatcagattg gttcgttaac ggagaaacct 1080
cagcatttaa ggttattcgg ccgtgaatct ttaacgtcta agtttaccca agcatttgtt 1140
gcaagacgct ggcaaagctt ttattttgaa gcagagacag ctgtttcgtt cttcccagaa 1200
aactttcagc aagccgcagg tcttgtgaat tattataata cggaaaactg gacagcactc 1260
caggtgacat atgatgagga acttggccgc acgcttgaac tatccgtctg tcaaaacctt 1320
gccttttctc agccgttgac acataaaatc atcattcctg acgaggtcac ttatgtctat 1380
ttaaaagtga ccgttcggaa agagacatat aaatattctt attcatttga tcagaaagag 1440
tggaaggaaa ttgatgtacc gtttgaatcc atccatttat ccgatgattt cattcgaggt 1500
gggggttttt ttacaggggc atttgtcggt atgcagtgcc aagatacgag cggcgagcgt 1560
cttcctgctg attttcacta ttttcgctat gaggaaacag acgaataa 1608
<210> 3
<211> 535
<212> PRT
<213> 野生酶(HJ14GH43)
<400> 3
Met Lys Ile Thr Asn Pro Val Leu Lys Gly Phe Asn Pro Asp Pro Ser
1 5 10 15
Ile Cys Arg Val Gly Glu Asp Tyr Tyr Met Ala Val Ser Thr Phe Glu
20 25 30
Trp Phe Pro Gly Val Gln Ile Tyr His Ser Lys Asp Leu Val His Trp
35 40 45
Arg Leu Ala Ala Arg Pro Leu Gln Lys Thr Ser Gln Leu Asp Met Lys
50 55 60
Gly Asn Pro Asp Ser Gly Gly Val Trp Ala Pro Cys Leu Ser Tyr Ala
65 70 75 80
Asp Gly Gln Phe Trp Leu Ile Tyr Ser Asp Ile Lys Val Val Asp Gly
85 90 95
Pro Phe Lys Asp Gly His Asn Tyr Leu Val Thr Ala Ser Glu Val Asp
100 105 110
Gly Asp Trp Ser Glu Pro Ile Leu Leu Asn Ser Ser Gly Phe Asp Pro
115 120 125
Ser Leu Phe His Asp His Ser Gly Lys Lys Tyr Val Leu Asn Met Leu
130 135 140
Trp Asp His Arg Glu Lys His His Ser Phe Ala Gly Ile Ala Leu Gln
145 150 155 160
Glu Tyr Ser Val Ala Glu Lys Lys Leu Ile Gly Gln Arg Lys Val Ile
165 170 175
Phe Lys Gly Thr Pro Ile Lys Leu Thr Glu Ala Pro His Leu Tyr His
180 185 190
Ile Gly Asp Tyr Tyr Tyr Leu Leu Thr Ala Glu Gly Gly Thr Arg Tyr
195 200 205
Glu His Ala Ala Thr Ile Ala Arg Ser Ser His Ile Glu Gly Pro Tyr
210 215 220
Glu Val His Pro Asp Asn Pro Ile Val Ser Ala Phe His Val Pro Glu
225 230 235 240
His Pro Leu Gln Lys Cys Gly His Ala Ser Ile Val Gln Thr His Thr
245 250 255
Asn Glu Trp Tyr Leu Ala His Leu Thr Gly Arg Pro Ile Gln Ser Ser
260 265 270
Lys Glu Ser Ile Phe Gln Gln Arg Gly Trp Cys Pro Leu Gly Arg Glu
275 280 285
Thr Ala Ile Gln Lys Leu Glu Trp Lys Asp Gly Trp Pro Tyr Val Val
290 295 300
Gly Gly Lys Glu Gly Thr Leu Glu Val Glu Ala Pro Lys Ile Glu Glu
305 310 315 320
Lys Val Phe Ala Pro Thr Tyr His Thr Val Asp Glu Phe Lys Glu Ser
325 330 335
Thr Leu Asn Arg His Phe Gln Thr Leu Arg Ile Pro Phe Thr Asp Gln
340 345 350
Ile Gly Ser Leu Thr Glu Lys Pro Gln His Leu Arg Leu Phe Gly Arg
355 360 365
Glu Ser Leu Thr Ser Lys Phe Thr Gln Ala Phe Val Ala Arg Arg Trp
370 375 380
Gln Ser Phe Tyr Phe Glu Ala Glu Thr Ala Val Ser Phe Phe Pro Glu
385 390 395 400
Asn Phe Gln Gln Ala Ala Gly Leu Val Asn Tyr Tyr Asn Thr Glu Asn
405 410 415
Trp Thr Ala Leu Gln Val Thr Tyr Asp Glu Glu Leu Gly Arg Thr Leu
420 425 430
Glu Leu Ser Val Cys Gln Asn Leu Ala Phe Ser Gln Pro Leu Thr His
435 440 445
Lys Ile Ile Ile Pro Asp Glu Val Thr Tyr Val Tyr Leu Lys Val Thr
450 455 460
Val Arg Lys Glu Thr Tyr Lys Tyr Ser Tyr Ser Phe Asp Gln Lys Glu
465 470 475 480
Trp Lys Glu Ile Asp Val Pro Phe Glu Ser Ile His Leu Ser Asp Asp
485 490 495
Phe Ile Arg Gly Gly Gly Phe Phe Thr Gly Ala Phe Val Gly Met Gln
500 505 510
Cys Gln Asp Thr Ser Gly Glu Arg Leu Pro Ala Asp Phe His Tyr Phe
515 520 525
Arg Tyr Glu Glu Thr Asp Glu
530 535
<210> 4
<211> 1608
<212> DNA
<213> 野生酶基因(hJ14GH43)
<400> 4
atgaagatta ccaatccagt gctcaaaggg tttaatcctg atccaagtat ttgccgtgta 60
ggagaagatt attatatggc cgtctctaca tttgaatggt ttccaggggt gcaaatttat 120
cattcaaagg atctcgtcca ttggcgtctt gctgcgcgtc cattgcaaaa aacgtcgcag 180
ctggatatga aggggaatcc tgactctggc ggggtatggg cgccgtgctt aagctatgct 240
gatgggcagt tttggcttat ttattcagat atcaaagtag tggatggccc atttaaagac 300
ggtcataatt atttggtcac ggcaagcgag gtggacggcg attggagtga accgatcctg 360
ctcaacagct ctggctttga tccatcttta ttccatgatc acagcgggaa gaaatacgtc 420
ttaaatatgc tgtgggatca tagggaaaag catcattcgt ttgcaggtat tgccttgcag 480
gaatatagtg tggctgaaaa gaagctcatc ggtcaaagga aggtcatttt taaaggcaca 540
ccgattaaac tgacagaagc gccgcatctg tatcatatcg gtgactacta ctatttatta 600
acggcagaag gaggtacccg gtatgagcat gcagcaacga tcgcccggtc ctcgcatatt 660
gaagggcctt atgaggttca tcctgataac ccgattgtaa gtgccttcca tgtgcctgaa 720
catccgcttc aaaaatgcgg gcatgcttca atcgttcaaa cgcatacaaa tgaatggtat 780
ctcgctcatc tcactggccg cccgattcaa tccagcaagg aatcgatttt tcaacagaga 840
gggtggtgcc ctttaggaag agaaacagcg atccaaaagc ttgaatggaa ggatggatgg 900
ccttatgttg taggcggaaa agaggggacg ctagaggttg aagcgccaaa gatcgaagaa 960
aaggtttttg caccaaccta tcatacagtc gatgaattta aagaatcaac tctaaataga 1020
cactttcaaa cattaagaat tccgtttacc gatcagattg gttcgttaac ggagaaacct 1080
cagcatttaa ggttattcgg ccgtgaatct ttaacgtcta agtttaccca agcatttgtt 1140
gcaagacgct ggcaaagctt ttattttgaa gcagagacag ctgtttcgtt cttcccagaa 1200
aactttcagc aagccgcagg tcttgtgaat tattataata cggaaaactg gacagcactc 1260
caggtgacat atgatgagga acttggccgc acgcttgaac tatccgtctg tcaaaacctt 1320
gccttttctc agccgttgac acataaaatc atcattcctg acgaggtcac ttatgtctat 1380
ttaaaagtga ccgttcggaa agagacatat aaatattctt attcatttga tcagaaagag 1440
tggaaggaaa ttgatgtacc gtttgaatcc atccatttat ccgatgattt cattcgaggt 1500
gggggttttt ttacaggggc atttgtcggt atgcagtgcc aagatacgag cggcgagcgt 1560
cttcctgctg attttcacta ttttcgctat gaggaaacag acgaataa 1608
<210> 5
<211> 535
<212> PRT
<213> 突变体(V322D)
<400> 5
Met Lys Ile Thr Asn Pro Val Leu Lys Gly Phe Asn Pro Asp Pro Ser
1 5 10 15
Ile Cys Arg Val Gly Glu Asp Tyr Tyr Met Ala Val Ser Thr Phe Glu
20 25 30
Trp Phe Pro Gly Val Gln Ile Tyr His Ser Lys Asp Leu Val His Trp
35 40 45
Arg Leu Ala Ala Arg Pro Leu Gln Lys Thr Ser Gln Leu Asp Met Lys
50 55 60
Gly Asn Pro Asp Ser Gly Gly Val Trp Ala Pro Cys Leu Ser Tyr Ala
65 70 75 80
Asp Gly Gln Phe Trp Leu Ile Tyr Ser Asp Ile Lys Val Val Asp Gly
85 90 95
Pro Phe Lys Asp Gly His Asn Tyr Leu Val Thr Ala Ser Glu Val Asp
100 105 110
Gly Asp Trp Ser Glu Pro Ile Leu Leu Asn Ser Ser Gly Phe Asp Pro
115 120 125
Ser Leu Phe His Asp His Ser Gly Lys Lys Tyr Val Leu Asn Met Leu
130 135 140
Trp Asp His Arg Glu Lys His His Ser Phe Ala Gly Ile Ala Leu Gln
145 150 155 160
Glu Tyr Ser Val Ala Glu Lys Lys Leu Ile Gly Gln Arg Lys Val Ile
165 170 175
Phe Lys Gly Thr Pro Ile Lys Leu Thr Glu Ala Pro His Leu Tyr His
180 185 190
Ile Gly Asp Tyr Tyr Tyr Leu Leu Thr Ala Glu Gly Gly Thr Arg Tyr
195 200 205
Glu His Ala Ala Thr Ile Ala Arg Ser Ser His Ile Glu Gly Pro Tyr
210 215 220
Glu Val His Pro Asp Asn Pro Ile Val Ser Ala Phe His Val Pro Glu
225 230 235 240
His Pro Leu Gln Lys Cys Gly His Ala Ser Ile Val Gln Thr His Thr
245 250 255
Asn Glu Trp Tyr Leu Ala His Leu Thr Gly Arg Pro Ile Gln Ser Ser
260 265 270
Lys Glu Ser Ile Phe Gln Gln Arg Gly Trp Cys Pro Leu Gly Arg Glu
275 280 285
Thr Ala Ile Gln Lys Leu Glu Trp Lys Asp Gly Trp Pro Tyr Val Val
290 295 300
Gly Gly Lys Glu Gly Thr Leu Glu Val Glu Ala Pro Lys Ile Glu Glu
305 310 315 320
Lys Asp Phe Ala Pro Thr Tyr His Thr Val Asp Glu Phe Lys Glu Ser
325 330 335
Thr Leu Asn Arg His Phe Gln Thr Leu Arg Ile Pro Phe Thr Asp Gln
340 345 350
Ile Gly Ser Leu Thr Glu Lys Pro Gln His Leu Arg Leu Phe Gly Arg
355 360 365
Glu Ser Leu Thr Ser Lys Phe Thr Gln Ala Phe Val Ala Arg Arg Trp
370 375 380
Gln Ser Phe Tyr Phe Glu Ala Glu Thr Ala Val Ser Phe Phe Pro Glu
385 390 395 400
Asn Phe Gln Gln Ala Ala Gly Leu Val Asn Tyr Tyr Asn Thr Glu Asn
405 410 415
Trp Thr Ala Leu Gln Val Thr Tyr Asp Glu Glu Leu Gly Arg Thr Leu
420 425 430
Glu Leu Ser Val Cys Gln Asn Leu Ala Phe Ser Gln Pro Leu Thr His
435 440 445
Lys Ile Ile Ile Pro Asp Glu Val Thr Tyr Val Tyr Leu Lys Val Thr
450 455 460
Val Arg Lys Glu Thr Tyr Lys Tyr Ser Tyr Ser Phe Asp Gln Lys Glu
465 470 475 480
Trp Lys Glu Ile Asp Val Pro Phe Glu Ser Ile His Leu Ser Asp Asp
485 490 495
Phe Ile Arg Gly Gly Gly Phe Phe Thr Gly Ala Phe Val Gly Met Gln
500 505 510
Cys Gln Asp Thr Ser Gly Glu Arg Leu Pro Ala Asp Phe His Tyr Phe
515 520 525
Arg Tyr Glu Glu Thr Asp Glu
530 535
<210> 6
<211> 1608
<212> DNA
<213> 突变体(v322d)
<400> 6
atgaagatta ccaatccagt gctcaaaggg tttaatcctg atccaagtat ttgccgtgta 60
ggagaagatt attatatggc cgtctctaca tttgaatggt ttccaggggt gcaaatttat 120
cattcaaagg atctcgtcca ttggcgtctt gctgcgcgtc cattgcaaaa aacgtcgcag 180
ctggatatga aggggaatcc tgactctggc ggggtatggg cgccgtgctt aagctatgct 240
gatgggcagt tttggcttat ttattcagat atcaaagtag tggatggccc atttaaagac 300
ggtcataatt atttggtcac ggcaagcgag gtggacggcg attggagtga accgatcctg 360
ctcaacagct ctggctttga tccatcttta ttccatgatc acagcgggaa gaaatacgtc 420
ttaaatatgc tgtgggatca tagggaaaag catcattcgt ttgcaggtat tgccttgcag 480
gaatatagtg tggctgaaaa gaagctcatc ggtcaaagga aggtcatttt taaaggcaca 540
ccgattaaac tgacagaagc gccgcatctg tatcatatcg gtgactacta ctatttatta 600
acggcagaag gaggtacccg gtatgagcat gcagcaacga tcgcccggtc ctcgcatatt 660
gaagggcctt atgaggttca tcctgataac ccgattgtaa gtgccttcca tgtgcctgaa 720
catccgcttc aaaaatgcgg gcatgcttca atcgttcaaa cgcatacaaa tgaatggtat 780
ctcgctcatc tcactggccg cccgattcaa tccagcaagg aatcgatttt tcaacagaga 840
gggtggtgcc ctttaggaag agaaacagcg atccaaaagc ttgaatggaa ggatggatgg 900
ccttatgttg taggcggaaa agaggggacg ctagaggttg aagcgccaaa gatcgaagaa 960
aaggattttg caccaaccta tcatacagtc gatgaattta aagaatcaac tctaaataga 1020
cactttcaaa cattaagaat tccgtttacc gatcagattg gttcgttaac ggagaaacct 1080
cagcatttaa ggttattcgg ccgtgaatct ttaacgtcta agtttaccca agcatttgtt 1140
gcaagacgct ggcaaagctt ttattttgaa gcagagacag ctgtttcgtt cttcccagaa 1200
aactttcagc aagccgcagg tcttgtgaat tattataata cggaaaactg gacagcactc 1260
caggtgacat atgatgagga acttggccgc acgcttgaac tatccgtctg tcaaaacctt 1320
gccttttctc agccgttgac acataaaatc atcattcctg acgaggtcac ttatgtctat 1380
ttaaaagtga ccgttcggaa agagacatat aaatattctt attcatttga tcagaaagag 1440
tggaaggaaa ttgatgtacc gtttgaatcc atccatttat ccgatgattt cattcgaggt 1500
gggggttttt ttacaggggc atttgtcggt atgcagtgcc aagatacgag cggcgagcgt 1560
cttcctgctg attttcacta ttttcgctat gaggaaacag acgaataa 1608

Claims (7)

1.一种耐硫酸铵的木糖苷酶突变体V322DH328DT329E，其特征在于，所述的突变体V322DH328DT329E由木糖苷酶序列AQM74402第322位的缬氨酸、第328位的组氨酸以及第329位的苏氨酸分别突变为天冬氨酸、天冬氨酸以及谷氨酸得到。
2.根据权利要求1所述的耐硫酸铵的木糖苷酶突变体V322DH328DT329E，其特征在于，所述的突变体V322DH328DT329E的氨基酸序列如SEQ ID NO.1所示。
3.权利要求1或2所述的突变体V322DH328DT329E的编码基因v322dh328dt329e，其特征在于，所述的编码基因的核苷酸序列如SEQ ID NO.2所示。
4.一种重组载体，其特征在于，包含权利要求3所述的编码基因。
5.一种重组菌，其特征在于，包含权利要求3所述的编码基因。
6.权利要求1所述的木糖苷酶突变体V322DH328DT329E的制备方法，其特征在于，包括以下步骤：
1)合成突变体V322DH328DT329E的编码基因v322dh328dt329e；
2)将(1)中合成的序列和表达载体pEasy-E1相连接，即可获得包含v322dh328dt329e的表达载体；
3)将连接产物转化大肠杆菌BL21(DE3)，获得表达V322DH328DT329E的重组菌株；
4)培养重组菌株，诱导木糖苷酶突变体V322DH328DT329E表达；
5)回收并纯化所表达的木糖苷酶突变体V322DH328DT329E。
7.权利要求1所述的突变体V322DH328DT329E在农业、制革和污水处理中的应用。
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WO2011079048A9 (en) * 2009-12-23 2011-08-25 Danisco Us Inc. Methods for improving the efficiency of simultaneous saccharification and fermentation reactions
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WO2011079048A9 (en) * 2009-12-23 2011-08-25 Danisco Us Inc. Methods for improving the efficiency of simultaneous saccharification and fermentation reactions
WO2016072448A1 (ja) * 2014-11-05 2016-05-12 東レ株式会社 エンドキシラナーゼ変異体、バイオマス分解用酵素組成物及び糖液の製造方法
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