CN110904078A - 一种耐硫酸钠和硫酸铵的木糖苷酶突变体v322r及其应用 - Google Patents

一种耐硫酸钠和硫酸铵的木糖苷酶突变体v322r及其应用 Download PDF

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CN110904078A
CN110904078A CN201911269845.XA CN201911269845A CN110904078A CN 110904078 A CN110904078 A CN 110904078A CN 201911269845 A CN201911269845 A CN 201911269845A CN 110904078 A CN110904078 A CN 110904078A
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周峻沛
黄遵锡
张蕊
李娜
韩楠玉
唐湘华
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Yunnan University YNU
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Abstract

本发明涉及基因工程及蛋白质改造技术领域，公开了一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R及其应用，突变体V322R的氨基酸序列如SEQ ID NO.1所示。V322R的最适pH为7.0；最适温度为25℃，在0℃、10℃和40℃分别具有18.5％、48.2％和26.0％的酶活；在3.0～30.0％(w/v)的Na2SO4和(NH4)2SO4中，V322R的活性和稳定性不仅没有下降，反而得到提高。本发明的耐硫酸钠和硫酸铵的木糖苷酶突变体V322R可应用于制革、造纸、污水处理等行业。

Description

一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R及其应用
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，具体为一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R及其应用。
背景技术
木聚糖是半纤维素中最丰富的一种多糖，其主链由木糖聚合而成，完全水解木聚糖需要多种酶的协同作用，包括内切木聚糖酶(endo-1,4-β-D-xylanase，EC 3.2.1.8)和木糖苷酶(β-D-xylosidase，EC3.2.1.37)等；内切木聚糖酶可随机地切割木聚糖的主链骨架，生成低聚木糖，而木糖苷酶可将低聚木糖水解为木糖(Collins et al.FEMS MicrobiologyReviews,2005,29:3～23.)。木糖可作为原料，经工程酵母发酵后生产乙醇、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，蛋白聚糖的糖链与核心蛋白间可通过三糖单元(β-1,3-半乳糖-β-1,3半乳糖-β-1,4木糖)与丝氨酸或苏氨酸以O-糖苷键连接，其也可被木糖苷酶降解(Takagaki et al.The Journal of Biological Chemistry,1990,265:854～860.)。
硫酸盐等盐类广泛存在于在自然界和各种生产实践中，包括污水、洗涤、制革、造纸等。例如，在皮革软化过程中，需要添加硫酸钠，并产生了含硫酸钠的废水。在皮革或裘革的制作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)。因此，在硫酸盐中具有良好活性和稳定性的酶能够具有更好的应用性，具有重要的开发价值。
发明内容
针对上述技术问题，本发明的目的旨在提供一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R，可应用于制革、造纸、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，设计了一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R，所述的突变体V322R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，V322R的第322位氨基酸为精氨酸，而AQM74402的第322位氨基酸为缬氨酸。
所述的突变体V322R的最适pH为7.0；最适温度为25℃，在0℃、10℃和40℃分别具有18.5％、48.2％和26.0％的酶活；V322R在20℃下稳定，在25℃时半衰期约为20min；在反应体系中加入3.0～30.0％(w/v)的Na2SO4，对V322R不仅没有抑制作用，反而使V322R的酶活提高10～42％；V322R在Na2SO4中极稳定，该酶经3.0～30.0％(w/v)的Na2SO4处理60min后，活性不仅没有下降，反而提高7～40％；在反应体系中加入3.0～30.0％(w/v)的(NH4)2SO4，对V322R不仅没有抑制作用，反而使V322R的酶活提高3～31％；V322R在(NH4)2SO4中极稳定，该酶经3.0～30.0％(w/v)的(NH4)2SO4处理60min后，活性不仅没有下降，反而提高9～27％。
本发明提供了所述的耐硫酸钠和硫酸铵的木糖苷酶突变体V322R的编码基因v322r，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一种包含木糖苷酶突变体V322R编码基因的重组载体。
本发明的另一目的在于提供一种包含木糖苷酶突变体V322R编码基因的重组菌。
另外，本发明所述的木糖苷酶突变体V322R在制革、造纸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钠和硫酸铵的木糖苷酶突变体V322R的制备方法，具体包括以下步骤：
1)合成突变体V322R的编码基因v322r(SEQ ID NO.2)；
2)将1)中合成的序列和表达载体pEasy-E1相连接，即可获得突变体V322R的表达载体；
3)将连接产物转化大肠杆菌BL21(DE3)，获得包含v322r的重组菌株；
4)培养重组菌株，诱导木糖苷酶突变体V322R表达；
5)回收并纯化所表达的木糖苷酶突变体V322R。
本发明的有益效果为：
与野生酶HJ14GH43和突变酶V322D相比，突变酶V322R在Na2SO4和(NH4)2SO4中的稳定性得到了增强。随着Na2SO4的浓度增高，HJ14GH43的稳定性逐渐变差，该酶经3.0～30.0％(w/v)的Na2SO4处理60min后，活性从85％降至47％；V322D在Na2SO4中比HJ14GH43稳定，V322D经3.0～10.0％(w/v)的Na2SO4处理60min后，活性不变，但V322D经15.0～30.0％(w/v)的Na2SO4处理60min后，活性从89％降至65％；V322R在Na2SO4中极稳定，V322R经3.0～30.0％(w/v)的Na2SO4处理60min后，活性不仅没有下降，反而提高7～40％。经20.0～30.0％(w/v)的(NH4)2SO4处理60min后，HJ14GH43的活性从79％降至38％，V322D的活性从79％降至60％；而V322R在(NH4)2SO4中极稳定，V322R经3.0～30.0％(w/v)的(NH4)2SO4处理60min后，活性不仅没有下降，反而提高9～27％。本发明的耐硫酸钠和硫酸铵的木糖苷酶突变体V322R可应用于制革、造纸、污水处理等行业。
附图说明
图1是野生酶HJ14GH43和突变酶V322D及V322R的SDS-PA GE分析，其中，M：蛋白质Marker；W：HJ14GH43；
图2是纯化的突变酶V322R的pH活性；
图3是纯化的突变酶V322R的pH稳定性；
图4是纯化的突变酶V322R的热活性；
图5是纯化的突变酶V322R的热稳定性；
图6是纯化的野生酶HJ14GH43和突变酶V322D及V322R在Na2SO4中的活性；
图7是纯化的野生酶HJ14GH43和突变酶V322D及V322R在Na2SO4中的稳定性；
图8是纯化的野生酶HJ14GH43和突变酶V322D及V322R在(NH4)2SO4中的活性；
图9是纯化的野生酶HJ14GH43和突变酶V322D及V322R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：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。
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V322R的编码基因v322r(SEQ ID NO.2)；
2)将(1)中合成的序列分别和表达载体pEasy-E1相连接，即可分别获得野生酶HJ14GH43和突变酶V322D及V322R的表达载体；
3)将连接产物分别转化大肠杆菌BL21(DE3)，获得分别包含hJ14GH43、v322d和v322r的重组菌株。
实施例2野生酶HJ14GH43和突变酶V322D及V322R的制备
将含hJ14GH43、v322d和v322r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pH7.0Tris～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-NTA Agarose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V322R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V322R的性质测定
采用pNP法测定纯化的野生酶HJ14GH43和突变酶V322D及V322R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2mL1MNa2CO3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)纯化的突变酶V322R的pH活性和pH稳定性测定
pH活性和pH稳定性测定采用pNP法。酶的最适pH测定：将酶液置于20℃下和pH6.0～9.0的缓冲液中进行酶促反应。酶的pH稳定性测定：将酶液置于pH6.0～9.0的缓冲液中，在20℃下处理1h，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。缓冲液为：McIlvaine buffer(pH6.0～8.0)和0.1M glycine～NaOH(pH9.0)。以pNPX为底物，反应10min，测定纯化的突变酶V322R的酶学性质。
结果表明：突变酶V322R在pH6.0～9.0中具有活性，最适pH为7.0(图2)；突变酶V322R在pH7.0～8.0的缓冲液中保持稳定，经pH7.0～8.0缓冲液在20℃下处理1h，酶活剩余达89％以上(图3)。
2)纯化的突变酶V322R的热活性及热稳定性测定
热活性及热稳定性测定采用pNP法。酶的热活性测定：在pH7.0的缓冲液中，于0～40℃下进行酶促反应。酶的热稳定性测定：将同样酶量的酶液分别置于20℃、25℃和30℃，处理0～60min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，测定纯化的突变酶V322R的酶学性质。
结果表明：突变酶V322R的最适温度为25℃，在0℃、10℃和40℃分别具有18.5％、48.2％和26.0％的酶活(图4)；V322R在20℃下稳定，在25℃时半衰期约为20min，在30℃时半衰期小于10min(图5)。
3)纯化的野生酶HJ14GH43和突变酶V322D及V322R在Na2SO4中的活性及稳定性
酶在Na2SO4中的活性及稳定性测定采用pNP法。酶在Na2SO4中的活性测定：在酶促反应体系中加入3.0～30.0％(w/v)Na2SO4，于pH7.0及20℃下进行酶促反应。以pNPX为底物，反应10min，测定纯化的HJ14GH43以及突变酶V322D及V322R的酶学性质。
结果表明：在反应体系中加入3.0～30.0％(w/v)的Na2SO4，对HJ14GH43、V322D和V322R不仅没有抑制作用，反而使HJ14GH43、V322D和V322R的酶活提高10～50％(图6)。
酶在Na2SO4中的稳定性测定：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以及突变酶V322D及V322R的酶学性质。
结果表明：随着Na2SO4的浓度增高，HJ14GH43的稳定性逐渐变差，该酶经3.0～30.0％(w/v)的Na2SO4处理60min后，活性从85％降至47％；V322D在Na2SO4中比HJ14GH43稳定，V322D经3.0～10.0％(w/v)的Na2SO4处理60min后，活性不变，但V322D经15.0～30.0％(w/v)的Na2SO4处理60min后，活性从89％降至65％；V322R在Na2SO4中极稳定，其稳定性远优于野生酶HJ14GH43和突变酶V322D，V322R经3.0～30.0％(w/v)的Na2SO4处理60min后，活性不仅没有下降，反而提高7～40％(图7)。
4)纯化的野生酶HJ14GH43和突变酶V322D及V322R在(NH4)2SO4中的活性及稳定性
酶在(NH4)2SO4中的活性及稳定性测定采用pNP法。酶在(NH4)2SO4中的活性测定：在酶促反应体系中加入3.0～30.0％(w/v)(NH4)2SO4，于pH7.0及20℃下进行酶促反应。以pNPX为底物，反应10min，测定纯化的HJ14GH43以及突变酶V322D及V322R的酶学性质。
结果表明：在反应体系中加入3.0～30.0％(w/v)的(NH4)2SO4，对HJ14GH43、V322D和V322R不仅没有抑制作用，反而可提高HJ14GH43、V322D和V322R的酶活(图8)。
酶在(NH4)2SO4中的稳定性测定：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以及突变酶V322D及V322R的酶学性质。
结果表明：HJ14GH43和V322D在3.0～15.0％(w/v)的(NH4)2SO4中保持稳定，但是经20.0～30.0％(w/v)的(NH4)2SO4处理60min后，HJ14GH43的活性从79％降至38％，V322D的活性从79％降至60％；V322R在(NH4)2SO4中极稳定，其稳定性远优于野生酶HJ14GH43和突变酶V322D，V322R经3.0～30.0％(w/v)的(NH4)2SO4处理60min后，活性不仅没有下降，反而提高9～27％(图9)。
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
<110> 云南师范大学
<120> 一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R及其应用
<160> 6
<170> SIPOSequenceListing 1.0
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<213> 突变体V322R(mutant V322R)
<400> 1
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cattcaaagg atctcgtcca ttggcgtctt gctgcgcgtc cattgcaaaa aacgtcgcag 180
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ccgattaaac tgacagaagc gccgcatctg tatcatatcg gtgactacta ctatttatta 600
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gcaagacgct ggcaaagctt ttattttgaa gcagagacag ctgtttcgtt cttcccagaa 1200
aactttcagc aagccgcagg tcttgtgaat tattataata cggaaaactg gacagcactc 1260
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gccttttctc agccgttgac acataaaatc atcattcctg acgaggtcac ttatgtctat 1380
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tggaaggaaa ttgatgtacc gtttgaatcc atccatttat ccgatgattt cattcgaggt 1500
gggggttttt ttacaggggc atttgtcggt atgcagtgcc aagatacgag cggcgagcgt 1560
cttcctgctg attttcacta ttttcgctat gaggaaacag acgaataa 1608
<210> 3
<211> 535
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<210> 4
<211> 1608
<212> DNA
<213> 野生酶基因hJ14GH43(Wild enzyme gene HJ14GH43)
<400> 4
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<210> 5
<211> 535
<212> PRT
<213> 突变体V322D(mutant V322D)
<400> 5
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<210> 6
<211> 1608
<212> DNA
<213> 突变体v322d(mutant v322d)
<400> 6
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cattcaaagg atctcgtcca ttggcgtctt gctgcgcgtc cattgcaaaa aacgtcgcag 180
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gatgggcagt tttggcttat ttattcagat atcaaagtag tggatggccc atttaaagac 300
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ctcaacagct ctggctttga tccatcttta ttccatgatc acagcgggaa gaaatacgtc 420
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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
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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)

1.一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R，其特征在于，所述的突变体V322R由木糖苷酶序列AQM74402第322位的缬氨酸突变为精氨酸得到。
2.根据权利要求1所述的一种耐硫酸钠和硫酸铵的木糖苷酶突变体V322R，其特征在于，所述的突变体V322R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V322R的编码基因v322r，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V322R的制备方法，其特征在于，包括以下步骤：
1)合成突变体V322R的编码基因；
2)将(1)中合成的序列和表达载体pEasy-E1相连接，即可获得突变体V322R的表达载体；
3)将连接产物转化大肠杆菌BL21(DE3)，获得包含v322r的重组菌株；
4)培养重组菌株，诱导木糖苷酶突变体V322R表达；
5)回收并纯化所表达的木糖苷酶突变体V322R。
7.权利要求1所述的突变体V322R在制革、造纸和污水处理中的应用。
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