葡萄糖氧化酶GOD突变体及其基因和应用的制作方法

本发明属于农业生物技术领域，具体涉及葡萄糖氧化酶GOD突变体及其基因和应用。

背景技术：

葡萄糖氧化酶(glucose oxidase，GOD)是一种黄素蛋白，其能够以分子氧为电子受体，高度专一的将β-D-葡萄糖氧化成葡萄糖酸内酯和过氧化氢，因此又称为需氧脱氢酶。催化反应过程大致分为两个部分：还原反应，GOD将β-D-葡萄糖氧化成葡萄糖酸内酯，而葡萄糖酸内酯能够在非酶促反应下转化成葡萄糖酸，随后GOD中的辅因子黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)被还原成FADH2；氧化反应，还原态的GOD-FADH2通过与分子氧反应被重新氧化成GOD-FAD，并生成H2O2。GOD的应用范围十分广泛，横跨了化学、制药、食品、临床诊断、生物技术等众多领域。尤其是在畜牧业领域，GOD的出现改变了传统的畜禽预防保健单一针对病原菌的模式。基于反应过程中需要消耗氧气，产生葡萄糖酸和过氧化氢的特性，GOD在保护肠道方面并存了五项生理功效：①保护肠道上皮细胞完整；②改善肠道酸性消化环境；③控制肠道病菌生长繁殖；④保持肠道菌群生态平衡；⑤解除肠道霉菌毒素中毒。

目前，工业化生产GOD的菌株主要来源于黑曲霉和青霉。这些野生型霉菌菌株生产GOD的产量较低，异源表达后可显著提高表达量。尽管如此，在GOD的实际应用过程中，对GOD的热稳定性和催化活性提出了更高的要求。

技术实现要素：

为了解决现有的葡萄糖氧化酶热稳定性、催化活性有待提高的问题，本发明提供葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4和GOD-M5，其热稳定性及酶活力较野生葡萄糖氧化酶均有提高。

本发明的目的是提供葡萄糖氧化酶GOD突变体。

本发明的再一目的是提供编码上述葡萄糖氧化酶GOD突变体的基因。

本发明的再一目的是提供包含上述葡萄糖酶GOD突变体基因的重组载体。

本发明的再一目的是提供包含上述葡萄糖酶GOD突变体基因的重组菌株。

本发明的再一目的是提供制备上述葡萄糖酶GOD突变体的基因工程方法。

本发明的再一目的是提供上述葡萄糖酶GOD突变体的应用。

根据本发明的具体实施方式，野生型葡萄糖氧化酶GOD的氨基酸序列如SEQ ID NO.1所示：

GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVELATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYNFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*

根据本发明的具体实施方式，将野生型葡萄糖氧化酶GOD氨基酸序列的第82位Glu突变为Cys，获得葡萄糖氧化酶突变体GOD-M1；将突变体GOD-M1的第418位Val突变为Glu，获得突变体GOD-M2；将突变体GOD-M2的第508位Asn突变为His，获得突变体GOD-M3；将突变体GOD-M3的第32位Thr突变为Val，获得突变体GOD-M4；将突变体GOD-M4的第313位Asp突变为Lys，获得突变体GOD-M5。

根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M1的氨基酸序列如SEQ ID NO.2所示：

GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGVASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYNFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*

根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M2的氨基酸序列如SEQ ID NO.3所示：

GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYNFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*

根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M3的氨基酸序列如SEQ ID NO.4所示：

GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTTAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*

根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M4的氨基酸序列如SEQ ID NO.5所示：

GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTVAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIDTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*

根据本发明的具体实施方式，葡萄糖氧化酶突变体GOD-M5的氨基酸序列如SEQ ID NO.6所示：

GIEASLLTDPKEVAGRTVDYIIAGGGLTGLTVAARLTENPDITVLVIESGSYESDRGPIIEDLNAYGDIFGSSVDHAYETVCLATNNQTALIRSGNGLGGSTLVNGGTWTRPHKAQVDSWETVFGNEGWNWDSVAAYSLQAERARAPNAKQIAAGHYFNASCHGINGTVHAGPRDTGDDYSPIVKALMSAVEDRGVPTKKDLGCGDPHGVSMFPNTLHEDQVRSDAAREWLLPNYQRPNLQVLTGQYVGKVLLSQNATTPRAVGVEFGTHKGNTHNVYAKHEVLLAAGSAVSPTILEYSGIGMKSILEPLGIKTVVDLPVGLNLQDQTTSTVRSRITSAGAGQGQAAWFATFNETFGDYTEKAHELLNTKLEQWAEEAVARGGFHNTTALLIQYENYRDWIVKDNVAYSELFLDTAGEASFDVWDLLPFTRGYVHILDKDPYLRHFAYDPQYFLNELDLLGQAAATQLARNISNSGAMQTYFAGETIPGDNLAYDADLRAWVEYIPYHFRPNYHGVGTCSMMPKEMGGVVDNAARVYGVQGLRVIDGSIPPTQMSSHVMTVFYAMALKIADAVLADYASMQ*

本发明提供了编码上述葡萄糖氧化酶GOD突变体的基因。

本发明提供了包含上述化葡萄糖氧化酶GOD突变体基因的重组载体。

本发明还提供了包含上述葡萄糖氧化酶GOD突变体基因的重组菌株，优选所述菌株为大肠杆菌、酵母菌(毕赤酵母细胞、啤酒酵母细胞或多型逊酵母细胞等)、芽孢杆菌或乳酸杆菌。其中，优选将重组表达质粒转化毕赤酵母细胞，得到重组菌株分别为GS115/GOD-M1，GS115/GOD-M2，GS115/GOD-M3，GS115/GOD-M4，GS115/GOD-M5。

根据本发明的具体实施方式，制备葡萄糖氧化酶GOD突变体的方法，包括如下步骤：

(1)用含有葡萄糖氧化酶GOD突变体基因的重组载体转化宿主细胞，得重组菌株；

(2)培养重组菌株，诱导重组葡萄糖氧化酶GOD突变体表达；

(3)回收并纯化所表达的葡萄糖氧化酶GOD突变体。

相对于野生型葡萄糖氧化酶GOD的比活229.6U/mg，本发明改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的酶活力分别提高到了352.5U/mg、366.8U/mg、379.8U/mg、392.1U/mg、381.2U/mg，提高幅度分别为54％、59.8％、65.4％、70.8％、66％。

在70℃下处理10min后，野生型葡萄糖氧化酶GOD的剩余酶活为14.5U/mg，改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为55.9U/mg、73.1U/mg、179.2U/mg、189.8U/mg、211.2U/mg，分别提高了2.6、4.0、11.4、12.1、13.6倍。

在80℃下处理2min后，野生型GOD的剩余酶活为4.5U/mg，改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为23.6U/mg、35.5U/mg、98.6U/mg、117.2U/mg、137.0U/mg，分别提高了4.2、6.9、20.9、25.0、29.4倍。

本发明提供了一种高酶活力与热稳定性提高的葡萄糖氧化酶GOD突变体，其能很好的满足食品、医药、饲料以及纺织工业等领域中应用的需求，适合于在食品、医药、饲料以及纺织工业等领域中应用，有着非常广阔的应用前景。

附图说明

图1显示野生型和各突变体在70℃下处理10min的稳定性；

图2显示野生型和各突变体在80℃下处理2min的稳定性。

具体实施方式

试验材料和试剂

1、菌株及载体：表达宿主Pichia pastoris GS115，表达质粒载体pPIC9。

2、酶类及其它生化试剂：点突变试剂盒及其它生化试剂都为生化试剂公司购得。

3、培养基：

(1)LB培养基：0.5％酵母提取物，1％蛋白胨，1％NaCl，pH 7.0；

(2)YPD培养基：1％酵母提取物，2％蛋白胨，2％葡萄糖；

(3)MD固体培养基：2％葡萄糖，1.5％琼脂糖，1.34％YNB，0.00004％Biotin；

(4)MM固体培养基：1.5％琼脂糖，1.34％YNB，0.00004％Biotin，0.5％甲醇；

(5)BMGY培养基：1％酵母提取物，2％蛋白胨，1％甘油(V/V)，1.34％YNB，0.00004％Biotin；

(6)BMMY培养基：1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％Biotin，0.5％甲醇(V/V)。

实施例1定点突变

野生型葡萄糖氧化酶基因god克隆于黑曲霉。

以来源于Aspergillus niger的葡萄糖氧化酶GOD作为母本，将氨基酸序列如SEQ ID NO.1所示的葡萄糖氧化酶GOD的第82位Glu突变为Cys，获得突变体GOD-M1；将突变体GOD-M1的第418位Val突变成Glu，获得突变体GOD-M2；将突变体GOD-M2的第508位Asn突变成His，获得突变体GOD-M3；将突变体GOD-M3的第32位Thr突变成Val，获得突变体GOD-M4；将突变体GOD-M4的第313位Asp突变成Lys，获得突变体GOD-M5。

通过定点突变PCR的方法引入突变位点，并对其进行测序验证。设计所用引物如表1所示：

表1.所述突变体特异性引物

实施例2制备葡萄糖氧化酶GOD突变体

将PCR产物中加入1μL DMT酶，混匀后于37℃孵育1h。取2-5μL DMT酶消化产物热击转化至感受态细胞中。测序验证正确后，将含有所述突变体基因的重组质粒分别转化毕赤酵母GS115感受态细胞，获得重组酵母菌株GS115/GOD-M1，GS115/GOD-M2，GS115/GOD-M3，GS115/GOD-M4，GS115/GOD-M5。

取含有重组质粒的GS115菌株，接种于300mL BMGY培养基的1L三角瓶中，置于30℃，220rpm摇床培养48h；后将培养液3000g离心5min，弃上清，沉淀用100mL含有0.5％甲醇的BMMY培养基重悬，并再次置于30℃，220rpm条件下诱导培养。每隔12h补加0.5mL甲醇，使菌液中的甲醇浓度保持在0.5％，同时取上清用于酶活性检测。

实施例3分析葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶的活性

一、采用邻联茴香胺法对葡萄糖氧化酶GOD酶活进行测定

具体方法如下：在pH6.0的条件下，3mL的反应体系包括2.5mL邻联茴香胺缓冲液(0.2mL的1％邻联茴香胺加入到25mL的0.1M磷酸缓冲液中)，300μL 18％葡萄糖溶液，100μL 0.03％辣根过氧化物酶，100μL适当的稀释酶液。在30℃下反应3min后，用2mL 2M H2SO4终止反应，并在540nm下测定吸光值。1个酶活单位(U)定义为在给定的条件下，单位时间内生成1μmol葡萄糖酸和过氧化氢所需的酶量。

二、测定葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶的酶活及热稳定性

1、测定葡萄糖氧化酶GOD突变体和野生型的酶活性:

将实施例2纯化的葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶GOD在pH 6.0、30℃下进行酶促反应以测定其酶活性。

野生型葡萄糖氧化酶GOD的比活为29.6U/mg，改造后的葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的酶活力分别提高到352.5U/mg、366.8U/mg、379.8U/mg、392.1U/mg、381.2U/mg，提高幅度分别为54％、59.8％、65.4％、70.8％、66％。

2、测定葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶GOD在70℃或80℃下的热稳定性

在0.1mol/L柠檬酸-磷酸氢二钠缓冲液(pH 6.0)缓冲液体系中，葡萄糖氧化酶GOD突变体和野生型葡萄糖氧化酶GOD分别于70℃下处理10min，80℃下处理2min，再在30℃下测定剩余酶活性。

如图1所示，70℃下处理10min后，野生型葡萄糖氧化酶GOD的剩余酶活为14.5U/mg，葡萄糖氧化酶突变体GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为55.9U/mg、73.1U/mg、179.2U/mg、189.8U/mg、211.2U/mg，分别提高了2.6、4.0、11.4、12.1、13.6倍。

如图2所示，在80℃下处理2min后，野生型葡萄糖氧化酶GOD的剩余酶活为4.5U/mg，改造后的葡萄糖氧化酶GOD-M1、GOD-M2、GOD-M3、GOD-M4、GOD-M5的剩余酶活分别为23.6U/mg、35.5U/mg、98.6U/mg、117.2U/mg、137.0U/mg，分别提高了4.2、6.9、20.9、25.0、29.4倍。

序列表

<110> 中国农业科学院饲料研究所

<120> 葡萄糖氧化酶GOD突变体及其基因和应用

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 581

<212> PRT

<213> 黑曲霉(Aspergillus niger)

<400> 1

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala

130 135 140

Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly

260 265 270

Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 2

<211> 581

<212> PRT

<213> 黑曲霉(Aspergillus niger)

<400> 2

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala

130 135 140

Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly

260 265 270

Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 3

<211> 581

<212> PRT

<213> 黑曲霉(Aspergillus niger)

<400> 3

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala

130 135 140

Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly

260 265 270

Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 4

<211> 581

<212> PRT

<213> 黑曲霉(Aspergillus niger)

<400> 4

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala

130 135 140

Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly

260 265 270

Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 5

<211> 581

<212> PRT

<213> 黑曲霉(Aspergillus niger)

<400> 5

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala

130 135 140

Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly

260 265 270

Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580

<210> 6

<211> 581

<212> PRT

<213> 黑曲霉(Aspergillus niger)

<400> 6

Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Glu Val Ala Gly Arg

1 5 10 15

Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Val

20 25 30

Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val Ile Glu

35 40 45

Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn

50 55 60

Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr

65 70 75 80

Val Cys Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn

85 90 95

Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro

100 105 110

His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly

115 120 125

Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala

130 135 140

Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala

145 150 155 160

Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr

165 170 175

Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu

180 185 190

Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp Pro His

195 200 205

Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser

210 215 220

Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu

225 230 235 240

Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn

245 250 255

Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly

260 265 270

Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly

275 280 285

Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys

290 295 300

Ser Ile Leu Glu Pro Leu Gly Ile Lys Thr Val Val Asp Leu Pro Val

305 310 315 320

Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser Arg Ile

325 330 335

Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe

340 345 350

Asn Glu Thr Phe Gly Asp Tyr Thr Glu Lys Ala His Glu Leu Leu Asn

355 360 365

Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe

370 375 380

His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp

385 390 395 400

Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala

405 410 415

Gly Glu Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly

420 425 430

Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe Ala Tyr

435 440 445

Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala

450 455 460

Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr

465 470 475 480

Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala

485 490 495

Asp Leu Arg Ala Trp Val Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn

500 505 510

Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly

515 520 525

Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val

530 535 540

Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr

545 550 555 560

Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Val Leu Ala Asp

565 570 575

Tyr Ala Ser Met Gln

580