一种麦芽糖淀粉酶突变体的制备及其应用的制作方法

本发明涉及一种麦芽糖淀粉酶突变体的制备及其应用，属于酶工程领域。

背景技术：

麦芽糖淀粉酶(maltogenicamylase或maltogenase，ec3.2.1.133)是糖苷水解酶gh-h系成员。目前，麦芽糖淀粉酶主要的细菌来源为嗜热脂肪芽孢杆菌(bacillusstearothermophilus)、蜡状芽孢杆菌(bacilluscereus)、枯草芽孢杆菌(bacillussubtilis)、地衣芽孢杆菌(bacilluslicheniformis)、嗜热放线菌(thermusvulgaris)以及栖热菌属(thermussp.)等。不同来源的麦芽糖淀粉酶，在性质上也有很大的区别。目前应用于麦芽糖浆制备和抗面包老化的麦芽糖淀粉酶，主要来源于嗜热脂肪芽孢杆菌。

麦芽糖是由两个葡萄糖单位经α-1，4糖苷键连接组成的还原性二糖，化学名称是4-o-d-六环葡萄糖基-d-六环葡萄糖。其甜度柔和，又因低粘度、低吸湿性及良好的热稳定性特点，可作为食品增甜剂取代葡萄糖和蔗糖，在食品工业领域具有巨大的应用潜力。工业上麦芽糖的制备，是以淀粉质为原料，经过α-淀粉酶，麦芽(或β-淀粉酶，真菌淀粉酶)水解工艺，制得一种以麦芽糖为主(40％-60％)的糖浆，若麦芽糖含量超过45％(最好在50％以上)，则被称为高麦芽糖浆。在食品工业中高麦芽糖浆的用途之一是制作糕点、糖果等产品。糖浆熬煮温度远高于饴糖，一般超过140℃。麦芽糖含量大于70％，甚至高达90％以上，则被称为超高麦芽糖浆。麦芽糖相比葡萄糖可避免血糖升高，对于应用于抗体、疫苗等的制备具有优于葡萄糖的应用优势。因此超高纯度的麦芽糖浆在医药领域的应用也引起了越来越多的关注。

当前麦芽糖生产工艺已较为成熟，使用α-淀粉酶和β-淀粉酶生产麦芽糖时，产物中麦芽糖含量可高达90％，葡萄糖、三糖、四糖以及部分低聚糖和糊精是主要的转化副产物。其中糊精和部分低聚糖，可通过乙醇沉淀去除。超高纯度麦芽糖的制备，则要通过色谱分离和结晶等方法获得。由于麦芽糖黏度大、难结晶，通常要求结晶原料中麦芽糖纯度在90％以上，因此色谱分离的纯度，对麦芽糖结晶起着至关重要的作用。色谱分离基本可去除葡萄糖和五糖及以上的小分子糖类，对麦芽糖纯度影响较小。但产物中的三糖和四糖由于与麦芽糖性质较为相近，往往成为分离纯化中的主要杂质，不仅直接降低了产品纯度，还给麦芽糖结晶性、糖浆粘度以及最终产品的水分含量带来很大不利影响，使麦芽糖最终收率大大降低。

麦芽糖淀粉酶具有小分子糖水解活性，可水解三糖、四糖等小分子糖，形成葡萄糖和麦芽糖，因此在超高麦芽糖生产中通常与α-淀粉酶、β-淀粉酶和普鲁兰酶等复配使用以降低副产物比例，使麦芽糖更利于结晶。据报道，来源于嗜热脂肪芽孢杆菌(bacillusstearothermophilus)的麦芽糖淀粉酶具有较高的最适反应温度和较低的最适ph反应条件，可满足较为苛刻的工业生产条件，将产物中麦芽糖比例提高至92％，在工业上有极大的应用优势。

技术实现要素：

本发明的第一个目的是提供一种麦芽糖淀粉酶的突变体，含有seqidno.1～8任一所示的序列。

本发明的第二个目的是提供一种提高麦芽糖淀粉酶转化效率的方法，所述方法是将ncbi登录号：aaa22233.1的麦芽糖淀粉酶为出发序列，分别对第198位和/或290位氨基酸进行突变。

在本发明的一种实施方式中，所述方法是将第198位的赖氨酸(lys)分别突变成谷氨酸(glu)和酪氨酸(tyr)，标记为k198e，k198y。

在本发明的一种实施方式中，所述方法是将麦芽糖淀粉酶中第290位的天冬氨酸(asp)分别突变成甘氨酸(g,ly)和苏氨酸(thr)。

在本发明的一种实施方式中，所述方法是将第198位的赖氨酸(lys)分别突变成谷氨酸(glu)和酪氨酸(tyr)，并将突变后的第290位的天冬氨酸(asp)分别突变成甘氨酸(g,ly)和苏氨酸(thr)。

本发明的第三个目的是提供一种制备所述麦芽糖淀粉酶突变体的方法，所述方法是将所述突变体在细胞中表达，并在培养基中培养，收集培养液，获得麦芽糖淀粉酶。

在本发明的一种实施方式中，所述方法是将所述突变体在细菌细胞中表达。

在本发明的一种实施方式中，所述方法是将所述突变体在真菌细胞中表达。

本发明的第四个目的是提供一种生产麦芽糖的方法，所述方法是以淀粉液为底物，以所述麦芽糖淀粉酶突变体为催化剂。

在本发明的一种实施方式中，所述转化是在50～65℃反应16～60h。

本发明还要求保护所述麦芽糖突变体在制备含麦芽糖的产品方面的应用。

有益效果：本发明构建的麦芽糖淀粉酶突变体在20u/g干淀粉的添加量下，二次糖化过程中突变体k198e/d290t可使麦芽糖含量在转化反应进行10h时提高至97％以上，三糖、四糖含量在反应5h时趋近于0。与野生型麦芽糖淀粉酶相比，具有更高的转化效率以及更少的转化副产物，具有重要的工业应用潜力。

附图说明

图1为野生型麦芽糖淀粉酶(wt)和突变体(k198e/d290t)生产麦芽糖过程中麦芽糖含量(a)、三糖含量(b)、四糖含量(c)变化。

具体实施方式

葡萄糖、麦芽糖、三糖和四糖的含量采用hplc分析。测定的色谱条件是：elite2000hplc色谱仪，elite自动进样器，色谱柱thermoaps-2hypersil13286(4.6mm×250mm)，hitachil-2490示差检测器；流动相为70％(v/v)乙腈水溶液，流速0.8ml/min；柱温40℃。

实施例1：野生麦芽糖淀粉酶的制备。

(1)麦芽糖淀粉酶重组菌的构建

根据ncbi上的amym氨基酸序列(ncbi编号：aaa22233.1)，对该序列进行密码子优化，采用化学全合成方法合成麦芽糖淀粉酶的基因序列amym。用于构建大肠杆菌表达载体的质粒是pet24a(+)。将pet24a(+)质粒和带有amym基因的质粒分别进行ncoⅰ和hindⅲ双酶切，酶切产物经胶回收后，用t4连接酶连接过夜，连接产物转化至大肠杆菌jm109感受态细胞，转化产物涂布于含100mg/l卡那霉素的lb平板，经37℃培养过夜，平板上挑取2个单菌落，接入lb液体培养基，8h后抽提质粒验证，结果正确，得到富集的amym/pet24a质粒。将质粒amym/pet24a转化大肠杆菌bl21(de3)感受态细胞，挑取转化子在lb液体培养基(含100mg/l卡那霉素)中37℃培养过夜，保存甘油管，命名为amym/pet24a/bl21(de3)。

(2)麦芽糖淀粉酶的表达与纯化

从甘油管接种amym/pet24a/bl21(de3)于lb液体培养基(含100mg/l卡那霉素)生长8h，按5％接种量将种子接入tb液体发酵培养基(含100mg/l卡那霉素)。大肠杆菌在37℃培养2h后，加入0.01mm终浓度的iptg进行诱导，并在25℃摇床继续培养发酵48h后，将发酵液于4℃、8000rpm离心10min除菌体，收集发酵上清。经测定，酶活可达4892u/ml。

在上清液中缓慢加入50％(nh4)2so4,4℃放置过夜，4℃、8000rpm离心20min，收集沉淀。用ph7.5的20mm柠檬酸缓冲液复溶沉淀后，在20mm柠檬酸缓冲液中透析过夜。期间更换2-3次缓冲液。通过0.22μm膜过滤后制成上样样品，采用avant蛋白纯化仪进行重组蛋白的纯化。阴离子交换色谱纯化步骤：(1)平衡：用5倍体积的20mm缓冲液平衡deae阴离子交换色谱柱；(2)上样：预先处理好的样品以1ml/min的流速上样；(3)洗脱：流速1ml/min进行梯度洗脱，检测波长为290nm，分步收集含麦芽糖淀粉酶活力的洗脱液。得到纯化的野生麦芽糖淀粉酶。

实施例2：麦芽糖淀粉酶突变体制备

(1)单突变

将麦芽糖淀粉酶中第198位的赖氨酸(lys)分别突变成谷氨酸(glu)和酪氨酸(tyr)，标记为k198e，k198y；将麦芽糖淀粉酶中第290位的天冬氨酸(asp)分别突变成甘氨酸(g,ly)和苏氨酸(thr)，分别标记为d290g，d290t。

引入k198e突变的定点突变引物为：

正向引物：5’-cgacgacgctaccgaaggttacttccacca-3’(下划线为突变碱基)

反向引物：5’-tggtggaagtaaccttcggtagcgtcgtcg-3’(下划线为突变碱基)

引入k198y突变的定点突变引物为：

正向引物：5’-cgacgacgctacctatggttacttccacca-3’(下划线为突变碱基)

反向引物：5’-tggtggaagtaaccataggtagcgtcgtcg-3’(下划线为突变碱基)

引入d290g突变的定点突变引物为：

正向引物：5’-gaatggtacggtgacggtccgggtaccgct-3’(下划线为突变碱基)

反向引物：5’-agcggtacccggaccgtcaccgtaccattc-3’(下划线为突变碱基)

引入d290t突变的定点突变引物为：

正向引物：5’-gaatggtacggtgacacgccgggtaccgct-3’(下划线为突变碱基)

反向引物：5’-agcggtacccggcgtgtcaccgtaccattc-3’(下划线为突变碱基)

利用上述引物，以amym/pet24a质粒为模板，进行pcr反应。反应均在50μl体系中进行，反应条件为：94℃预变性4min；随后进行30个循环(94℃10s，55℃10s，72℃7min20s)；72℃延伸10min；最后4℃保温。pcr产物经dpnⅰ(fermentas公司)消化，分别转化大肠杆菌jm109感受态细胞，转化产物涂布于含100mg/l卡那霉素的lb平板，经37℃培养过夜，平板上挑取2个单菌落，接入lb液体培养基，8h后抽提质粒，经测序均正确，保存甘油管。

(2)突变体酶的表达与纯化

突变体表达及纯化过程如实施例1所述。

(3)双突变

在单突变的基础上，进行组合突变，分别构建突变体k198e/d290g，k198y/d290g，k198e/d290t，k198y/d290t。

实施例3：麦芽糖淀粉酶酶活分析

(1)酶活单位定义

采用3，5-二硝基水扬酸法(dns法)测定麦芽糖淀粉酶活时，每分钟催化产生1μmol还原糖所需的酶量作为一个活力单位。

(2)酶活力测定步骤

预热：取2ml的0.5％可溶性淀粉溶液(50mmph5.5柠檬酸缓冲液)于试管中，置于60℃水浴锅中预热10min。

反应：加入0.1ml样品酶液，振荡均匀，准确计时10min，加入3mldns振荡均匀，放入冰水中终止反应，沸水浴煮沸7min。冷却。

测量：向上述反应体系中加入蒸馏水并定容至15ml，混匀。在540nm波长下测定吸光值并计算酶活力。

野生型麦芽糖淀粉酶和突变体酶的比活力列于下表(将野生型麦芽糖淀粉酶的比活力设为100％，各突变体比活力相对于野生酶比活力的比值为相对比活力)：

表1野生型麦芽糖淀粉酶和突变体酶的比活力及相对比活力

实施例4：酶法生产麦芽糖

配制2l20％(w/v)马铃薯淀粉溶液，调节ph至5.5，加入30u/g干淀粉的酸性α-淀粉酶(购自杰能科)，经喷射液化处理后加入24u/g干淀粉的普鲁兰酶(购自杰能科)和10u/g干淀粉的β-淀粉酶(从红薯中提取)，60℃搅拌处理24h，进行一次糖化。

经过一次糖化，反应体系中葡萄糖、麦芽糖、三糖和四糖含量分别为0.26％，89.73％，9.21％和0.8％。

然后，向一次糖化反应体系中分别加入20u/g干淀粉的野生型麦芽糖淀粉酶和突变体酶k198e、k198e/d290g、k198e/d290t。60℃搅拌，进行二次糖化。反应至25h取样测定产物组成。

反应体系中各组分含量如表2所示：

表2不同麦芽糖淀粉酶的糖化效果(反应20h)

对反应过程中各物质含量进行监测，结果如图1所示，突变体k198e/d290t可使麦芽糖含量在转化反应进行10h时提高至97％以上，三糖、四糖含量在反应5h时趋近于0。

虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

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alaglypheserleualaaspleuserglngluasnglythrileala

225230235240

glntyrleuthraspalaalavalglnleuvalalahisglyleuarg

245250255

ileaspalavallyshispheasnserglypheserlysserleuala

260265270

asplysleutyrglnlyslysaspilepheleuvalglyglutrptyr

275280285

glyaspaspproglythralaasnhisleuglulysvalargtyrala

290295300

asnasnserglyvalasnvalleuasppheaspleuasnthrvalile

305310315320

argasnvalpheglythrphethrglnthrmettyraspleuasnasn

325330335

metvalasnglnthrglyasnglutyrlystyrlysgluasnleuile

340345350

thrpheileaspasnhisaspmetserargpheleuservalasnser

355360365

lysasnlysalaasnleuhisglnargleuleuserpheserleuarg

370375380

glyvalargproproiletyrtyrglythrgluglntyrmetalagly

385390395400

glyasnaspprotyrasnargglymetmetproalapheaspthrthr

405410415

thrthralaphelysgluvalserthrleualaglyleuargargasn

420425430

asnalaalaileglntyrglythrthrthrglnargtrpileasnasn

435440445

aspvaltyriletyrgluarglysphepheasnaspvalvalleuval

450455460

alaileasnargasnthrglnsersertyrserileserglyleugln

465470475480

thralaleuproasnglysertyralaasptyrleuserglyleuleu

485490495

glyglyasnglyileservalserasnglyservalalaserphethr

500505510

leualaproglyalavalservaltrpglntyrserthrseralaser

515520525

alaproglnileglyservalalaproasnmetglyileproglyasn

530535540

valvalthrileaspglylysglypheglythrthrglnglythrval

545550555560

thrpheglyglyvalthralathrvallyssertrpthrserasnarg

565570575

ilegluvaltyrvalproasnmetalaalaglyleuthraspvallys

580585590

valthralaglyglyvalserserasnleutyrsertyrasnileleu

595600605

serglythrglnthrservalvalphethrvallysseralapropro

610615620

thrasnleuglyasplysiletyrleuthrglyasnileprogluleu

625630635640

glyasntrpserthraspthrserglyalavalasnasnalaglngly

645650655

proleuleualaproasntyrproasptrpphetyrvalpheserval

660665670

proalaglylysthrileglnphelysphepheilelysargalaasp

675680685

glythrileglntrpgluasnglyserasnhisvalalathrthrpro

690695700

thrglyalathrglyasnilethrvalthrtrpglnasn

705710715

<210>3

<211>717

<212>prt

<213>人工序列

<400>3

metlyslyslysthrleuserleuphevalglyleumetleuleuile

151015

glyleuleupheserglyserleuprotyrasnproasnalaalaglu

202530

alaserserseralaservallysglyaspvaliletyrglnileile

354045

ileaspargphetyraspglyaspthrthrasnasnasnproalalys

505560

sertyrglyleutyraspprothrlysserlystrplysmettyrtrp

65707580

glyglyaspleugluglyvalargglnlysleuprotyrleulysgln

859095

leuglyvalthrthriletrpleuserprovalleuasnasnleuasp

100105110

thrleualaglythraspasnthrglytyrhisglytyrtrpthrarg

115120125

aspphelysglnileglugluhispheglyasntrpthrthrpheasp

130135140

thrleuvalasnaspalahisglnasnglyilelysvalilevalasp

145150155160

phevalproasnhisserthrprophelysalaasnaspserthrphe

165170175

alagluglyglyalaleutyrasnasnglythrtyrmetglyasntyr

180185190

pheaspaspalathrlysglytyrphehishisasnglyaspileser

195200205

asntrpaspaspargtyrglualaglntrplysasnphethrasppro

210215220

alaglypheserleualaaspleuserglngluasnglythrileala

225230235240

glntyrleuthraspalaalavalglnleuvalalahisglyleuarg

245250255

ileaspalavallyshispheasnserglypheserlysserleuala

260265270

asplysleutyrglnlyslysaspilepheleuvalglyglutrptyr

275280285

glyglyaspproglythralaasnhisleuglulysvalargtyrala

290295300

asnasnserglyvalasnvalleuasppheaspleuasnthrvalile

305310315320

argasnvalpheglythrphethrglnthrmettyraspleuasnasn

325330335

metvalasnglnthrglyasnglutyrlystyrlysgluasnleuile

340345350

thrpheileaspasnhisaspmetserargpheleuservalasnser

355360365

lysasnlysalaasnleuhisglnargleuleuserpheserleuarg

370375380

glyvalargproproiletyrtyrglythrgluglntyrmetalagly

385390395400

glyasnaspprotyrasnargglymetmetproalapheaspthrthr

405410415

thrthralaphelysgluvalserthrleualaglyleuargargasn

420425430

asnalaalaileglntyrglythrthrthrglnargtrpileasnasn

435440445

aspvaltyriletyrgluarglysphepheasnaspvalvalleuval

450455460

alaileasnargasnthrglnsersertyrserileserglyleugln

465470475480

thralaleuproasnglysertyralaasptyrleuserglyleuleu

485490495

glyglyasnglyileservalserasnglyservalalaserphethr

500505510

leualaproglyalavalservaltrpglntyrserthrseralaser

515520525

alaproglnileglyservalalaproasnmetglyileproglyasn

530535540

valvalthrileaspglylysglypheglythrthrglnglythrval

545550555560

thrpheglyglyvalthralathrvallyssertrpthrserasnarg

565570575

ilegluvaltyrvalproasnmetalaalaglyleuthraspvallys

580585590

valthralaglyglyvalserserasnleutyrsertyrasnileleu

595600605

serglythrglnthrservalvalphethrvallysseralapropro

610615620

thrasnleuglyasplysiletyrleuthrglyasnileprogluleu

625630635640

glyasntrpserthraspthrserglyalavalasnasnalaglngly

645650655

proleuleualaproasntyrproasptrpphetyrvalpheserval

660665670

proalaglylysthrileglnphelysphepheilelysargalaasp

675680685

glythrileglntrpgluasnglyserasnhisvalalathrthrpro

690695700

thrglyalathrglyasnilethrvalthrtrpglnasn

705710715

<210>4

<211>717

<212>prt

<213>人工序列

<400>4

metlyslyslysthrleuserleuphevalglyleumetleuleuile

151015

glyleuleupheserglyserleuprotyrasnproasnalaalaglu

202530

alaserserseralaservallysglyaspvaliletyrglnileile

354045

ileaspargphetyraspglyaspthrthrasnasnasnproalalys

505560

sertyrglyleutyraspprothrlysserlystrplysmettyrtrp

65707580

glyglyaspleugluglyvalargglnlysleuprotyrleulysgln

859095

leuglyvalthrthriletrpleuserprovalleuasnasnleuasp

100105110

thrleualaglythraspasnthrglytyrhisglytyrtrpthrarg

115120125

aspphelysglnileglugluhispheglyasntrpthrthrpheasp

130135140

thrleuvalasnaspalahisglnasnglyilelysvalilevalasp

145150155160

phevalproasnhisserthrprophelysalaasnaspserthrphe

165170175

alagluglyglyalaleutyrasnasnglythrtyrmetglyasntyr

180185190

pheaspaspalathrlysglytyrphehishisasnglyaspileser

195200205

asntrpaspaspargtyrglualaglntrplysasnphethrasppro

210215220

alaglypheserleualaaspleuserglngluasnglythrileala

225230235240

glntyrleuthraspalaalavalglnleuvalalahisglyleuarg

245250255

ileaspalavallyshispheasnserglypheserlysserleuala

260265270

asplysleutyrglnlyslysaspilepheleuvalglyglutrptyr

275280285

glythraspproglythralaasnhisleuglulysvalargtyrala

290295300

asnasnserglyvalasnvalleuasppheaspleuasnthrvalile

305310315320

argasnvalpheglythrphethrglnthrmettyraspleuasnasn

325330335

metvalasnglnthrglyasnglutyrlystyrlysgluasnleuile

340345350

thrpheileaspasnhisaspmetserargpheleuservalasnser

355360365

lysasnlysalaasnleuhisglnargleuleuserpheserleuarg

370375380

glyvalargproproiletyrtyrglythrgluglntyrmetalagly

385390395400

glyasnaspprotyrasnargglymetmetproalapheaspthrthr

405410415

thrthralaphelysgluvalserthrleualaglyleuargargasn

420425430

asnalaalaileglntyrglythrthrthrglnargtrpileasnasn

435440445

aspvaltyriletyrgluarglysphepheasnaspvalvalleuval

450455460

alaileasnargasnthrglnsersertyrserileserglyleugln

465470475480

thralaleuproasnglysertyralaasptyrleuserglyleuleu

485490495

glyglyasnglyileservalserasnglyservalalaserphethr

500505510

leualaproglyalavalservaltrpglntyrserthrseralaser

515520525

alaproglnileglyservalalaproasnmetglyileproglyasn

530535540

valvalthrileaspglylysglypheglythrthrglnglythrval

545550555560

thrpheglyglyvalthralathrvallyssertrpthrserasnarg

565570575

ilegluvaltyrvalproasnmetalaalaglyleuthraspvallys

580585590

valthralaglyglyvalserserasnleutyrsertyrasnileleu

595600605

serglythrglnthrservalvalphethrvallysseralapropro

610615620

thrasnleuglyasplysiletyrleuthrglyasnileprogluleu

625630635640

glyasntrpserthraspthrserglyalavalasnasnalaglngly

645650655

proleuleualaproasntyrproasptrpphetyrvalpheserval

660665670

proalaglylysthrileglnphelysphepheilelysargalaasp

675680685

glythrileglntrpgluasnglyserasnhisvalalathrthrpro

690695700

thrglyalathrglyasnilethrvalthrtrpglnasn

705710715

<210>5

<211>717

<212>prt

<213>人工序列

<400>5

metlyslyslysthrleuserleuphevalglyleumetleuleuile

151015

glyleuleupheserglyserleuprotyrasnproasnalaalaglu

202530

alaserserseralaservallysglyaspvaliletyrglnileile

354045

ileaspargphetyraspglyaspthrthrasnasnasnproalalys

505560

sertyrglyleutyraspprothrlysserlystrplysmettyrtrp

65707580

glyglyaspleugluglyvalargglnlysleuprotyrleulysgln

859095

leuglyvalthrthriletrpleuserprovalleuasnasnleuasp

100105110

thrleualaglythraspasnthrglytyrhisglytyrtrpthrarg

115120125

aspphelysglnileglugluhispheglyasntrpthrthrpheasp

130135140

thrleuvalasnaspalahisglnasnglyilelysvalilevalasp

145150155160

phevalproasnhisserthrprophelysalaasnaspserthrphe

165170175

alagluglyglyalaleutyrasnasnglythrtyrmetglyasntyr

180185190

pheaspaspalathrtyrglytyrphehishisasnglyaspileser

195200205

asntrpaspaspargtyrglualaglntrplysasnphethrasppro

210215220

alaglypheserleualaaspleuserglngluasnglythrileala

225230235240

glntyrleuthraspalaalavalglnleuvalalahisglyleuarg

245250255

ileaspalavallyshispheasnserglypheserlysserleuala

260265270

asplysleutyrglnlyslysaspilepheleuvalglyglutrptyr

275280285

glyglyaspproglythralaasnhisleuglulysvalargtyrala

290295300

asnasnserglyvalasnvalleuasppheaspleuasnthrvalile

305310315320

argasnvalpheglythrphethrglnthrmettyraspleuasnasn

325330335

metvalasnglnthrglyasnglutyrlystyrlysgluasnleuile

340345350

thrpheileaspasnhisaspmetserargpheleuservalasnser

355360365

lysasnlysalaasnleuhisglnargleuleuserpheserleuarg

370375380

glyvalargproproiletyrtyrglythrgluglntyrmetalagly

385390395400

glyasnaspprotyrasnargglymetmetproalapheaspthrthr

405410415

thrthralaphelysgluvalserthrleualaglyleuargargasn

420425430

asnalaalaileglntyrglythrthrthrglnargtrpileasnasn

435440445

aspvaltyriletyrgluarglysphepheasnaspvalvalleuval

450455460

alaileasnargasnthrglnsersertyrserileserglyleugln

465470475480

thralaleuproasnglysertyralaasptyrleuserglyleuleu

485490495

glyglyasnglyileservalserasnglyservalalaserphethr

500505510

leualaproglyalavalservaltrpglntyrserthrseralaser

515520525

alaproglnileglyservalalaproasnmetglyileproglyasn

530535540

valvalthrileaspglylysglypheglythrthrglnglythrval

545550555560

thrpheglyglyvalthralathrvallyssertrpthrserasnarg

565570575

ilegluvaltyrvalproasnmetalaalaglyleuthraspvallys

580585590

valthralaglyglyvalserserasnleutyrsertyrasnileleu

595600605

serglythrglnthrservalvalphethrvallysseralapropro

610615620

thrasnleuglyasplysiletyrleuthrglyasnileprogluleu

625630635640

glyasntrpserthraspthrserglyalavalasnasnalaglngly

645650655

proleuleualaproasntyrproasptrpphetyrvalpheserval

660665670

proalaglylysthrileglnphelysphepheilelysargalaasp

675680685

glythrileglntrpgluasnglyserasnhisvalalathrthrpro

690695700

thrglyalathrglyasnilethrvalthrtrpglnasn

705710715

<210>6

<211>717

<212>prt

<213>人工序列

<400>6

metlyslyslysthrleuserleuphevalglyleumetleuleuile

151015

glyleuleupheserglyserleuprotyrasnproasnalaalaglu

202530

alaserserseralaservallysglyaspvaliletyrglnileile

354045

ileaspargphetyraspglyaspthrthrasnasnasnproalalys

505560

sertyrglyleutyraspprothrlysserlystrplysmettyrtrp

65707580

glyglyaspleugluglyvalargglnlysleuprotyrleulysgln

859095

leuglyvalthrthriletrpleuserprovalleuasnasnleuasp

100105110

thrleualaglythraspasnthrglytyrhisglytyrtrpthrarg

115120125

aspphelysglnileglugluhispheglyasntrpthrthrpheasp

130135140

thrleuvalasnaspalahisglnasnglyilelysvalilevalasp

145150155160

phevalproasnhisserthrprophelysalaasnaspserthrphe

165170175

alagluglyglyalaleutyrasnasnglythrtyrmetglyasntyr

180185190

pheaspaspalathrgluglytyrphehishisasnglyaspileser

195200205

asntrpaspaspargtyrglualaglntrplysasnphethrasppro

210215220

alaglypheserleualaaspleuserglngluasnglythrileala

225230235240

glntyrleuthraspalaalavalglnleuvalalahisglyleuarg

245250255

ileaspalavallyshispheasnserglypheserlysserleuala

260265270

asplysleutyrglnlyslysaspilepheleuvalglyglutrptyr

275280285

glyglyaspproglythralaasnhisleuglulysvalargtyrala

290295300

asnasnserglyvalasnvalleuasppheaspleuasnthrvalile

305310315320

argasnvalpheglythrphethrglnthrmettyraspleuasnasn

325330335

metvalasnglnthrglyasnglutyrlystyrlysgluasnleuile

340345350

thrpheileaspasnhisaspmetserargpheleuservalasnser

355360365

lysasnlysalaasnleuhisglnargleuleuserpheserleuarg

370375380

glyvalargproproiletyrtyrglythrgluglntyrmetalagly

385390395400

glyasnaspprotyrasnargglymetmetproalapheaspthrthr

405410415

thrthralaphelysgluvalserthrleualaglyleuargargasn

420425430

asnalaalaileglntyrglythrthrthrglnargtrpileasnasn

435440445

aspvaltyriletyrgluarglysphepheasnaspvalvalleuval

450455460

alaileasnargasnthrglnsersertyrserileserglyleugln

465470475480

thralaleuproasnglysertyralaasptyrleuserglyleuleu

485490495

glyglyasnglyileservalserasnglyservalalaserphethr

500505510

leualaproglyalavalservaltrpglntyrserthrseralaser

515520525

alaproglnileglyservalalaproasnmetglyileproglyasn

530535540

valvalthrileaspglylysglypheglythrthrglnglythrval

545550555560

thrpheglyglyvalthralathrvallyssertrpthrserasnarg

565570575

ilegluvaltyrvalproasnmetalaalaglyleuthraspvallys

580585590

valthralaglyglyvalserserasnleutyrsertyrasnileleu

595600605

serglythrglnthrservalvalphethrvallysseralapropro

610615620

thrasnleuglyasplysiletyrleuthrglyasnileprogluleu

625630635640

glyasntrpserthraspthrserglyalavalasnasnalaglngly

645650655

proleuleualaproasntyrproasptrpphetyrvalpheserval

660665670

proalaglylysthrileglnphelysphepheilelysargalaasp

675680685

glythrileglntrpgluasnglyserasnhisvalalathrthrpro

690695700

thrglyalathrglyasnilethrvalthrtrpglnasn

705710715

<210>7

<211>717

<212>prt

<213>人工序列

<400>7

metlyslyslysthrleuserleuphevalglyleumetleuleuile

151015

glyleuleupheserglyserleuprotyrasnproasnalaalaglu

202530

alaserserseralaservallysglyaspvaliletyrglnileile

354045

ileaspargphetyraspglyaspthrthrasnasnasnproalalys

505560

sertyrglyleutyraspprothrlysserlystrplysmettyrtrp

65707580

glyglyaspleugluglyvalargglnlysleuprotyrleulysgln

859095

leuglyvalthrthriletrpleuserprovalleuasnasnleuasp

100105110

thrleualaglythraspasnthrglytyrhisglytyrtrpthrarg

115120125

aspphelysglnileglugluhispheglyasntrpthrthrpheasp

130135140

thrleuvalasnaspalahisglnasnglyilelysvalilevalasp

145150155160

phevalproasnhisserthrprophelysalaasnaspserthrphe

165170175

alagluglyglyalaleutyrasnasnglythrtyrmetglyasntyr

180185190

pheaspaspalathrtyrglytyrphehishisasnglyaspileser

195200205

asntrpaspaspargtyrglualaglntrplysasnphethrasppro

210215220

alaglypheserleualaaspleuserglngluasnglythrileala

225230235240

glntyrleuthraspalaalavalglnleuvalalahisglyleuarg

245250255

ileaspalavallyshispheasnserglypheserlysserleuala

260265270

asplysleutyrglnlyslysaspilepheleuvalglyglutrptyr

275280285

glythraspproglythralaasnhisleuglulysvalargtyrala

290295300

asnasnserglyvalasnvalleuasppheaspleuasnthrvalile

305310315320

argasnvalpheglythrphethrglnthrmettyraspleuasnasn

325330335

metvalasnglnthrglyasnglutyrlystyrlysgluasnleuile

340345350

thrpheileaspasnhisaspmetserargpheleuservalasnser

355360365

lysasnlysalaasnleuhisglnargleuleuserpheserleuarg

370375380

glyvalargproproiletyrtyrglythrgluglntyrmetalagly

385390395400

glyasnaspprotyrasnargglymetmetproalapheaspthrthr

405410415

thrthralaphelysgluvalserthrleualaglyleuargargasn

420425430

asnalaalaileglntyrglythrthrthrglnargtrpileasnasn

435440445

aspvaltyriletyrgluarglysphepheasnaspvalvalleuval

450455460

alaileasnargasnthrglnsersertyrserileserglyleugln

465470475480

thralaleuproasnglysertyralaasptyrleuserglyleuleu

485490495

glyglyasnglyileservalserasnglyservalalaserphethr

500505510

leualaproglyalavalservaltrpglntyrserthrseralaser

515520525

alaproglnileglyservalalaproasnmetglyileproglyasn

530535540

valvalthrileaspglylysglypheglythrthrglnglythrval

545550555560

thrpheglyglyvalthralathrvallyssertrpthrserasnarg

565570575

ilegluvaltyrvalproasnmetalaalaglyleuthraspvallys

580585590

valthralaglyglyvalserserasnleutyrsertyrasnileleu

595600605

serglythrglnthrservalvalphethrvallysseralapropro

610615620

thrasnleuglyasplysiletyrleuthrglyasnileprogluleu

625630635640

glyasntrpserthraspthrserglyalavalasnasnalaglngly

645650655

proleuleualaproasntyrproasptrpphetyrvalpheserval

660665670

proalaglylysthrileglnphelysphepheilelysargalaasp

675680685

glythrileglntrpgluasnglyserasnhisvalalathrthrpro

690695700

thrglyalathrglyasnilethrvalthrtrpglnasn

705710715

<210>8

<211>717

<212>prt

<213>人工序列

<400>8

metlyslyslysthrleuserleuphevalglyleumetleuleuile

151015

glyleuleupheserglyserleuprotyrasnproasnalaalaglu

202530

alaserserseralaservallysglyaspvaliletyrglnileile

354045

ileaspargphetyraspglyaspthrthrasnasnasnproalalys

505560

sertyrglyleutyraspprothrlysserlystrplysmettyrtrp

65707580

glyglyaspleugluglyvalargglnlysleuprotyrleulysgln

859095

leuglyvalthrthriletrpleuserprovalleuasnasnleuasp

100105110

thrleualaglythraspasnthrglytyrhisglytyrtrpthrarg

115120125

aspphelysglnileglugluhispheglyasntrpthrthrpheasp

130135140

thrleuvalasnaspalahisglnasnglyilelysvalilevalasp

145150155160

phevalproasnhisserthrprophelysalaasnaspserthrphe

165170175

alagluglyglyalaleutyrasnasnglythrtyrmetglyasntyr

180185190

pheaspaspalathrgluglytyrphehishisasnglyaspileser

195200205

asntrpaspaspargtyrglualaglntrplysasnphethrasppro

210215220

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