本发明属于农业生物技术领域,具体涉及活性黑5的应用及霉菌毒素降解酶的筛选方法。
背景技术:
霉菌毒素是真菌产生的次级代谢产物,主要污染储存的粮油食品和饲料,严重危害人畜健康。根据其结构特征,霉菌毒素可分为芳香环和非芳香环两大类,芳香环类包括黄曲霉毒素、玉米赤霉烯酮、桔霉素、赭曲霉素、棒曲霉素和单端孢霉烯族毒素等;非芳香环类仅包括伏马毒素。其中黄曲霉毒素、玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇(呕吐毒素)是最为常见和危害最大的霉菌毒素。因此,亟需建立简单、有效且环境友好的霉菌毒素的脱毒方法。
目前,被霉菌毒素污染的饲料的脱毒方法主要包括物理法、化学法、吸附法和生物法等。物理和化学脱毒法存在操作困难、效果不稳定、营养成分损失大以及影响饲料适口性等缺点。吸附法虽简单易行,但存在用量大、不够经济,容易引起二次污染等缺点。微生物脱毒法具有作用条件温和,对原料的感官性状、适口性等影响极小、增加原料营养价值等优点,被认为是最佳脱毒方法。生物脱毒作用主要是指降解酶通过酶促反应将毒素转化成低毒或者无毒产物,其中降解酶包括氧化酶如漆酶、锰过氧化物酶和水解酶(如酯酶)等。
生物脱毒技术实现规模化应用的过程中,寻找和筛选能降解霉菌毒素的菌株,并对其所产胞外降解酶进行特性研究及对降解酶基因进行克隆和表达,是霉菌毒素生物降解研究领域重要的突破点和发展方向。因此,建立饲料毒素降解酶高通量筛选方法是生物脱毒技术推广的关键所在。现有霉菌毒素降解酶筛选过程中操作者接触霉菌毒素,对操作者所产生的潜在危害。而在寻找无毒替代底物的过程中,无毒底物或者无法正确反映霉菌毒素的降解率,或者无毒底物只能反映一种或两种霉菌毒素的降解率,从而无法满足工业中的实际需求。
技术实现要素:
本发明的目的在于提供活性黑5在霉菌毒素筛选中的应用。
本发明的再一目的在于提供一种霉菌毒素降解酶的筛选方法。
根据本发明具体实施方式的霉菌毒素降解酶的筛选方法,所述方法包括在有机酸体系中以活性黑5作为检测底物检测待测蛋白酶对活性黑5降解率的步骤,其中,如对活性黑5具有降解作用,则判断所述待测蛋白酶为霉菌毒素降解酶。
根据本发明具体实施方式的霉菌毒素降解酶的筛选方法,所述待测蛋白酶包括锰过氧化物酶。
根据本发明具体实施方式的霉菌毒素降解酶的筛选方法,所述有机酸体系包括丙二酸-丙二酸钠缓冲液
根据本发明具体实施方式的霉菌毒素降解酶的筛选方法,丙二酸-丙二酸钠缓冲液的浓度为0.2m,丙二酸-丙二酸钠缓冲液的的ph为5.0。
根据本发明具体实施方式的霉菌毒素降解酶的筛选方法,所述有机酸体系还包括硫酸锰溶液,硫酸镁溶液的浓度为40mm。
根据本发明具体实施方式的霉菌毒素降解酶的筛选方法,所述有机酸体系还包括过氧化氢溶液,过氧化氢溶液的浓度为4mm。
本发明选用染料活性黑5作为霉菌毒素的无毒替代物,操作简单、通量高,避免了霉菌毒素降解酶筛选过程中操作者接触霉菌毒素所产生的潜在危害,而且成本投资低、适用范围广,可广泛用于饲料毒素降解酶领域。
附图说明
图1显示重组锰过氧化物酶ilmnp5、ilmnp6在不同体系下对黄曲霉毒素b1和玉米赤霉烯酮的降解作用,其中,
a显示丙二酸-丙二酸钠、醋酸-醋酸钠和乳酸-乳酸钠体系下ilmnp5、ilmnp6对黄曲霉毒素b1的作用情况,
b显示丙二酸-丙二酸钠、醋酸-醋酸钠和乳酸-乳酸钠体系下ilmnp5、ilmnp6对玉米赤霉烯酮的作用情况,
c显示丙二酸体系下添加超氧化物歧化酶和芦丁ilmnp5对黄曲霉毒素b1的作用情况,
d显示丙二酸体系下添加超氧化物歧化酶和芦丁ilmnp5对玉米赤霉烯酮的作用情况,
e显示丙二酸体系下添加超氧化物歧化酶和芦丁ilmnp6对黄曲霉毒素b1的作用情况,
f显示丙二酸体系下添加超氧化物歧化酶和芦丁ilmnp6对玉米赤霉烯酮的作用情况;
图2显示丙二酸体系下添加超氧化物歧化酶和芦丁对降解染料活性黑5的影响,其中,a显示ilmnp5对活性黑5的作用情况,b显示ilmnp6对活性黑5的作用情况;
图3显示不同来源的锰过氧化物酶的序列相似性比较和进化关系情况;
图4显示不同来源的锰过氧化物酶对不同毒素的降解率与对活性黑5降解率之间相关性关系。
具体实施方式
试验材料和试剂
1、菌株:产锰过氧化物酶的大肠杆菌工程菌株。
2、生化试剂:黄曲霉毒素b1、玉米赤霉烯酮、呕吐毒素、伏马菌素、活性黑5、超氧化物歧化酶、芦丁;色谱纯乙腈、三氟乙酸、tris、丙二酸、丙二酸钠、醋酸、醋酸钠、柠檬酸、柠檬酸钠、乳酸、乳酸钠、丁二酸、丁二酸钠、硫酸锰和过氧化氢。
3、培养基:
(1)大肠杆菌培养基lb(1%蛋白胨、0.5%酵母提取物、1%nacl,ph7.0)。
ilmnp1氨基酸序列如下:
mafktilafvalataalaapssrvtcspgrvvsngacckwfdvlddiqenlfdggvcgeevheslrltfhdaigfslsaeregkfggggadgsimafaeietnfhanngvdeiveaqrpfaikhkvsfgdfiqfagavgvsnclggprlefmagrsnisraapdltvpepsdsvdkilarmgdagfsssevvdllishtvaaqdhvdptipgtpfdstpsefdpqffvetllkgtlfpgngsnvgelqsplrgefrlqsdallardprtacewqsfvnnqrlmvtkfeavmsklavlghnprdlvdcsevipvppraktnvavlpagktradvqaacaatpfptlqtapgpatsivpv
ilmnp2氨基酸序列如下:
mafkhlvvalsivlslgvaqaaitkrvacpdgkntatnaaccslfairddiqanlfdggecgeevhesfrltfhdaigtgsfggggadgsiivfddietnfhanngvdeiideqkpfiarhnitpgdfiqfagavgvsncpgaprldfflgrpnpvaaapdktvpepfdtvdsilarfkdaggftpaeivallgshtiaaadhvdptipgtpfdstpevfdtqvfvevqlrgtlfpgtggnqgevqsplrgeirlqsdhdlardsrtacewqsfvnnqaklqsafkaafkklsvlghninnlidcsevipeppnvkvkpatfpagithadveqacattpfptlatdpgpatsvapvpps
ilmnp4氨基酸序列如下:
mtfkallalltvtsavlaapqdvtaankvscgggrvaghaqcckwydvlddiqknlfdggecgeevheslrltfhdaigfslsaqregkfggggadgsimafaeietkfhanngvdeiieaqrpfalnhsvsfgdfiqfagavgvsncgggprlqflagrsnsskaapdgtvpepfdstdkilahmgdagfspsevvdllashsvaaqdhvdasipgtpfdstpstfdaqffvetllkgtlfpgngsnqgevqsplhgefrlqsdfelardsrtacewqsfitdhnsmvrkfeaamaklavlghdprtlidcsdvipqpkgaksnvavlpagkhradiqaschqtpfptlktapgpetsippvpps
ilmnp5氨基酸序列如下:
mafkqlvatlslallahgavvrrvtcpdgvntatnaaccslfavrddiqqnlfdngqcgedvhesfrlsfhdaigispkiaatgqfggggadgsiilfeeietnfhanigvdeivdeqkpfiarhnitpgdfiqfaaavgvsncpgaprldfflgrpaatqpapdktvpepfdtvdtilerfadagnftpaevvallvshtiaaadevdptipgtpfdstpevfdsqffvetqlrgtgfpgtagnqgevesplagelrlqsdselardsrtacewqsfvgnqqkiqtafkaafqkmavlgvdtskmvdcselipvppelkitaahfpagktnadveqacastpfptlstdpgpatsvapvpps
ilmnp6氨基酸序列如下:
mafkqlvaaltvalslgvaqgaitrrvacpdgvntatnaaccslfairddiqqnlfdggecgeevhesfrltfhdaigigsnggggadgsiavfedietafhanngvdeiideqkpflarhnitpgdfiqfagavgvsncpgaprldfflgrpnpvapapdktvpepfdtvdsilarfadaggfspaevvallgshtiaaadhvdptipgtpfdstpevfdtqvflevqlrgtlfpgtggnqgevesplrgeirlqsdhdlardsrtacewqsfvnnqvklqtafkaafkklavlghdvnnmvdcsevipeppnvkikaatfpagqtnadveqacastpfptlatdpgpatsvapvpps
pcmnp1氨基酸序列如下:
mafgsllafvalaaitraaptaesavcpdgtrvtnaaccafiplaqdlqetlfqgdcgedahevirltfhdaiaisqslgpqagggadgsmlhfptiepnfsansgiddsvnnllpfmqkhdtisaadlvqfagavalsncpgaprlefmagrpnttipaveglipepqdsvtkilqrfedagnfspfevvsllashtvaradkvdetidaapfdstpftfdtqvflevllkgtgfpgsnnntgevmsplplgsgsdtgemrlqsdfalardertacfwqsfvneqefmaasfkaamaklailghsrsslidcsdvvpvpkpavnkpatfpatkgpkdldtltckalkfptltsdpgatetliphcsnggmscpgvqfdgpa
csmnp氨基酸序列如下:
maftsfvalaalvgiasaaptticpdgtrvsnhaccafiplaedlqktifmndcgedahevirltfhdavaisrklgpkagggadgsmllfptvepnfsanngiddsvnnlipfmarhptvsagdlvqfagavalsncpgaprleflagrpnhtiaaidglipepqddvtkilerfddaggftpfevvsllashtvaradkvdetidaapfdstpftfdtqvflevllkgvgfpgtdnntgevasplpkgsgndtgemrlqsdfalardprtacfwqgfvdeqefmaesfkaamaklailghnrasltdcsdvvpiprpavkkpasfpattgpkdleltcraerfptltvdrgavqaliphcsnggqdcpsvqfdgpa
nfmnp氨基酸序列如下:
mafnfaailafvslaavtsaapsgttcsngvvvpdavccdfvplasalqsevlmgdcgedahelvrlifhdaiaisqsmgpsagggadgsmlifptvepaffpnlgiadsvnnlipflsqfptisagdlvqfagavaisncpgapqleflagrsnatapaidglipepqddvtkilarfadagnftpdevvallashsiaradhvdptldaapfdstpfdfdtqvflevllkgtgfpgldnntgevssplpvtdgtdvgelrlqsdfvlardertacawqsfvneqqamadafkeavkklavlghsrsdlvdcsavvpvpkpatgtpatfpastgpqdleltcttvpfptlstapgaqqtliphcsdgtmtcnsvqfdgpatnfggadds
实施例1确定锰过氧化物酶对霉菌毒素的降解机制
1.制备重组锰过氧化物酶
分别取产锰过氧化物酶ilmnp1、ilmnp2、ilmnp4、ilmnp5、ilmnp6及pcmnp1的大肠杆菌工程菌株,接种于50mllb培养基中,37℃220rpm振荡培养12h,以2%比例转接于200mllb培养基中,37℃220rpm振荡培养2h,至od600达到0.6-0.8,加入终浓度1mm的诱导剂iptg诱导4h后,离心收集菌体。
ilmnp1、ilmnp2、ilmnp4、ilmnp5、ilmnp6、pcmnp1采取溶菌酶法裂解菌体,8m尿素溶解包涵体蛋白后进行变复性后获得活性蛋白。
取分别产锰过氧化物酶csmnp及nfmnp的大肠杆菌菌株,接种于50mllb培养基中,37℃220rpm振荡培养12h后,按2%比例转接于200mllb培养基中,37℃220rpm振荡培养约2h(od590≈0.5)。冰水浴处理后于10℃220rpm培养30min后加入60μl浓度1m的诱导剂iptg,于10℃220rpm继续培养6h。之后的9h培养过程中每小时分别加入33μl浓度1m的cacl2和330μl浓度10g/l的hemin(hemin溶于0.1mnaoh中),而后10℃220rpm继续培养10h后离心收集菌体。采取超声法裂解菌体,利用镍柱进行亲和层析纯化获得单一的csmnp及nfmnp蛋白。
2重组锰过氧化物酶ilmnp5、ilmnp6于不同有机酸体系下降解黄曲霉毒素b1和玉米赤霉烯酮
按如下反应体系:50μl有机酸缓冲液(0.2m,ph5.0),20μl去离子水,20μl黄曲霉毒素b1(afb1)溶液或玉米赤霉烯酮(zen)溶液,5μl硫酸锰(40mm),100μl锰过氧化酶(1000u/l),5μl过氧化氢(4mm)。所用的有机酸缓冲液分别为丙二酸-丙二酸钠、醋酸-醋酸钠和乳酸-乳酸钠,以未加入锰过氧化物酶的体系作为对照,每个锰过氧化物酶设3个重复。于30℃下反应9h,采用高效液相色谱(hplc)分析黄曲霉毒素b1及玉米赤霉烯酮的降解率,具体实验结果见图1。
如图1中的a所示,在醋酸和乳酸有机酸体系下,黄曲霉毒素b1不能被锰过氧化物酶降解,但丙二酸体系下黄曲霉毒素b1能被锰过氧化物酶有效降解。基于不同有机酸的机制差异:乙酸不能螯合三价锰离子;乳酸螯合三价锰离子,但不生成自由基;丙二酸螯合三价锰离子,同时会生成自由基。重组锰过氧化物酶依靠螯合的三价锰与丙二酸反应生成的自由基实现对黄曲霉毒素b1的降解。如图1中b所示,玉米赤霉烯酮在丙二酸体系下也能得到有效降解。
3确认自由基在反应中的作用
按如下反应体系:50μl丙二酸缓冲液(0.2m,ph5.0),20μl去离子水,50μl超氧化物歧化酶,20μl黄曲霉毒素b1溶液或玉米赤霉烯酮溶液,5μl硫酸锰(40mm),50μl锰过氧化酶(2000u/l),5μl过氧化氢(4mm)。以未加入锰过氧化物酶的有机酸体系作为对照,每个锰过氧化物酶设3个重复。于30℃下反应9h,采用高效液相色谱(hplc)分析黄曲霉毒素b1及玉米赤霉烯酮的降解率,具体实验结果见图1。
如图1中的c、d、e、f所示,重组锰过氧化物酶降解黄曲霉毒素b1、玉米赤霉烯酮体系中添加超氧化物歧化酶或芦丁后,锰过氧化物酶对黄曲霉毒素b1及玉米赤霉烯酮的降解率均明显降低。由于超氧化物歧化酶和芦丁能有效的消除超氧阴离子和烷基过氧自由基,因此,表明超氧阴离子自由基参与锰过氧化物酶对黄曲霉毒素b1及玉米赤霉烯酮的降解。
实施例2锰过氧化物酶ilmnp5、ilmnp6对染料活性黑5的作用
按如下反应体系:50μl丙二酸缓冲液(0.2m,ph5.0),30μl去离子水,50μl超氧化物歧化酶或芦丁,10μl活性黑5溶液,5μl硫酸锰(40mm),50μl锰过氧化酶(2000u/l),5μl过氧化氢(4mm)。以未加入锰过氧化物酶的有机酸体系作为对照,每个锰过氧化物酶设3个重复。于30℃下反应9h,采用分光光度计测定活性黑5的降解率,具体实验结果见图2。
如图2所示,在丙二酸体系下,添加超氧歧化酶或芦丁对锰过氧化物酶降解活性黑5具有明显抑制作用,说明超氧阴离子和烷基过氧自由基参与锰过氧化物酶降解染料活性黑5。
实施例3考察不同来源的锰过氧化物酶对活性黑5、霉菌毒素的作用关系
不同来源的锰过氧化物酶分别选择ilmnp1、ilmnp2、ilmnp4、ilmnp5、ilmnp6、pcmnp1、csmnp和nfmnp。
毒素降解反应按如下体系:50μl丙二酸缓冲液(0.2m,ph5.0),20μl去离子水,20μl霉菌毒素溶液,5μl硫酸锰(40mm),100μl锰过氧化酶(1000u/l),5μl过氧化氢(4mm)。以未加入锰过氧化物酶的体系作为对照,每个锰过氧化物酶设3个重复。于30℃下反应72h,采用高效液相色谱(hplc)分析对黄曲霉毒素b1(afb1)、玉米赤霉烯酮(zen)、呕吐毒素(don)、伏马菌素b1(fb1)的降解率。
活性黑5降解反应按如下体系:50μl丙二酸缓冲液(0.2m,ph5.0),20μl去离子水,20μl染料活性黑5溶液,5μl硫酸锰(40mm),100μl锰过氧化酶(1000u/l),5μl过氧化氢(4mm)。以未加入锰过氧化物酶的体系作为对照,每个锰过氧化物酶设3个重复。于30℃下反应9h,采用分光光度计测定活性黑5在od556的值,降解率(%)=[(ai-at)/ai]×100。具体实验结果如表1、图3、图4所示。
表1丙二酸体系下霉菌毒素的降解率
如表1、图3所示,进化关系较远、序列一致性较低的锰过氧化物酶均能够在丙二酸体系下对黄曲霉毒素b1、玉米赤霉烯酮、呕吐毒素及伏马菌素b1发挥明显的降解效果。
实验结果表明,锰过氧化物酶对霉菌毒素作用72h后,降解率达到最大值,锰过氧化物酶对染料活性黑5作用9h后,降解率已达最大值,即锰过氧化物酶对两种底物的作用均达到最大效果,因此,选择作用霉菌72小时与活性黑5作用9小时,这两个时间点进行对比。
如图4所示,锰过氧化物酶对不同毒素作用72h后的降解率与对染料活性黑5作用9h后的降解率均存在较高正相关性,r2介于0.710-0.945之间,表明染料活性黑5可作为饲料霉菌毒素降解酶的初筛底物。
序列表
<110>中国农业科学院饲料研究所
<120>活性黑5的应用及霉菌毒素降解酶的筛选方法
<160>8
<170>siposequencelisting1.0
<210>1
<211>358
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>1
metalaphelysthrileleualaphevalalaleualathralaala
151015
leualaalaproserserargvalthrcysserproglyargvalval
202530
serasnglyalacyscyslystrppheaspvalleuaspaspilegln
354045
gluasnleupheaspglyglyvalcysglyglugluvalhisgluser
505560
leuargleuthrphehisaspalaileglypheserleuseralaglu
65707580
arggluglylyspheglyglyglyglyalaaspglyserilemetala
859095
phealagluilegluthrasnphehisalaasnasnglyvalaspglu
100105110
ilevalglualaglnargprophealailelyshislysvalserphe
115120125
glyasppheileglnphealaglyalavalglyvalserasncysleu
130135140
glyglyproargleugluphemetalaglyargserasnileserarg
145150155160
alaalaproaspleuthrvalprogluproseraspservalasplys
165170175
ileleualaargmetglyaspalaglyphesersersergluvalval
180185190
aspleuleuileserhisthrvalalaalaglnasphisvalasppro
195200205
thrileproglythrpropheaspserthrproserglupheasppro
210215220
glnphephevalgluthrleuleulysglythrleupheproglyasn
225230235240
glyserasnvalglygluleuglnserproleuargglygluphearg
245250255
leuglnseraspalaleuleualaargaspproargthralacysglu
260265270
trpglnserphevalasnasnglnargleumetvalthrlyspheglu
275280285
alavalmetserlysleualavalleuglyhisasnproargaspleu
290295300
valaspcyssergluvalileprovalproproargalalysthrasn
305310315320
valalavalleuproalaglylysthrargalaaspvalglnalaala
325330335
cysalaalathrpropheprothrleuglnthralaproglyproala
340345350
thrserilevalproval
355
<210>2
<211>359
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>2
metalaphelyshisleuvalvalalaleuserilevalleuserleu
151015
glyvalalaglnalaalailethrlysargvalalacysproaspgly
202530
lysasnthralathrasnalaalacyscysserleuphealailearg
354045
aspaspileglnalaasnleupheaspglyglyglucysglygluglu
505560
valhisgluserpheargleuthrphehisaspalaileglythrgly
65707580
serpheglyglyglyglyalaaspglyserileilevalpheaspasp
859095
ilegluthrasnphehisalaasnasnglyvalaspgluileileasp
100105110
gluglnlyspropheilealaarghisasnilethrproglyaspphe
115120125
ileglnphealaglyalavalglyvalserasncysproglyalapro
130135140
argleuaspphepheleuglyargproasnprovalalaalaalapro
145150155160
asplysthrvalproglupropheaspthrvalaspserileleuala
165170175
argphelysaspalaglyglyphethrproalagluilevalalaleu
180185190
leuglyserhisthrilealaalaalaasphisvalaspprothrile
195200205
proglythrpropheaspserthrprogluvalpheaspthrglnval
210215220
phevalgluvalglnleuargglythrleupheproglythrglygly
225230235240
asnglnglygluvalglnserproleuargglygluileargleugln
245250255
serasphisaspleualaargaspserargthralacysglutrpgln
260265270
serphevalasnasnglnalalysleuglnseralaphelysalaala
275280285
phelyslysleuservalleuglyhisasnileasnasnleuileasp
290295300
cyssergluvalileprogluproproasnvallysvallysproala
305310315320
thrpheproalaglyilethrhisalaaspvalgluglnalacysala
325330335
thrthrpropheprothrleualathraspproglyproalathrser
340345350
valalaprovalproproser
355
<210>3
<211>366
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>3
metthrphelysalaleuleualaleuleuthrvalthrseralaval
151015
leualaalaproglnaspvalthralaalaasnlysvalsercysgly
202530
glyglyargvalalaglyhisalaglncyscyslystrptyraspval
354045
leuaspaspileglnlysasnleupheaspglyglyglucysglyglu
505560
gluvalhisgluserleuargleuthrphehisaspalaileglyphe
65707580
serleuseralaglnarggluglylyspheglyglyglyglyalaasp
859095
glyserilemetalaphealagluilegluthrlysphehisalaasn
100105110
asnglyvalaspgluileileglualaglnargprophealaleuasn
115120125
hisservalserpheglyasppheileglnphealaglyalavalgly
130135140
valserasncysglyglyglyproargleuglnpheleualaglyarg
145150155160
serasnserserlysalaalaproaspglythrvalprogluprophe
165170175
aspserthrasplysileleualahismetglyaspalaglypheser
180185190
prosergluvalvalaspleuleualaserhisservalalaalagln
195200205
asphisvalaspalaserileproglythrpropheaspserthrpro
210215220
serthrpheaspalaglnphephevalgluthrleuleulysglythr
225230235240
leupheproglyasnglyserasnglnglygluvalglnserproleu
245250255
hisglyglupheargleuglnseraspphegluleualaargaspser
260265270
argthralacysglutrpglnserpheilethrasphisasnsermet
275280285
valarglyspheglualaalametalalysleualavalleuglyhis
290295300
aspproargthrleuileaspcysseraspvalileproglnprolys
305310315320
glyalalysserasnvalalavalleuproalaglylyshisargala
325330335
aspileglnalasercyshisglnthrpropheprothrleulysthr
340345350
alaproglyprogluthrserileproprovalproproser
355360365
<210>4
<211>363
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>4
metalaphelysglnleuvalalathrleuserleualaleuleuala
151015
hisglyalavalvalargargvalthrcysproaspglyvalasnthr
202530
alathrasnalaalacyscysserleuphealavalargaspaspile
354045
glnglnasnleupheaspasnglyglncysglygluaspvalhisglu
505560
serpheargleuserphehisaspalaileglyileserprolysile
65707580
alaalathrglyglnpheglyglyglyglyalaaspglyserileile
859095
leupheglugluilegluthrasnphehisalaasnileglyvalasp
100105110
gluilevalaspgluglnlyspropheilealaarghisasnilethr
115120125
proglyasppheileglnphealaalaalavalglyvalserasncys
130135140
proglyalaproargleuaspphepheleuglyargproalaalathr
145150155160
glnproalaproasplysthrvalproglupropheaspthrvalasp
165170175
thrileleugluargphealaaspalaglyasnphethrproalaglu
180185190
valvalalaleuleuvalserhisthrilealaalaalaaspgluval
195200205
aspprothrileproglythrpropheaspserthrprogluvalphe
210215220
aspserglnphephevalgluthrglnleuargglythrglyphepro
225230235240
glythralaglyasnglnglygluvalgluserproleualaglyglu
245250255
leuargleuglnseraspsergluleualaargaspserargthrala
260265270
cysglutrpglnserphevalglyasnglnglnlysileglnthrala
275280285
phelysalaalapheglnlysmetalavalleuglyvalaspthrser
290295300
lysmetvalaspcyssergluleuileprovalproprogluleulys
305310315320
ilethralaalahispheproalaglylysthrasnalaaspvalglu
325330335
glnalacysalaserthrpropheprothrleuserthraspprogly
340345350
proalathrservalalaprovalproproser
355360
<210>5
<211>359
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>5
metalaphelysglnleuvalalaalaleuthrvalalaleuserleu
151015
glyvalalaglnglyalailethrargargvalalacysproaspgly
202530
valasnthralathrasnalaalacyscysserleuphealailearg
354045
aspaspileglnglnasnleupheaspglyglyglucysglygluglu
505560
valhisgluserpheargleuthrphehisaspalaileglyilegly
65707580
serasnglyglyglyglyalaaspglyserilealavalphegluasp
859095
ilegluthralaphehisalaasnasnglyvalaspgluileileasp
100105110
gluglnlyspropheleualaarghisasnilethrproglyaspphe
115120125
ileglnphealaglyalavalglyvalserasncysproglyalapro
130135140
argleuaspphepheleuglyargproasnprovalalaproalapro
145150155160
asplysthrvalproglupropheaspthrvalaspserileleuala
165170175
argphealaaspalaglyglypheserproalagluvalvalalaleu
180185190
leuglyserhisthrilealaalaalaasphisvalaspprothrile
195200205
proglythrpropheaspserthrprogluvalpheaspthrglnval
210215220
pheleugluvalglnleuargglythrleupheproglythrglygly
225230235240
asnglnglygluvalgluserproleuargglygluileargleugln
245250255
serasphisaspleualaargaspserargthralacysglutrpgln
260265270
serphevalasnasnglnvallysleuglnthralaphelysalaala
275280285
phelyslysleualavalleuglyhisaspvalasnasnmetvalasp
290295300
cyssergluvalileprogluproproasnvallysilelysalaala
305310315320
thrpheproalaglyglnthrasnalaaspvalgluglnalacysala
325330335
serthrpropheprothrleualathraspproglyproalathrser
340345350
valalaprovalproproser
355
<210>6
<211>382
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>6
metalapheglyserleuleualaphevalalaleualaalailethr
151015
argalaalaprothralagluseralavalcysproaspglythrarg
202530
valthrasnalaalacyscysalapheileproleualaglnaspleu
354045
glngluthrleupheglnglyaspcysglygluaspalahisgluval
505560
ileargleuthrphehisaspalailealaileserglnserleugly
65707580
proglnalaglyglyglyalaaspglysermetleuhispheprothr
859095
ilegluproasnpheseralaasnserglyileaspaspservalasn
100105110
asnleuleuprophemetglnlyshisaspthrileseralaalaasp
115120125
leuvalglnphealaglyalavalalaleuserasncysproglyala
130135140
proargleugluphemetalaglyargproasnthrthrileproala
145150155160
valgluglyleuileprogluproglnaspservalthrlysileleu
165170175
glnargphegluaspalaglyasnpheserprophegluvalvalser
180185190
leuleualaserhisthrvalalaargalaasplysvalaspgluthr
195200205
ileaspalaalapropheaspserthrprophethrpheaspthrgln
210215220
valpheleugluvalleuleulysglythrglypheproglyserasn
225230235240
asnasnthrglygluvalmetserproleuproleuglyserglyser
245250255
aspthrglyglumetargleuglnseraspphealaleualaargasp
260265270
gluargthralacysphetrpglnserphevalasngluglngluphe
275280285
metalaalaserphelysalaalametalalysleualaileleugly
290295300
hisserargserserleuileaspcysseraspvalvalprovalpro
305310315320
lysproalavalasnlysproalathrpheproalathrlysglypro
325330335
lysaspleuaspthrleuthrcyslysalaleulyspheprothrleu
340345350
thrseraspproglyalathrgluthrleuileprohiscysserasn
355360365
glyglymetsercysproglyvalglnpheaspglyproala
370375380
<210>7
<211>378
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>7
metalaphethrserphevalalaleualaalaleuvalglyileala
151015
seralaalaprothrthrilecysproaspglythrargvalserasn
202530
hisalacyscysalapheileproleualagluaspleuglnlysthr
354045
ilephemetasnaspcysglygluaspalahisgluvalileargleu
505560
thrphehisaspalavalalaileserarglysleuglyprolysala
65707580
glyglyglyalaaspglysermetleuleupheprothrvalglupro
859095
asnpheseralaasnasnglyileaspaspservalasnasnleuile
100105110
prophemetalaarghisprothrvalseralaglyaspleuvalgln
115120125
phealaglyalavalalaleuserasncysproglyalaproargleu
130135140
glupheleualaglyargproasnhisthrilealaalaileaspgly
145150155160
leuileprogluproglnaspaspvalthrlysileleugluargphe
165170175
aspaspalaglyglyphethrprophegluvalvalserleuleuala
180185190
serhisthrvalalaargalaasplysvalaspgluthrileaspala
195200205
alapropheaspserthrprophethrpheaspthrglnvalpheleu
210215220
gluvalleuleulysglyvalglypheproglythraspasnasnthr
225230235240
glygluvalalaserproleuprolysglyserglyasnaspthrgly
245250255
glumetargleuglnseraspphealaleualaargaspproargthr
260265270
alacysphetrpglnglyphevalaspgluglngluphemetalaglu
275280285
serphelysalaalametalalysleualaileleuglyhisasnarg
290295300
alaserleuthraspcysseraspvalvalproileproargproala
305310315320
vallyslysproalaserpheproalathrthrglyprolysaspleu
325330335
gluleuthrcysargalagluargpheprothrleuthrvalasparg
340345350
glyalavalglnalaleuileprohiscysserasnglyglyglnasp
355360365
cysproservalglnpheaspglyproala
370375
<210>8
<211>390
<212>prt
<213>大肠杆菌(escherichiacoli)
<400>8
metalapheasnphealaalaileleualaphevalserleualaala
151015
valthrseralaalaproserglythrthrcysserasnglyvalval
202530
valproaspalavalcyscysaspphevalproleualaseralaleu
354045
glnsergluvalleumetglyaspcysglygluaspalahisgluleu
505560
valargleuilephehisaspalailealaileserglnsermetgly
65707580
proseralaglyglyglyalaaspglysermetleuilepheprothr
859095
valgluproalaphepheproasnleuglyilealaaspservalasn
100105110
asnleuilepropheleuserglnpheprothrileseralaglyasp
115120125
leuvalglnphealaglyalavalalaileserasncysproglyala
130135140
proglnleuglupheleualaglyargserasnalathralaproala
145150155160
ileaspglyleuileprogluproglnaspaspvalthrlysileleu
165170175
alaargphealaaspalaglyasnphethrproaspgluvalvalala
180185190
leuleualaserhisserilealaargalaasphisvalaspprothr
195200205
leuaspalaalapropheaspserthrpropheasppheaspthrgln
210215220
valpheleugluvalleuleulysglythrglypheproglyleuasp
225230235240
asnasnthrglygluvalserserproleuprovalthraspglythr
245250255
aspvalglygluleuargleuglnseraspphevalleualaargasp
260265270
gluargthralacysalatrpglnserphevalasngluglnglnala
275280285
metalaaspalaphelysglualavallyslysleualavalleugly
290295300
hisserargseraspleuvalaspcysseralavalvalprovalpro
305310315320
lysproalathrglythrproalathrpheproalaserthrglypro
325330335
glnaspleugluleuthrcysthrthrvalpropheprothrleuser
340345350
thralaproglyalaglnglnthrleuileprohiscysseraspgly
355360365
thrmetthrcysasnservalglnpheaspglyproalathrasnphe
370375380
glyglyalaaspaspser
385390