CN109486794B - 一种酶活提高的甲壳素酶突变体 - Google Patents

一种酶活提高的甲壳素酶突变体 Download PDF

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CN109486794B
CN109486794B CN201811484682.2A CN201811484682A CN109486794B CN 109486794 B CN109486794 B CN 109486794B CN 201811484682 A CN201811484682 A CN 201811484682A CN 109486794 B CN109486794 B CN 109486794B
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刘龙
潘梦妍
吕雪芹
堵国成
李江华
陈坚
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Abstract

本发明公开了一种酶活提高的甲壳素酶突变体，属于基因工程和酶工程技术领域。本发明通过定点突变的方式，将甲壳素酶分子催化结构域附近的第43位半胱氨酸突变为天冬氨酸，第273位苯丙氨酸突变为色氨酸、第336位谷氨酸突变为精氨酸，显著提高了菌株表达甲壳素酶的酶活力，其中单突变株F273W和E336R较出发菌株酶活分别提高了1.37倍和1.22倍。改造后的菌株产酶能力和催化效率都得到了提高，通过HPLC检测纯化后突变体中几丁寡糖的含量发现，催化3h，F273W突变株产糖量为1.65g/L，其中GlcNAc为0.12g/L，(GlcNAc)2为1.53g/L，相比野生菌株提高了49.7％。

Description

一种酶活提高的甲壳素酶突变体
技术领域
本发明涉及一种酶活提高的甲壳素酶突变体，属于酶工程和基因工程技术领域。
背景技术
甲壳素酶(EC 3.2.1.14.)又称几丁质酶，能催化不溶性的甲壳素糖链β-1,4糖苷键的断裂，生成水溶性的几丁寡糖。由于甲壳素酶既可作为生物防治剂，用于开发转基因植物，达到抗病的目的；也可用于原生质分离、细胞化学定位以及生产单细胞蛋白，因此，其在农业、生物技术、食品、卫生方面均有很重要的应用。而甲壳素酶的水解产物几丁寡糖能够提高机体免疫力、抑制肿瘤细胞生长、在人体肠道内活化增殖双歧杆菌、抗菌防腐、保湿，因此，其在医药、食品、化妆品等工业中也具有广阔的应用前景。
甲壳素酶分为3种类型，内切甲壳素酶、外切甲壳素酶和β-N乙酰氨基葡萄糖苷酶。这三种类型的酶均能以甲壳素为底物，将其切割成不同聚合度的几丁寡糖。内切甲壳素酶能随机切割甲壳素糖链中的β-1,4糖苷键，释放N-乙酰几丁寡糖；外切甲壳素酶从甲壳素糖链的非还原末端进行连续切割，释放(GlcNAc)2；N-乙酰氨基葡萄糖苷酶能够水解(GlcNAc)2释放出GlcNAc。
甲壳素酶广泛存在于动物、植物和微生物(包括细菌、真菌、粘菌、原生动物、藻类等)中。动物和植物中的甲壳素酶含量低，提取困难，而微生物法生产甲壳素酶具有易培养、操作、成本低等优点，成为广大学者研究的重点。目前，研究产甲壳素酶的微生物主要包括Paenibacillus barengoltzii、Marine Bacterium(Alteromonas sp.Strain 0-7)、Streptomyces thermoviolaceus OPC-520和Bacillus cereus等，但野生菌株中的甲壳素酶产量很低，仅有0.83-1.13U/mL。
目前，已经有研究通过异源表达、酶分子改造等技术手段试图提高甲壳素酶表达量，例如，在大肠杆菌或毕赤酵母中异源表达甲壳素酶基因以及通过定点突变或以随机突变的方式改造甲壳素酶的底物结合结构域和催化结构域以提高酶活，但是，在大肠杆菌中异源表达甲壳素酶基因易形成包涵体；在大肠杆菌以及毕赤酵母中异源表达甲壳素酶基因时，甲壳素酶为胞内分泌，因此，提取甲壳素酶需破壁导致酶活损失；在毕赤酵母中异源表达甲壳素酶基因操作复杂、培养周期长；以随机突变的方式改造甲壳素酶的底物结合结构域和催化结构域以提高酶活具有随机突变的不确定性，因此，会导致筛选困难，上述技术均并不能够真正运用于工业生产。
因此，急需找到一种新的可克服表达量低、易形成包涵体、破壁导致酶活损失等缺陷、可大幅度提高甲壳素酶表达量的方法。
发明内容
本发明的第一个目的是提供一种甲壳素酶突变体，含如SEQ ID NO.2-6所示的氨基酸序列。
本发明的第二个目的是提供编码上述甲壳素酶突变体的基因。
在本发明的一种实施方式中，编码所述甲壳素酶突变体C43D、F273W、E336R、C43D/F273W、C43D/F273W/E336R的基因的核苷酸序列分别如SEQ ID NO.10-14所示
本发明的第三个目的是提供含上述基因的载体。
本发明的第四个目的是提供表达上述甲壳素酶突变体的细胞。
本发明的第五个目的是提供一种基因工程菌，是以枯草芽孢杆菌为宿主，表达上述甲壳素酶突变体。
在本发明的一种实施方式中，是以Bacillus.subtilis WB600为宿主，以pP43NMK为表达载体。
本发明的第六个目的是提供一种提高甲壳素酶酶活的方法，是将氨基酸序列如SEQ ID NO.1所示的甲壳素酶第43位的半胱氨酸突变为天冬氨酸(氨基酸序列如SEQ IDNO.2所示)；
或，将第273位的苯丙氨酸突变为色氨酸(氨基酸序列如SEQ ID NO.3所示)；
或，将第336位的谷氨酸突变为精氨酸(氨基酸序列如SEQ ID NO.4所示)；
或，将第43位的半胱氨酸突变为天冬氨酸，并将第273位的苯丙氨酸突变为色氨酸(氨基酸序列如SEQ ID NO.5所示)；
或，将第43位的半胱氨酸突变为天冬氨酸，并将第273位的苯丙氨酸突变为色氨酸，并将第336位的谷氨酸突变为精氨酸(氨基酸序列如SEQ ID NO.6所示)。
本发明的第七个目的是提供上述基因工程菌在发酵领域的应用。
本发明的第八个目的是提供一种生产上述甲壳素酶突变体的方法，是将上述基因工程菌接种到LB液体培养基中，35-40℃培养7-10h后，转接到TB培养基中，接种量为2-5％，35-40℃培养12-14h。
本发明的第九个目的是提供上述的甲壳素酶突变体在农业、生物、化妆品、食品或卫生领域的应用。
本发明通过定点突变的方式，将甲壳素酶分子催化结构域附近的第43位氨基酸(半胱氨酸)突变为天冬氨酸，第273位氨基酸(苯丙氨酸)突变为色氨酸、第336位氨基酸(谷氨酸)突变为精氨酸，显著提高了菌株表达甲壳素酶的酶活力，其中单突变株F273W和E336R较出发菌株酶活分别提高了1.37倍和1.22倍。改造后的菌株产酶能力和催化效率都得到了提高，通过HPLC检测纯化后突变体中几丁寡糖的含量发现，催化3h，F273W突变株产糖量为1.65g/L，其中GlcNAc为0.12g/L，(GlcNAc)2为1.53g/L，相比野生菌株提高了49.7％。
附图说明
图1：突变体酶活。
图2：纯化后突变体的蛋白电泳图，M：marker；1：C43D；2：穿透液；3：20％B；4：F273W；5：穿透液；6：15％B；7：20％B；8：E336R；9：穿透液；10：15％B。
图3：突变体催化后产物含量。
具体实施方式
LB培养基：胰蛋白胨10g/L、酵母粉5g/L、NaCl 10g/L，pH 7.0。
TB培养基：蛋白胨12g/L、酵母膏24g/L、甘油5g/L、KH2PO4 17mmol/L、K2HPO472mmol/L。
采用分光光度法测定甲壳素酶酶活，1个单位甲壳素酶酶活定义为：在60℃反应条件下，每小时释放1μmol还原糖所需的酶量为一个酶活单位(U/mL)。酶活测定条件：60℃条件下，0.1mL 1％的胶体几丁质，0.3mL磷酸氢二钠-柠檬酸盐缓冲液(pH5.0)和0.1mL发酵上清液，保温15min，100℃加热5min终止反应，加入1mLDNS试剂，沸水浴加热10min后迅速冷却，用去离子水定容至5mL。利用分光光度计在540nm处测定吸光值，通过GlcNAc绘制标准曲线，根据标准曲线计算酶活。
实施例1产甲壳素酶菌株构建
化学合成甲壳素酶基因片段chisb(核苷酸序列如SEQ ID NO.9所示)。PCR条件为：98℃3min，30个循环(98℃10s、55℃15s、72℃1min)，72℃5min。以pP43NMK为模板，p43-F和p43-R为引物(见表1)进行全质粒PCR扩增，获得线性化pP43NMK载体。PCR条件为：98℃5min，25个循环(98℃10s、55℃15s、72℃4min 30s)，72℃5min。以上扩增产物经电泳检验后，采用胶回收试剂盒对PCR产物进行纯化和回收。通过一步克隆试剂盒clonExpressTM One StepCloning Kit(Vazyme Biotech Co.,Ltd.Nanjing,China)，将甲壳素酶基因片段与线性化载体pP43NMK进行融合，得到重组质粒pP43NMK-chisb。
将融合的重组质粒pP43NMK-chisb转化至感受态E.coli JM109，用氨苄青霉素LB平板，挑取阳性菌落。37℃摇床过夜培养后提取质粒，转化子由天霖(无锡)有限责任公司进行测序。
表1引物
Figure BDA0001894201480000031
Figure BDA0001894201480000041
实施例2甲壳素酶生产菌株的验证
将实施例1中测序正确的质粒pP43NMK-chisb，转化Bacillus.subtilis WB600。挑选转化子接种到LB培养基中，37℃，培养8h；转接到TB培养基中，接种量为2％，37℃培养12h，收集发酵上清液，检测发酵上清酶活，结果显示，甲壳素酶被分泌到胞外，酶活力为28.98U/mL(结果如图1所示)。
实施例3突变株的获得
利用定点突变试剂盒(购自TaKaRa，货号：KM101)，设计引物(见表2)，以已构建的pP43NMK-chisb为模板，进行PCR，将甲壳素酶分子催化结构域附近的第43位的半胱氨酸突变为天冬氨酸；第273位苯丙氨酸突变为色氨酸；或将第336位谷氨酸突变为精氨酸，分别命名为C43D、F273W、E336R。PCR反应条件为98℃5min，30个循环(98℃10s、55℃15s、72℃4min30s)，72℃5min。采用胶回收试剂盒对PCR产物进行纯化和回收，电泳检验回收产物。产物转化E.coli JM109，由天霖(无锡)有限责任公司进行测序，转化子命名为pP43NMK-chisb-C43D、pP43NMK-chisb-F273W、pP43NMK-chisb-E336R。
以测序正确的菌株pP43NMK-chisb-C43D的质粒为模板，将甲壳素酶分子第273位苯丙氨酸进行复合突变，突变为色氨酸，命名为C43D/F273W，转化子由天霖(无锡)有限责任公司进行测序，转化子命名为pP43NMK-chisb-C43D/F273W。
以测序正确的菌株pP43NMK-chisb-C43D/F273W的质粒为模板，将甲壳素酶分子第336位谷氨酸进行复合突变，突变为精氨酸，命名为C43D/F273W/E336R，转化子由天霖(无锡)有限责任公司进行测序，转化子命名为pP43NMK-chisb-C43D/F273W/E336R。
表2定点突变引物序列
引物名称 引物序列(5'-3')
C43D-F GCATTTGCCGACATTGACTGGGAGGGACGC
C43D-R GTCAATGTCGGCAAATGCATAGTTTATATGAG
F273W-F CCGAACGCAGAAACGTGGAATATTGAGAGC
F273W-R CCACGTTTCTGCGTTCGGCACCCCTGCCTC
E336R-F CGATTTTTCTGATTTGAGAAAGAACTATATC
E336R-R TCTCAAATCAGAAAAATCGAACACTCCTTT
实施例4甲壳素酶生产菌株的验证
将测序正确的质粒转化Bacillus.subtilis WB600，挑选转化子接种到LB液体培养基中，37℃，培养8h，转接到TB培养基中，接种量为2％，37℃，培养12h。收集发酵上清液，检测发酵上请酶活，结果如图1所示。实验结果表明与出发菌株比较，酶活力有所提高，WT、C43D、F273W、E336R、C43D/F273W、C43D/F273W/E336R酶活力分别为28.98U/mL、32.43U/mL、39.74U/mL、32.46U/mL、34.96U/mL、30.42U/mL；其中单突变株F273W和E336R的酶活较出发菌株分别提高了1.37倍和1.22倍；复合突变株C43D/F273W酶活较出发菌株提高了1.14倍。
实施例5突变体的纯化
按照实施例4中描述的操作方法，收集发酵12h后的上清。利用甲壳素酶基因chisb的C端所带的His标签进行镍柱纯化。纯化所需缓冲液为A液：40mM Tris-HCl、150mM NaCl(pH7.5)；B液：40mM Tris-HCl、150mM NaCl、500mM咪唑(pH 7.5)。纯化结果如图2所示。研究结果表明，三株突变体菌株均能用Ni柱进行纯化，C43D突变株用20％B液可以完全洗脱，穿透液中没有目的蛋白；F273W和E336R突变株用15％B液可以完全洗脱，穿透液中无目的蛋白。
实施例6突变体的催化活性检测
将纯化后的酶液与终浓度为2g/L的底物胶体甲壳素在60℃下反应3h，100℃加热5min终止反应，14000rpm离心10min获得上清液，用于HPLC检测。反应体系如下(1mL)：450μL缓冲液(磷酸氢二钠-柠檬酸缓冲液，pH 5.0)，300μL胶体甲壳素，250μL纯酶液。检测结果如图3所示。产量分别为：出发菌株WT：0.09g/L GlcNAc和0.74g/L(GlcNAc)2；C43D：0.09g/LGlcNAc和1.15g/L(GlcNAc)2；F273W：0.12g/L GlcNAc和1.53g/L(GlcNAc)2；E336R：0.17g/LGlcNAc和1.31g/L(GlcNAc)2。
对比例
以已构建的pP43NMK-chisb为模板，采用相同的方法，分别将甲壳素酶分子的第57位天冬酰胺和第308位脯氨酸突变为组氨酸，突变引物见表3。命名为N57H(氨基酸序列如SEQ ID NO.7所示，核苷酸序列如SEQ ID 15所示)和P308H(氨基酸序列如SEQ ID NO.8所示，核苷酸序列如SEQ ID 16所示)，转化子由天霖(无锡)有限责任公司进行测序，转化子命名为pP43NMK-chisb-N57H，pP43NMK-chisb-P308H。N57H和P308H突变菌株的酶活相比出发菌株均下降，分别为13.2U/mL和8.7U/mL。
表3突变引物
引物名称 引物序列(5'-3')
N57H-F GATCCTACAGGGCCACACCCACAGAAATGG
N57H-F GTGTGGCCCTGTAGGATCAGGATTCCCATG
P308H-F TGGAGTAATTGCGAACATGCTGATAACGGT
P308H-R ATGTTCGCAAATTACTCCAACCTCTACCGT
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。
SEQUENCE LISTING
<110> 江南大学
<120> 一种酶活提高的甲壳素酶突变体
<160> 30
<170> PatentIn version 3.3
<210> 1
<211> 563
<212> PRT
<213> Bacillus sp. DAU101
<400> 1
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Cys Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Phe Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Glu
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys
<210> 2
<211> 563
<212> PRT
<213> 人工合成
<400> 2
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Asp Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Phe Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Glu
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys
<210> 3
<211> 563
<212> PRT
<213> 人工合成
<400> 3
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Cys Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Trp Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Glu
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys
<210> 4
<211> 563
<212> PRT
<213> 人工合成
<400> 4
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Cys Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Phe Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Arg
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys
<210> 5
<211> 563
<212> PRT
<213> 人工合成
<400> 5
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Asp Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Trp Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Glu
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys
<210> 6
<211> 563
<212> PRT
<213> 人工合成
<400> 6
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Asp Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro Asn Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Trp Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Arg
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys
<210> 7
<211> 569
<212> PRT
<213> 人工合成
<400> 7
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Asp Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro His Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Trp Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu Pro Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Arg
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys His His His His His His
565
<210> 8
<211> 569
<212> PRT
<213> 人工合成
<400> 8
Ser Ser Asp Lys Ser Tyr Lys Ile Ile Gly Tyr Tyr Pro Ser Trp Gly
1 5 10 15
Ala Tyr Gly Arg Asp Phe Gln Val Trp Asp Met Asp Ala Ser Lys Val
20 25 30
Ser His Ile Asn Tyr Ala Phe Ala Asp Ile Asp Trp Glu Gly Arg His
35 40 45
Gly Asn Pro Asp Pro Thr Gly Pro His Pro Gln Lys Trp Ser Cys Gln
50 55 60
Asp Glu Asn Gly Val Ile Asp Val Pro Asn Gly Ser Ile Val Met Gly
65 70 75 80
Asp Pro Trp Ile Asp Val Gln Lys Ser Asn Ala Gly Asp Thr Trp Asp
85 90 95
Glu Pro Ile Arg Gly Asn Phe Lys Gln Leu Leu Lys Leu Lys Lys Asn
100 105 110
His Pro His Leu Lys Thr Phe Ile Ser Val Gly Gly Trp Ser Trp Ser
115 120 125
Asn Arg Phe Ser Asp Val Ala Ala Asp Pro Ala Ala Arg Glu Asn Phe
130 135 140
Ala Ala Ser Ala Val Asp Phe Leu Arg Lys Tyr Gly Phe Asp Gly Val
145 150 155 160
Asp Leu Asp Trp Glu Tyr Pro Val Ser Gly Gly Leu Pro Gly Asn Ser
165 170 175
Thr Arg Pro Glu Asp Lys Arg Asn Tyr Thr Leu Leu Leu Gln Asp Val
180 185 190
Arg Glu Lys Leu Asp Ala Ala Glu Ala Lys Asp Gly Lys Lys Tyr Leu
195 200 205
Leu Thr Ile Ala Ser Gly Ala Ser Pro Glu Tyr Val Ser Asn Thr Glu
210 215 220
Leu Asp Lys Ile Ala Glu Thr Val Asp Trp Ile Asn Ile Met Thr Tyr
225 230 235 240
Asp Phe Asn Gly Gly Trp Gln Ser Ile Ser Ala His Asn Ala Pro Leu
245 250 255
Phe Tyr Asp Pro Lys Ala Lys Glu Ala Gly Val Pro Asn Ala Glu Thr
260 265 270
Trp Asn Ile Glu Ser Thr Val Lys Arg Tyr Lys Glu Ala Gly Val Lys
275 280 285
Ala Asp Lys Leu Val Leu Gly Thr Pro Phe Tyr Gly Arg Gly Trp Ser
290 295 300
Asn Cys Glu His Ala Asp Asn Gly Glu Tyr Gln Lys Cys Gly Pro Val
305 310 315 320
Lys Glu Gly Thr Trp Glu Lys Gly Val Phe Asp Phe Ser Asp Leu Arg
325 330 335
Lys Asn Tyr Ile Asn Lys Asn Gly Tyr Lys Arg Tyr Trp Asn Asp Arg
340 345 350
Ala Lys Val Pro Phe Leu Tyr Asn Ala Glu Asn Gly Asn Phe Ile Thr
355 360 365
Tyr Asp Asp Glu Glu Ser Tyr Gly Tyr Lys Thr Asp Leu Ile Gln Ser
370 375 380
Asn Gly Leu Ser Gly Ala Met Phe Trp Asp Phe Ser Gly Asp Ser Asn
385 390 395 400
Gln Thr Leu Leu Asn Lys Leu Ala Ala Asp Leu Gly Phe Ala Pro Gly
405 410 415
Gly Gly Asn Pro Glu Pro Pro Ala Ser Ala Pro Gly Asn Leu Arg Val
420 425 430
Thr Glu Lys Thr Ala Thr Ser Ile Ser Leu Val Trp Asp Ala Pro Ser
435 440 445
Asp Gly Ala Asn Ile Ala Glu Tyr Val Leu Ser Tyr Glu Gly Gly Ala
450 455 460
Val Ser Val Lys Asp Thr Ser Ala Thr Ile Gly Gln Leu Lys Pro Asn
465 470 475 480
Thr Thr Tyr Ser Phe Thr Val Ser Ala Lys Asp Ala Asp Gly Lys Leu
485 490 495
His Thr Gly Pro Thr Ile Glu Ala Thr Thr Asn Ser Asp Gln Thr Cys
500 505 510
Gly Tyr Asn Glu Trp Lys Asp Thr Ala Val Tyr Thr Gly Gly Asp Arg
515 520 525
Val Val Phe Asn Gly Lys Val Tyr Glu Ala Lys Trp Trp Thr Lys Gly
530 535 540
Glu Gln Pro Asp Gln Ala Gly Glu Ser Gly Val Trp Lys Leu Ile Gly
545 550 555 560
Asp Cys Lys His His His His His His
565
<210> 9
<211> 1710
<212> DNA
<213> Bacillus sp. DAU101
<400> 9
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacatttgct gggagggacg ccatgggaat cctgatccta cagggccaaa cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgttta atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acctgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttggagaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 10
<211> 1710
<212> DNA
<213> 人工合成
<400> 10
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacattgact gggagggacg ccatgggaat cctgatccta cagggccaaa cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgttta atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acctgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttggagaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 11
<211> 1710
<212> DNA
<213> 人工合成
<400> 11
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacatttgct gggagggacg ccatgggaat cctgatccta cagggccaaa cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgtgga atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acctgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttggagaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 12
<211> 1710
<212> DNA
<213> 人工合成
<400> 12
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacatttgct gggagggacg ccatgggaat cctgatccta cagggccaaa cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgttta atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acctgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttgagaaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 13
<211> 1710
<212> DNA
<213> 人工合成
<400> 13
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacattgact gggagggacg ccatgggaat cctgatccta cagggccaaa cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgtgga atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acctgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttggagaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 14
<211> 1710
<212> DNA
<213> 人工合成
<400> 14
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacattgact gggagggacg ccatgggaat cctgatccta cagggccaaa cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgtgga atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acctgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttgagaaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 15
<211> 1710
<212> DNA
<213> 人工合成
<400> 15
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacatttgct gggagggacg ccatgggaat cctgatccta cagggccaca cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgttta atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acctgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttggagaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 16
<211> 1710
<212> DNA
<213> 人工合成
<400> 16
agttccgaca agtcttataa gataataggt tattaccctt cttggggtgc ctacggacgg 60
gattttcagg tgtgggatat ggatgcgtct aaggtgtctc atataaacta tgcatttgcc 120
gacatttgct gggagggacg ccatgggaat cctgatccta cagggccaaa cccacagaaa 180
tggagctgtc aagacgagaa tggcgtgatt gatgtgccta atggatcaat cgtgatgggt 240
gacccgtgga ttgatgtcca aaaaagcaac gcaggagata catgggatga gccgattaga 300
ggaaatttca aacaactcct caagttgaag aaaaatcatc cgcaccttaa gacgtttatc 360
tctgtggggg gttggagttg gtcaaataga ttttctgatg tagcggctga cccggcagca 420
cgtgaaaact tcgccgccag cgcagtggac tttctgcgga agtacggttt tgacggcgtt 480
gacttggact gggaataccc agtcagtggg ggtctccctg gcaactcaac aagaccagaa 540
gacaaacgta attacacgtt attgcttcag gatgtgcggg agaaattgga cgccgctgaa 600
gcaaaagatg gtaaaaagta cctcctcacg atcgcgagtg gggctagccc ggaatatgta 660
agcaatacag aattggataa gatcgctgaa acggtcgact ggattaatat tatgacatat 720
gacttcaacg ggggatggca gtcaattagc gcgcataacg ctccactgtt ctatgacccg 780
aaggcaaaag aggcaggggt gccgaacgca gaaacgttta atattgagag cacggtgaag 840
cgctacaagg aagctggcgt gaaggcggat aagctggtac tcggaacccc tttttacggt 900
agaggttgga gtaattgcga acatgctgat aacggtgagt atcagaagtg tggtccagta 960
aaagagggta cctgggaaaa aggagtgttc gatttttctg atttggagaa gaactatatc 1020
aacaaaaatg gctataagcg ctactggaac gatcgggcaa aggttccgtt cttatataac 1080
gctgagaacg ggaactttat tacctatgac gatgaggaaa gctatggcta caagacggac 1140
ttgatccaat caaacggact gtccggggcg atgttttggg atttctcagg agatagcaac 1200
cagaccttac ttaacaaatt agccgctgat ttgggatttg ctccgggcgg tggtaatcct 1260
gaaccgccag caagtgcacc tgggaatctc cgtgtcacag agaagacagc cacttctatc 1320
agtcttgttt gggacgctcc aagtgatggg gctaacatag ccgagtacgt attatcttac 1380
gagggtggag ctgtgagcgt taaggacaca tcagctacaa taggtcagct gaaacctaat 1440
acaacgtact cttttactgt ctcagccaaa gatgctgatg ggaagctgca cacggggccg 1500
acgatcgaag ccaccactaa ctcagatcag acctgtggct ataatgaatg gaaggatact 1560
gcagtttaca ccgggggtga tagagttgtc tttaacggaa aagtgtacga agccaagtgg 1620
tggacaaagg gagaacagcc tgaccaggct ggcgagtcag gcgtttggaa gttaataggc 1680
gactgcaagc accaccacca ccaccactaa 1710
<210> 17
<211> 22
<212> DNA
<213> 人工合成
<400> 17
tgatgaaagc ttggcgtaat ca 22
<210> 18
<211> 24
<212> DNA
<213> 人工合成
<400> 18
agctgaggca tgtgttacaa aaac 24
<210> 19
<211> 30
<212> DNA
<213> 人工合成
<400> 19
gcatttgccg acattgactg ggagggacgc 30
<210> 20
<211> 32
<212> DNA
<213> 人工合成
<400> 20
gtcaatgtcg gcaaatgcat agtttatatg ag 32
<210> 21
<211> 30
<212> DNA
<213> 人工合成
<400> 21
ccgaacgcag aaacgtggaa tattgagagc 30
<210> 22
<211> 30
<212> DNA
<213> 人工合成
<400> 22
ccacgtttct gcgttcggca cccctgcctc 30
<210> 23
<211> 31
<212> DNA
<213> 人工合成
<400> 23
cgatttttct gatttgagaa agaactatat c 31
<210> 24
<211> 30
<212> DNA
<213> 人工合成
<400> 24
tctcaaatca gaaaaatcga acactccttt 30
<210> 25
<211> 30
<212> DNA
<213> 人工合成
<400> 25
gatcctacag ggccacaccc acagaaatgg 30
<210> 26
<211> 30
<212> DNA
<213> 人工合成
<400> 26
gtgtggccct gtaggatcag gattcccatg 30
<210> 27
<211> 30
<212> DNA
<213> 人工合成
<400> 27
tggagtaatt gcgaacatgc tgataacggt 30
<210> 28
<211> 30
<212> DNA
<213> 人工合成
<400> 28
tggagtaatt gcgaacatgc tgataacggt 30
<210> 29
<211> 49
<212> DNA
<213> 人工合成
<400> 29
ttgtaacaca tgcctcagct agttccgaca agtcttataa gataatagg 49
<210> 30
<211> 41
<212> DNA
<213> 人工合成
<400> 30
attacgccaa gctttcatca ttagtggtgg tggtggtggt g 41

Claims (10)

1.一种甲壳素酶突变体，其特征在于，所述突变体氨基酸序列如SEQ ID NO.2-6任一所示。
2.编码权利要求1所述的甲壳素酶突变体的基因。
3.含有权利要求2所述基因的载体。
4.表达权利要求1所述甲壳素酶突变体的细胞。
5.一种基因工程菌，其特征在于，是以枯草芽孢杆菌为宿主，表达权利要求1所述的甲壳素酶突变体。
6.如权利要求5所述的基因工程菌，其特征在于，是以Bacillus.subtilis WB600为宿主，以pP43NMK为表达载体。
7.一种提高甲壳素酶酶活的方法，其特征在于，将氨基酸序列如SEQ ID NO.1所示的甲壳素酶第43位的半胱氨酸突变为天冬氨酸；
或，将第273位的苯丙氨酸突变为色氨酸；
或，将第336位的谷氨酸突变为精氨酸；
或，将第43位的半胱氨酸突变为天冬氨酸，并将第273位的苯丙氨酸突变为色氨酸；
或，将第43位的半胱氨酸突变为天冬氨酸，并将第273位的苯丙氨酸突变为色氨酸，并将第336位的谷氨酸突变为精氨酸。
8.权利要求5或6所述基因工程菌在发酵领域的应用。
9.一种生产权利要求1所述甲壳素酶突变体的方法，其特征在于，将权利要求5或6所述的基因工程菌接种到LB液体培养基中，35-40℃培养7-10h后，转接到TB培养基中，接种量为2-5％，35-40℃培养12-14h。
10.权利要求1所述的甲壳素酶突变体在农业、生物、化妆品、食品或卫生领域制备含有几丁寡糖产品中的应用。
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