CN109295031A - 一种抗真菌蛋白β-1,3-葡聚糖酶和含有该基因的工程菌及其应用 - Google Patents
一种抗真菌蛋白β-1,3-葡聚糖酶和含有该基因的工程菌及其应用 Download PDFInfo
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- CN109295031A CN109295031A CN201811236660.4A CN201811236660A CN109295031A CN 109295031 A CN109295031 A CN 109295031A CN 201811236660 A CN201811236660 A CN 201811236660A CN 109295031 A CN109295031 A CN 109295031A
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Abstract
本发明公开了一种β‑1,3‑葡聚糖酶基因、含有该基因的工程菌及其在抗真菌方面的应用。本发明提供了一种抗真蛋白β‑1,3‑葡聚糖酶基因，其核苷酸序列为：SEQ ID NO.1。所编码的β‑1,3‑葡聚糖酶氨基酸序列为：SEQ ID NO.2。利用该基因构建的毕赤酵母工程菌株能高效表达β‑1,3‑葡聚糖酶，该酶以昆布多糖为底物时其比活力高达10.8 U/mg。该β‑1,3‑葡聚糖酶能有效的水解真菌的细胞壁，如安琪酵母和稻瘟病菌，进而抑制稻瘟孢子的萌发和附着孢的形成，展现出良好的抗真菌活性。利用基因生产的酶制剂可用于农业上植物病原真菌的生物防治，在解决实际问题的同时还可以取得可观的经济效益。
Description
技术领域
本发明属于应用工业微生物领域，公开了一种新的抗真菌蛋白β-1,3-葡聚糖酶、含有该基因的工程菌及其在抗真菌方面的应用。
背景技术
β-葡聚糖(β-Glucan)是一类非淀粉性多糖(NSP)，由D-葡萄糖单元通过的β-糖苷键连接的同聚糖。广泛分布于植物、藻类、细菌、真菌及酵母等细胞壁中。β-1,3-葡聚糖酶是一类能特异性作用于β-1,3-葡聚糖中的β-1,3-糖苷键的糖苷水解酶，又称为海带多糖酶或昆布多糖酶(Laminarinase)。β-1,3-葡聚糖酶可以水解植物病原真菌细胞壁最外层的β-1,3-葡聚糖，导致细胞壁骨架受损，从而抑制病原真菌的生长与增殖。因此，近年来β-1,3-葡聚糖酶是研究最多、应用最广泛的抗真菌蛋白之一，其抗真菌具有广谱性、持久性的特点。
β-1,3-葡聚糖酶的来源广泛，在自然界中的真菌、细菌、古菌、放线菌、藻类、昆虫、低等动物、植物甚至病毒中都存在。目前，微生物来源的β-1,3-葡聚糖酶已经在工业酶制剂中得到广泛的应用，包括来源于真菌的有哈茨木酶(Trichoderma harzianum)、无根根霉菌(Rhizopus arrhizus)、香菇(Lentinula edodes)、米曲霉(Aspergillus oryzae)等，来源于细菌的有枯草芽孢杆菌(Bacillus subtilis)、类芽孢杆菌(Paenibacillus sp.)、链霉菌(Streptomyces sp.)等，来源于古菌的有Pyrococcus furiosus。Liangwu Sun等还从小球藻病毒(Chlorella virus PBCV-1)克隆到一个属于GH16家族的β-1,3-葡聚糖酶A94L，并对其酶学性质进行了研究。
β-1,3-葡聚糖酶的应用范围广泛，包括在真菌细胞壁的结构分析、原生质体的制备等科学研究方面；植物病原真菌的防治等农业及生态保护方面及酵母提取物的制备、饲养业和酿造业等工业生产方面。目前许多体外抑菌试验表明，无论内切型还是外切型的β-1,3-葡聚糖酶对病原真菌细胞壁都具有不同程度的水解作用。Fridlender等从Pseudomonas cepacia中纯化到一个内切型的β-1,3-葡聚糖酶，该酶对立枯丝核菌(Rhizoctonia solani)、齐整小核菌(Sclerotium rolfsii)、腐霉(Pythium ultimwn)等菌丝的生长具有良好的抑制作用，生防效果分别达到85%、48%和71%。Donzelli等从生防菌哈茨木酶(T. harzianum)中克隆到一个具有生防作用的外切型的β-1,3-葡聚糖酶GLUC78基因。另外，外源β-1,3-葡聚糖酶基因可以通过转基因技术导入到农作物中，从而提高农作物对病原真菌的抗性。β-1,3-葡聚糖酶还可以运用于酵母细胞壁葡聚糖结构的分析、酵母原生质体的制备、酵母功能性多糖提取等。同时，在饲料中添加β-1,3-葡聚糖酶和植酸酶等，可以消除饲料中抗营养作用，提高家禽对营养物质的消化利用以及改善调节家禽肠道微生态菌等作用。
植物病害是我国农作物优质高产的重要制约因素之一，严重威胁着我国粮食安全。在植物病害中，70%-80%的病害是病原真菌侵染所致，真菌病害导致农作物产量和品质下降，且侵染过程中所分泌的有害毒素和代谢物威胁着农产品安全。据估算，真菌病害已让水稻、小麦、玉米、马铃薯和黄豆等5大粮食作物的产量在全球范围内每年减少1.25亿吨，其中，单是对水稻、小麦和玉米的危害，就给全球农业带来每年600亿美元的经济损失。目前我国重大农作物真菌病害的防治主要通过化学防治、作物轮作及生物防治等方式，其中化学防治是主要的病害防治方式。针对目前杀菌剂的大量使用所造成的环境污染和农药残留等安全问题，绿色生物防治研究是植物病害控制的重要方向，其中抗真菌蛋白作为生物防治的重要资源已成为研究者关注的对象。因此筛选抗真菌微生物资源、发掘性能优良的抗真菌蛋白、解析抗菌蛋白的作用机制，可为植物真菌病害防治关键技术的研发奠定基础，从而进一步为我国农作物优质高产、降低生产成本、减少污染性化学农药使用提供技术保障。
发明内容
本发明的目的在于提供一种抗真菌蛋白β-1,3-葡聚糖酶。
本发明的另一目的是提供含有该β-1,3-葡聚糖酶基因的基因工程菌株。
本发明的又一目的是提供该基因及其编码蛋白质的应用。
一种β-1,3-葡聚糖酶基因，其核苷酸序列为：SEQ ID NO.1，全长（从起始密码子到终止密码子）为1242 bp，G+C%高达为69.8%，编码413个氨基酸，蛋白质的理论分子量为45.6kDa。本发明基因是一个新型β-1,3-葡聚糖酶基因，与来源于嗜热古菌P. furiosus的β-1,3-葡聚糖酶pfLamA的同源性最高，仅为38%。
本发明所述的β-1,3-葡聚糖酶基因编码的β-1,3-葡聚糖酶蛋白质,其氨基酸序列为：SEQ ID NO.2。
本发明所述的β-1,3-葡聚糖酶最适反应pH为7.0，最适反应温度为45 ℃。在50 ℃孵育30 min后还残留80%的原始酶活；在pH值为5-9的缓冲液中4℃孵育24 h后，依然残留40%以上的酶活力，表现出较好的稳定性。本发明所述的β-1,3-葡聚糖酶是一种新型多功能葡聚糖酶。与现有技术公开的β-1,3-葡聚糖酶相比，β-1,3-葡聚糖酶LamC除了可以水解β-1,3-糖苷键，对β-1,4、β-1,6的葡聚糖糖苷键及β-1,4-木聚糖糖苷键都具有水解活性，具有广底物谱特性。同时，重组β-1,3-葡聚糖酶具有显著裂解酵母细胞和灰霉孢子细胞壁的作用，展现出良好的抗真菌活性。
含本发明所述β-1,3-葡聚糖酶基因的重组质粒。
所述的重组质粒优选将所述β-1,3-葡聚糖酶基因克隆到pEFaA中所得。
含本发明所述的重组质粒的重组微生物。
所述的重组微生物，优选以毕赤酵母（Pichia pastoris GS115）为宿主菌。
本发明所述β-1,3-葡聚糖酶蛋白质在水解酵母细胞壁方面的应用。
本发明所述β-1,3-葡聚糖酶作为抗菌蛋白在农业抗植物病原真菌方面的应用。
有益效果
1. 本发明以粘细菌菌株EGB为材料，参考EGB基因组序列信息并结合PCR扩增，成功获得β-1,3-葡聚糖酶基因序列。该基因全长（从起始密码子到终止密码子）为1242 bp，G+C含量为69.8%，编码413个氨基酸。
2. 通过PCR技术扩增末端含XhoI和XbaI酶切位点的β-1,3-葡聚糖酶基因片段，命名为lamC。将它连接到毕赤酵母P. pastoris GS115高表达载体pEFaA（购自Novegen公司）的XhoI和XbaI酶切位点上，转化表达宿主菌P. pastoris GS115（购自Invitrogen公司），进行甲醇诱导表达，诱导表达量为164 mg/L。
3. 本发明对β-1,3-葡聚糖酶基因表达的产物，通过DNS方法对β-1,3-葡聚糖酶进行酶活性测定，该β-1,3-葡聚糖酶能有效的作用于昆布多糖、羧甲基纤维素、木聚糖和石耳素等多糖。在以昆布多糖为底物时的比活力高达10.8 U/mg。
4. 利用该基因构建的工程菌株能高效表达β-1,3-葡聚糖酶，在以安琪酵母细胞为目标时，经12 h 处理后，经简单染色显微观察发现，大部分酵母细胞出现细胞壁破裂，细胞空洞等显现象。
5、利用该基因构建的工程菌株能高效表达β-1,3-葡聚糖酶，在以稻瘟病菌Guy11孢子为目标底物时。经重组β-1,3-葡聚糖酶LamC处理后，孢子发生明显的变形，结构变得不完整，且无附着胞的形成，孢子萌发受阻。以上现象表明重组酶LamC对稻瘟病菌具有较好的生防效果。
附图说明
图1 β-1,3-葡聚糖酶基因的PCR扩增电泳图；其中：1：DL5000核酸Marker；2：β-1,3-葡聚糖酶基因PCR扩增；
图2 β-1,3-葡聚糖酶基因克隆的策略图；
图3 β-1,3-葡聚糖酶基因在P. pastoris GS115（pEFaA）中高效表达实验方案图；
图4重组β-1,3-葡聚糖酶的SDS-PAGE电泳图；其中：M:标准蛋白Marker；1：重组蛋白纯酶；
图5 β-1,3-葡聚糖酶酶学性质，其中：a：β-1,3-葡聚糖酶最适温度；b：温度稳定性；c：β-1,3-葡聚糖酶最适pH；d：pH稳定性；
图6 β-1,3-葡聚糖酶对安琪酵母细胞完整性的影响，其中：CK：PBS缓冲液对照处理；1：β-1,3-葡聚糖酶处理安琪酵母细胞6 h；2：β-1,3-葡聚糖酶处理安琪酵母细胞12 h。
图7 β-1,3-葡聚糖酶对稻瘟病菌Guy11孢子萌发的影响，其中：Guy11：PBS缓冲液稻瘟病菌Guy11孢子的对照处理；热失活处理后的LamC表示稻瘟菌孢子与经热失活处理的酶液孵育后的孢子萌发情况；LamC组表示稻瘟菌孢子与正常活性的酶液孵育后的孢子萌发情况。
具体实施方式
以下实施例中，酶活测定方法为：取一定量的酶液加至1 mL含有10 mg/mL昆布多糖的20 mM PBS（pH 7.0）中，于45 ℃反应20 min后加入2 mL DNS进行测活，以DNS法测定产物葡萄糖的生成量。酶活单位定义为每分钟产生1 μmol葡萄糖当量所需的酶量（mg）即为一个单位（1 U）。
实施例1 β-1,3-葡聚糖酶基因的克隆及表达载体的构建
1.1 β-1,3-葡聚糖酶基因的PCR扩增
参考粘细菌Corallococcussp.EGB（CCTCC NO：M2012528，来源于中国典型培养物保藏中心）基因组序列并结合NCBI基因组信息进行ORF预测，以全长序列设计β-1,3-葡聚糖酶基因引物，以EGB菌的基因组DNA为模板，对β-1,3-葡聚糖酶基因进行PCR扩增，得到β-1,3-葡聚糖酶基因，所用引物为F和R，结果见图1。具体过程参照图2。
F: ctcgagAAAAGAGAGGCTTCGCGGGACGCGGCGCCTC (Xho I)（SEQ ID NO. 3）
R: tctgagTTAATGATGATGATGATGATGGCGCCACTGGTAGGCGCGC (Xba I)（SEQ ID NO. 4）
PCR扩增体系为：
| 体系成分 | 体积 |
| PrimeStar polymerase | 0.8µL |
| 2x GC Buffer | 25µL |
| dNTP | 8µL |
| 正向引物F（SEQ ID NO. 3） | 1µL |
| 反向引物R（SEQ ID NO. 4） | 1µL |
| EGB基因组DNA | 2µL |
| ddH<sub>2</sub>O | 12.2µL |
| 总体积 | 50µL |
PCR扩增条件：
| 步骤 | 温度 | 时间 | 循环数 |
| 预变性 | 94℃ | 5min | 1 |
| 变性 | 94℃ | 30s | |
| 退火 | 60℃ | 30s | 30 |
| 延伸 | 72℃ | 1min30s | |
| 终末延伸 | 72℃ | 10min | 1 |
| 保温 | 10℃ | 10min | 1 |
PCR扩增产物进行0.75％的琼脂糖凝胶电泳切胶回收。
1.2 E.coliDH5α普通感受态的制备
从-80 ℃保存的菌种中划一环菌体E. coli DH5α划线接于SOB平板上，37 ℃培养16~20 h，用无菌牙签挑取直径在2 mm左右的菌落，挑接种至含25 mL SOB培养基的250 mL三角瓶中，37 ℃以250 r∙min-1转速振荡培养6~8 h，分别接种2 mL，4 mL，10 mL培养液于装有250 mL SOB液体培养基的1 L三角瓶中，于18 ℃摇床中以200 rpm转速振荡培养过夜。通过分光光度计测定菌体的密度（OD600 nm），取用OD约等于0.45的培养液，将培养物先置于冰上10 min，4 ℃，4,000 rpm离心10 min，倒弃液体，将离心管倒扣在吸水纸上2 min流干剩余培养基，加80mL预冷的0.1mM CaCl2转化缓冲液在冰水浴里轻轻悬起菌体，于4 ℃，4,000rpm离心10 min，倒弃上层缓冲液并在吸水纸上扣干，再次用20 mL预冷的0.1mM CaCl2转化缓冲液重悬细菌沉淀，加1.5 mL DMSO。轻轻混匀细菌悬液，置于冰上10 min，按每管200 μL分装于1.5 mL无菌离心管中，置液氮中速冻，即为高效感受态细胞，可转存至-80 ℃冰箱待用。
1.3 酶连转化
经PCR 产生的β-1,3-葡聚糖酶DNΑ片段与pMD19-T Vector（TaKaRa）按摩尔比3:1 混
合，在连接酶液作用下，16℃水浴过夜。酶连体系如下：
| 体系成分 | 体积 |
| pMD19-T simple Vector（TaKaRa） | 1µL |
| β-1,3-葡聚糖酶基因PCR DNA产物 | 3µL |
| 10xT4 Ligase Buffer（TaKaRa） | 1µL |
| T4连接酶（TaKaRa） | 0.5µL |
| ddH<sub>2</sub>O | 4.5µL |
| 总体积 | 10µL |
将10 μL 酶连产物加入到200μl 在冰上融化后的E.coli DH5α感受态细胞中，冰浴30min，在42 ℃水浴锅中热激90 s 后。快速转移到冰浴中冷却3 min，向每管中加入800 μL液体LB 培养基，37℃摇床80-90 rpm 温育45 min，复苏细胞。4000 rpm 离心3 min，剩余200 μL感受态细胞涂布于含100 mg/L 氨苄青霉素的LB琼脂平板上，平板倒置于37 ℃培养箱培养。
1.4 目的基因质粒的提取与测序
挑取1.3中的单菌落含氨苄青霉素的LB培养基中培养过夜，6000 rpm离心1 min收集菌体，利用质粒提取试剂盒提取质粒，送上海英潍捷基生物有限公司测序。结果该基因全长（从起始密码子到终止密码子）为1239 bp，G+C含量为69.19%，序列为SEQ ID NO.1；该基因编码413个氨基酸，其氨基酸序列为SEQ ID NO.2。
1.5 将1.4 中提取的重组质粒用Xho I 和Xba I 双酶切
体系如下：
| 体系成分 | 体积 |
| <i>Xho</i> I | 2µL |
| <i>Xba</i> I | 2µL |
| 10×M Buffer | 5µL |
| 重组质粒DNA | 25µL |
| ddH<sub>2</sub>O | 16µL |
| 总体积 | 50µL |
将反应体系放置于37℃水浴锅中过夜反应，后跑1%琼脂糖凝胶核酸电泳回收酶切产物。
1.6 将表达载体pEFαA（购于Novegen）用Xho I 和Xba I双酶切（参考1.5）
1.7 酶连转化
体系如下：
| 体系成分 | 体积 |
| pEFαA双酶切产物 | 2 µL |
| 目的基因双酶切产物 | 3 µL |
| 10 x T4 Ligase Buffer | 1 µL |
| T4连接酶 | 0.5 µL |
| ddH<sub>2</sub>O | 3.5 µL |
| 总体积 | 10 µL |
将体系放置于16 ℃水浴锅中过夜反应得含有目的基因的重组质粒pEFαA-lamC。将酶连产物转化到E.coli DH5α中后，涂布于含有终浓度为25 µg/mL的Zeocin的LLB培养基平板上，挑取转化子质粒并酶切验证，测序正确后保存于终浓度为15 %甘油的-80 ℃低温冰箱中。
1.8重组质粒pEFαA-lamC的线性化
采用限制性内切酶NdeI对正确的重组质粒进行线性化，将体系放置于37 ℃水浴条件下反应8 h后回收酶切产物。
反应体系如下：
| 体系成分 | 体积 |
| <i>Nde</i>I | 3 µL |
| 10 x H Buffer | 5 µL |
| 重组质粒DNA | 30 µL |
| ddH<sub>2</sub>O | 12 µL |
| 总体积 | 50 µL |
本实验采用乙醇沉淀法纯化和回收线性化产物，具体方法如下：
1. 加入预冷的1/10体积的3 M醋酸钠(pH 5.2)和2倍体积的无水乙醇，轻轻混匀后放置于-20 ℃下20 min；
2. 然后于4 ℃条件下12000 rpm，离心10 min，弃上清液；
3. 向离心管中加入2倍体积的预冷70 %乙醇，12000 rpm，离心3 min，重复一次，弃上清；
4. 待乙醇挥发干净后，加入30 µL无菌水溶解。
1.9P.pastorisGS115感受态细胞制备
从接种在新鲜的YPD斜面上的P.pastorisGS115（购自中国典型培养物保藏中心）挑取单菌落；接种单菌落至50 mL YPD培养基的250 mL三角瓶中，28 ℃，200 rpm，培养过夜至OD600值在1.0-1.5之间；吸取培养液按5%接种量接种至50 mL YPD培养基的250 mL三角瓶中，28℃，200 rpm，培养至OD600值在0.3-0.5之间；4500 rpm，离心5 min，收集菌体；将菌体与新鲜配制的8 mL酵母细胞感受态母液轻轻混匀后，室温放置30 min，每10 min轻轻摇匀一次；4500 rpm，离心5 min，收集菌体；用2 mL的预冷的1 M山梨醇重悬，然后于4 ℃条件下，4000 rpm，离心5 min，收集菌体，重复三次；然后用100 µL预冷的1 M山梨醇重悬，分装至1.5 mL离心管中，每管80 µL，于-80 ℃下保存。
1.10电转化
将0.2 cm电转化杯放置于冰上5 min，打开电转化仪，调整所需电转化参数：电压1.5kV，电阻250 Ω，电容25 µF；向制备好的酵母感受态细胞中加入1-3 µg 1.6中线性化的DNA片段，轻轻混匀后放置于冰上5 min后转移至电转杯中；按照仪器使用说明书进行电转操作；电转化后立刻向电转杯中加入1 mL预冷的1 M山梨醇，转入1.5 mL离心管中，放置在30℃培养箱中静置培养1 h；室温下4000 rpm下离心3 min，弃上清，收集菌体后用200 µLYPDS重悬；将100 µL细胞悬浮液涂布在含有100 µg/mL Zeocin的YPD平板上；将平板放置在30 ℃下培养3-4天，待菌体长出。
将平板上长出的单菌落挑至含有100 µg/mL Zeocin的YPD平板上，待长出后使用目的基因的特征引物进行菌落PCR验证,以检测目的基因是否整合到酵母染色体中；25 µL反应体系，退火温度60 ℃，延伸时间1 min 45s。PCR产物用0.75 %琼脂糖凝胶电泳检测DNA片段大小。
其中整合有β-1,3-葡聚糖酶基因即为基因工程菌株P. pastoris GS115（pEFαA-lamC）。
实施例2. β-1,3-葡聚糖酶基因在P. pastoris GS115（pEFaA）中的高效表达
2.1 β-1,3-葡聚糖酶LamC的表达
将重组微生物菌株P. pastoris GS115 (pEFαA-lamC)划线平板培养，挑取单菌落至50mL液体YPD三角瓶中，28 ℃，200 rpm培养24 h；然后在室温下4500 rpm，离心10 min，弃去上清液，将菌体用25 mL BMMY培养基重悬，开始诱导酵母细胞的表达；继续28 ℃条件下，200 rpm培养，每隔24 h补加甲醇至终浓度为0.5 % (v/v)，共培养96 h；待培养结束后，离心收集发酵上清液即为β-1,3-葡聚糖酶粗酶液。取适量β-1,3-葡聚糖酶粗酶液加至1ml 含有0.5％的昆布多糖的Tris-HCl 缓冲液中，于50℃反应10 min 后，通过DNS检测还原糖的生成情况。检测目的蛋白的酶活，结果发现该β-1,3-葡聚糖酶在毕赤酵母中实现了高表达。
2.2 β-1,3-葡聚糖酶LamC的纯化
取P.pastoris GS115 (pEFαA-lamC)经过96小时诱导表达的发酵上清，采用盐析法先对发酵酶液进行浓缩。按0-80%的硫酸铵饱和梯度进行分级沉淀。操作步骤：将发酵酶液量好体积，倒入烧杯中，烧杯内放置磁性转子，将烧杯置于冰上，打开磁力搅拌器。按粗酶液的体积缓慢加入硫酸铵，使饱和度达到80%，4℃，13,000 rpm 离心20 min，沉淀以少量不含硫酸铵的50 mM Tris-HCl pH7.0的缓冲液重悬，测定蛋白含量和酶活。重悬后的酶液于4℃下在50 mM Tris-HCl pH7.0缓冲液中透析过夜。将透析后的酶液上样至预先以两倍柱体积的50 mM Tris-HCl pH7.0平衡的Ni2+-NTA 亲和层析柱，4℃孵育1 h，以5倍柱体积的50 mMTris-HCl（pH7.0，50 mM 咪唑）洗去杂蛋白，然后以50 mM Tris-HCl（pH7.0，100 mM、300 mM咪唑）进行梯度洗脱，经酶活测定以及SDS-PAGE 电泳，收集合并重组酶纯度最高的洗脱液，4℃，以截留分子量10 KDa的透析袋在50 mM Tris-HCl（pH 7.0）缓冲液中透析过夜去除咪唑，获得β-1,3-葡聚糖酶的纯酶液。将最后纯化的重组蛋白进行SDS-PAGE检测，结果如图4所示，在考马斯亮蓝染色胶显示单一的目的条带，达到电泳纯化标准，且与预测的分子量44.6 kDa一致。取适量β-1,3-葡聚糖酶纯酶液以0.5％的昆布多糖为底物时，β-1,3-葡聚糖酶的比活力为10.8 U/mg。
实施例3. β-1,3-葡聚糖酶LamC酶学性质的研究
3.1温度对酶活力的影响
最适反应温度的测定：在不同温度（20℃、30℃、40℃、45℃、50℃、55℃、60℃、70℃），pH7.0的条件下测定重组酶纯化酶的活性，将最高酶活力设定为100%（图5 a）。热稳定性的测定：将重组酶纯化酶液在20℃、30℃、40℃、45℃、50℃、60℃、70℃，pH7.0下保温30 min和1 h,随后置于冰上迅速冷却，各自测定残余酶活力，以未保温的酶活力为100%（图5 b）。重组酶LamC的最适反应温度为45℃，在20~60℃范围酶活力保持在60%以上的。
3.2 pH对酶活力的影响
最适反应pH的测定：在不同的pH值的缓冲液体系中测定酶活力，缓冲液分别为：柠檬酸缓冲液(pH 3-6); 20 mM Tris-HCl缓冲液(pH 6-9); 20 mM Gly-NaOH缓冲液(pH 9-10)。45℃下测定重组酶纯化酶液的活性，将最高活力设定为100%（图5 c）。pH稳定性的测定：将重组酶置于不同pH值的缓冲液中，4℃放置24 h后，加入到测活体系中，45℃，反应20 min后各自测定其残余活力，以pH 7.0的酶活力为100%（图5 d）。LamC的最适pH值为7.0，该酶在pH值为5.0-8.0时酶活力维持在80%以上，表现出较宽的pH适应性。同时，其在pH值为6.0-8.0的缓冲液中稳定最好，在此pH区间的缓冲液中孵育24 h后依然维持70%的原始酶活力。
以昆布多糖、羧甲基纤维素、木聚糖和石耳素为底物，对表达的β-1,3-葡聚糖酶进行底物特异性进行分析（如表1），结果发现该β-1,3-葡聚糖酶对昆布多糖、羧甲基纤维素、木聚糖和石耳素等底物均具有活性，对右旋糖苷没有活性，表明β-1,3-葡聚糖酶LamC可以作用于多种类型β-糖苷键，包括β-1,3、β-1,4和β-1,6的葡聚糖糖苷键及β-1,4-木聚糖糖苷键。其中以昆布多糖为底物时活性最高，β-1,3-葡聚糖酶的比活力为10.8 U/mg。
表1
| 底物 | 主要键型 | 比酶活 | 相对酶活(100%) |
| 昆布多糖 | β-1,3, β-1,6 (Glc) | 10.8±1.2 | 100 |
| 羧甲基纤维素 | β-1,4 (Glc) | 4.5±0.3 | 41.6 |
| 木聚糖 | β-1,4 (Xyl) | 6.1±0.7 | 56.4 |
| 石耳素 | β-1,6 (Glc) | 1.9±0.1 | 17.6 |
| 右旋糖苷 | α-1,6 (Glc) | 0 | 0 |
实施例4. β-1,3-葡聚糖酶LamC在水解安琪酵母细胞壁的应用
大多植物病害真菌细胞壁的主要成分是β-1,3-葡聚糖。β-1,3-葡聚糖酶（EC3.2.1.39）作为一种重要的病程相关(PR)蛋白已经被广泛研究，β-1,3-葡聚糖酶能催化β-1,3-葡聚糖多聚体的水解，可作为抗菌蛋白破坏真菌细胞壁，从而抑制真菌的生长增殖或杀死病原菌。
利用该基因构建的工程菌株能高效表达β-1,3-葡聚糖酶，在以安琪酵母细胞为底物时，置于28℃处理12和24 h。随后离心用ddH2O重悬洗涤两次，最后用等体积的ddH2O重悬并取10μL用于显微观察(图6)。经简单染色显微观察发现，大部分酵母细胞出现细胞壁破裂，细胞空洞等显现象。表明β-1,3-葡聚糖酶LamC具有水解真菌细胞壁的作用。
实施例5. β-1,3-葡聚糖酶LamC在稻瘟病菌生防的应用
酶对稻瘟孢子抑制和攻击作用的测定：将供试稻瘟病菌Guy11繁殖后配成108个/mL的孢子悬液，与重组酶液按1:1等量混合后，置于28℃处理2 h，吸取20 μL滴加至疏水膜上，28℃黑暗保湿培养6 h，以正常的稻瘟菌孢子和经热失活处理的酶液处理作为对照。显微镜下观察分生孢子萌发、芽管及附着孢形成情况。
将经过重组酶LamC处理的稻瘟病菌Guy11孢子，吸取20 μL滴加至疏水膜上，28℃黑暗保湿培养6 h后，显微镜下观察分生孢子萌发、芽管及附着孢形成情况（图7）。结果见图4-2-3，未经处理的和经失活处理的重组酶LamC处理的稻瘟孢子几乎全部萌发并形成附着管。而重组酶LamC处理组对稻瘟菌孢子的芽管和附着胞的形成都造成显著的抑制作用，与对照相比可以明显的观察到孢子发生变形，结构变得不完整，无附着胞的形成。以上现象表明重组酶LamC对稻瘟病菌具有一定的生防活性。
序列表
<110> 南京工业大学
<120> 一种抗真菌蛋白β-1,3-葡聚糖酶和含有该基因的工程菌及其应用
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Claims (10)
1.β-1,3-葡聚糖酶基因，其特征在于,其核苷酸序列如SEQ ID NO.1所示。
2.权利要求1所述的β-1,3-葡聚糖酶基因编码的β-1,3-葡聚糖酶蛋白质,其氨基酸序列如SEQ ID NO.2所示。
3.含权利要求1所述β-1,3-葡聚糖酶基因的重组质粒。
4.根据权利要求3所述的重组质粒，其特征在于，所述的重组质粒是将权利要求1所述β-1,3-葡聚糖酶基因克隆到pEFaA中所得。
5.含权利要求3所述的重组质粒的重组微生物。
6.根据权利要求5所述的重组微生物，其特征在于，以毕赤酵母GS115为宿主菌。
7.权利要求1所述的β-1,3-葡聚糖酶基因在水解酵母细胞壁中的应用。
8.根据权利要求7所述的应用，其特征在于，所述的酵母为安琪酵母细胞。
9.权利要求2所述的β-1,3-葡聚糖酶作为抗菌蛋白在农业抗植物病原真菌方面的应用。
10.根据权利要求9所述的应用，其特征在于，所述的农业抗植物病原真菌为稻瘟病菌Guy11。
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