CN115094075A - 一种海藻酸裂解酶高产菌株及其应用 - Google Patents
一种海藻酸裂解酶高产菌株及其应用 Download PDFInfo
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- CN115094075A CN115094075A CN202210888128.0A CN202210888128A CN115094075A CN 115094075 A CN115094075 A CN 115094075A CN 202210888128 A CN202210888128 A CN 202210888128A CN 115094075 A CN115094075 A CN 115094075A
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- Prior art keywords
- alginate lyase
- strain
- pichia pastoris
- algf
- alginate
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Abstract
本发明涉及基因工程技术领域,具体涉及一种海藻酸裂解酶高产菌株及其应用。所述菌株为毕赤酵母ALGF‑71(Pichia pastoris ALGF‑71),能显著提高海藻酸裂解酶的表达量,其摇瓶发酵上清液中海藻酸裂解酶酶活高达3442U/ml,比出发菌提高了55%,取得了意料不到的技术效果。所述突变菌株可作为海藻酸裂解酶的发酵生产菌株,有利于降低该酶的生产成本,促进其在工业领域中的广泛应用。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种海藻酸裂解酶高产菌株及其应用。
背景技术
海藻酸裂解酶是一种重要的工业酶,广泛应用于寡糖制备、生物能源生产和
制药工程等领域。海藻酸裂解酶大多数来自海洋,例如海藻类,软体动物,微生物和海洋细菌。根据其剪切方式的不同分为内切型海藻酸裂解酶和外切型海藻酸裂解酶;根据底物特异性可以分为poly M特异性、poly G特异性以及对Poly M和Poly G都具有活性的Poly MG裂解酶。海藻酸裂解酶通过β-消除反应断裂海藻酸分子间的1,4-糖苷键,内切型海藻酸裂解酶切割海藻酸聚合物的糖苷键生成的主要产物为不饱和寡糖(二糖、三糖和四糖),并在其非还原端的C4和C5之间形成双键,而外切型藻酸盐裂解酶产生不饱和单糖。海藻酸裂解酶得到的最终产物是不饱和单糖,不饱和单糖通过非酶促反应转变为4-脱氧-L-赤-5-己糖酮糖醛酸(DEH)。
研究发现,海藻酸裂解酶和抗生素联合使用可有效治疗囊性纤维化,含有抗生素如庆大霉素的海藻酸裂解酶能够增加呼吸道中粘液的杀伤力,因此海藻酸裂解酶可以被用来治疗细菌粘液生物膜依赖性疾病。海藻酸裂解酶也可以被用来制备藻类的原生质体进行基因工程改造和墨角藻细胞壁的研究。同时,海藻酸裂解酶在生物技术的应用中具有重要的作用,具有不同底物特异性的海藻酸裂解酶可以用来产生许多具有生物功能的寡糖。此外,海藻酸裂解酶也可以被用于海藻酸精细结构的确定和特定模式海藻酸的制备,具有活性的海藻酸裂解酶可以进一步利用乙酰化的海藻酸联合抗生素进行囊性纤维化的治疗。
近年来,随着海藻酸及其寡糖越来越多地应用于化工、医药、农业、分子生物学、海洋生物学等领域,海藻酸裂解酶日益成为海洋生物资源开发的竞争热点。发展大型海藻饲料可充分利用海域资源,保护水生生物资源。同时,大型海藻饲料富含生物活性物质,可促进养殖业和健康发展,为人民提供营养丰富的健康食品,因此利用海藻酸裂解酶生产加工大型海藻饲料具有广阔的发展前景。随着酶的发展,具有高活性和广泛底物特异性的新酶的分离和特性描述将会提高和扩大海藻酸裂解酶的应用,以便生产出可以应用到许多领域的具有新颖结构和生物活性的寡糖。有研究表明,海藻酸裂解酶在酵母菌株中进行表达,利用其具有甲醇诱导的强启动子,表达后通过加工、折叠使异源蛋白分泌到胞外,有利于产生活性蛋白,从而简化纯化过程,降低生产成本,为实现海藻酸裂解酶的工业化生产提供了可能性。
发明内容
本发明为解决现有技术问题,提供了一种海藻酸裂解酶高产菌株及其应用。所述菌株是通过紫外诱变方法筛选获得的突变菌,海藻酸裂解酶产量得到显著提高,有利于降低该酶的生产成本,促进其广泛应用。
本发明一方面提供了一种海藻酸裂解酶基因,其核苷酸序列为SEQ ID NO:1,编码的氨基酸序列为SEQ ID NO:2。
本发明一方面提供了一种重组质粒,其携带有上述海藻酸裂解酶基因。
本发明还提供了一种毕赤酵母工程菌,其携带有上述重组质粒。
本发明还提供了一种毕赤酵母突变菌株,是以上述毕赤酵母工程菌为出发菌,通过紫外诱变的方法获得的。
所述突变菌株为毕赤酵母ALGF-71(Pichia pastoris ALGF-71)保藏于中国武汉武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20221119。
本发明还提供了一种发酵生产海藻酸裂解酶的方法,是以上述毕赤酵母突变菌为发酵菌株。
有益效果
本发明提供的突变菌株毕赤酵母ALGF-71,能显著提高海藻酸裂解酶的表达量,其摇瓶发酵上清液中海藻酸裂解酶酶活高达3442U/ml,比出发菌提高了55%,取得了意料不到的技术效果。所述突变菌株可作为海藻酸裂解酶的发酵生产菌株,有利于降低该酶的生产成本,促进其在工业领域中的广泛应用。
具体实施方式
本发明用到了遗传工程和分子生物学领域使用的常规技术和方法,例如MOLECULAR CLONING: A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001)和CURRENTPROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003)中所记载的方法。这些一般性参考文献提供了本领域技术人员已知的定义和方法。但是,本领域的技术人员可以在本发明所记载的技术方案的基础上,采用本领域其它常规的方法、实验方案和试剂,而不限于本发明具体实施例的限定。
菌株与载体:大肠杆菌DH5α本公司保藏,毕赤酵母GS115、载体pPIC9k、G418购自Invitrogen公司。
酶与试剂:DNA聚合酶、T4连接酶、限制性内切酶购自Takara公司,质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司,其他化学试剂为国产或进口分析纯试剂。
实施例中所述海藻酸裂解酶酶活检测方法如下:
(1)海藻酸裂解酶酶活单位的定义
在40℃、pH7.5的条件下,每分钟降解底物海藻酸钠产生不饱和键,在235nm下,吸光度每增加0.1,为一个酶活单位U。
(2)试剂
磷酸缓冲液(0.05M,pH7.5)
1)配制0.05M磷酸二氢钠溶液
称取3.9g二水合磷酸二氢钠,去离子水溶解后,定容至500ml。
2)配制0.05m磷酸氢二钠溶液
称取17.91g十二水合磷酸氢二钠,去离子水溶解后,定容至1000ml。
3)混合配制0.05M磷酸缓冲液
将2中的磷酸氢二钠溶液放置于2L烧杯中,用1中的磷酸二氢钠溶液调节其pH至7.5。
底物:0.3%海藻酸钠溶液
取100ml小烧杯,加入约80ml磷酸缓冲液;称取0.3g海藻酸钠,磁力搅拌条件下均匀加入到小烧杯中,搅拌至溶解;用磷酸缓冲液定容至100ml,配制当天使用。
终止液:0.06mol/L的磷酸终止液。根据不同的磷酸浓度进行计算。
(3)海藻酸裂解酶酶活测定步骤
取三支15mm×150mm试管,加入1.8ml底物,40℃水浴预热5min,加入准备好的酶液0.2ml,准确计时,涡旋振荡,40℃保温10min,将试管从水浴中取出并立即加入2ml磷酸终止液,涡旋振荡,将试管放置在水浴锅外的试管架上。
空白:取15mm*150mm试管,加入1.8ml底物,40℃水浴预热5min,加入0.2ml缓冲液,准确计时,涡旋振荡,40℃保温10min,将试管从水浴中取出并立即加入2ml磷酸终止液,涡旋振荡,将试管放置在水浴锅外的试管架上。
比色:每个样品的空白和酶反应终止后,立即在235nm比色,并记录吸光值A0和A样。
计算:X=(A0-A样)×2×N/(t×0.1)。
X——酶活力,U/ml或U/g;
2——加入2ml磷酸终止液的体积系数;
t(min)——酶促反应时间(在酶反应的线性范围内);
0.1——体系系数,即将吸光值增长单位换算为0.1;
N——稀释倍速;
经简化:酶活力(U/ml)=吸光值差值×2×N。
下面结合具体实施方式,对本发明做进一步阐述。
实施例1、海藻酸裂解酶基因ALGF的克隆
将GenBank号为AEB69783.1的海藻酸裂解酶基因命名为ALGF,分析其氨基酸序列,去除其自身的信号肽,再根据毕赤酵母的密码子偏好性,对其进行密码子优化,由华大基因公司进行全基因合成。海藻酸裂解酶ALGF基因的核苷酸序列为SEQ ID NO:1,编码的氨基酸序列为SEQ ID NO:2。
以ALGF基因第一个核苷酸到全基因终止密码子序列设计 PCR 扩增引物克隆海藻酸裂解酶ALGF基因片段,引物和反应条件如下:
上游引物1(F):GCGCGAATTCCAAGATAAAAAATCTAAATCTAAAA(划线处为EcoR I的酶切位点);
下游引物1(R):TAAAGCGGCCGCTTAATGAGTAACTTGCAAAGAATAA(划线处为Not I的酶切位点)。
PCR条件为:94℃变性5min;然后94℃变性30s,56℃复性30s,72℃延伸1min,35个循环后,72℃保温10min。ALGF基因全长801bp。其中上、下游引物中分别引入EcoR I和Not I的酶切位点,如下划线所示。
实施例2、重组表达海藻酸裂解酶的毕赤酵母工程菌的构建
1、重组质粒的构建
将克隆得到的ALGF基因成熟蛋白编码序列经限制性内切酶EcoR I和Not I双酶切后插入毕赤酵母表达载体pPIC9K的多克隆位点,位于α-因子信号肽序列的下游,得到重组质粒pPIC9K-ALGF,并对该质粒进行测序鉴定。测序验证正确的转化子转接到LB+Amp液体培养基(0.5%酵母提取物,1%蛋白胨,1%NaCl,100μg/mL氨苄青霉,pH7.0)中,37℃过夜培养,提质粒,即为重组酵母表达质粒pPIC9K-ALGF。
2、转化与筛选
将重组酵母表达质粒pPIC9K-ALGF用Sal I进行线性化,线性化产物用柱纯化试剂盒纯化后,通过电穿孔法转化毕赤酵母GS115,涂布MD平板(1.34% YNB,4×10-5%生物素,1%甘油、2%琼脂糖)。在MD平板上生长出的菌落即为毕赤酵母工程菌株,然后涂布含不同浓度遗传霉素G418的YPD平板(1%酵母提取物,2%蛋白胨,2%葡萄糖)上筛选多拷贝的转化子。
3、摇瓶发酵验证
挑取单个多拷贝转化子分别接入BMGY培养基(2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34% YNB,4×10-5%生物素,1%甘油)中,30℃、220rpm振荡培养24小时后,再转入BMMY培养基(2%蛋白胨,1%酵母提取物,100 mM磷酸钾缓冲液(pH6.0),1.34%YNB,4×10-5%生物素,0.5%甲醇)中,30℃、220rpm振荡培养,每24小时添加0.5%的甲醇。诱导表达4d后,离心取上清液,进行海藻酸裂解酶酶活力测定。
结果显示,在摇瓶条件下,上述构建得到的毕赤酵母工程菌发酵上清液中海藻酸裂解酶酶活最高达到2220U/ml。将酶活水平最高的工程菌命名为毕赤酵母ALGF(Pichia pastoris ALGF)。
实施例3、海藻酸裂解酶高产菌株的诱变筛选
诱变育种的基本原理是基因突变,主要包括染色体畸变和基因突变。诱变育种是利用各种被称为诱变剂的物理因素和化学试剂处理微生物细胞,提高基因突变频率,再通过适当的筛选方法获得所需要的高产优质菌种的育种方法。
紫外诱变导致的突变随机性很强,需要进行多轮紫外诱变,筛选的工作量较大,但因为紫外诱变所需设备简单、费用少,并且可在短时间内获得大量突变体,因此,它现在仍是一种常用的诱变选育方法。
申请人以毕赤酵母ALGF为出发菌株,通过紫外诱变方法对其进行遗传学改造,进一步提高其海藻酸裂解酶的产量。
将毕赤酵母ALGF接种于YPD平板,30℃培养2-3天,使用无菌水洗菌体,制成悬浮液,稀释至1×106个/mL,紫外灯(40W)照射2-10min,距离约22cm,致死率达到90%以上,涂布平板,30℃培养48h。
前三轮紫外诱变共获得了约270个突变菌单菌落,将各个单菌落分别接种于装有200ul BMGY液体培养基的96孔板,30℃、250rpm振荡培养1天后,离心去掉上层培养基,再加入200ul BMMY培养基,30℃、250rpm振荡培养2天,每天添加0.5%的甲醇。诱导表达2天后,离心取上清液,测定海藻酸裂解酶的活力,筛选出酶活力得到显著提高的突变菌株。
结果显示,前三轮紫外诱变筛选获得的突变菌中,没有一株突变菌发酵上清液中海藻酸裂解酶酶活高于出发菌。申请人又按照上述方法继续进行了23轮诱变筛选,最终获得1株海藻酸裂解酶产量显著高于出发菌的突变菌株,命名毕赤酵母ALGF-71(Pichia pastoris ALGF-71)。该突变菌在摇瓶条件下发酵,其上清液中海藻酸裂解酶酶活力高达3442U/ml,比出发菌提高了55%,取得了意料不到的技术效果。
申请人已于2022年7月15日将毕赤酵母ALGF-71(Pichia pastoris ALGF-71)保藏于中国武汉 武汉大学的中国典型培养物保藏中心,保藏编号为CCTCC NO:M 20221119。
序列表
<110> 青岛蔚蓝生物集团有限公司
潍坊康地恩生物科技有限公司
<120> 一种海藻酸裂解酶高产菌株及其应用
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tctgttgttt tttatgcttt tccatctggt gttactactg ctaatactca ttattctaga 240
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Claims (6)
1.一种海藻酸裂解酶基因,其特征在于,所述海藻酸裂解酶基因的核苷酸序列为SEQID NO:1。
2.一种重组质粒,其特征在于,所述的重组质粒携带有权利要求1所述的海藻酸裂解酶基因。
3.一种毕赤酵母工程菌,其特征在于,所述的毕赤酵母工程菌携带有权利要求 2所述的重组质粒。
4.一种毕赤酵母突变菌,其特征在于,所述的突变菌是以权利要求3所述的毕赤酵母工程菌为出发菌,通过紫外诱变的方法获得的。
5.如权利要求4所述的毕赤酵母突变菌,其特征在于,所述突变菌的保藏编号为CCTCCNO:M 20221119。
6.一种生产海藻酸裂解酶的方法,其特征在于,所述方法是以权利要求5所述的毕赤酵母突变菌为发酵菌株。
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