CN109776686B - 一种热稳性提高的融合型脂肪酶及其制备方法和应用 - Google Patents
一种热稳性提高的融合型脂肪酶及其制备方法和应用 Download PDFInfo
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- CN109776686B CN109776686B CN201910235250.6A CN201910235250A CN109776686B CN 109776686 B CN109776686 B CN 109776686B CN 201910235250 A CN201910235250 A CN 201910235250A CN 109776686 B CN109776686 B CN 109776686B
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Abstract
本发明属于生物工程技术领域,公开了一种热稳性提高的融合型脂肪酶及其制备方法和应用,融合型脂肪酶的氨基酸序列如SEQ ID NO.4或SEQ ID NO.5所示,融合型脂肪酶由编码梳棉状嗜热丝孢菌脂肪酶氨基酸序列中N端融合了一段由30~32个氨基酸组成的双亲短肽,所述的双亲短肽的氨基酸序列为SEQ ID NO.1或SEQ ID NO.2。两种融合型脂肪酶具有较理想耐热特性,因此特别适合工业化大规模生产。
Description
技术领域
本发明属于生物工程技术领域,涉及两种融合型脂肪酶,具体为一种热稳性提高的融合型脂肪酶及其制备方法和应用。
背景技术
脂肪酶是一类重要的水解酶,能够催化甘油三酯上酯键的水解。在特定的条件下还可以催化酸解、醇解、转酯、氨解、间接氧化还原等许多反应。以脂肪酶整体来看,它们具有底物多样性;具体到某一种酶的时候,又表现出很强的底物特异性。所有这些特性使得脂肪酶成为了重要的生物催化剂,被广泛应用于食品、化工、洗涤剂、医药、化妆品等领域。
但是目前我国脂肪酶产品种类比较单一,能够产生重大工业价值的酶种有限,市售的酶制剂纯度也有待提高,对于有重要生产使用价值的酶种应该大力研究和开发。其中热稳定、高活力的脂肪酶更是具有无可比拟的开发和应用优势。疏棉状嗜热丝孢菌(Thermomyces lanuginosus)是一种分布广泛、生长上限温度较高的真菌。它能够产生热稳定的、具有重要工业价值的脂肪酶。但是野生菌产生脂肪酶需要培养温度高(80℃),造成在实际生产中菌体培养条件苛刻,产品成本增加的问题,同时限制了脂肪酶在工业生产的应用。
梳棉状嗜热丝孢菌脂肪酶工业生产损失量比较大,生产成本也会大幅度提高。若能将梳棉状嗜热丝孢菌脂肪酶的热稳定性提高,则其生产成本也会随之下降。因此,本发明通过对梳棉状嗜热丝孢菌脂肪酶基因上的改造使其成为耐热性比较高的脂肪酶。
发明内容
本发明的目的在于提供提供一种融合型脂肪酶及其应用,旨在解决梳棉状嗜热丝孢菌脂肪酶的耐热性较差和在工业生产上不理想的问题。
本发明具体通过以下技术方案实现:
一种热稳性提高的融合型脂肪酶,所述的融合型脂肪酶由梳棉状嗜热丝孢菌脂肪酶在N端融合双亲短肽构成,所述的双亲短肽由30~32个氨基酸组成,所述的双亲短肽为双亲短肽1或双亲短肽2,其氨基酸序列分别为SEQ ID NO.1和SEQ ID NO.2。
所述的梳棉状嗜热丝孢菌脂肪酶氨基酸序列如SEQ ID NO.3所示。
具体的,所述的融合型脂肪酶由梳棉状嗜热丝孢菌脂肪酶氨基酸序列SEQ IDNO.3中在N端融合了一段由30~32个氨基酸组成的双亲短肽1或双亲短肽2而获得。
本发明所述的融合型脂肪酶的氨基酸序列如SEQ ID NO.4或SEQ ID NO.5所示。所述的融合型脂肪酶的编码基因如SEQ ID NO.6或SEQ ID NO.7所示。
所述的融合型脂肪酶的酶促反应最适pH值为9.0;最适温度为50℃;在pH7-pH12、37℃条件下,pH耐受1小时,残活还在50%,在pH9-pH12、37℃条件下,pH耐受1小时,残活还在80%以上,脂肪酶融合双亲短肽1在80℃耐受45min残活在50%以上;脂肪酶融合双亲短肽2在80℃耐受35min残活在50%以上。
在本发明的另一方面,所述的氨基酸序列SEQ ID NO.4、SEQ ID NO.5经修饰、缺失或添加一或几个氨基酸获得氨基酸序列,且保持只有90%的同源性的序列也在本发明的保护范围内。
在本发明的另一方面,与所述的编码基因SEQ ID NO.6、SEQ ID NO.7编码相同蛋白质,但因遗传密码的简并性而与SEQ ID NO.6、SEQ ID NO.7所示的核苷酸序列或其互补序列不同的核苷酸序列也在本发明的保护范围内。
在本发明的另一方面,本发明还包括携带有编码基因序列为S SEQ ID NO.6或SEQID NO.7的脂肪酶突变体的质粒。
本发明一并提供上述所述的融合型脂肪酶的构建方法,包括以下步骤:
1)设计引物F1和R1、F2和R2进行无模板PCR,从而获得双亲短肽1、双亲短肽2;再设计引物F3和R3、F3和R4分别将双亲短肽1、双亲短肽2进行PCR扩增;
2)以梳棉状嗜热丝孢菌脂肪酶基因连接到载体上的重组质粒为模板,设计引物F4和R5、F5和R5分别进行PCR扩增脂肪酶1、脂肪酶2;
3)以F3、R5为引物,分别以双亲短肽1与脂肪酶1、双亲短肽2与脂肪酶2为模版进行融合PCR扩增;
4)将融合PCR扩增产物与载体进行重组;
5)将5μL重组产物于50μL DH5α感受态细胞中,经冰浴冷却、热激,冷却后,加500μLLB培养基,37℃180转培养1h,离心保留部分清液悬浮沉淀,取全部菌液涂板,37℃过夜培养;
6)进行阳性克隆子筛选验证,挑取单菌落于相应抗性的LB培养基中,培养2-3h后PCR鉴定,将筛选出的阳性克隆子测序。
所述的引物序列具体如下:
F1:GCTGAAGCTGAAGCTAAAGCTAAAGCTGAAGCTGAAGCTAAAGCTAAACCAACTCCACC;
R1:AGTTGGAGTTGGAGTAGTTGGTGGAGTTGGAGTAGTTGGTGGAGTTGGTTTAGCTTTAG;
F2:GCTGAAGCTGAAGCTAAAGCTAAAGCTGAAGCTGAACCGAAAGTCAGCCCGGAGGCTGT;
R2:GAGCTCAGCCTCCTTCTTAACAGCCTCCGGGCTGACTTTCGGTTCAGCTTCAGCTTTAG;
F3:GAGGCTGAAGCTTACGTAGAATTCGCTGAAGCTGAAGCTAAAGC;
R3:TCTTCTAATAGGACTAGTTGGAGTTGGAGT;
F4:AAGGAGGCTGAGCTCAGTCCTATTAGAAGA;
R4:TCTTCTAATAGGACTGAGCTCAGCCTCCTT;
F5:AAGGAGGCTGAGCTCAGTCCTATTAGAAGA;
R5:TCTAAGGCGAATTAATTCGCGGCCGCAAGACATGTTCCAATTAAACCG。
在本发明的另一方面,本发明还提供了所述的融合型脂肪酶的基因的工程菌,所述的工程菌含有具有SEQ ID NO.6或SEQ ID NO.7所示基因的载体。
所述的工程菌通过将SEQ ID NO.6或SEQ ID NO.7所示的基因克隆到表达载体上,然后进行细胞转化,获得重组基因工程菌。
所述的编码基因EQ ID NO.6或SEQ ID NO.7的表达载体选自pPIC9K、pPIC9、pPICZaA\B\C、pPICZA\B\C或PGAPZaA\B\C。
在本发明另一方面,本发明所述的融合型脂肪酶在饲料添加剂中的应用也在本发明的保护范围之内。
本发明的有益效果为:
本发明提供的融合型脂肪酶的基因除与pPIC9K构建重组质粒外,还可以与pPIC9、pPICZaA\B\C、pPICZA\B\C、PGAPZaA\B\C等表达载体构建重组质粒,转化相应宿主菌中,通过在平板中加入G418、Zeocin等抗生素,筛选获得脂肪酶基因工程菌,然后通过发酵获得新的脂肪酶。使其成为耐热性高的脂肪酶,这两种脂肪酶与野生型脂肪酶在高温下耐受经实验证明,80℃耐受25min脂肪酶融合双亲短肽1相对酶活大约还有69.87%,脂肪酶融合双亲短肽2相对酶活大约还有58.58%,而野生型脂肪酶仅仅剩余50.12%。在80℃时野生型脂肪酶相对酶活剩余一半时的时间大约为25min,而脂肪酶融合双亲短肽1相对酶活剩余一半时的时间大约为45min,脂肪酶融合双亲短肽2相对酶活剩余一半时的时间大约为35min。这两种脂肪酶的耐热性有了明显的提高,工业化生产中损失率有一定的降低。
附图说明
图1是本发明实施例提供的融合型脂肪酶的构建方法流程图;
图2是本发明实施例提供的最适pH的测定曲线图;
图3是本发明实施例提供的最适温度曲线图;
图4是本发明实施例提供的pH耐受曲线图;
图5是本发明实施例提供的80℃耐受曲线图。
具体实施方式
下面将结合本发明具体的实施例,对本发明技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
通过对对梳棉状嗜热丝孢菌脂肪酶的蛋白质空间结构进行模拟分析,对其分别融合了双亲短肽1,双亲短肽2。具体实施方案为以梳棉状嗜热丝孢菌脂肪酶基因为模板,通过基因的融合PCR方法,进行梳棉状嗜热丝孢菌脂肪酶基因与双亲短肽融合,获得了两种融合型脂肪酶基因,将两种融合型脂肪酶基因与pPIC9、pPICZaA\B\C、pPICZA\B\C、PGAPZaA\B\C等载体相连构建重组质粒,转入相应宿主菌(GS115或X33、SMD1168、PICHIAPINK)中进行异源表达,发酵可以获得两种脂肪酶。该两种脂肪酶能在酸性环境中很好发生作用,并且具有较理想耐热特性,适合耐高温制粒,因此适合工业上生产。
本发明实施例的脂肪酶融合后的氨基酸序列如SEQ ID NO.4或SEQ ID NO.5所示。
1、实验材料和试剂:
实验菌株和载体:基因来源菌株:由本实验室筛选并保存的梳棉状嗜热丝孢菌脂肪酶;表达宿主菌及载体:GS115和pPIC9K均购于Novagen公司;脂肪酶重组质粒由实验室构建;宿主菌:DH5α感受态细胞购于北京全式金公司。
主要试剂:DNAMarker、蛋白Marker(TaKaRa公司);Plasmid Mini Kit I(Omega公司)。琼脂糖购自Tiangen生化科技(北京)有限公司;核酸染料购自百泰克生物公司;限制性核酸内切酶、PCR扩增酶均购自TaKaRa公司。
实验仪器:离心机(Eppendorf);PCR扩增仪(Bio-Rad);核酸电泳仪(Bio-Rad);蛋白电泳仪(Amersham Bioscience);凝胶成像仪(Bio-Rad)。
主要培养基:YPD、LB、酵母发酵培养基(FA与FB)均按照“Invitrogen公司操作手册”的推荐方法来配制。
2、脂肪酶酶活力的测定
在一定条件下每分钟水解p-NP底物而生成1μmoL的对硝基苯酚所需要的酶量,即一个酶活单位,以U表示。
1)实验仪器:恒温水浴锅;pH仪器;酶标仪(Bio-Rad)等。
2)实验材料:对硝基苯酚棕榈酸酯(p-NPC16)(Sigma公司)。
3)溶液配制:
pH缓冲液:0.1mol/L一水合柠檬酸缓冲液和0.1mol/L磷酸缓冲液(pH2-7);
0.1mol/L Tris-HCl缓冲液(pH7-9);
0.1mol/L甘氨酸-NaOH缓冲液(pH9-12);
底物溶液:10mmol/L对硝基苯酚棕榈酸酯(p-NPC16)。
4)采用对硝基苯酚法(p-nitrophenol):总体系为500μL,其中含有50mmol/L缓冲液420μL,10mmol/L底物p-NP30μL和稀释的酶液50μL。底物和缓冲液混合后在反应温度下预热2min,加入稀释酶液混匀,反应5min加入50μL1.0mol/L的SDS终止反应,并加入500μL1.0mol/L的Na2CO3显色;在波长为405nm下测定其OD值。
实施例1脂肪酶融合双亲短肽的制备
如图1所示,本发明实施例的两种脂肪酶的获得方法包括以下步骤:
(1)RCR扩增:设计引物(F1,R1)、(F2,R2)进行无模板PCR,从而获得双亲短肽1、双亲短肽2;再设计引物分别将双亲短肽1(F3,R3)、双亲短肽2(F3,R4)进行PCR扩增;以梳棉状嗜热丝孢菌脂肪酶基因连接到载体上的重组质粒为模板,设计引物(F4,R5)、(F5,R5)进行PCR扩增脂肪酶基因1、脂肪酶基因2;
(2)以F3、R5为引物,分别用双亲短肽1与梳棉状嗜热丝孢菌脂肪酶基因1、双亲短肽2与梳棉状嗜热丝孢菌脂肪酶基因2为模版分别进行融合PCR扩增;
(3)重组质粒构建:将融合PCR产物与载体37℃进行重组30min。
(4)转化:加入5μL突变后的产物于50μL DH5α感受态细胞中,轻弹混匀,冰浴30min;42℃准确热激45s后立即置于冰上冷却l0min;加500μL LB培养基,180转,37℃培养lh;7000rpm离心3min,弃上部分清液,保留100-150μL上清轻弹悬浮菌体,取全部菌液涂板,37℃过夜培养。
(5)阳性克隆子验证:挑取单菌落于500μL相应抗性的LB培养基中,200rpm培养2-3h后PCR鉴定;将筛选出的阳性克隆子送出测序,测序结果与原序列比对。
(6)找融合正确的重组质粒;将突变质粒转入毕赤酵母GS115或X33、SMD1168、PICHIAPINK中进行表达,发酵并进行对比测酶活,研究酶学及应用特性。
其中,上述方法中设计得到引物具体如下:
双亲短肽1、双亲短肽2的氨基酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。
梳棉状嗜热丝孢菌脂肪酶基因融合双亲短肽1、2,按上实验方法进行融合后送华大基因公司测序,结果如序列SEQ ID NO.4和SEQ ID NO.5所示,对应脂肪酶核苷酸序列如序列SEQ ID NO.6、SEQ ID NO.7,转化的酵母菌株具有脂肪酶活性,各选取一株发酵酶活性单位高的菌株进行发酵获得酶液进行酶学性质测定。
实施例2两种脂肪酶最适pH测定
将缓冲液pH调成2、3、4、5、6、7、8、9、10、11、12,将酶液稀释到适应的倍数,依照脂肪酶活测定方法在37℃测出最适pH之后在最大值两侧补半点继续检测最适值(例如最适pH为9,则再取pH8、8.5、9、9.5和10按照脂肪酶活测定方法进行测定)。
脂肪酶酶促反应最适pH值结果如图2所示。脂肪酶融合双亲短肽1、2与野生型脂肪酶最适均为9。无明显变化。
实施例3脂肪酶最适温度测定
依照脂肪酶活测定方法测定,在上述最适pH的条件下,将反应物放在不同温度下反应0℃、10℃、20℃、30℃、40℃、50℃、80℃、70℃、80℃,测出最适温度后,在最大值两侧补半点(例如最适温度是50℃,则补充、40℃、45℃、50℃、55℃按照脂肪酶活测定方法测定)。
脂肪酶酶促反应最适温度值如图3所示,脂肪酶基因融合双亲短肽1、2与野生型脂肪酶最适温度分别为50℃。
实施例4脂肪酶pH耐受测定
将缓冲液调节到不同pH:2、3、4、5、6、7、8、9、10、11、12,用这些不同的缓冲液稀释酶液,从放入酶液之时开始计时,稀释好的酶液放入37℃水浴锅中耐受1小时放后在冰上,立即按照脂肪酶活测定方法在最适pH和最适温度下进行反应。对照组的酶液是未耐受过的酶液。
由图4可知,三种脂肪酶的耐受曲线趋势是相同的,在pH为4-9之间37℃耐受1小时相对酶活有了明显的升高。
实施例5脂肪酶温度耐受测定
将酶液稀释到相应倍数,然后放入不同温度:80℃下耐受1min、3min、5min、7min、10min、15min、20min、25min、30min、35min、45min、60min之后按照脂肪酶活测定方法在最适pH和最适温度下反应。对照实验组酶液是未温度耐受过的酶液。
高温下脂肪酶的温度耐受情况如图5所示,随着温度的升高,相对酶活不断降低,随着时间的增加,相对酶活也逐渐降低。无论在任何温度和时间下,融合后的相对酶活都高于突变前的80℃耐受25min脂肪酶融合双亲短肽1相对酶活大约还有69.87%,脂肪酶融合双亲短肽2相对酶活大约还有58.58%,而野生型脂肪酶仅仅剩余50.12%。在80℃时野生型脂肪酶相对酶活剩余一半时的时间大约为25min,而脂肪酶融合双亲短肽1相对酶活剩余一半时的时间大约为45min,脂肪酶融合双亲短肽2相对酶活剩余一半时的时间大约为35min。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
序列表
<110> 云南师范大学
<120> 一种热稳性提高的融合型脂肪酶及其制备方法和应用
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> PRT
<213> 双亲短肽(Amphiphilic short peptide)
<400> 1
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Pro Thr Pro Pro Thr Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr
20 25 30
<210> 2
<211> 30
<212> PRT
<213> 双亲短肽(Amphiphilic short peptide)
<400> 2
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Pro Lys Val Ser Pro Glu Ala Val Lys Lys Glu Ala Glu Leu
20 25 30
<210> 3
<211> 274
<212> PRT
<213> 脂肪酶(Lipase)
<400> 3
Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn
1 5 10 15
Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp
20 25 30
Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu
35 40 45
Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly
50 55 60
Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu
65 70 75 80
Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly
85 90 95
Asn Leu Asn Phe Lys Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys
100 105 110
Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr
115 120 125
Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg
130 135 140
Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala
145 150 155 160
Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr
165 170 175
Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val
180 185 190
Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val
195 200 205
Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Arg His Ser Ser Pro Glu
210 215 220
Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile
225 230 235 240
Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn
245 250 255
Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr
260 265 270
Cys Leu
<210> 4
<211> 306
<212> PRT
<213> 融合型脂肪酶1(Fusion lipase)
<400> 4
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Ala Lys Ala Lys
1 5 10 15
Pro Thr Pro Pro Thr Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr
20 25 30
Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn
35 40 45
Leu Phe Ala Gln Tyr Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp
50 55 60
Ala Pro Ala Gly Thr Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu
65 70 75 80
Val Glu Lys Ala Asp Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly
85 90 95
Val Gly Asp Val Thr Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu
100 105 110
Ile Val Leu Ser Phe Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly
115 120 125
Asn Leu Asn Phe Lys Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys
130 135 140
Arg Gly His Asp Gly Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr
145 150 155 160
Leu Arg Gln Lys Val Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg
165 170 175
Val Val Phe Thr Gly His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala
180 185 190
Gly Ala Asp Leu Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr
195 200 205
Gly Ala Pro Arg Val Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val
210 215 220
Gln Thr Gly Gly Thr Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val
225 230 235 240
Pro Arg Leu Pro Pro Arg Glu Phe Gly Tyr Arg His Ser Ser Pro Glu
245 250 255
Tyr Trp Ile Lys Ser Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile
260 265 270
Val Lys Ile Glu Gly Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn
275 280 285
Ile Pro Asp Ile Pro Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr
290 295 300
Cys Leu
305
<210> 5
<211> 300
<212> PRT
<213> 融合型脂肪酶2(Fusion lipase)
<400> 5
Ala Glu Ala Glu Ala Lys Ala Lys Ala Glu Ala Glu Pro Lys Val Ser
1 5 10 15
Pro Glu Ala Val Lys Lys Glu Ala Glu Leu Ser Pro Ile Arg Arg Glu
20 25 30
Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr Ser
35 40 45
Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr Asn
50 55 60
Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp Ala
65 70 75 80
Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr Gly
85 90 95
Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe Arg
100 105 110
Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Lys Leu
115 120 125
Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly Phe
130 135 140
Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val Glu
145 150 155 160
Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly His
165 170 175
Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg Gly
180 185 190
Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val Gly
195 200 205
Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr Leu
210 215 220
Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro Arg
225 230 235 240
Glu Phe Gly Tyr Arg His Ser Ser Pro Glu Tyr Trp Ile Lys Ser Gly
245 250 255
Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly Ile
260 265 270
Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro Ala
275 280 285
His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu
290 295 300
<210> 6
<211> 918
<212> DNA
<213> 编码基因(Coding gene)
<400> 6
gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacca 60
actactccaa ctccaccaac tactccaact ccaactagtc ctattagaag agaggtctcg 120
caggatctgt ttaaccagtt caatctcttt gcacagtatt ctgcagccgc atactgcgga 180
aaaaacaatg atgccccagc tggtacaaac attacgtgca cgggaaatgc ctgcccagag 240
gtagagaagg ccgatgcaac gtttctctac tcgtttgaag actctggagt gggagatgtc 300
accggattcc ttgctctcga caacacgaac aaattgatcg tcctctcttt cagaggatct 360
agatccattg agaactggat cggaaatctt aacttcaagt tgaaagagat caatgacatt 420
tgctccggat gcagaggaca tgacggtttc acttcgtcct ggagatctgt agccgatacg 480
ttaagacaga aggtggagga tgctgtgaga gagcatccag actatagagt ggtgtttacc 540
ggacatagct tgggtggtgc attggcaact gttgccggag cagacctgag aggaaatggt 600
tatgatatcg acgtgttttc atatggagcc cctagagtcg gaaacagagc ttttgcagag 660
ttcctgaccg tacagaccgg aggaacactc tacagaatta cccacaccaa tgatattgtc 720
cctagactcc ctccacgcga gttcggttac agacattcta gcccagagta ctggatcaaa 780
tctggaaccc ttgtcccagt caccagaaac gatatcgtga agattgaagg aatcgatgcc 840
accggaggaa acaaccagcc taacattcct gatatccctg cccacctatg gtacttcggt 900
ttaattggaa catgtctt 918
<210> 7
<211> 900
<212> DNA
<213> 编码基因(Coding gene)
<400> 7
gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgtt 60
aagaaggagg ctgagctcag tcctattaga agagaggtct cgcaggatct gtttaaccag 120
ttcaatctct ttgcacagta ttctgcagcc gcatactgcg gaaaaaacaa tgatgcccca 180
gctggtacaa acattacgtg cacgggaaat gcctgcccag aggtagagaa ggccgatgca 240
acgtttctct actcgtttga agactctgga gtgggagatg tcaccggatt ccttgctctc 300
gacaacacga acaaattgat cgtcctctct ttcagaggat ctagatccat tgagaactgg 360
atcggaaatc ttaacttcaa gttgaaagag atcaatgaca tttgctccgg atgcagagga 420
catgacggtt tcacttcgtc ctggagatct gtagccgata cgttaagaca gaaggtggag 480
gatgctgtga gagagcatcc agactataga gtggtgttta ccggacatag cttgggtggt 540
gcattggcaa ctgttgccgg agcagacctg agaggaaatg gttatgatat cgacgtgttt 600
tcatatggag cccctagagt cggaaacaga gcttttgcag agttcctgac cgtacagacc 660
ggaggaacac tctacagaat tacccacacc aatgatattg tccctagact ccctccacgc 720
gagttcggtt acagacattc tagcccagag tactggatca aatctggaac ccttgtccca 780
gtcaccagaa acgatatcgt gaagattgaa ggaatcgatg ccaccggagg aaacaaccag 840
cctaacattc ctgatatccc tgcccaccta tggtacttcg gtttaattgg aacatgtctt 900
<210> 8
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacc 59
<210> 9
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agttggagtt ggagtagttg gtggagttgg agtagttggt ggagttggtt tagctttag 59
<210> 10
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
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gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgt 59
<210> 11
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
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gagctcagcc tccttcttaa cagcctccgg gctgactttc ggttcagctt cagctttag 59
<210> 12
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gaggctgaag cttacgtaga attcgctgaa gctgaagcta aagc 44
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tcttctaata ggactagttg gagttggagt 30
<210> 14
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
aaggaggctg agctcagtcc tattagaaga 30
<210> 15
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tcttctaata ggactgagct cagcctcctt 30
<210> 16
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aaggaggctg agctcagtcc tattagaaga 30
<210> 17
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
tctaaggcga attaattcgc ggccgcaaga catgttccaa ttaaaccg 48
Claims (8)
1.一种热稳性提高的融合型脂肪酶,其特征在于,所述的融合型脂肪酶由梳棉状嗜热丝孢菌脂肪酶在N端融合双亲短肽构成,所述的双亲短肽由30~32个氨基酸组成,所述的双亲短肽为双亲短肽1或双亲短肽2,其氨基酸序列分别为SEQ ID NO.1和SEQ ID NO.2;
所述的融合型脂肪酶的氨基酸序列如SEQ ID NO.4或SEQ ID NO.5所示。
2.编码权利要求1所述的融合型脂肪酶的DNA分子。
3.根据权利要求2所述的DNA分子,其特征在于,所述的DNA分子的核苷酸序列如SEQ IDNO.6或SEQ ID NO.7所示。
4.权利要求1所述的融合型脂肪酶的制备方法,其特征在于,包括以下步骤:
1)设计引物F1和R1、F2和R2进行无模板PCR,从而获得双亲短肽1、双亲短肽2;再设计引物F3和R3、F3和R4分别将双亲短肽1、双亲短肽2进行PCR扩增;
2)以梳棉状嗜热丝孢菌脂肪酶基因连接到载体上的重组质粒为模板,设计引物F4和R5、F5和R5分别进行PCR扩增脂肪酶1、脂肪酶2;
3)以F3、R5为引物,分别以双亲短肽1与脂肪酶1、双亲短肽2与脂肪酶2为模版进行融合PCR扩增;
4)将融合PCR扩增产物与载体进行重组;
5)将5μL重组产物于50μLDH5α感受态细胞中,经冰浴冷却、热激,冷却后,加500μLLB培养基,37℃180转培养1h,离心保留部分清液悬浮沉淀,取全部菌液涂板,37℃过夜培养;
6)进行阳性克隆子筛选验证,挑取单菌落于相应抗性的LB培养基中,培养2-3h后PCR鉴定,将筛选出的阳性克隆子测序。
5.根据权利要求4所述的制备方法,其特征在于,F1、R1、F2、R2、F3、R3、F4、R4、F5、R5的核苷酸序列分别如SEQ ID NO.8~17所示。
6.一种工程菌,其特征在于,所述的工程菌含有具有SEQ ID NO.6或SEQ ID NO.7所示基因的载体。
7.根据权利要求6所述的工程菌,其特征在于,所述的载体选自pPIC9K、pPIC9、pPICZaA\B\C、pPICZA\B\C或PGAPZaA\B\C。
8.权利要求1所述的融合型脂肪酶在饲料添加剂中的应用。
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