CN109750014B - Fusion type rhizopus chinensis lipase with improved heat stability and application thereof - Google Patents

Fusion type rhizopus chinensis lipase with improved heat stability and application thereof Download PDF

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CN109750014B
CN109750014B CN201910235208.4A CN201910235208A CN109750014B CN 109750014 B CN109750014 B CN 109750014B CN 201910235208 A CN201910235208 A CN 201910235208A CN 109750014 B CN109750014 B CN 109750014B
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黄遵锡
姜占宝
苗华彪
韩楠玉
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Yunnan Normal University
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Abstract

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华根霉脂肪酶及其应用,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,融合型脂肪酶由编码华根霉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.2或SEQ ID NO.3所示;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。The invention belongs to the technical field of bioengineering, and discloses a fusion-type Rhizopus sinensis lipase with improved thermal stability and its application. The amino acid sequence of the fusion-type lipase is as shown in SEQ ID NO.4 or SEQ ID NO.5. shows that the fusion type lipase is encoded by the N-terminus of the amino acid sequence of Rhizopus sinensis lipase fused with a short amphipathic peptide consisting of 30 to 32 amino acids, and the amino acid sequence of the amphipathic short peptide is SEQ ID NO.2 or SEQ ID NO.2. As shown in ID NO.3; the two fusion-type lipases have relatively ideal heat-resistant properties, so they are particularly suitable for industrialized large-scale production.

Description

一种热稳性提高的融合型华根霉脂肪酶及其应用A kind of fusion type rhizopus sinolina lipase and its application that a kind of thermostability improves

技术领域technical field

本发明属于生物工程技术领域,涉及融合型脂肪酶,尤其一种热稳性提高的融合型华根霉脂肪酶及其应用。The invention belongs to the technical field of bioengineering, and relates to a fusion type lipase, in particular to a fusion type Rhizopus sinensis lipase with improved thermal stability and application thereof.

背景技术Background technique

脂肪酶能逐步地将甘油三酯水解成脂肪酸、二酸甘油酯、单酸甘油酯及甘油;同时它还能催化酯的酸解、醇解、氨解以及转酯化和酯合成等反应,因其有独特的催化活性而被广泛地应用到食品加工、油脂加工、皮革加工、生物能源以及饲料添加等过程中,并且脂肪酶在再生能源的开发和环境保护中也起着至关重要的作用。虽然在工业生产中已经有很多种来源于根霉的脂肪酶可供选择,但天然根霉脂肪酶大多为中温脂肪酶,热稳定性不高,高温条件下易失活,而油脂加工反应一般都需要在较高温度下进,天然根霉脂肪酶不耐高温这一缺陷使得工业生产成本大幅度提高,也限制了脂肪酶在工业生产的应用。Lipase can gradually hydrolyze triglycerides into fatty acids, diglycerides, monoglycerides and glycerol; at the same time, it can also catalyze the reactions of acidolysis, alcoholysis, aminolysis, transesterification and ester synthesis. Because of its unique catalytic activity, it is widely used in food processing, oil processing, leather processing, bioenergy and feed addition, and lipase also plays a vital role in the development of renewable energy and environmental protection effect. Although many kinds of lipases derived from Rhizopus have been available in industrial production, most of the natural Rhizopus lipases are mesophilic lipases with low thermal stability and easy inactivation under high temperature conditions, while the oil processing reaction is generally All need to be carried out at a higher temperature, and the defect of the high temperature resistance of the natural rhizopus lipase has greatly increased the cost of industrial production, and also limited the application of lipase in industrial production.

华根霉脂肪酶的热稳定性比较差,工业生产损失量比较大,因此生产成本也会大幅度提高。若能将华根霉脂肪酶的热稳定性提高,则其生产成本也会随之下降。因此,本发明通过对华根霉脂肪酶基因上的改造使其成为一种耐热性比较高的脂肪酶。The thermal stability of Rhizopus sinica lipase is relatively poor, and the industrial production loss is relatively large, so the production cost will also be greatly increased. If the thermal stability of Rhizopus sinica lipase can be improved, its production cost will also decrease thereupon. Therefore, the present invention makes it a lipase with relatively high heat resistance by modifying the Rhizopus chinensis lipase gene.

发明内容Contents of the invention

本发明的目的在于提供一种融合型脂肪酶及其应用,旨在解决华根霉脂肪酶的耐热性较差和在工业生产上不理想的问题。The object of the present invention is to provide a fusion type lipase and its application, aiming at solving the problems of poor heat resistance and unsatisfactory industrial production of Rhizopus sinensis lipase.

本发明具体通过以下技术方案实现:The present invention is specifically realized through the following technical solutions:

一种热稳性提高的融合型华根霉脂肪酶,是由华根霉脂肪酶的氨基酸序列分别在N端融合了一段由30~32个氨基酸组成的双亲短肽,其中华根霉脂肪酶氨基酸序列如SEQID NO.1所示。A fusion-type Rhizopus sinensis lipase with improved thermostability is composed of an amphipathic short peptide composed of 30-32 amino acids fused to the amino acid sequence of Rhizopus sinensis lipase at the N-terminus, wherein the Rhizopus sinensis lipase The amino acid sequence is shown in SEQID NO.1.

具体的,所述的双亲短肽为双亲短肽1或双亲短肽2,所述的融合型脂肪酶由华根霉脂肪酶氨基酸序列SEQ ID NO.1中分别在N端融合了一段由30~32个氨基酸组成的双亲短肽1、双亲短肽2而获得。Specifically, the parental short peptide is parental short peptide 1 or parental short peptide 2, and the fusion type lipase is fused with a segment consisting of 30 Amphiparental short peptide 1 and amphiparental short peptide 2 composed of ~32 amino acids were obtained.

所述的双亲短肽1和双亲短肽2的氨基酸序列分别如SEQ ID NO.2和SEQ ID NO.3所示。The amino acid sequences of the amphipathic short peptide 1 and the amphipathic short peptide 2 are respectively shown in SEQ ID NO.2 and SEQ ID NO.3.

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。The amino acid sequence of the fusion lipase of the present invention is shown in SEQ ID NO.4 or SEQ ID NO.5.

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40℃;在pH4-pH10、37℃条件下,pH耐受1小时,残活还在50%,在pH7-pH10、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60℃耐受45min残活都在50%以上;脂肪酶融合双亲短肽2在60℃耐受30min残活都在50%以上。The optimum pH value of the enzymatic reaction of the fusion lipase is 9.0; the optimum temperature is 40°C; under the conditions of pH4-pH10, 37°C, the pH can be tolerated for 1 hour, and the residual activity is still 50%. -Under the conditions of pH 10 and 37°C, the pH is tolerated for 1 hour, and the residual activity is still more than 80%. The lipase-fused parent short peptide 1 has a residual activity of more than 50% at 60°C for 45 minutes; the lipase-fused parent short peptide 2 At 60°C for 30 minutes, the residual activity was above 50%.

在本发明的另一方面,所述的氨基酸序列SEQ ID NO.4或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。In another aspect of the present invention, the amino acid sequence of SEQ ID NO.4 or SEQ ID NO.5 is modified, deleted or added with one or several amino acids to obtain an amino acid sequence that maintains only 90% homology Also within the protection scope of the present invention.

本发明一并提供上述融合型脂肪酶的构建方法,包括以下步骤:The present invention also provides a method for constructing the above-mentioned fusion lipase, comprising the following steps:

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;1) Design primers F1 and R1, F2 and R2 for template-free PCR to obtain parental short peptide 1 and parental short peptide 2; then design primers F3 and R3, F3 and R4 to carry out parental short peptide 1 and parental short peptide 2 respectively PCR amplification;

2)以华根霉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;2) Using the recombinant plasmid in which the Rhizopus sinensis lipase gene is connected to the carrier as a template, design primers F4 and R5, F5 and R5 to amplify lipase 1 and lipase 2 by PCR respectively;

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。3) F3 and R5 were used as primers, and the parental short peptide 1 and lipase 1, and the parental short peptide 2 and lipase 2 were used as templates for fusion PCR amplification, respectively.

4)将融合PCR扩增产物与载体进行重组;4) recombining the fusion PCR amplification product with the vector;

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;5) Put 5 μL of the recombinant product in 50 μL DH5α competent cells, cool in an ice bath, heat shock, add 500 μL LB medium after cooling, and culture at 180°C at 37°C for 1 hour, centrifuge to retain part of the supernatant to suspend the precipitate, and take all the bacterial liquid to smear plates, cultured overnight at 37°C;

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送出测序,测序结果与原序列比对。6) Carry out screening and verification of positive clones, pick a single colony in the corresponding resistant LB medium, culture for 2-3 hours and then identify by PCR, send the screened positive clones for sequencing, and compare the sequencing results with the original sequence.

上述引物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。The nucleotide sequences of the above primers F1, R1, F2, R2, F3, R3, F4, R4, F5 and R5 are respectively shown in SEQ ID NO.8-17.

本发明所述的编码基因SEQ ID NO.4、SEQ ID NO.5的表达载体选自pPIC9K、pPIC9、pPICZaA\B\C、pPICZA\B\C或PGAPZaA\B\C。The expression vectors of the encoding genes SEQ ID NO.4 and SEQ ID NO.5 of the present invention are selected from pPIC9K, pPIC9, pPICZaA\B\C, pPICZA\B\C or PGAPZaA\B\C.

在本发明另一方面,本发明提供的两种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。In another aspect of the present invention, the application of the two fusion lipases provided by the present invention in feed additives is also within the protection scope of the present invention.

本发明的有益效果为:The beneficial effects of the present invention are:

本发明提供的融合型脂肪酶,融合后的基因除与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,60℃耐受20min脂肪酶融合双亲短肽1相对酶活大约还有67.98%,脂肪酶融合双亲短肽2相对酶活大约还有59.65%,而野生型脂肪酶仅仅剩余50.68%。在60℃时野生型脂肪酶相对酶活剩余一半时的时间大约为20min,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30min。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。The fusion lipase provided by the present invention, in addition to constructing recombinant plasmids with pPIC9K, the fused gene can also construct recombinant plasmids with expression vectors such as pPIC9, pPICZaA\B\C, pPICZA\B\C, PGAPZaA\B\C, In transforming the corresponding host bacteria, antibiotics such as G418 and Zeocin are added to the plate to screen and obtain lipase genetically engineered bacteria, and then new lipase is obtained by fermentation. This makes it a lipase with high heat resistance. These two lipases are resistant to high temperatures compared with wild-type lipase. It has been proved by experiments that the relative enzyme activity of lipase fusion parent short peptide 1 is about 67.98% after 60°C tolerance for 20 minutes. , the relative enzymatic activity of the lipase fusion parent short peptide 2 is about 59.65%, while that of the wild-type lipase is only 50.68%. At 60°C, the remaining half of the relative enzymatic activity of wild-type lipase is about 20 minutes, while the remaining half of the relative enzymatic activity of lipase fusion parent short peptide 1 is about 45 minutes, and the relative enzymatic activity of lipase fusion parent short peptide 2 is about 20 minutes. The time for the remaining half is about 30 minutes. The heat resistance of these two lipases has been significantly improved, and the loss rate in industrial production has been reduced to a certain extent.

附图说明Description of drawings

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;Fig. 1 is the flow chart of the construction method of fusion type lipase provided by the embodiment of the present invention;

图2是本发明实施例提供的最适pH的测定曲线图;Fig. 2 is the determination curve figure of optimum pH that the embodiment of the present invention provides;

图3是本发明实施例提供的最适温度曲线图;Fig. 3 is the optimal temperature curve figure that the embodiment of the present invention provides;

图4是本发明实施例提供的pH耐受曲线图;Fig. 4 is the pH tolerance curve figure that the embodiment of the present invention provides;

图5是本发明实施例提供的60℃耐受曲线图。Fig. 5 is a 60°C tolerance curve diagram provided by an embodiment of the present invention.

具体实施方式detailed description

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。The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

通过对对华根霉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。具体实施方案为以华根霉脂肪酶基因为模板,通过基因的融合PCR方法,进行华根霉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。Through the simulation analysis of the protein spatial structure of Rhizopus sinensis lipase, it was fused with the parental short peptide 1 and the parental short peptide 2 respectively. The specific embodiment is to use the Rhizopus sinorifica lipase gene as a template, and carry out the fusion of the Rhizopus sinensis lipase gene and the parental short peptide by the fusion PCR method of the gene, obtain two kinds of fusion type lipase genes, and combine the two kinds of fusion type fat The enzyme gene was connected with pPIC9, pPICZaA\B\C, pPICZA\B\C, PGAPZaA\B\C and other vectors to construct recombinant plasmids, which were transferred into corresponding host bacteria (GS115 or X33, SMD1168, PICHIAPINK) for heterologous expression, and fermentation Two lipases are available. The two lipases can function well in an acidic environment, and have relatively ideal heat-resistant properties, are suitable for high-temperature-resistant granulation, and are therefore suitable for industrial production.

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。The fused amino acid sequence of the lipase in the embodiment of the present invention is shown in SEQ ID NO.4 or SEQ ID NO.5.

1、实验材料和试剂:1. Experimental materials and reagents:

基因来源菌株:由本实验室筛选并保存的华根霉Rhizopus chinensis CCTCCM201021;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。Gene source strain: Rhizopus chinensis CCTCCM201021 screened and preserved by our laboratory; expression host bacteria and vectors: GS115 and pPIC9K were purchased from Novagen; lipase recombinant plasmid was constructed by the laboratory; host bacteria: DH5α competent cells were purchased from In Beijing Quanshijin Company.

主要试剂:DNA Marker、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。Main reagents: DNA Marker, Protein Marker (TaKaRa Company); Plasmid Mini Kit I (Omega Company). Agarose was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; nucleic acid dyes were purchased from Biotech Biological Company; restriction endonucleases and PCR amplification enzymes were purchased from TaKaRa Company.

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)。Experimental equipment: centrifuge (Eppendorf); PCR amplification instrument (Bio-Rad); nucleic acid electrophoresis instrument (Bio-Rad); protein electrophoresis instrument (Amersham Bioscience); gel imager (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。Main media: YPD, LB, and yeast fermentation media (FA and FB) were all prepared in accordance with the recommended methods in the "Invitrogen Company Operation Manual".

2、脂肪酶酶活力的测定2. Determination of lipase enzyme activity

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。Under certain conditions, the amount of enzyme needed to hydrolyze the p-NP substrate to generate 1 μmoL of p-nitrophenol per minute, that is, one enzyme activity unit, expressed in U.

1)实验仪器:恒温水浴锅;pH仪器;酶标仪(Bio-Rad)等。1) Experimental instruments: constant temperature water bath; pH instrument; microplate reader (Bio-Rad), etc.

2)实验材料:对硝基苯酚棕榈酸酯(p-NPC16)(Sigma公司)。2) Experimental material: p-nitrophenol palmitate (p-NPC16) (Sigma Company).

3)溶液配制:3) Solution preparation:

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);pH buffer: 0.1mol/L citrate monohydrate buffer and 0.1mol/L phosphate buffer (pH2-7);

0.1mol/L Tris-HCl缓冲液(pH7-9);0.1mol/L Tris-HCl buffer (pH7-9);

0.1mol/L甘氨酸-NaOH缓冲液(pH9-12)。0.1mol/L glycine-NaOH buffer (pH9-12).

底物溶液:10mmol/L对硝基苯酚棕榈酸酯(p-NPC16)。Substrate solution: 10 mmol/L p-nitrophenol palmitate (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-NP 30μL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50μL 1.0mol/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。4) Using the p-nitrophenol method (p-nitrophenol): the total system is 500 μL, which contains 420 μL of 50 mmol/L buffer solution, 30 μL of 10 mmol/L substrate p-NP and 50 μL of diluted enzyme solution. After mixing the substrate and buffer, preheat at the reaction temperature for 2 minutes, add diluted enzyme solution and mix well, add 50 μL 1.0 mol/L SDS for 5 minutes to stop the reaction, and add 500 μL 1.0 mol/L Na 2 CO 3 for color development; The OD value was measured at a wavelength of 405 nm.

实施例1脂肪酶融合双亲短的制备The preparation of embodiment 1 lipase fusion parent short

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:As shown in Figure 1, the obtaining method of two kinds of lipases of the embodiment of the present invention comprises the following steps:

(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;以华根霉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;(1) RCR amplification: Design primers (F1, R1) and (F2, R2) for template-free PCR to obtain parental short peptide 1 and parental short peptide 2; then design primers to synthesize parental short peptide 1 (F3, R3 ), parental short peptide 2 (F3, R4) for PCR amplification; using the recombinant plasmid of Rhizopus chinensis lipase gene connected to the carrier as a template, design primers (F4, R5) (F5, R5) for PCR amplification of fat Enzyme gene 1, lipase gene 2;

(2)以F3、R5为引物,分别用双亲短肽1与华根霉脂肪酶基因1、双亲短肽2与华根霉脂肪酶基因2为模版分别进行融合PCR扩增;(2) Using F3 and R5 as primers, respectively use parental short peptide 1 and Rhizopus sinensis lipase gene 1, parental short peptide 2 and Rhizopus sinensis lipase gene 2 as templates to carry out fusion PCR amplification respectively;

(3)重组质粒构建:将融合PCR产物与载体37℃进行重组30min;(3) Recombinant plasmid construction: recombine the fusion PCR product and vector at 37°C for 30 minutes;

(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;(4) Transformation: add 5 μL of the mutated product to 50 μL DH5α competent cells, flick and mix well, and place on ice for 30 min; heat shock at 42°C for 45 s and immediately cool on ice for 10 min; add 500 μL LB medium, 180 Transform and incubate at 37°C for 1 hour; centrifuge at 7,000rpm for 3 minutes, discard the supernatant, retain 100-150 μL of the supernatant and lightly flick the suspended bacteria, take all the bacterial liquid to spread on the plate, and culture overnight at 37°C;

(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;(5) Verification of positive clones: Pick a single colony in 500 μL of correspondingly resistant LB medium, culture it at 200 rpm for 2-3 hours, and then identify it by PCR; send the screened positive clones for sequencing, and compare the sequencing results with the original sequence;

(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。(6) Find the recombinant plasmid with correct fusion; transfer the mutant plasmid into Pichia pastoris GS115 or X33, SMD1168, PICHIAPINK for expression, ferment and compare enzyme activity to study enzymology and application characteristics.

其中,上述方法中设计得到引物具体如下:Wherein, the primers designed in the above method are specifically as follows:

Figure GDA0003737475680000081
Figure GDA0003737475680000081

双亲短肽1、双亲短肽2的氨基酸序列分别如SEQ ID NO.2和SEQ ID NO.3所示。The amino acid sequences of the amphipathic short peptide 1 and the amphipathic short peptide 2 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

华根霉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,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。The lipase gene of Rhizopus sinica was fused with parental short peptides 1 and 2, and the fusion was carried out according to the above experimental method, and then sent to Huada Gene Company for sequencing. The results were as shown in the sequences of SEQ ID NO.4 and SEQ ID NO.5, and the transformed yeast strain had lipase Activity, one strain with high fermentation enzyme activity unit was selected for fermentation to obtain enzyme liquid for enzymatic property determination.

实施例2脂肪酶最适pH测定Embodiment 2 lipase optimum pH is measured

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)。Adjust the pH of the buffer solution to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, dilute the enzyme solution to an appropriate multiple, and measure the optimum pH at 37°C according to the lipase activity assay method. After the pH, add half points on both sides of the maximum value and continue to detect the optimum value (for example, if the optimum pH is 9, then take pH8, 8.5, 9, 9.5 and 10 to measure according to the lipase activity assay method).

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。The results of the optimal pH value of the lipase enzymatic reaction are shown in Figure 2. The optimal value of lipase fusion parent short peptide 1, 2 and wild-type lipase was 9, and there was no significant change.

实施例3脂肪酶最适温度测定Embodiment 3 lipase optimum temperature is measured

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℃、60℃、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40℃,则补充30℃、35℃、40℃、45℃、50℃按照脂肪酶活测定方法测定)。According to the assay method of lipase activity, under the conditions of the above-mentioned optimum pH, the reactants were placed at different temperatures to react at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, after measuring the optimum temperature, add half points on both sides of the maximum value (for example, if the optimum temperature is 40°C, add 30°C, 35°C, 40°C, 45°C, 50°C to determine the lipase activity method) .

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40℃。The optimum temperature for the enzymatic reaction of lipase is shown in Figure 3. The optimum temperature for lipase gene fusion parent short peptide 1 and 2 and wild-type lipase is 40°C, respectively.

实施例4脂肪酶pH耐受测定Example 4 Lipase pH Tolerance Determination

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。Adjust the buffer to different pH: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, dilute the enzyme solution with these different buffer solutions, and start timing from the time the enzyme solution is put in, The diluted enzyme solution was placed in a water bath at 37°C for 1 hour, then placed on ice, and immediately reacted at the optimum pH and temperature according to the lipase activity assay method. The enzyme solution of the control group is the enzyme solution that has not been tolerated.

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。It can be seen from Figure 4 that the resistance curves of the three lipases have the same tendency, and the relative enzyme activity of the three lipases is significantly increased when the pH is between 4 and 9 at 37°C for 1 hour.

实施例5脂肪酶温度耐受测定Embodiment 5 lipase temperature tolerance assay

将酶液稀释到相应倍数,然后放入不同温度:60℃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。Dilute the enzyme solution to the corresponding multiple, and then put it into different temperatures: 60°C for 1min, 3min, 5min, 7min, 10min, 15min, 20min, 25min, 30min, 35min, 45min, 60min, then follow the lipase activity assay method React at optimum pH and optimum temperature. The enzyme solution of the control experiment group is the enzyme solution without temperature tolerance.

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,60℃耐受20min融合型脂肪酶1相对酶活大约还有67.98%,融合型脂肪酶2相对酶活大约还有59.65%,而野生型脂肪酶仅仅剩余50.68%。在60℃时野生型脂肪酶相对酶活剩余一半时的时间大约为20min,而融合型脂肪酶1相对酶活剩余一半时的时间大约为45min,融合型脂肪酶2相对酶活剩余一半时的时间大约为30min。The temperature tolerance of lipase at high temperature is shown in Figure 5. As the temperature increases, the relative enzyme activity decreases continuously, and as time increases, the relative enzyme activity also gradually decreases. No matter at any temperature and time, the relative enzyme activity after fusion is higher than that before mutation, and the relative enzyme activity of fusion lipase 1 is about 67.98%, and the relative activity of fusion lipase 2 is about 67.98%. 59.65%, while only 50.68% of wild-type lipase remained. At 60°C, the remaining half of the relative enzymatic activity of wild-type lipase is about 20 minutes, while the remaining half of the relative enzymatic activity of fusion-type lipase 1 is about 45 minutes, and the remaining half of the relative enzymatic activity of fusion-type lipase 2 is about 20 minutes. The time is about 30 minutes.

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。Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variants, the scope of the invention is defined by the appended claims and their equivalents.

序列表sequence listing

<110> 云南师范大学<110> Yunnan Normal University

<120> 一种热稳性提高的融合型华根霉脂肪酶及其应用<120> A fusion-type Rhizopus chinensis lipase with improved thermostability and its application

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<400> 6<400> 6

gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacca 60gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacca 60

actactccaa ctccaccaac tactccaact ccaactgttc ctgttgctgg tcataaaggt 120actactccaa ctccaccaac tactccaact ccaactgttc ctgttgctgg tcataaaggt 120

tcagtcaagg caactaatgg tactgacttc caactccctc ctctcatctc tagcagatgt 180tcagtcaagg caactaatgg tactgacttc caactccctc ctctcatctc tagcagatgt 180

actcctcctt cccatcctga aaccacaggt gatcctgatg ccgaagctta ctatattaac 240actcctcctt cccatcctga aaccacaggt gatcctgatg ccgaagctta ctatattaac 240

aagagcgttc aatggtacca agctcacggt ggtaactaca ctgctcttat caagagagat 300aagagcgttc aatggtacca agctcacggt ggtaactaca ctgctcttat caagagagat 300

actgaaaccg tcggtggtat gaccttggat ttgcctgaga accctcctcc tattcctgcc 360actgaaaccg tcggtggtat gaccttggat ttgcctgaga accctcctcc tattcctgcc 360

acgtccactg ctcctagctc tgattcaggt gaagttgtca cagccactgc tgctcaaatc 420acgtccactg ctcctagctc tgattcaggt gaagttgtca cagccactgc tgctcaaatc 420

aaagagctca ctaactacgc tggtgttgct gctactgctt actgtagaag tgtcgttcca 480aaagagctca ctaactacgc tggtgttgct gctactgctt actgtagaag tgtcgttcca 480

ggtaccaagt gggactgtaa gcaatgtctc aagtatgttc ctgatggtaa gcttatcaag 540ggtaccaagt gggactgtaa gcaatgtctc aagtatgttc ctgatggtaa gcttatcaag 540

accttcactt ctcttctcac tgataccaat ggttttatct tgagaagtga tgctcaaaag 600accttcactt ctcttctcac tgataccaat ggttttatct tgagaagtga tgctcaaaag 600

accatctatg ttactttcag aggtactaat tccttcagaa gcgctattac tgacatggtc 660accatctatg ttactttcag aggtactaat tccttcagaa gcgctattac tgacatggtc 660

ttcaccttta ctgattattc tcctgtcaag ggtgccaaag ttcacgctgg tttcctttcc 720ttcaccttta ctgattattc tcctgtcaag ggtgccaaag ttcacgctgg tttcctttcc 720

tcatacaacc aagttgtcaa agactacttc cctgtcgttc aagaccaatt gaccgcttac 780tcatacaacc aagttgtcaa agactacttc cctgtcgttc aagaccaatt gaccgcttac 780

cctgactata aggtcatcgt caccggtcac tctctcggtg gtgcccaagc cttgctcgct 840cctgactata aggtcatcgt caccggtcac tctctcggtg gtgcccaagc cttgctcgct 840

ggtatggatc tctaccaaag agaaaagaga ttatctccta agaacttgag catctatact 900ggtatggatc tctaccaaag agaaaagaga ttatctccta agaacttgag catctatact 900

gttggttgtc ctagagtcgg taacaatgca ttcgcttact acgtcgacag caccggaatt 960gttggttgtc ctagagtcgg taacaatgca ttcgcttact acgtcgacag caccggaatt 960

cctttccaca gaaccgttca caagagagat atcgtccctc atgttcctcc tcaagccttc 1020cctttccaca gaaccgttca caagagagat atcgtccctc atgttcctcc tcaagccttc 1020

ggttatcttc accctggtgt cgaatcttgg atcaaggaag accctgctga tgttcaaatc 1080ggttatcttc accctggtgt cgaatcttgg atcaaggaag accctgctga tgttcaaatc 1080

tgtacttcca acattgaaac caaacaatgc agtaactcta tcgttccttt cacctctatc 1140tgtacttcca acattgaaac caaacaatgc agtaactcta tcgttccttt cacctctatc 1140

gctgatcact taacctactt tggtattaac gaaggaagct gtttg 1185gctgatcact taacctactt tggtattaac gaaggaagct gtttg 1185

<210> 7<210> 7

<211> 1167<211> 1167

<212> DNA<212>DNA

<213> Coding gene<213> Coding gene

<400> 7<400> 7

gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgtt 60gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgtt 60

aagaaggagg ctgagctcgt tcctgttgct ggtcataaag gttcagtcaa ggcaactaat 120aagaaggagg ctgagctcgt tcctgttgct ggtcataaag gttcagtcaa ggcaactaat 120

ggtactgact tccaactccc tcctctcatc tctagcagat gtactcctcc ttcccatcct 180ggtactgact tccaactccc tcctctcatc tctagcagat gtactcctcc ttcccatcct 180

gaaaccacag gtgatcctga tgccgaagct tactatatta acaagagcgt tcaatggtac 240gaaaccacag gtgatcctga tgccgaagct tactatatta acaagagcgt tcaatggtac 240

caagctcacg gtggtaacta cactgctctt atcaagagag atactgaaac cgtcggtggt 300caagctcacg gtggtaacta cactgctctt atcaagagag atactgaaac cgtcggtggt 300

atgaccttgg atttgcctga gaaccctcct cctattcctg ccacgtccac tgctcctagc 360atgaccttgg atttgcctga gaaccctcct cctattcctg ccacgtccac tgctcctagc 360

tctgattcag gtgaagttgt cacagccact gctgctcaaa tcaaagagct cactaactac 420tctgattcag gtgaagttgt cacagccact gctgctcaaa tcaaagagct cactaactac 420

gctggtgttg ctgctactgc ttactgtaga agtgtcgttc caggtaccaa gtgggactgt 480gctggtgttg ctgctactgc ttactgtaga agtgtcgttc caggtaccaa gtgggactgt 480

aagcaatgtc tcaagtatgt tcctgatggt aagcttatca agaccttcac ttctcttctc 540aagcaatgtc tcaagtatgt tcctgatggt aagcttatca agaccttcac ttctcttctc 540

actgatacca atggttttat cttgagaagt gatgctcaaa agaccatcta tgttactttc 600actgatacca atggttttat cttgagaagt gatgctcaaa agaccatcta tgttactttc 600

agaggtacta attccttcag aagcgctatt actgacatgg tcttcacctt tactgattat 660agaggtacta attccttcag aagcgctatt actgacatgg tcttcacctt tactgattat 660

tctcctgtca agggtgccaa agttcacgct ggtttccttt cctcatacaa ccaagttgtc 720tctcctgtca agggtgccaa agttcacgct ggtttccttt cctcatacaa ccaagttgtc 720

aaagactact tccctgtcgt tcaagaccaa ttgaccgctt accctgacta taaggtcatc 780aaagactact tccctgtcgt tcaagaccaa ttgaccgctt accctgacta taaggtcatc 780

gtcaccggtc actctctcgg tggtgcccaa gccttgctcg ctggtatgga tctctaccaa 840gtcaccggtc actctctcgg tggtgcccaa gccttgctcg ctggtatgga tctctaccaa 840

agagaaaaga gattatctcc taagaacttg agcatctata ctgttggttg tcctagagtc 900agagaaaaga gattatctcc taagaacttg agcatctata ctgttggttg tcctagagtc 900

ggtaacaatg cattcgctta ctacgtcgac agcaccggaa ttcctttcca cagaaccgtt 960ggtaacaatg cattcgctta ctacgtcgac agcaccggaa ttcctttcca cagaaccgtt 960

cacaagagag atatcgtccc tcatgttcct cctcaagcct tcggttatct tcaccctggt 1020cacaagagag atatcgtccc tcatgttcct cctcaagcct tcggttatct tcaccctggt 1020

gtcgaatctt ggatcaagga agaccctgct gatgttcaaa tctgtacttc caacattgaa 1080gtcgaatctt ggatcaagga agaccctgct gatgttcaaa tctgtacttc caacattgaa 1080

accaaacaat gcagtaactc tatcgttcct ttcacctcta tcgctgatca cttaacctac 1140accaaacaat gcagtaactc tatcgttcct ttcacctcta tcgctgatca cttaacctac 1140

tttggtatta acgaaggaag ctgtttg 1167tttggttatta acgaaggaag ctgtttg 1167

<210> 8<210> 8

<211> 59<211> 59

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 8<400> 8

gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacc 59gctgaagctg aagctaaagc taaagctgaa gctgaagcta aagctaaacc aactccacc 59

<210> 9<210> 9

<211> 59<211> 59

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 9<400> 9

agttggagtt ggagtagttg gtggagttgg agtagttggt ggagttggtt tagctttag 59agttggagtt ggagtagttg gtggagttgg agtagttggt ggagttggtt tagctttag 59

<210> 10<210> 10

<211> 59<211> 59

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 10<400> 10

gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgt 59gctgaagctg aagctaaagc taaagctgaa gctgaaccga aagtcagccc ggaggctgt 59

<210> 11<210> 11

<211> 59<211> 59

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 11<400> 11

gagctcagcc tccttcttaa cagcctccgg gctgactttc ggttcagctt cagctttag 59gagctcagcc tccttcttaa cagcctccgg gctgactttc ggttcagctt cagctttag 59

<210> 12<210> 12

<211> 44<211> 44

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 12<400> 12

gaggctgaag cttacgtaga attcgctgaa gctgaagcta aagc 44gaggctgaag cttacgtaga attcgctgaa gctgaagcta aagc 44

<210> 13<210> 13

<211> 30<211> 30

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 13<400> 13

accagcaaca ggaacagttg gagttggagt 30accagcaaca ggaacagttg gagttggagt 30

<210> 14<210> 14

<211> 30<211> 30

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 14<400> 14

accagcaaca ggaacgagct cagcctcctt 30accagcaaca ggaacgagct cagcctcctt 30

<210> 15<210> 15

<211> 30<211> 30

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 15<400> 15

actccaactc caactgttcc tcttgctggt 30actccaactc caactgttcc tcttgctggt 30

<210> 16<210> 16

<211> 30<211> 30

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 16<400> 16

gttcctgttg ctggtcataa aggttcagtc 30gttcctgttg ctggtcataa aggttcagtc 30

<210> 17<210> 17

<211> 44<211> 44

<212> DNA<212>DNA

<213> Artificial Sequence<213> Artificial Sequence

<400> 17<400> 17

tctaaggcga attaattcgc ggccgcctac aaacagcttc cttc 44tctaaggcga attaattcgc ggccgcctac aaacagcttc cttc 44

Claims (2)

1. Fusion type rhizopus chinensis (with improved heat stability)Rhizopus chinensis ) The lipase is characterized in that the amino acid sequence of the fusion type rhizopus chinensis lipase is shown as SEQ ID NO. 5.
2. Use of the fused Rhizopus chinensis lipase of claim 1 in feed additives.
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