WO2016169306A1 - 糖基化改造提高脂肪酶表达的方法、突变酶及其应用 - Google Patents

糖基化改造提高脂肪酶表达的方法、突变酶及其应用 Download PDF

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WO2016169306A1
WO2016169306A1 PCT/CN2016/070236 CN2016070236W WO2016169306A1 WO 2016169306 A1 WO2016169306 A1 WO 2016169306A1 CN 2016070236 W CN2016070236 W CN 2016070236W WO 2016169306 A1 WO2016169306 A1 WO 2016169306A1
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lipase
mutant
glycosylation
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amino acid
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喻晓蔚
徐岩
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江南大学
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/845—Rhizopus

Definitions

  • the invention relates to a method for improving lipase expression by glycosylation modification, a mutant enzyme and application thereof, and belongs to the field of enzyme engineering.
  • Rhizopus oryzae lipase has a good 1,3-position specificity, preferentially catalyzes long-chain fatty acids, and has important applications in food, chemical and bioenergy. Rhizopus oryzae lipase has different applications using enzyme hydrolysis, synthesis and transesterification activities.
  • Rhizopus oryzae lipase Currently, the entire gene sequence of Rhizopus oryzae lipase has been published at NCBI.
  • the enzyme consists of a 26 amino acid signal sequence (presequsece), a 97 amino acid leader peptide sequence (prosequsence) and a 269 amino acid mature peptide sequence (mROL).
  • Presequsece pre-sequse
  • leader peptide plays an important role in the secretion and folding of Rhizopus oryzae lipase: the secretion of the enzyme must have the residues 20 to 37 of the pre-sequence, and the folding to form the active lipase must have No. 38 To the 57th residue.
  • Rhizopus oryzae lipase is well expressed in E. coli, but these lipases can only be expressed in an inactive form.
  • P.pastoris is one of the most widely used and most efficient methods for the expression of foreign proteins.
  • the characteristics of genes, protein folding in the endoplasmic reticulum, transport of proteins from the endoplasmic reticulum to Golgi, and translation of proteins can affect many factors.
  • P. pastoris expresses the level of protein. It is a common occurrence that P. pastoris expresses protein to produce N-glycosylation. Glycosylation can stabilize the mature conformation of proteins, affect the activity and thermal stability of proteins, and also play an important role in the correct folding, transport and localization of proteins. effect. Among them, N-glycosylation plays a crucial role in many protein research reports, and it is necessary for the secretion of proteins, etc., and people have relatively deep research. Often, changes in the function of a protein are often due to changes in the structure of the protein, and the addition of a large sugar chain to the protein can greatly alter the structure of the protein.
  • N-glycosylation has an important influence on the secretion level and activity of P. pastoris expressed protein.
  • Kohno et al. constructed a P. pastoris genetically engineered strain expressing Kex2 locus mutant lipase, and found that the untreated RNL has higher thermal stability and the thermal stability of RNL based on the length of the sugar chain bound to the lipase. no effect.
  • Ito et al. studied the effect of N-glycosylation of ovalbumin on the amount of protein secreted, demonstrating that 292 N-glycosylation is necessary for the secretion and folding of this protein. Boivin et al.
  • N-glycosylation studied the effect of N-glycosylation on the secretion level and activity of the thrombolytic agent DSPA ⁇ 1 expressed by P. pastoris, and found that the N-glycans (N117 and N362) of the two sites of the protein secreted proteins and enzymes. Activity plays an important role.
  • N-glycosylation study of Rhizopus arrhizus lipase also proved that it plays a key role in the secretion of enzymes, but N-glycosylation has a negative effect on the acquisition of its enzyme activity.
  • a lipase mutant having improved enzyme activity and expression amount is obtained by performing N-glycosylation mutation on a leader peptide of Rhizopus oryzae lipase.
  • the present invention provides a method for improving lipase expression based on N-glycosylation modification, and an improved expression of lipase and application thereof.
  • a first object of the invention is to provide an expression enhancing lipase mutant.
  • the mutant mutates the SAS and/or NT amino acid positions of its leader peptide to an N-glycosylation site based on Rhizopus oryzae lipase.
  • the mutant in one embodiment of the present invention, mutates the SAS amino acid position in the leader peptide sequence to an N-glycosylation site NGT, designated as proROLA, and the amino acid sequence of the mutant is SEQ ID NO.1 is shown.
  • the mutant in one embodiment of the present invention, mutates the NT amino acid position in the leader peptide sequence to an N-glycosylation site NLT, which is named proROLB, and the amino acid sequence of the mutant is SEQ ID Shown in NO.2.
  • the mutant in one embodiment of the present invention, mutates the SAS and NT amino acid positions in the leader peptide sequence to N-glycosylation sites NGT and NLT, respectively, and is named as proROLAB.
  • the amino acid sequence is shown in SEQ ID NO.
  • the mutant may also be hybridized under stringent conditions (Tm-10-15 ° C) to the amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 and encoding lipase activity. Protein molecule.
  • Rhizopus oryzae lipase in one embodiment of the present invention, the amino acid sequence before the N-glycosylation mutation occurs as shown in SEQ ID NO. 4, due to the lipase amino acid sequence derived from different Rhizopus oryzae strains There are only a few differences in amino acids, and therefore, the method is applicable not only to the sequence of SEQ ID NO. 4, but also to lipase sequences having an amino acid sequence similarity of 80% or more.
  • Rhizopus oryzae lipase in one embodiment of the present invention, the nucleotide sequence before the N-glycosylation mutation occurs is shown in SEQ ID NO.
  • a second object of the present invention is to provide an expression vector containing the lipase mutant.
  • the expression vector is a secretory vector.
  • the expression vector in one embodiment of the present invention, may be any one of the following: pGAPZ ⁇ , pPIC9, pPIC3K, pPIC9K, PAO815 or pPICZ ⁇ .
  • a third object of the present invention is to provide a genetically engineered bacterium containing the lipase mutant.
  • the genetically engineered bacteria in one embodiment of the present invention, is constructed by using Pichia pastoris as a host.
  • the genetically engineered bacteria in an embodiment of the present invention, is constructed by cloning a nucleotide sequence encoding the lipase mutant into an expression plasmid pGAPZ ⁇ , then linearizing the plasmid, and electroporating Competent cells of Pichia pastoris GS115.
  • a fourth object of the present invention is a method for increasing the expression of Aspergillus oryzae lipase by subjecting the SAS and/or NT amino acid positions of the leader peptide of Aspergillus oryzae lipase to N-glycosylation mutation.
  • amino acid sequence of the mutated lipase is represented by SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO.
  • the mutant lipase is expressed in Pichia pastoris using pGAPZ ⁇ as a vector.
  • the invention also claims the use of the lipase mutant in the fields of food, chemical or bioenergy, and the use of the genetically engineered bacteria in the production of an enzyme preparation.
  • the N-glycosylation-mutated Aspergillus oryzae lipase of the present invention not only does not affect the growth of the strain, but also has a significantly increased enzyme activity and secretion amount when it is progressively expressed in the engineered bacteria.
  • the extracellular protein concentrations of proROLA, proROLB and proROLAB were increased by 211%, 188% and 233%, respectively, compared with the non-glycosylated proROL; the mutants of proROLA, proROLB and proROLAB were separately shaken.
  • Figure 1 Schematic diagram of the mutation of the leader peptide
  • Figure 2 Growth curve of genetically engineered bacteria expressing lipase before and after mutation
  • FIG. 1 Protein secretion of genetically engineered strains
  • Figure 4 SDS-PAGE of expression of lipase by genetically engineered bacteria at different times; wherein M is a protein marker, and lanes 1-5 represent culture for 24h, 48h, 72h, 84h and 96h, respectively;
  • FIG. 5 Mutant enzyme activity at different fermentation times.
  • the present invention designs three N-glycosylation site mutants in the leader peptide of Rhizopus oryzae lipase ROL, that is, the SAS and NT amino acids are respectively transformed into N-glycosylation sites NGT and NLT, respectively, and the mutant enzymes are respectively named proROLA (amino acid sequence as shown in SEQ ID NO. 1) and proROLB (amino acid sequence as shown in SEQ ID NO. 2); naming of mutant enzymes that simultaneously transform both SAS and NT sites into N-glycosylation sites Is proROLAB (amino acid sequence as shown in SEQ ID NO. 3).
  • the specific mutation pattern of the leader peptide is shown in Figure 1.
  • primers NGT-A F/NGT-A R and NLT-BF/NLT-BR were designed to perform full-plasmid PCR, respectively, to construct the SAS and LT amino acids in the ROL gene.
  • Recombinant plasmids of NGT and NLT were named pGAPZ ⁇ -proROLA and pGAPZ ⁇ -proROLB.
  • the recombinant plasmid pGAPZ ⁇ -proROLAB was constructed by using the plasmid pGAPZ ⁇ -proROLA as a template and primer NLT-BF/NLT-BR whole plasmid PCR. The primers used are shown in Table 1.
  • Example 2 Comparison of growth of genetically engineered bacteria expressing mutant enzymes and wild enzymes
  • Rhizopus oryzae lipase mutants The growth of genetically engineered bacteria expressing Rhizopus oryzae lipase mutants was compared using GS115/pGAPZ ⁇ -proROL as a control.
  • Shake flask fermentation conditions positive transformants were streaked into YPD-G418 plates ((w/v): yeast extract 1%, glucose 2%, agar powder 2%, tryptone 2%, G4180.025%), cultured at 30 °C 3d, pick the monoclonal inoculation to 100mLYPD liquid medium ((w / v): yeast extract 1%, glucose 2%, tryptone 2%), 30 ° C 200rpm ⁇ min -1 shake flask culture, every 12h or 24h sampling.
  • the strain growth curve is shown in Figure 2. The results showed that the growth of each mutant strain was similar to that of the control. The strain grew rapidly within 48 hours. After 72 hours, the growth of each strain became slow and stabilized. It is indicated that the introduction of glycosylation sites in the Rhizopus oryzae lipase gene does not affect the growth of each strain.
  • the present invention compares lipase secretion of genetically engineered bacteria expressing mutant enzymes and wild enzymes.
  • the genetically engineered bacteria were fermented and cultured.
  • the specific culture conditions were as follows: the positive transformants were streaked into YPD-G418 plates, cultured at 30 ° C for 3 days, and the monoclonal seeds were inoculated into 100 mLYPD liquid medium, and cultured at 30 ° C 200 rpm ⁇ min -1 shake flask. , sampling every 12h or 24h.
  • the protein concentration of Rhizopus oryzae lipase proROL without introducing a glycosylation site was the lowest, and the extracellular protein concentration of Rhizopus oryzae lipase proROLAB which introduced two glycosylation sites was slightly higher.
  • the lipases proROLA and proROLB, which only introduce a glycosylation site initially determined that the introduced glycosylation site was glycosylated and affected the secretion level of Rhizopus oryzae lipase.
  • the results showed that the extracellular protein concentrations of proROLA, proROLB and proROLAB were increased by 211%, 188% and 233%, respectively, compared with proROL without glycosylation.
  • Figure 4 shows the SDS-PAGE plots for different sampling times. Since the secreted Rhizopus oryzae lipase leader peptide is partially cleaved (the KR site in Figure 1 is the specific restriction site for the excision signal peptide), the 28 leader peptide amino acids are retained, and the leader peptide fragment introduced into the glycosylation site is introduced. It was also excised, so the molecular weight of the secreted expressed Rhizopus oryzae lipase was consistent.
  • the lipase-free proROL was not secreted from 24h to 96h, and the amount of lipase released into the glycosylation site increased with time, reached the maximum at 84h, and the mutant enzymes proROLA and proROLAB The amount of secretion was slightly higher than the lipase proROLB during the same period of time.
  • Example 4 Effect of N-glycosylation on extracellular enzyme activity of Rhizopus oryzae lipase under shake flask conditions
  • the constructed genetically engineered bacteria were subjected to shake flask fermentation to produce enzymes, and the lipase activities of different fermentation time were measured.
  • the positive transformants were streaked into YPD-G418 plates, cultured at 30 ° C for 3 d, and the monoclonal clones were inoculated into 100 mL YPD liquid medium, and cultured at 30 ° C, 200 rpm ⁇ min -1 shake flask, and sampled every 12 h or 24 h.
  • Enzyme activity is defined as the amount of enzyme used to produce 1 ⁇ mol of pNP per minute under standard reaction conditions is a lipase hydrolase active international unit. The method for measuring the enzyme activity was carried out by the pNPP method.
  • Example 5 Effect of N-glycosylation on extracellular enzyme activity of Rhizopus oryzae lipase under fermenter conditions
  • the constructed genetically engineered bacteria were fermented in a 30 L fermentor to produce an enzyme, and the lipase activity of the fermentation time of 84 h was measured.
  • the positive transformant was streaked into YPD-G418 plate, cultured at 30 °C for 3 days, and the monoclonal seed was inoculated into 200 mL YPD liquid medium. When the OD value reached 2-6, it was inoculated into a 30 L fermentor for fermentation and fermentation.
  • the glucose solution was added during the process, the concentration was controlled to be about 2%, the ammonia was added to control pH 5.5, the temperature was 28 ° C, and the lipase activity was measured after 84 h of fermentation.
  • Enzyme activity is defined as the amount of enzyme used to produce 1 ⁇ mol of pNP per minute under standard reaction conditions. Solving enzymes in international units. The method for measuring the enzyme activity was carried out by the pNPP method.

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Abstract

提供了糖基化改造提高脂肪酶表达的方法、突变酶及其应用。具体是将米根霉脂肪酶的前导肽序列进行N-糖基化突变，将SAS和/或NT氨基酸分别改造为N-糖基化位点NGT和/或NLT，得到的突变体酶proROLA、proROLB、proROLAB的胞外蛋白浓度分别比未进行糖基化的proROL提高了211％、188％、233％，发酵罐培养时酶活分别达到8210U·mL -1、8457U·mL -1和9366U·mL -1，而未突变的proROL的胞外酶活几乎为零。

Description

糖基化改造提高脂肪酶表达的方法、突变酶及其应用 技术领域
本发明涉及糖基化改造提高脂肪酶表达的方法、突变酶及其应用，属于酶工程领域。
背景技术
米根霉脂肪酶具有良好的1,3-位置特异性，优先催化中长链脂肪酸，在食品、化工及生物能源等方面都有很重要的应用。利用酶的水解、合成及转酯等活性，米根霉脂肪酶有不同的应用。
目前，米根霉脂肪酶的全基因序列已经在NCBI上公布。该酶包括26个氨基酸信号序列(presequsece)、97个氨基酸前导肽序列(prosequsence)和269个氨基酸成熟肽序列(mROL)所组成。已有研究表明前导肽对米根霉脂肪酶的分泌和折叠起着十分重要的作用：酶的分泌必需有前序列的20号到37号残基，而经折叠形成活性脂肪酶必需有38号到57号残基。米根霉脂肪酶在大肠杆菌中有很好的表达，但这些脂肪酶只能以无活性的形式表达。
P.pastoris是应用广泛、最高效的外源蛋白表达工具之一，基因的特性、内质网中的蛋白质折叠、蛋白从内质网到高尔基体的转运以及蛋白的翻译等许多因素都能影响P.pastoris表达蛋白的水平。P.pastoris表达蛋白产生N-糖基化是一种常见情况，糖基化能够稳定蛋白质的成熟构象，影响蛋白的活性及热稳定性，以及对蛋白质的正确折叠、运输、定位也起着重要作用。其中N-糖基化在许多蛋白质研究报道中都有着至关重要的作用，对蛋白的分泌等作用是必需的，人们对其研究也相对深入。通常，蛋白质的功能发生改变往往是由于蛋白的结构发生了变化，而蛋白质上增加一个大的糖链能极大的改变蛋白质的结构。
据研究报道N-糖基化对P.pastoris表达蛋白的分泌水平与活性等具有重要影响。Kohno等构建表达Kex2位点突变脂肪酶的P.pastoris基因工程菌，发现前导肽未被处理的RNL的热稳定性更高，且结合到脂肪酶上的糖链长度大小对RNL的热稳定性无影响。Ito等研究卵清蛋白的N-糖基化对蛋白分泌量的影响，证明292位N-糖基化对该蛋白的分泌折叠是必须的。Boivin等研究N-糖基化对P.pastoris表达的溶栓剂DSPAα1分泌水平和活性的影响，结果发现该蛋白的两个位点的N-糖链(N117和N362)对蛋白的分泌和酶活性具有重要作用。此外，根毛霉脂肪酶的N-糖基化研究也证明其对酶的分泌有关键作用，但N-糖基化对其酶活的获得却有消极作用。
本发明通过对米根霉脂肪酶的前导肽进行N-糖基化突变，获得了酶活和表达量提高的脂肪酶突变体。
发明内容
为了克服现有米根霉脂肪酶表达量和酶活较低的缺陷，本发明提供一种基于N-糖基化改造提高脂肪酶表达的方法，以及表达提高的脂肪酶及其应用。
本发明的第一个目的是提供一种表达提高脂肪酶突变体。
所述突变体，是在米根霉脂肪酶的基础上，将其前导肽的SAS和/或NT氨基酸位点突变为N-糖基化位点。
所述突变体，在本发明的一种实施方式中，是将前导肽序列中的SAS氨基酸位点突变成N-糖基化位点NGT，命名为proROLA，突变体的氨基酸序列如SEQ ID NO.1所示。
所述突变体，在本发明的一种实施方式中，是将前导肽序列中的NT氨基酸位点突变成N-糖基化位点NLT，命名为proROLB，突变体的氨基酸序列如SEQ ID NO.2所示。
所述突变体，在本发明的一种实施方式中，是将前导肽序列中的SAS、NT氨基酸位点分别突变成N-糖基化位点NGT、NLT，命名为proROLAB，突变体的氨基酸序列如SEQ ID NO.3所示。
所述突变体，还可以是在严格条件下(Tm－10～15℃)与SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示氨基酸序列杂交且编码具有脂肪酶活性的蛋白质分子。
所述米根霉脂肪酶，在本发明的一种实施方式中，发生N-糖基化突变前的氨基酸序列如SEQ ID NO.4所示，由于不同米根霉菌株来源的脂肪酶氨基酸序列仅有个别氨基酸的差异，因此，该方法不仅适用于SEQ ID NO.4序列，而且对氨基酸序列相似性达到80％以上的脂肪酶序列均适用于本专利。
所述米根霉脂肪酶，在本发明的一种实施方式中，发生N-糖基化突变前的核苷酸序列如SEQ ID NO.9所示。
本发明的第二个目的是提供含有所述脂肪酶突变体的表达载体。
所述表达载体为分泌型载体。
所述表达载体，在本发明的一种实施方式中，可以是以下任意一种：pGAPZα、pPIC9、pPIC3K、pPIC9K、PAO815或pPICZα。
本发明的第三个目的是提供含有所述脂肪酶突变体的基因工程菌。
所述基因工程菌，在本发明的一种实施方式中，是以毕赤酵母为宿主构建的。
所述基因工程菌，在本发明的一种实施方式中，其构建方法是：将编码所述脂肪酶突变体的核苷酸序列克隆到表达质粒pGAPZα上，然后线性化质粒，并电转入毕赤酵母GS115的感受态细胞中。
本发明的第四个目的是一种提高米曲霉脂肪酶表达的方法，是将米曲霉脂肪酶的前导肽的SAS和/或NT氨基酸位点进行N-糖基化突变。
所述方法，在本发明的一种实施方式中，突变后的脂肪酶的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示。
所述方法，在本发明的一种实施方式中，是将突变后的脂肪酶以pGAPZα为载体，在毕赤酵母中进行表达。
本发明还要求保护所述脂肪酶突变体在食品、化工或生物能源等领域的应用，以及所述基因工程菌在酶制剂生产中的应用。
本发明的有益效果：
本发明的经过N-糖基化突变的米曲霉脂肪酶，在工程菌中进步表达时，不仅不会影响菌株的生长，而且具有显著提高的酶活和分泌量。摇瓶培养96h后，proROLA、proROLB、proROLAB的胞外蛋白浓度分别比未进行糖基化的proROL提高了211％、188％、233％；突变体proROLA、proROLB以及proROLAB的摇瓶发酵酶活分别达到150U·mL-1、175U·mL-1和200U·mL-1，30L发酵罐发酵酶活分别达到8210U·mL-1、8457U·mL-1和9366U·mL-1，而未突变的proROL的胞外酶活几乎为零。
附图说明
图1：前导肽突变示意图；
图2：表达突变前后脂肪酶的基因工程菌的生长曲线；
图3：基因工程菌株的蛋白分泌情况；
图4：不同时间下基因工程菌表达脂肪酶的SDS-PAGE；其中，M为蛋白marker，泳道1-5分别代表培养24h、48h、72h、84h和96h；
图5：不同发酵时间下的突变体酶活。
具体实施方式
实施例1：引入N-糖基化的突变酶及基因工程的构建
本发明在米根霉脂肪酶ROL的前导肽中设计3种N-糖基化位点突变体，即将SAS和NT氨基酸分别改造为N-糖基化位点NGT和NLT，突变酶分别命名为proROLA(氨基酸序列如SEQ ID NO.1所示)和proROLB(氨基酸序列如SEQ ID NO.2所示)；将SAS和NT两个位点同时改造为N-糖基化位点的突变酶命名为proROLAB(氨基酸序列如SEQ ID NO.3所示)。前导肽具体突变方式如图1所示。
具体构建方法如下：
(1)以含有SEQ ID NO.4所示氨基酸序列的米曲霉脂肪酶基因proROL的载体pPIC9K-proROL、pGAPZα为模板，同时用限制性内切酶EcoR I、Not I双酶切，胶回收目的基因proROL和pGAPZα载体片段，用T4DNA连接酶连接目的基因和载体，构建重组质粒 pGAPZα-proROL。
(2)以重组质粒pGAPZα-proROL为模板，设计引物NGT-A F/NGT-A R和NLT-BF/NLT-BR分别进行全质粒PCR，构建分别将ROL基因中SAS、LT氨基酸突变成NGT、NLT的重组质粒，命名为pGAPZα-proROLA和pGAPZα-proROLB。构建完成后，再以质粒pGAPZα-proROLA为模板，用引物NLT-BF/NLT-BR全质粒PCR构建重组质粒pGAPZα-proROLAB。所用引物如表1所示。
表1 引物
Figure PCTCN2016070236-appb-000001
(3)将上述构建的重组表达质粒pGAPZα-proROL、pGAPZα-proROLA、pGAPZα-proROLB及pGAPZα-proROLAB分别用限制性内切酶AvrII酶切线性化，胶回收目的片段，电转入酵母感受态GS115细胞，设计引物进行基因组PCR验证阳性菌株，即得到基因工程菌GS115/pGAPZα-proROL、GS115/pGAPZα-proROLA、GS115/pGAPZα-proROLB、GS115/pGAPZα-proROLAB。经测定，proROL、proROLA、proROLB、proROLAB基因在酵母基因组中均为单拷贝。
实施例2：表达突变酶与野生酶的基因工程菌的生长比较
以GS115/pGAPZα-proROL为对照，比较了表达米根霉脂肪酶突变体的基因工程菌的生长情况。
摇瓶发酵条件：将阳性转化子划线YPD-G418平板((w/v)：酵母膏1％，葡萄糖2％，琼脂粉2％，胰蛋白胨2％，G4180.025％)，30℃培养3d，挑单克隆接种至100mLYPD液体培养基((w/v)：酵母膏1％，葡萄糖2％，胰蛋白胨2％)中，30℃ 200rpm·min-1摇瓶培养，每隔12h或24h取样。
菌株生长曲线如图2所示。结果表明，各突变菌株与对照生长情况相似，48h以内菌株生长迅速，72h后各菌株生长变得缓慢，趋于平稳。说明在米根霉脂肪酶基因中引入糖基化位点后不会影响各菌株的生长。
实施例3：N-糖基化对米根霉脂肪酶分泌水平的影响
本发明比较了表达突变酶和野生酶的基因工程菌的脂肪酶分泌情况。
将各基因工程菌进行发酵培养，具体培养条件：将阳性转化子划线YPD-G418平板，30℃培养3d，挑单克隆接种至100mLYPD液体培养基中，30℃ 200rpm·min-1摇瓶培养，每隔12h或24h取样。
结果如图3所示，未引入糖基化位点的米根霉脂肪酶proROL的蛋白浓度最低，而引入两个糖基化位点的米根霉脂肪酶proROLAB的胞外蛋白浓度略高于只引入一个糖基化位点的脂肪酶proROLA和proROLB，初步判断引入的糖基化位点发生了糖基化，且影响了米根霉脂肪酶的分泌水平。结果显示，发酵培养96h后，proROLA、proROLB、proROLAB的胞外蛋白浓度分别比未进行糖基化的proROL提高了211％、188％、233％。
如图4为不同取样时间的SDS-PAGE图。由于分泌的米根霉脂肪酶前导肽被部分切割(图1中KR位点为切除信号肽的特异性酶切位点)，保留28个前导肽氨基酸，引入糖基化位点的前导肽片段也被切除了，因此分泌表达的米根霉脂肪酶分子量均一致。未引入糖基化位点脂肪酶proROL从24h-96h都几乎未分泌，其余引入糖基化位点脂肪酶的分泌量都随着时间延长而增加，84h达到最大，且突变酶proROLA和proROLAB的分泌量在相同时间段都略高于脂肪酶proROLB。
实施例4：摇瓶条件下N-糖基化对米根霉脂肪酶胞外酶活的影响
将构建的基因工程菌进行摇瓶发酵培养产酶，同时测定不同发酵时间的脂肪酶酶活。
具体条件：将阳性转化子划线YPD-G418平板，30℃培养3d，挑单克隆接种至100mL YPD液体培养基中，30℃ 200rpm·min-1摇瓶培养，每隔12h或24h取样。
酶活力的定义为：每分钟在标准反应条件下产生1μmol pNP所用的酶量为一个脂肪酶水解酶活国际单位。酶活力的测定方法用pNPP法。
结果如图5所示。发酵过程中proROL的胞外酶活几乎为零，而其他三种引入N-糖基化位点的脂肪酶突变体的胞外酶活随着发酵时间的延长酶活不断增加，84h酶活达到最大值。突变体proROLA、proROLB以及proROLAB的酶活最高值分别达到150U·mL-1、175U·mL-1和200U·mL-1。
实施例5：发酵罐条件下N-糖基化对米根霉脂肪酶胞外酶活的影响
将构建的基因工程菌进行30L发酵罐发酵培养产酶，同时测定84h发酵时间的脂肪酶酶活。
具体条件：将阳性转化子划线YPD-G418平板，30℃培养3d，挑单克隆接种至200mL YPD液体培养基中，OD值达到2-6时，接种于30L发酵罐中进行发酵培养，发酵过程中流加葡萄糖溶液，控制浓度为2％左右，流加氨水控制pH5.5，温度28℃，发酵84h之后测定脂肪酶酶活力。
酶活力的定义为：每分钟在标准反应条件下产生1μmol pNP所用的酶量为一个脂肪酶水 解酶活国际单位。酶活力的测定方法用pNPP法。
发酵结果显示，proROL的胞外酶活几乎为零，而其他三种引入N-糖基化位点的脂肪酶突变体proROLA、proROLB以及proROLAB的胞外酶活在84h酶活分别达到8210U·mL-1、8457U·mL-1和9366U·mL-1。
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。
Figure PCTCN2016070236-appb-000002
Figure PCTCN2016070236-appb-000003
Figure PCTCN2016070236-appb-000004
Figure PCTCN2016070236-appb-000005
Figure PCTCN2016070236-appb-000006
Figure PCTCN2016070236-appb-000007
Figure PCTCN2016070236-appb-000008
Figure PCTCN2016070236-appb-000009
Figure PCTCN2016070236-appb-000010
Figure PCTCN2016070236-appb-000011

Claims (10)

  1. 一种脂肪酶突变体，其特征在于，所述突变体是在米根霉脂肪酶的基础上，将其前导肽的SAS和/或NT氨基酸位点突变为N-糖基化位点；所述突变体是以下任意一种：
    (1)氨基酸序列是SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的序列；
    (2)在严格条件下与(1)限定的氨基酸序列杂交且编码具有脂肪酶活性的蛋白质分子。
  2. 根据权利要求1所述的脂肪酶突变体，其特征在于，所述米根霉脂肪酶突变前的氨基酸序列是SEQ ID NO.4所示的序列。
  3. 含有权利要求1-2任一所述脂肪酶突变体的表达载体。
  4. 根据权利要求3所述的表达载体，其特征在于，所述表达载体为分泌型载体。
  5. 根据权利要求3所述的表达载体，其特征在于，所述表达载体为以下任意一种：pGAPZα，pPIC9，pPIC3K，pPIC9K，PAO815和pPICZα。
  6. 含有权利要求1-2任一所述脂肪酶突变体的基因工程菌。
  7. 一种权利要求6所述基因工程菌的构建方法，其特征在于，所述方法是将编码权利要求2所述脂肪酶突变体的核苷酸序列克隆到表达质粒pGAPZα上，然后线性化质粒，并电转入毕赤酵母GS115的感受态细胞中。
  8. 一种提高米曲霉脂肪酶表达的方法，其特征在于，所述方法是将米曲霉脂肪酶的前导肽进行N-糖基化突变，突变后脂肪酶的氨基酸序列是SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示的序列。
  9. 权利要求1所述脂肪酶突变体在食品、化工或生物能源方面的应用。
  10. 权利要求6所述基因工程菌在酶制剂生产中的应用。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116606836A (zh) * 2023-07-05 2023-08-18 广东博创佳禾科技有限公司 一种酶活、热稳定性提高的脂肪酶突变体
CN117660411A (zh) * 2023-11-30 2024-03-08 浙江容锐科技有限公司 基于n-糖基化改造的热稳定性脂肪酶突变体及其应用
CN117887688A (zh) * 2024-03-07 2024-04-16 合肥工业大学 一种高活力、高稳定性的脂肪酶突变体及其编码基因和应用

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104762277B (zh) * 2015-04-22 2017-07-14 江南大学 糖基化改造提高脂肪酶表达的方法、突变酶及其应用
CN106967737A (zh) * 2017-03-22 2017-07-21 东莞泛亚太生物科技有限公司 用于生产具有高活性脂肪酶的基因
CN108841807B (zh) * 2018-06-29 2021-09-17 广东丰绿源生物医药科技有限公司 一种热稳定性提高的脂肪酶突变体
CN108841805B (zh) * 2018-06-29 2021-07-06 深圳市夏盟生物科技有限公司 一种热稳定性提高的脂肪酶突变体
CN108841806B (zh) * 2018-06-29 2021-07-06 深圳市夏盟生物科技有限公司 一种热稳定性提高的脂肪酶突变体
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CN109929822B (zh) * 2019-04-10 2021-04-06 浙江工业大学 一种米曲霉脂肪酶突变体及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101857856A (zh) * 2010-05-25 2010-10-13 中国科学院武汉病毒研究所 一种枯草芽孢杆菌脂肪酶及制备方法和应用
CN102363786A (zh) * 2011-10-17 2012-02-29 中国水产科学研究院黄海水产研究所 一种适冷性海洋酵母Bohai Sea-9145低温碱性脂肪酶基因、氨基酸序列及重组脂肪酶
CN104762277A (zh) * 2015-04-22 2015-07-08 江南大学 糖基化改造提高脂肪酶表达的方法、突变酶及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101857856A (zh) * 2010-05-25 2010-10-13 中国科学院武汉病毒研究所 一种枯草芽孢杆菌脂肪酶及制备方法和应用
CN102363786A (zh) * 2011-10-17 2012-02-29 中国水产科学研究院黄海水产研究所 一种适冷性海洋酵母Bohai Sea-9145低温碱性脂肪酶基因、氨基酸序列及重组脂肪酶
CN104762277A (zh) * 2015-04-22 2015-07-08 江南大学 糖基化改造提高脂肪酶表达的方法、突变酶及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank [O] 12 February 2004 (2004-02-12), XP055323713, Database accession no. AAF32408.1 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116606836A (zh) * 2023-07-05 2023-08-18 广东博创佳禾科技有限公司 一种酶活、热稳定性提高的脂肪酶突变体
CN117660411A (zh) * 2023-11-30 2024-03-08 浙江容锐科技有限公司 基于n-糖基化改造的热稳定性脂肪酶突变体及其应用
CN117887688A (zh) * 2024-03-07 2024-04-16 合肥工业大学 一种高活力、高稳定性的脂肪酶突变体及其编码基因和应用

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