WO2016197296A1 - 一种催化活性和比酶活提高的脯氨酰内肽酶突变体 - Google Patents

一种催化活性和比酶活提高的脯氨酰内肽酶突变体 Download PDF

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WO2016197296A1
WO2016197296A1 PCT/CN2015/080962 CN2015080962W WO2016197296A1 WO 2016197296 A1 WO2016197296 A1 WO 2016197296A1 CN 2015080962 W CN2015080962 W CN 2015080962W WO 2016197296 A1 WO2016197296 A1 WO 2016197296A1
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mutant
amino acid
prolyl endopeptidase
aspergillus oryzae
activity
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PCT/CN2015/080962
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French (fr)
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喻晓蔚
徐岩
康超
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江南大学
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Priority to CN201580003335.1A priority Critical patent/CN106795520B/zh
Priority to PCT/CN2015/080962 priority patent/WO2016197296A1/zh
Publication of WO2016197296A1 publication Critical patent/WO2016197296A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

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  • the invention relates to a prolyl endopeptidase mutant with improved catalytic activity and specific enzyme activity, and belongs to the field of enzyme engineering.
  • Proline (Pro) is the only amino acid with an imine structure whose cyclic structure affects the spatial conformation when forming peptide bonds with other amino acids.
  • the proline-specific endoprotease has strong specificity for the position and bonding configuration of Pro in the polypeptide, and can specifically hydrolyze the proline carboxy terminal peptide bond in the small molecular weight polypeptide. Therefore, it can be applied to the fields of food industry, medicine, peptide industry, etc. At the same time, it can be used as a molecular enzyme tool enzyme for protein sequence determination, peptide mapping, specific site digestion, peptide chain modification and processing. Wait.
  • the proline-specific endoprotease not only shows the unparalleled superiority in the application of the high-quality protein hydrolysate to release the bitter peptide directly related to the inhibition of bitterness, but also does not directly inhibit the bitterness.
  • the enzyme can also be used in beer brewing to solve the problem of cold turbidity in the actual production of beer.
  • barley proteins are rich in proline-rich sequences, and in their non-germinated form, cereal proteins are extremely difficult to degrade into the free amino acids necessary to produce a suitable fermentable wort.
  • AO-PEP Aspergillus oryzae prolyl endopeptidase having proline-specific endoprotease activity from Aspergillus oryzae, but there is still room for improvement in its expression ability. It has been reported that the flexibility of the loop loop of the irregular region near the central region of the enzyme activity plays a key role in the entry of the substrate into the enzyme active center and substrate specificity. At the same time, the special arrangement of the amino acid His, which is close to the active catalytic site before and after the charged amino acid Arg, plays a key role in the catalytic mechanism of human proline carboxypeptidase (PDBID: 3n2z).
  • the present invention provides an Aspergillus oryzae prolyl endopeptidase mutant having improved catalytic activity.
  • a first object of the present invention is to provide a prolyl endopeptidase mutant which is based on a parental Aspergillus oryzae prolyl endopeptidase, which catalyzes the vicinity of a catalytic activity center or a catalytic key site.
  • the amino acid was obtained by mutation.
  • the mutation may be a mutation in any one of the following ways: (1) replacing the hydrophilic amino acid Thr of T370 and T516 on the loop ring near the catalytic active center with other small molecular weight amino acids or hydrophobic amino acids; (2) Cyto508 and/or Gln512 mutations in the vicinity of histidine His507, which catalyze the key site, are mutated into His508 and/or Arg512; (3) the combination of the above two methods (1) and (2), that is, simultaneously mutating the amino acids on the loop loop and Catalyzes amino acids near histidine His507.
  • the above mutation mode makes the active pocket and surface area of the substrate expand, and the difficulty of the substrate entering the active pocket is reduced, thereby improving the catalytic efficiency of the enzyme.
  • the mutation on the loop loop in one embodiment of the invention, mutates Thr to Cys or Ala.
  • amino acid sequence of the parent Aspergillus oryzae prolyl endopeptidase is the sequence shown in SEQ ID NO.
  • the parent Aspergillus oryzae prolyl endopeptidase in one embodiment of the present invention, is the sequence shown in SEQ ID NO.
  • the mutant in one embodiment of the present invention, is any one of the following: T370A, T370C, T516A, T516C, C508H, C508H/Q512R, T516A/T370A, T516A/T370C, T516A/C508H or T516A/C508H /Q512R.
  • the mutant in one embodiment of the present invention, is obtained by a method of site-directed mutagenesis.
  • An expression vector comprising the mutant of claim 1.
  • the expression vector may be any one of pPIC9K, pPIC9, pPIC Z ⁇ , pGAPZ ⁇ , pPIC3.5, pPIC3.5K, pYAM75P6.
  • the invention also provides a genetically engineered bacterium expressing the mutant.
  • the genetically engineered bacteria may be Pichia pastoris, and the Pichia pastoris may be any one of the following: GS115, KM71, SMD1168, X33.
  • the genetically engineered bacterium in one embodiment of the present invention, is constructed by ligating the parental Aspergillus oryzae prolyl endopeptidase gene AO-PEP having the gene sequence as shown in SEQ ID NO. 2 to the pPIC9K vector
  • the pPIC9K-AO-PEP was obtained, and then pPIC9K-AO-PEP was mutated to a recombinant plasmid containing the mutant sequence by site-directed mutagenesis, and the plasmid was linearized and transformed into Pichia pastoris to obtain a genetically engineered strain expressing the mutant. .
  • the present invention also provides a method for increasing the catalytic activity or specific activity of Aspergillus oryzae prolyl endopeptidase.
  • the amino acid sequence is mutated according to the parental Aspergillus oryzae prolyl endopeptidase represented by SEQ ID NO. 1, and the amino acid near the catalytic active center is mutated. Catalytic activity or ratio Prolyl endopeptidase mutant with increased enzyme activity.
  • the mutant in one embodiment of the invention, is T370A, T370C, T516A, T516C, C508H, C508H/Q512R, T516A/T370A, T516A/T370C, T516A/C508H or T516A/C508H/Q512R.
  • the invention also claims the use of vectors and mutants containing the mutants in the food industry, the peptide industry or in the preparation of pharmaceuticals and the like.
  • the mutant of the present invention is named after the amino acid sequence shown in SEQ ID NO. 1 and the "amino acid substituted with the original amino acid position" is used to indicate the mutant.
  • T370A represents that the amino acid at position 370 is replaced by a threonine (Thr, T) of the parent with alanine (Ala, A), and for example, T516A/C508H represents a simultaneous mutation of amino acids at positions 516 and 508.
  • the mutant obtained by the present invention has a markedly improved catalytic efficiency
  • the crude enzyme solution obtained by expressing the mutant in Pichia pastoris has a markedly enhanced specific activity.
  • the composite mutants C508H/Q512R, T516A/T370A, T516A/T370C, T516A/C508H/Q512R have a catalytic efficiency higher than the parent by more than 32% and an increase of more than 28% (up to 45.9%).
  • Figure 1 Aspergillus oryzae prolyl endopeptidase homology modeling of the loos loop near the active center.
  • the supernatant of the v 1 cell was taken, and the solution of v 2 citric acid/disodium hydrogen phosphate buffer (pH 5.0) and v 3 250 ⁇ mol/l of ZG ly-Pro-pNA was added, shaken, and reacted at 37 ° C for 10 min.
  • the absorbance of the solution at 410 nm before and after the reaction was measured.
  • the amount of enzyme that decomposes ZG ly-Pro-pNA to release 1 ⁇ mol of pNA per minute is defined as an enzyme unit (U/ml).
  • V-reaction system total volume (mL) v 1 , the sum of v 2 and v 3 ; v 1 - the volume of the enzyme solution (mL); v 2 - the volume of the buffer ( mL); v 3 - volume of substrate (mL); r-mole extinction wash (cm 2 / ⁇ mol); b- cuvette optical path (cm).
  • the flexibility of the loop loop in the irregular region near the active center of the enzyme plays a key role in the entry of the substrate into the enzyme active center and substrate specificity, that is, the loop loop is more flexible and the catalytic activity of the enzyme is also increased.
  • the loopA and loopB loops in the simulated structure of Aspergillus oryzae prolyl endopeptidase are close to the active pocket surface (Fig. 1), and before the substrate enters the active site, this The two loop loops act to block the substrate from entering the active center, so these amino acids are likely to be key amino acids. Therefore, it is possible to try to replace the hydrophilic amino acid Thr on the loop ring with other small molecular weight amino acids or hydrophobic amino acids, so that the difficulty of the substrate entering the active pocket is reduced, thereby increasing the enzyme. Catalytic efficiency.
  • the structure of the PRCP enzyme in the protease family forms a special histidine catalytic active site arrangement (His455, His456, Arg460), which contains two histidines, forming an apparent charge system, which has an important influence on its catalytic activity. .
  • His455, His456, Arg460 histidine catalytic active site arrangement
  • AO-PEP AO- was designed. Cys508 and Gln512 of PEP were mutated to the corresponding His508 and Arg512.
  • the site of site-directed mutagenesis of Aspergillus oryzae prolyl endopeptidase should be based on two sites (T370 and T516) in the loops region that may be important for enhancing enzyme activity, and the catalytic histidine arrangement. Sites (Cys508 and Gln512).
  • the recombinant mutant expressing bacteria was constructed as follows:
  • a histidine-tagged recombinant plasmid containing a parental Aspergillus oryzae prolyl endopeptidase a parental Aspergillus oryzae prolyl endopeptidase gene (amino acid sequence such as SEQ ID NO. 1, nucleotide The sequence was cloned into pPIC9K as shown in SEQ ID NO. 2 to obtain the recombinant plasmid pPIC9K-AO-PEP, and then PCR was carried out by using pPIC9K-AO-PEP as a template and adding 6*His tag F and R as primers. The target fragment was ligated with SnaBI and NotI and pPIC9K, and the expression plasmid containing the 6*his tag was named pPIC9K-AO-PEP-6his.
  • the initial concentration of the enzyme solution and its renaturation were carried out by precipitation with saturated ammonium sulfate and dialysis, followed by adsorption purification of the nickel column.
  • the nickel column purification procedure is as follows: the system is cleaned with 5 column volumes of ultrapure water, and the nickel chelate chromatography is equilibrated with 10 column volumes of 20 mM sodium phosphate balance solution (pH 7.0) containing 0.5 mol/l NaCl and 20 mmol/l imidazole.
  • the crude enzyme solution (0.22 ⁇ m filtration) was applied to the column, and then washed with 15 column volumes of a 20 mM sodium phosphate balance solution (pH 7.0) containing 0.5 mol/l of NaCl and 20 mmol/l of imidazole until the baseline was stable.
  • the extract was eluted with a 20 mM sodium phosphate eluate (pH 7.0) containing 0.5 mol/l NaCl, 500 mmol/l imidazole, and the active peak was collected. After dialysis for 48 hours at 4 ° C, ultrafiltration (30 kDa) was concentrated to obtain a purified enzyme.
  • the present invention compares the enzyme activity and specific enzyme activity of the recombinant supernatant and the purified enzyme before and after the mutation, and the results are shown in Table 2.
  • the results showed that the specific activities of the crude enzymes of T370A, T370C, T516A, T516C, C508H, C508H/Q512R, T516A/T370A, T516A/T370C, T516A/C508H or T516A/C508H/Q512R were higher than those of recombinant AO-PEP, respectively. 7.7%, 1.9%, 22.6%, 9.1%, 20.2%, 28%, 37.6%, 32.3%, 45.9%, 39.6%, indicating that these mutation sites have a positive effect on the improvement of enzyme activity.
  • the kinetic parameters of prolyl endopeptidase before and after mutation were determined as follows: the activity of prolyl endopeptidase was determined at 37 °C with different concentrations of Z-Gly-Pro-pNA as substrate. A 410 , with time as the abscissa and A 410 as the ordinate. The initial velocity of the substrate with different concentrations is obtained from the slope of the straight line. According to the Lineweaver-Burk double reciprocal method, the reciprocal of the substrate with different concentrations is plotted as the abscissa, and the reciprocal of the corresponding reaction velocity is plotted for the ordinate. The kinetic parameters of the prolyl endopeptidase were calculated.
  • the treatment results showed that at the end of forced aging, the bottom of the blank sample had obvious sludge-like sediments.
  • the bottom of the sample bottle with the enzyme dosage of 5U was basically no sedimentation, and the wine body was clearer than the blank, indicating that the sensitive protein content of the fermentation broth was The trend is reduced and the non-biological stability is improved.

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Abstract

提供了一种催化活性和比酶活提高的脯氨酰内肽酶突变体，属于酶工程领域。通过将亲本米曲霉脯氨酰内肽酶的催化活性中心附近loop环上的亲水性氨基酸Thr370和/或Thr516用其他小分子量氨基酸或者疏水性氨基酸替换，或者将催化关键位点组氨酸His507附近的Cys508和/或Gln512突变成His508和/或Arg512，得到了一系列突变体。

Description

一种催化活性和比酶活提高的脯氨酰内肽酶突变体 技术领域
本发明涉及一种催化活性和比酶活提高的脯氨酰内肽酶突变体，属于酶工程领域。
背景技术
脯氨酸(Pro)是唯一具有亚氨结构的氨基酸，其环状结构影响了与其它氨基酸形成肽键时的空间构象。脯氨酸特异性内切蛋白酶对Pro在多肽中的位置和成键的构型都有很强的专一性，且能够特异性水解小分子量多肽中脯氨酸羧基端肽键。因此，可以应用于食品工业、医药、多肽工业等领域；同时可以作为一种分子生物学的工具酶，应用于蛋白质序列测定，肽谱分析、特异位点的酶切、肽链的修饰以及加工等。
特别地，在食品工业方面，由于蛋白质水解产物可以为特定人群提供营养补充，因此有广泛的市场效应。来自乳清产品特别受欢迎的原因在于其极好的口感及良好的可溶性。但是，目前这些酪蛋白水解产物还未获得广泛的流行，缺乏市场渗透力的主要原因在于酪蛋白水解过程中所产生的苦味，这种出现苦味的情况代表了一个技术问题，目前在食品工业上还没有解决。而脯氨酸残基在肽的羧基末端的高发生率与低的苦味相关，并且证明了羧基末端脯氨酸残基预期的高发生率只能用高浓度的脯氨酸特异性内切蛋白酶来实现。因此，在食品工业中，脯氨酸特异性内切蛋白酶不仅在解除直接与抑制苦味相关的苦味肽获得优质的蛋白质水解产物应用显示出了无以伦比的优越性，还在不直接与抑制苦味相关，比如将减少食物蛋白质的变应原性；减缓所得生面团制作面包的腐败；生成富含脯氨酸的肽作为各种食物或营养产品的理想添加剂，以增强蛋白质利用。尤其引人注目的是，该酶还可以用于啤酒酿造，解决啤酒实际生产过程中的冷混浊问题。主要是因为大麦蛋白质中富含脯氨酸序列很丰富，且在他们非发芽的形式时谷类蛋白质是极其难以降解成生产适当的可发酵的麦芽汁所必需的游离氨基酸。
前期，发明人已经从米曲霉中获得了具有脯氨酸特异性内切蛋白酶活性的米曲霉脯氨酰内肽酶(AO-PEP)，但是其表达能力还有进一步的提升空间。已有报道表明，酶活性中心区域附近的不规则区loop环的柔性对底物进入酶活性中心以及底物特异性起着关键作用。同时，靠近带电荷氨基酸Arg前后的活性催化位点氨基酸His的特殊排列方式对人源脯氨酸羧肽酶(PDBID：3n2z)的催化机制起着关键作用。因此，本发明通过对AO-PEP的活性中心及底物结合口袋进行定点突变，发现活性催化位点组氨酸排列方式和loop环柔性对AO-PEP催化性能具有显著影响，并得到了一系列催化活性和比酶活提高的脯氨酰内肽酶突变体。
发明内容
为了克服现有米曲霉脯氨酰内肽酶催化效率、比酶活较低的缺陷，本发明提供一种催化活性提高的米曲霉脯氨酰内肽酶突变体。
本发明的第一个目的是提供一种脯氨酰内肽酶突变体，所述突变体是以亲本米曲霉脯氨酰内肽酶为基础，将其催化活性中心附近或催化关键位点的氨基酸进行突变得到的。
所述突变，可以是以下任意一种方式的突变：(1)将催化活性中心附近loop环上的T370和T516的亲水性氨基酸Thr用其他小分子量氨基酸或者疏水性氨基酸替换；(2)将催化关键位点组氨酸His507附近的Cys508和/或Gln512突变成His508和/或Arg512；(3)上述(1)、(2)两种方式的结合，即同时突变loop环上的氨基酸和催化组氨酸His507附近的氨基酸。以上突变方式使得底物活性口袋及表面积扩大，底物进入活性口袋的难度降低，从而提高酶的催化效率。
所述loop环上的突变，在本发明的一种实施方式中，是将Thr突变为Cys或Ala。
所述亲本米曲霉脯氨酰内肽酶的氨基酸序列是SEQ ID NO.1所示的序列。
所述亲本米曲霉脯氨酰内肽酶，在本发明的一种实施方式中，其核苷酸序列是SEQ ID NO.2所示的序列。
所述突变体，在本发明的一种实施方式中，是以下任意一种：T370A、T370C、T516A、T516C、C508H、C508H/Q512R、T516A/T370A、T516A/T370C、T516A/C508H或T516A/C508H/Q512R。
所述突变体，在本发明的一种实施方式中，是通过定点突变的方法得到的。
含有权利要求1所述突变体的表达载体。
所述表达载体可以是pPIC9K、pPIC9、pPIC Zα、pGAPZα、pPIC3.5、pPIC3.5K、pYAM75P6中的任意一种。
本发明还提供一种表达所述突变体的基因工程菌。
所述基因工程菌可以是毕赤酵母菌，毕赤酵母可以是以下任意一种：GS115、KM71、SMD1168、X33。
所述基因工程菌，在本发明的一种实施方式中，按以下方法构建：将基因序列如SEQ ID NO.2所示的亲本米曲霉脯氨酰内肽酶基因AO-PEP连接到pPIC9K载体得到pPIC9K-AO-PEP，然后通过定点突变的方法将pPIC9K-AO-PEP突变成含有突变体序列的重组质粒，线性化质粒后转化到毕赤酵母中，即得到表达突变体的基因工程菌。
本发明还提供一种提高米曲霉脯氨酰内肽酶催化活性或比酶活的方法。
所述方法，在本发明的一种实施方式中，是以氨基酸序列如SEQ ID NO.1所示的亲本米曲霉脯氨酰内肽酶为基础，将其催化活性中心附近的氨基酸进行突变，即得到催化活性或比 酶活提高的脯氨酰内肽酶突变体。
所述突变体，在本发明的一种实施方式中，是T370A、T370C、T516A、T516C、C508H、C508H/Q512R、T516A/T370A、T516A/T370C、T516A/C508H或T516A/C508H/Q512R。
本发明还要求保护含有所述突变体的载体以及突变体在食品工业、多肽工业或制备药物等方面的应用。
本发明的突变体命名，是以SEQ ID NO.1所示氨基酸序列为基准，采用“原始氨基酸位置替换的氨基酸”来表示突变体。比如T370A代表将位置370的氨基酸由亲本的苏氨酸(Thr，T)替换为丙氨酸(Ala，A)，又比如T516A/C508H代表第516位和508位的氨基酸同时发生了突变。
本发明得到的突变体具有显著提高的催化效率，突变体在毕赤酵母中表达得到的粗酶液具有显著提高的比活力。尤其是复合突变体C508H/Q512R、T516A/T370A、T516A/T370C、T516A/C508H/Q512R，其催化效率比亲本提高了32％以上，比酶活提高了28％以上(最高可达45.9％)。
附图说明
图1：米曲霉脯氨酰内肽酶同源建模靠近活性中心的loos环。
具体实施方式
脯氨酰内肽酶酶活的测定
取v1菌体上清液，加入v2柠檬酸/磷酸氢二钠缓冲液(pH5.0)和v3250μmol/l的Z-G ly-Pro-pNA溶液，摇匀，37℃分别反应10min。测定反应前后溶液在410nm处的吸光度。在上述条件下，每分钟分解Z-G ly-Pro-pNA释放1μmol pNA的酶量，定义为一个酶活单位(U/ml)。通过△A410计算脯氨酰内肽酶酶活，公式如下：U/ml＝(△A/min)*(V/rvb)。
其中：△A/min-吸光度变化；V-反应体系总体积(mL)＝v1，v2与v3之和；v1-酶液的体积(mL)；v2-缓冲液的体积(mL)；v3-底物的体积(mL)；r-摩尔消光洗漱(cm2/μmol)；b-比色杯光程(cm)。
实施例1：突变位点的选择
根据已有报道，酶活性中心附近的不规则区loop环的柔性对底物进入酶活性中心以及底物特异性起着关键作用，即loop环柔性增大，酶的催化活性也随之增大，结合同源建模获得的三维结构，分析得出米曲霉脯氨酰内肽酶模拟结构中的loopA和loopB环都靠近活性口袋表面(图1)，并且底物进入活性位点之前，这两个loop环起着阻隔底物进入活性中心的作用，因此这些氨基酸有可能是关键的氨基酸。因此可以尝试把loop环上的亲水性氨基酸Thr用其他小分子量氨基酸或者疏水性氨基酸替换，使得底物进入活性口袋的难度降低，从而提高酶 的催化效率。
蛋白酶家族中的PRCP酶的结构中形成特殊的组氨酸催化活性位点排列方式(His455，His456，Arg460)，其中含有两个组氨酸，形成表观电荷系统，对其催化活性具有重要影响。通过对AO-PEP二级结构以及三级结构进行分析，发现在仅有保守位点His507与PRCP酶的His455相对应，因此模拟PRCP酶的组氨酸催化活性位点排列方式，设计将AO-PEP的Cys508和Gln512突变成对应的His508以及Arg512。
综合上述分析得出米曲霉脯氨酰内肽酶定点突变的位点应该立足于可能对提高酶活性比较重要的loops区域上的两个位点(T370和T516)以及催化组氨酸排列方式的位点(Cys508和Gln512)。
实施例2：表达突变体的重组菌的构建
按以下方法构建表达突变体重组菌：
(1)含有亲本米曲霉脯氨酰内肽酶的带有组氨酸标签的重组质粒的构建：将亲本米曲霉脯氨酰内肽酶基因(氨基酸序列如SEQ ID NO.1、核苷酸序列如SEQ ID NO.2所示)克隆到pPIC9K得到重组质粒pPIC9K-AO-PEP，然后以pPIC9K-AO-PEP为模板，以添加6*His标签的F和R为引物进行PCR，筛选获得正确的目的片段，用SnaBI和NotI双酶切目的片段和pPIC9K，连接，即得到含6*his标签的表达质粒命名为pPIC9K-AO-PEP-6his。
(2)以pPIC9K-AO-PEP-6his为模板，采用表1中的引物，利用一步法质粒PCR介导的定点突变方法分别构建得到T370A、T370C、T516A、T516C、C508H、C508H/Q512R六个突变体的表达质粒，再以突变重组质粒T537A为模板分别构建T516A/T370A、T516A/T370C、T516A/C508H或T516A/C508H/Q512R四个复合突变重组质粒。
(3)所有突变体都经过酶切、测序验证正确，然后转化毕赤酵母表达宿主GS115。得到的正确的重组毕赤酵母经过诱导表达，发酵液经过离心，测定上清液活性，突变体都具有脯氨酰内肽酶活力。纯化后的突变体达到电泳纯，所有突变体分子量大小均没有明显差异，和野生型AO-PEP类似，约为66kDa。
表1 引物
Figure PCTCN2015080962-appb-000001
Figure PCTCN2015080962-appb-000002
实施例3：重组菌的发酵表达及突变体纯化
按以下方法进行重组菌的发酵表达及突变体纯化。
(1)重组菌的发酵表达：
挑取重组毕赤酵母单菌落接种装有25ml BMGY培养基的250ml三角瓶，30℃培养至对数期OD600＝2-6(16-18h)；离心收集菌体，弃上清，菌体转至装有50ml BMMY培养基的500ml三角瓶，置于摇床28℃ 250r/min继续培养，每隔24h取样，并补加100％甲醇使其终浓度达到1％，诱导培养4天，离心收集菌体，进行酶活测定以及SDS-PAGE电泳分析。
(2)酶的纯化：
取60ml发酵液，8,000r/min离心5min，收集上清液，上清即为粗酶液。首先采用饱和硫酸铵沉淀以及透析进行酶液的初步浓缩及其复性后，接着进行镍柱的吸附纯化。镍柱纯化步骤如下：用5个柱体积超纯水清洗系统，再用10个柱体积含0.5mol/l NaCl，20mmol/l咪唑的20mM磷酸钠平衡液(pH 7.0)平衡镍螯合层析柱后，将粗酶液(0.22μm过滤)上样过柱，之后用15个柱体积含0.5mol/l NaCl，20mmol/l咪唑的20mM磷酸钠平衡液(pH 7.0)洗涤至基线平稳，再用含0.5mol/l NaCl，500mmol/l咪唑的20mM磷酸钠洗脱液(pH 7.0)洗脱，收集活性峰，4℃透析48h后，超滤(30kDa)浓缩，即得到纯化酶。
本发明比较了突变前后重组菌发酵上清及纯化后的酶活、比酶活，结果如表2所示。结果显示T370A、T370C、T516A、T516C、C508H、C508H/Q512R、T516A/T370A、T516A/T370C、T516A/C508H或T516A/C508H/Q512R的粗酶液比活力分别比重组AO-PEP的比活力提高了7.7％，1.9％，22.6％，9.1％，20.2％，28％，37.6％，32.3％、45.9％、39.6％，说明这些突变位点对酶活的提高是具有积极作用的。
表2 突变体的酶活和比酶活的比较
Figure PCTCN2015080962-appb-000003
Figure PCTCN2015080962-appb-000004
实施例4：突变前后的反应动力学参数比较
按以下方法测定了突变前后脯氨酰内肽酶的动力学参数：以不同浓度的Z-Gly-Pro-pNA为底物在37℃测定脯氨酰内肽酶活力，反应不同时间取样，测定A410，以时间为横坐标，A410为纵坐标作图。由直线的斜率得出不同浓度底物相应的初速度，按照Lineweaver-Burk双倒数法作图，以不同浓度底物的倒数为横坐标，以其相应的反应速度的倒数为纵坐标作图，计算脯氨酰内肽酶的动力学参数。
结果如表3所示，其中AO-PEP代表亲本米曲霉脯氨酰内肽酶。结果显示，从催化效率(kcat/Km)可以看出，突变酶蛋白对底物的催化效率都有了部分提高。
表3 突变体酶蛋白动力学参数
Figure PCTCN2015080962-appb-000005
实施例5 利用米曲霉脯氨酰内肽酶提高啤酒非生物稳定性
啤酒发酵液强制老化试验：将样品放入恒温水浴中，60℃处理24h、0℃处理24h为1个循环，共7个循环。测定酶处理后的啤酒浊度，强制老化后浊度值(EBC)越低代表效果越好，结果如表4所示。
表4 啤酒发酵液7个强制老化循环后浊度的测定
Figure PCTCN2015080962-appb-000006
处理结果表明，强制老化结束时，空白样品的瓶底有明显的淤泥状沉淀物，加酶量为5U的样品瓶底基本无沉淀，酒体明显比空白清亮，说明发酵液的敏感蛋白含量有降低的趋势，非生物稳定性有所提高。
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。

Claims (10)

  1. 一种脯氨酰内肽酶突变体，其特征在于，所述突变体是以亲本米曲霉脯氨酰内肽酶为基础，将其催化活性中心附近或催化关键位点的氨基酸进行突变得到的；所述亲本米曲霉脯氨酰内肽酶的氨基酸序列是SEQ ID NO.1所示的序列。
  2. 根据权利要求1所述的突变体，其特征在于，所述突变体发生了以下任意一种方式的突变：(1)将催化活性中心附近loop环上的T370和T516的亲水性氨基酸Thr用其他小分子量的氨基酸或者疏水性氨基酸替换；(2)将催化关键位点组氨酸His507附近的Cys508和/或Gln512突变成His508和/或Arg512；(3)上述(1)、(2)两种方式的结合，即同时突变loop环上的氨基酸和催化组氨酸His507附近的氨基酸。
  3. 根据权利要求1或2所述的突变体，其特征在于，所述突变体是以下任意一种：T370A、T370C、T516A、T516C、C508H、C508H/Q512R、T516A/T370A、T516A/T370C、T516A/C508H或T516A/C508H/Q512R。
  4. 含有权利要求1所述突变体的表达载体。
  5. 根据权利要求4所述的表达载体，其特征在于，所述表达载体为分泌型载体。
  6. 表达权利要求1所述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，所述方法是将基因序列如SEQ ID NO.2所示的亲本米曲霉脯氨酰内肽酶基因连接到pPIC9K载体得到pPIC9K-AO-PEP，然后通过定点突变的方法将pPIC9K-AO-PEP突变成含有突变体序列的重组质粒，线性化质粒后转化到毕赤酵母中，即得到表达突变体的基因工程菌。
  8. 一种提高米曲霉脯氨酰内肽酶催化活性或比酶活的方法，其特征在于，所述方法是以氨基酸序列如SEQ ID NO.1所示的亲本米曲霉脯氨酰内肽酶为基础，将其催化活性中心附近的氨基酸进行突变，即得到催化活性或比酶活提高的脯氨酰内肽酶突变体。
  9. 根据权利要求8所述的方法，其特征在于，所述突变体是T370A、T370C、T516A、T516C、C508H、C508H/Q512R、T516A/T370A、T516A/T370C、T516A/C508H或T516A/C508H/Q512R。
  10. 权利要求1所述突变体在食品工业、多肽工业或制备药物方面的应用。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1155906A (zh) * 1994-06-23 1997-07-30 贾帕特有限公司 耐热脯氨酰内肽酶
EP0967285B1 (en) * 1991-07-24 2007-09-12 Novartis AG expression vector for prolylendopeptidase from Flavobacterium meningosepticum
AU2008202554B2 (en) * 2001-02-23 2011-03-10 Dsm Ip Assets B.V. Novel genes encoding novel proteolytic enzymes
CN103602605A (zh) * 2013-11-14 2014-02-26 江南大学 一种高效表达米曲霉脯氨酰内肽酶的基因工程菌及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6156554A (en) * 1994-06-23 2000-12-05 Ciba Geigy Japan Limited Heat-stable prolylendopeptidase

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0967285B1 (en) * 1991-07-24 2007-09-12 Novartis AG expression vector for prolylendopeptidase from Flavobacterium meningosepticum
CN1155906A (zh) * 1994-06-23 1997-07-30 贾帕特有限公司 耐热脯氨酰内肽酶
AU2008202554B2 (en) * 2001-02-23 2011-03-10 Dsm Ip Assets B.V. Novel genes encoding novel proteolytic enzymes
CN103602605A (zh) * 2013-11-14 2014-02-26 江南大学 一种高效表达米曲霉脯氨酰内肽酶的基因工程菌及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [O] 3 March 2011 (2011-03-03), "serine peptidase, family S28 [Aspergillus oryzae RIB40]", XP055334511, Database accession no. XP001825996 *

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