WO2017005213A1 - 一种重组赖氨酸特异性酶的制备方法 - Google Patents

一种重组赖氨酸特异性酶的制备方法 Download PDF

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WO2017005213A1
WO2017005213A1 PCT/CN2016/089324 CN2016089324W WO2017005213A1 WO 2017005213 A1 WO2017005213 A1 WO 2017005213A1 CN 2016089324 W CN2016089324 W CN 2016089324W WO 2017005213 A1 WO2017005213 A1 WO 2017005213A1
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lysine
specific enzyme
piv
recombinant
ppic9k
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肖海鹏
李利佳
林树珊
李宇晟
杨彬
陈小锋
李文佳
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Sunshine Lake Pharma Co Ltd
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    • C12R2001/84—Pichia

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  • the invention relates to the field of genetic engineering technology. Specifically, the present invention provides an efficient preparation method of a recombinant lysine-specific endoprotease (Lys-C), including construction of an engineered strain, expression and purification of a recombinant lysine-specific enzyme.
  • Lys-C lysine-specific endoprotease
  • Lysine-specific endoprotease is an alkaline protease with a molecular weight of approximately 26 kD and is a member of the serine protease family. As a high-purity protease, it can specifically hydrolyze the carboxy terminus of Lys (except when arginine is attached), and can produce more enzymatic peptides than Trypsin, which can be used for peptide mass spectrometry or MS/MS spectral matching. Protein identification, therefore has a good application prospect in proteomic analysis.
  • Lys-C can also be used for precursor cleavage (such as insulin precursors) in the industrial production of recombinant proteins, so Lys-C is currently expressed in the field of genetic engineering pharmaceuticals.
  • precursor cleavage such as insulin precursors
  • Lys-C enzyme-producing bacterium, hydrolyzed bacterium, and Pseudomonas aeruginosa.
  • the lysine-specific enzyme is mainly produced by the production of the enzyme-producing bacillus, followed by the isolation of the culture medium of the bacterium Achromobacter.
  • Naturally extracted Lys-C is extremely expensive, such as Promega's Lys-C market price of about 370 yuan / ⁇ g.
  • the enzyme-producing bacterium is the main producing strain of Lys-C, the production efficiency is not high, the fermentation cycle is long, and the extraction process is complicated, which greatly limits the wide application of Lys-C.
  • the naturally-derived lysine-specific enzyme is not only limited, but also has high cost, low yield, and is easily contaminated by other impurities, thereby causing degradation of the target protein.
  • the production method of high activity rLys-C has important application and research value for the development of genetic engineering drugs and biological research.
  • the present invention provides a method for preparing a recombinant lysine-specific enzyme, relates to the design of a recombinant lysine-specific enzyme gene sequence, a recombinant expression vector containing the gene, and genetic engineering. Construction of bacteria and preparation of recombinant lysine-specific enzymes. It provides an effective way for the industrial production of recombinant lysine-specific enzymes.
  • the invention provides a highly efficient expression of a recombinant lysine-specific enzyme, the DNA sequence of which is SEQ ID NO: 1.
  • the amino acid of the above recombinant lysine-specific enzyme has the sequence of SEQ ID NO: 2.
  • a recombinant vector comprising the pPIC9k-PIV of the sequence SEQ ID NO: 1.
  • Another aspect of the present invention provides an engineered bacterium which efficiently expresses a recombinant lysine-specific enzyme, which is a Pichia pastoris having the sequence of SEQ ID NO: 1.
  • the engineered bacteria is GS115/pPIC9k-PIV.
  • the technical scheme of the present invention also provides a construction method for efficiently expressing recombinant lysine-specific enzyme engineering bacteria, comprising the following steps:
  • a method for producing a recombinant lysine-specific enzyme by using the above genetically engineered bacteria comprising the steps of culturing a genetically engineered bacteria and obtaining a recombinantly expressed recombinant lysine-specific enzyme.
  • a method for producing a recombinant lysine-specific enzyme by using the above genetically engineered bacteria comprising the steps of:
  • lysine-specific enzyme-producing strains Inoculate the strain into the primary culture medium and incubate at 28-30 ° C until the OD 600 is 15-20, transfer to the secondary medium, 28-30 The culture was shaken at °C, and methanol was added for induction every 8-12 hours. After fermentation for 12-120 hours, the supernatant was centrifuged, and the supernatant was centrifuged;
  • Fermentation production the re-screened strain is inoculated into the fermentation medium, and cultured at 28-30 ° C until the dissolved oxygen amount reaches 70-80%, and glycerin is added at 2-4 ml/min for 9-12 hours, and added.
  • the methanol solution containing 1% to 1.2% PTM1 was induced, and the fermentation broth was taken every 4-12 hours after 60-72 hours for enzyme activity detection;
  • the inoculum amount of the strain in the fermentation medium in step b) is 1-5%.
  • the pH of the methanol induction in step a) is from 4.0 to 8.0; the pH of the medium in the step b) of methanol induction is from 5.5 to 6.0.
  • the purification is selected from the group consisting of exchange chromatography, ultrafiltration or lyophilization.
  • the numbers in the present invention are approximate, regardless of whether or not the words "about” or “about” are used.
  • the numerical values may vary by 1%, 2%, 5%, 7%, 8%, 10%, etc.
  • any has N +/- 1%, N +/- 2%, N +/- 3%, N +/- 5%, N +/- 7%, N +/- 8% or N+
  • the number of /-10% value will be explicitly disclosed, where "+/-" means plus or minus.
  • the invention improves the gene of the recombinant lysine-specific enzyme and adopts the expression product of Pichia pastoris, which provides an effective way for the industrial production of the recombinant lysine-specific enzyme.
  • Previously used enzyme-producing bacterium, hydrolyzed Achromobacter, and Pseudomonas aeruginosa expression products it overcomes the shortcomings of low production efficiency, long fermentation cycle, and complicated extraction process, and reduces production cost. It is also fast, simple and stable, and has high activity of rLys-C. It has important application and research value for the development of genetic engineering drugs and biological research.
  • Figure 1 is a schematic diagram of the recombinant expression plasmid pPIC9k-PIV
  • FIG. 2 is a schematic diagram showing the results of the enzyme activity determination of the supernatant of the fermentation broth at different induction times of PIV;
  • the lysine-specific enzyme nucleic acid sequence was optimized according to the yeast codon optimization strategy, and 6 was added at its C-terminus. ⁇ His tag, with the XhoI site at the 5' end of the PIV gene sequence and the codon AAAAGA corresponding to the Kex2 protease cleavage site Lys-Arg, and the TGA stop codon and NotI site at the 3' end to obtain the coding gene PIV Finally, it was handed over to the gene synthesis company for whole gene synthesis (pUC57-PIV).
  • the intermediate vector 18T- ⁇ treated with XhoI and NotI sites was used as a vector fragment, and the fragment obtained by double digestion of pUC57-PIV by XhoI and NotI was ligated, and the positive plasmid 18T- ⁇ -PIV was obtained.
  • the obtained positive plasmid 18T- ⁇ -PIV was digested with BamHI and NotI, and the target gene fragment ( ⁇ -PIV) was recovered, and the vector fragment recovered by double digestion of pPIC9k by BamHI and NotI was ligated to obtain the final recombinant expression. Plasmid pPIC9k-PIV, the results are shown in Figure 1. Sequencing was performed using the 5' AOX1 and 3' AOX1 primers, and the sequencing results showed that the cloned gene fragment was consistent with the theory.
  • Example 2 Pichia pastoris GS115 produces lysine-specific enzyme
  • the expression plasmid pPIC9k-PIV was linearized by SacI cleavage site, and the yeast host bacteria competent state was prepared according to the method of Invitrogen EasySele Pichia Expression Kit, and electrotransformed into Pichia pastoris GS115 strain, and coated on MD plate. Single colonies were grown for 3-4 days at 30 °C.
  • BMGY medium mass percentage, yeast powder 1%, peptone 2%, YNB 1.34%, Glycerol 1%, biotin 0.004%, 100 mM phosphate buffer
  • OD 600 is about 15-20, transferred to a 500 mL cone containing 50 mL of BMMY medium (mass percentage, yeast powder 1%, peptone 2%, YNB 1.34%, biotin 0.004%, 100 mM phosphate buffer)
  • BMMY medium mass percentage, yeast powder 1%, peptone 2%, YNB 1.34%, biotin 0.004%, 100 mM phosphate buffer
  • the cells were cultured at 30 ° C in a 220 rpm shaker, and 1% by volume of methanol was added every 12 hours to induce expression, and the cells were cultured for 96 hours.
  • the culture solution was taken, centrifuged (12,000 rpm, 5 min, 4 ° C), and the supernatant was taken. The enzyme activity of the fermentation broth of different single colonies was measured, and the strain with higher enzyme activity was screened, and then the second round of rescreening was performed.
  • the specific method is as follows: a high-yield strain obtained by rescreening (shake flask fermentation enzyme activity 0.817 U/mL) is subjected to 50 L fermentor fermentation.
  • the inoculum was 5%, and transferred to 20 L of basic medium (glycerol 40 g/L, K 2 SO 4 17.5 g/L, MgSO 4 14 g/L, KOH 3.2 g/L, CaSO 40.8 g/L, PTM13ml/L, pH 6.0) in a 50L fermenter, cultured for 16-18h, the dissolved oxygen increased from nearly 0% to 70-80%, glycerol was added at 2mL/min for 10 hours, induction was started, and the induced pH was optimized. 5.5-6.0, induced by adding 1.2% PTM1 methanol for 60-72h during stable induction period, fermented for 121h, and the fermentation broth was taken every 4h for enzyme activity detection. The results are shown in Figure 2.
  • the supernatant of the fermentation broth was replaced by an ultrafiltration system, the replacement solution was 30 mM Tris-HCl, the membrane package was selected to have a molecular weight of 5 k, and the area was 0.1 m 2 .
  • the ultrafiltered solution was equilibrated with pH 5.0 and 0.1 M NaAc buffer.
  • the cation chromatography packing was determined according to the penetration of the small test.
  • the pH of the sample was adjusted to pH 5.0 and the conductance was 7.0 ms/cm.
  • the cells were washed with pH 5.0, 0.1 M NaAc buffer, followed by elution with 0.1 M NaAc and 0.3 M NaCl buffer (pH 6.0), finally with 0.5 M NaOH solution, and finally lyophilized to determine the enzyme activity. See Table 1.
  • Solution preparation Tris-HCL buffer (100 mM, pH 8.5): 2.28 g of Tris was weighed and dissolved in 200 mg of ultrapure water, and the pH was adjusted to 8.5 with HCl.
  • Lys-C substrate solution Take a 5mg substrate (N-p-Tosyl-Gly-Pro-Lys 4-nitroaniline acetate) and dissolve directly with 3.939mL of ultrapure water.
  • ⁇ molar extinction coefficient (nitroaniline is 9.620mL / ⁇ mol / cm)
  • Enzyme activity definition The amount of enzyme required to catalyze the hydrolysis of 1 ⁇ mol of substrate per minute is defined as one enzyme unit.

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Abstract

一种在毕赤酵母中重组表达赖氨酸特异性内切蛋白酶（Lysine-specific endoprotease）的制备方法，其包括根据酵母密码子偏好性优化赖氨酸特异性内切蛋白酶的基因序列，构建含该优化基因的重组表达载体和基因工程菌，并发酵生产重组赖氨酸特异性内切蛋白酶。

Description

一种重组赖氨酸特异性酶的制备方法 技术领域
本发明涉及基因工程技术领域。具体地，本发明提供了一种重组赖氨酸特异性酶(Lysine-specific endoprotease，Lys-C)的高效制备方法，包括工程菌的构建、重组赖氨酸特异性酶的表达与纯化。
背景技术
赖氨酸特异性酶(Lysine-specific endoprotease)是一种碱性蛋白酶，分子量约为26 kD，属于丝氨酸蛋白酶家族成员之一。作为一种高纯度蛋白酶，它能特异水解Lys的羧基端(与精氨酸相连时除外)，可以产生比Trypsin多的酶解肽段，可用于通过肽质谱指纹印迹或MS/MS光谱匹配进行蛋白鉴定，因此在蛋白质组学分析中有着很好的应用前景。另外，因其高度的专一性和高酶切活性，Lys-C也可用于重组蛋白工业生产中的前体切割(如胰岛素前体)，因此Lys-C目前在基因工程制药领域成为蛋白表达下游纯化的重要工具酶，尤其适用于生物工程制药业及基因工程、生物化学、分子生物学研究。
目前市场上的赖氨酸特异性酶大多为天然菌种的表达产物，已知有三种微生物可合成Lys-C：产酶溶杆菌，水解无色杆菌，铜绿假单胞菌。赖氨酸特异性酶主要由产酶溶杆菌表达生产，其次为水解无色杆菌培养液中分离得到。天然提取的Lys-C价格极为昂贵，如Promega的Lys-C市价约为370元/μg。同时产酶溶杆菌作为Lys-C的主要产生菌，生产效率不高，发酵周期较长，且提取工艺比较复杂，这些大大限制了Lys-C的广泛应用。
目前天然提取的赖氨酸特异性酶不仅有限，而且成本高、得率低，也容易被其它杂质污染，从而导致目的蛋白降解。同时国内目前还未有赖氨酸特异性酶重组生产的企业，而进口rLys-c的价格较昂贵，大量购买进口rLys-c将会大大提高药物生产成本，因此，建立一种快速、简便、稳定、活性高rLys-C的生产方法，对于基因工程药物的开发以及生物学研究具有重要的应用与研究价值。
发明内容
本发明为了克服上述现有技术的缺陷和不足，提供了一种重组赖氨酸特异性酶制备方法，涉及重组赖氨酸特异性酶基因序列的设计、含有该基因的重组表达载体和基因工程菌的构建及重组赖氨酸特异性酶的制备方法。为重组赖氨酸特异性酶的工业生产提供了有效途径。
本发明一方面提供了一种高效表达重组赖氨酸特异性酶，该酶的DNA序列为SEQ ID NO：1。
上述重组赖氨酸特异性酶的氨基酸，其序列为SEQ ID NO：2。
一种重组载体，含有序列SEQ ID NO：1的pPIC9k-PIV。
本发明的技术方案另一方面提供了一种高效表达重组赖氨酸特异性酶的工程菌，所述工程菌为含序列SEQ ID NO：1的毕赤酵母菌。
在本发明的一些实施方式中，所述的工程菌为GS115/pPIC9k-PIV。
本发明的技术方案同时提供了一种高效表达重组赖氨酸特异性酶工程菌的构建方法，包括以下步骤：
1)获得优化的DNA序列SEQ ID NO：1，在其C端添加6×His标签，并在基因序列5’端和3’端分别添加合适酶切位点与相Kex2位点，最终进行全基因合成，获得PIV；
2)将连接有前导肽α因子的PIV插入质粒pPIC9k中，得重组质粒pPIC9k-PIV；
3)将上述所得质粒pPIC9k-PIV进行Sac线性化，并电转至毕赤酵母GS115菌株中，经培养与酶活检测，筛选得高产菌株GS115/pPIC9k-PIV。
一种利用上述基因工程菌生产重组赖氨酸特异性酶的方法，包括以下步骤，培养基因工程菌，并获得重组表达的重组赖氨酸特异性酶。
一种利用上述基因工程菌生产重组赖氨酸特异性酶的方法，其特征在于，包括以下步骤：
a)赖氨酸特异性酶高产菌株的筛选：将菌株接种至一级培养基中，在28-30℃培养至OD600为15-20时，转接至二级培养基中，28-30℃下震荡培养，每8-12h补加甲醇进行诱导表达，发酵12-120h后停止，离心培养液取上清；
b)发酵生产：将复筛后的菌株接种至发酵培养基中，在28-30℃下培养至溶氧量达70-80％时，以2-4ml/min加入甘油9～12小时，加入含1％～1.2％PTM1的甲醇溶液进行诱导，60-72h后每隔4-12h取发酵液进行酶活检测；
c)将上述培养液经分离、纯化后得到赖氨酸特异性酶蛋白的蛋白冻干粉。
在本发明的一些实施方式中，步骤b)发酵培养基中菌株的接种量为1-5％。
在本发明的一些实施方式中步骤a)中甲醇诱导时的pH值为4.0-8.0；步骤b)中甲醇诱导时培养基的pH值为5.5-6.0。
在本发明的一些实施方式中，纯化选自交换层析、超滤或冻干。
除非明确地说明与此相反，否则，本发明引用的所有范围包括端值。例如，“步骤a)中甲醇诱导时的pH值为4.0-8.0”，表示步骤a中甲醇诱导时的pH值为4.0≤pH≤8.0。
本发明中的数字均为近似值，无论有否使用“大约”或“约”等字眼。数字的数值有可能会出现1％、2％、5％、7％、8％、10％等差异。每当公开一个具有N值的数字时，任何具有N+/-1％，N+/-2％，N+/-3％，N+/-5％，N+/-7％，N+/-8％或N+/-10％值的数字会被明确地公开，其中“+/-”是指加或减。
本发明在对重组赖氨酸特异性酶的基因进行改进后，并采用毕赤酵母菌表达产物，为重组赖氨酸特异性酶的工业生产提供了有效途径。与目前常用的产酶溶杆菌，水解无色杆菌，铜绿假单胞菌表达产物相比，克服了生产效率不高，发酵周期较长，且提取工艺比较复杂等缺点，在降低生产成本的同时还具有快速、简便、稳定等特点，且生产的rLys-C活性高，对于基因工程药物的开发以及生物学研究具有重要的应用与研究价值。
附图说明
图1为重组表达质粒pPIC9k-PIV的示意图；
图2为PIV不同诱导时间发酵液上清酶活测定结果示意图；
具体实施方式
下面结合具体实施例对本发明作进一步具体详细描述，但本发明的实施方式不限于此，对于未特别注明的工艺参数，可参照常规技术进行。
实施例1重组表达质粒pPIC9k-PIV的构建
1.重组赖氨酸特异性酶基因(PIV)的全合成
根据GenBank公布的赖氨酸特异性酶基因序列(AY062882)和氨基酸序列(AAL47683.1)，按照酵母密码子优化策略，对赖氨酸特异性酶核酸序列进行优化，同时在其C端添加6×His标签，并在PIV基因序列5’端带有XhoI位点和Kex2蛋白酶酶切位点Lys-Arg对应的密码子AAAAGA，3’端添加TGA终止密码子和NotI位点，获得编码基因PIV，最终交给基因合成公司进行全基因合成(pUC57-PIV)。
2.重组表达质粒pPIC9k-PIV的构建
利用XhoI和NotI位点处理过的中间载体18T-α为载体片段，与pUC57-PIV经XhoI和NotI双酶切回收得到的片段进行连接，筛选得到阳性质粒18T-α-PIV。得到的阳性质粒18T-α-PIV经BamHI和NotI双酶切后，回收目的基因片段(α-PIV)，与pPIC9k经BamHI和NotI双酶切后回收的载体片段进行连接，筛选得到最终重组表达质粒pPIC9k-PIV，结果见图1。利用5’AOX1和3’AOX1引物进行测序，测序结果显示克隆基因片段与理论一致。
实施例2毕赤酵母GS115生产赖氨酸特异性酶
1.pPIC9k-PIV电转化
利用SacI酶切位点对表达质粒pPIC9k-PIV进行线性化，按照Invitrogen公司EasySele Pichia Expression Kit中的方法制备酵母宿主菌感受态，并电转至毕赤酵母GS115菌株中，涂布于MD平板上，30℃下培养3-4天长出单菌落。
2.赖氨酸特异性酶高产菌株的筛选
挑选大小适中且较为饱满的单菌落接种至BMGY培养基(质量百分比，酵母粉1％、蛋白胨2％、YNB1.34％、Glycerol1％、生物素0.004％、100mM磷酸盐缓冲液)，30℃过夜培养，OD600约为15-20，转接至含有50mL BMMY培养基(质量百分比，酵母粉1％、蛋白胨2％、YNB1.34％、生物素0.004％、100mM磷酸盐缓冲液)的500mL锥形瓶中，于220rpm摇床中30℃培养，每12h补加1％体积的甲醇进行诱导表达，共培养96h。发酵完后取培养液，离心(12000rpm，5min，4℃)，取上清。测定不同单菌落的发酵液酶活，筛选酶活较高的菌株，再进行第二轮复筛。
3.赖氨酸特异性酶的发酵生产
具体方法为：以复筛得到的高产菌株(摇瓶发酵酶活0.817U/mL)进行50L发酵罐发酵。种子液接种量为5％，转接至装有20L基础培养基(甘油40g/L、K2SO417.5g/L、MgSO414g/L、KOH 3.2g/L、CaSO40.8g/L、PTM13ml/L，pH为6.0)的50L发酵罐中，培养16-18h，待溶氧从接近0％上升到70-80％，按2mL/min加入甘油10小时，开始诱导，优化诱导的pH为5.5-6.0，稳定诱导期内加入含1.2％PTM1的甲醇诱导60-72h，共发酵121h，每隔4h取发酵液进行酶活检测，结果见图2。
实施例3赖氨酸特异性酶的纯化与活性检测
1.赖氨酸特异性酶的纯化
发酵液上清经超滤系统置换缓冲液，置换溶液为30mMTris-HCl，膜包选择为5k分子量，面积为0.1m2，超滤后的溶液经pH5.0，0.1M NaAc缓冲液平衡过的阳离子层析填料，上样量根据小试穿透情况确定，上样的pH调为pH 5.0，电导7.0ms/cm。并用pH5.0，0.1M NaAc缓冲液进行冲洗，紧接着用0.1M NaAc和0.3MNaCl缓冲液(pH 6.0)进行洗脱，最后用0.5M NaOH溶液进行再生，最后进行冻干，测定酶活，见表1。
2.赖氨酸特异性酶的酶活测定
1)溶液配制：Tris-HCL缓冲液(100mM，pH8.5)：称取Tris2.428g溶于200mg超纯水中，用HCl调pH至8.5。Lys-C底物溶液：取一支5mg装底物(N-p-Tosyl-Gly-Pro-Lys 4-硝基苯胺醋酸盐)直接用3.939mL超纯水溶解即得。
2)实验过程：取Tris-HCL缓冲液180μL，加入样品10μL，底物溶液10μL，混匀后用酶标仪，于405nm处，每隔20S读一次数，共30min，反应温度30℃，绘制动力学曲线，取反应初始阶段呈线性部分求酶活。
3)计算公式：
Figure PCTCN2016089324-appb-000001
△A/t：动力学曲线斜率(取反应初始阶段呈线性的部分)
Tv：反应体系的体积
Sv：加入样品的体积
b：光径为1cm
N：样品稀释倍数
ξ：摩尔消光系数(硝基苯胺为9.620mL/μmol/cm)
4)酶活定义：每分钟催化1μmol底物水解所需的酶量定义为一个酶单位。
表1赖氨酸特异性酶纯化富集后酶活测定结果
Figure PCTCN2016089324-appb-000002
Figure PCTCN2016089324-appb-000003
Figure PCTCN2016089324-appb-000004
Figure PCTCN2016089324-appb-000005
Figure PCTCN2016089324-appb-000006

Claims (10)

  1. 一种重组赖氨酸特异性酶，其特征在于，其核苷酸序列为SEQ ID NO：1。
  2. 根据权利要求1所述的重组赖氨酸特异性酶，其特征在于，其氨基酸序列为SEQ ID NO：2。
  3. 一种重组载体，其特征在于，所述载体为含有序列SEQ ID NO：1的pPIC9k-PIV。
  4. 一种高效表达重组赖氨酸特异性酶的基因工程菌，其特征在于，所述工程菌为含序列SEQ ID NO：1的毕赤酵母菌。
  5. 根据权利要求4所述的基因工程菌，其特征在于，所述的基因工程菌为GS115/pPIC9k-PIV。
  6. 一种高效表达重组赖氨酸特异性酶工程菌的构建方法，其特征在于，包括以下步骤：
    1)获得优化的DNA序列SEQ ID NO：1，在其C端添加6×His标签，并在基因序列5’端和3’端分别添加合适酶切位点与Kex2位点，最终进行全基因合成，获得PIV；
    2)将连接有前导肽α因子的PIV插入质粒pPIC9k中，得重组质粒pPIC9k-PIV；
    3)将上述所得质粒pPIC9k-PIV进行Sac线性化，并电转至毕赤酵母GS115菌株中，经培养与酶活检测，筛选得高产菌株GS115/pPIC9k-PIV。
  7. 一种生产权利要求1所述重组赖氨酸特异性酶的方法，其特征在于，培养如权利要求4～5中任一项所述的基因工程菌，获得重组表达的所述重组赖氨酸特异性酶。
  8. 一种生产权利要求1所述重组赖氨酸特异性酶的方法，其特征在于，包括以下步骤：
    a)赖氨酸特异性酶高产菌株的筛选：将菌株接种至一级培养基中，在28-30℃培养至OD600为15-20时，转接至二级培养基中，在28-30℃下震荡培养，每8-12h补加甲醇进行诱导表达，发酵12-120h后停止，离心培养液并取上清；
    b)发酵生产：将复筛后的菌株接种至发酵培养基中，在28-30℃下培养至溶氧量达70-80％时，以2-4ml/min加入甘油9～12小时，加入含1％～1.2％PTM1的甲醇溶液进行诱导，60-72h后每隔4-12h取发酵液进行酶活检测；
    c)将上述培养液经分离、纯化后得到赖氨酸特异性酶蛋白的蛋白冻干粉。
  9. 根据权利要求8所述的方法，其特征在于，步骤b)发酵培养基中菌株的接种量为1-5％。
  10. 根据权利要求8所述的方法，其特征在于，步骤a)中甲醇诱导时的pH值为4.0-8.0；步骤b)中甲醇诱导时培养基的pH值为5.5-6.0。
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