WO2023125831A1 - 一种适合自动化操作的从发酵液中收集菌体的方法 - Google Patents
一种适合自动化操作的从发酵液中收集菌体的方法 Download PDFInfo
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- the invention belongs to the technical field of post-treatment of biochemical engineering, and in particular relates to a method for recovering bacteria from fermentation liquid of bacteria and fungi, which is suitable for automatic operation.
- Escherichia coli is an important genetically engineered host bacterium in modern biotechnology. It has the advantages of clear genetic background, fast growth, simple genetic manipulation, and easy production process amplification. It is widely used in laboratory or industrial scale. Production of plasmid DNA, recombinant proteins, enzymes and other biological products.
- E. coli As a host cell to produce plasmid DNA, it is necessary to recover the E. coli cells from the culture medium, and then extract the desired plasmid DNA from the E. coli cells.
- High-speed centrifugation is usually used for the preparation of plasmid DNA at the microgram to milligram level in the laboratory.
- the number of samples processed in batches is limited, and more manual operations are required, and the throughput and scale are difficult to scale up. , suitable for a large number of processing of the same sample, but there are risks such as membrane clogging, damage, pollution, etc., the cost of consumables is high, and it is difficult to achieve multi-sample, high-throughput processing.
- both the centrifugation method and the membrane filtration method rely on more manual operations, and when the number of samples to be processed in batches increases, equipment and personnel input need to be multiplied accordingly. Therefore, it is urgent to establish a method for collecting E. coli cells suitable for high-throughput and automated processing.
- Chemical flocculation can be used to separate bacterial cells from culture media and is a low-cost alternative to centrifugation and membrane filtration.
- the published Chinese patent (CN109439584A) shows that chitosan and sodium alginate can concentrate Escherichia coli fermentation broth through flocculation to achieve the enrichment effect of bacteria.
- this method only studies the recovery of E. coli cells, and does not study the effect of sedimentation on cell integrity, product and more importantly subsequent plasmid recovery.
- Chitosan is a linear aminopolysaccharide prepared by deacetylation of chitin in hot lye
- chemical name is ⁇ -(1,4)-2-amino-2-deoxy-D-glucan
- molecular formula For (C6H11NO4)n the structural formula is as follows:
- Chitosan is insoluble in water and organic solvents, but soluble in dilute acid solutions. Chitosan is insoluble in water, which makes its use complicated, so its application in flocculation and collection of bacteria has not been widely realized.
- this area still needs to develop a kind of simple operation, has no influence on subsequent plasmid production yield, and does not rely on centrifuge or membrane filtration, the bacterium collection method that can be realized automatically, this is to the high-throughput automatic preparation of plasmid DNA and The rapid production of proteins required for late-stage biopharmaceutical development is of great significance.
- the purpose of the invention of the present invention is to provide a kind of thalline of improved recovery bacterial and fungal cell fermentation culture, and keep its plasmid DNA, RNA integrity and the high-efficiency method of protein and nucleic acid product extraction.
- the present invention provides a kind of method of collecting thalline from bacterium or fungal fermentation broth, it is characterized in that, the method comprises steps:
- step b) in the above-mentioned method is omitted, so in this aspect a method of collecting bacterial sediment or fermentation culture supernatant from bacterial or fungal fermentation culture liquid is provided, the method Include steps:
- a preferred embodiment of this aspect also includes the step d) of recovering the bacteria after step c).
- the bacteria are selected from Bacillus, Escherichia coli, Lactobacillus, Lactococcus, preferably Escherichia coli or Bacillus subtilis;
- the fungi are selected from yeast, preferably baker's yeast, feed yeast, Saccharomyces cerevisiae, Pichia Yeast; Candida; Mold, more preferably Rhizopus, Aspergillus, most preferably Saccharomyces cerevisiae and Pichia pastoris.
- the bacterium is Escherichia coli, and/or the fungus is Pichia pastoris.
- the amount of carboxylated chitosan mixed in step a) is 1%-32% by weight of the fermentation broth, preferably 1-16% by weight, more preferably 4%-8% by weight, most preferably 8% by weight. %weight.
- the amount of sodium alginate added in step b) is 1%-16% by weight of the fermentation broth, preferably 1%-8% by weight, more preferably 4%-8% by weight, most preferably 8% weight.
- the amount of carboxylated chitosan and sodium alginate added in the fermentation broth is respectively 8% by weight of the fermentation broth, or the amount of carboxylated chitosan added in the fermentation broth The amount is 8% by weight of the fermentation medium, and no sodium alginate is added.
- the fermentation broth is preferably selected from Escherichia coli fermentation broth and Pichia pastoris fermentation broth.
- the mass ratio of added carboxylated chitosan to sodium alginate is 1:4-4:1, preferably 1:2-2:1, more preferably 1:1.
- the carboxylated chitosan is selected from amino groups (-NH 2 ), or hydroxyl groups (-OH), or shells in which amino groups (-NH 2 ) and hydroxyl groups (-OH) are modified by carboxylation groups.
- the straight chain or branched hydrocarbon group preferably has 1 to 6 carbon atoms.
- the strains of Escherichia coli include E.coli TOP10, E.coli JM109, E.coli HB101, E.coli DH5 ⁇ , E.coli BL21 (DE3), and other Escherichia coli K strains and B Derivative strains of strains.
- the strain of yeast comprises Saccharomyces cerevisiae, Pichia pastoris.
- the recovery of step d) is selected from pouring the culture medium or an automated robotic arm to pour the culture medium to obtain the bacteria, using physical or chemical methods to break the bacteria to release DNA or RNA in the bacteria, and magnetic beads to adsorb DNA or RNA , the cleaning solution is used to clean and purify the DNA or RNA, preferably pouring, more preferably manually pouring the culture medium or an automated robotic arm pouring the culture medium.
- protein can also be extracted from the bacterial cells after the bacterial cells are obtained.
- the culture supernatant recovered in step d) is obtained by pouring the culture medium or an automated robotic arm to pour the culture medium, and then purifying the DNA or protein or small molecule metabolites in the supernatant .
- the E. coli fermentation medium is selected from LB broth medium, and the yeast fermentation medium is selected from YPD medium.
- the advantage of the method for the thalline of reclaiming bacterium and fungal fermentation culture of the present invention is as follows:
- the fermentation supernatant can be removed by pouring to obtain sedimentation bacteria. This process can be automated with a robotic arm.
- the bacterial sediment obtained by sedimentation is relatively dispersed and does not form flocs, which is beneficial to the lysis of the bacterial cells and the subsequent extraction of intracellular plasmid DNA, RNA and protein.
- the fermentation broth After the fermentation broth is treated with carboxylated chitosan and sodium alginate, it can also be directly separated by pouring to obtain the fermentation supernatant, which can be used for subsequent biochemical separation processes to obtain target products (including but not limited to protein , alcohols, acids, esters, ketones and other biological metabolites).
- target products including but not limited to protein , alcohols, acids, esters, ketones and other biological metabolites.
- reagents, cells, and instrumentation used in the present invention are commonly commercially available and publicly available.
- Figure 1 shows the agarose gel electrophoresis patterns of the plasmid DNA extracted from the bacteria collected by centrifugation (lane 1) and the plasmid DNA extracted from the bacteria collected by carboxylated chitosan (lane 2).
- the carboxylated chitosan used in the specific examples herein is selected from amino (-NH 2 ), or hydroxyl (-OH), or amino (-NH 2 ) and hydroxyl (-OH) modified by carboxylated groups Chitosan, wherein the carboxylation group is a C1-C6 straight chain or branched chain hydrocarbon group containing 1-3 carboxyl substitutions, preferably containing 3 carboxyl groups, more preferably 2 carboxyl groups, most preferably 1 carboxyl group;
- the linear or branched hydrocarbon group preferably has 1 to 6 carbon atoms.
- Carboxylated chitosan has good water solubility.
- Carboxylated chitosan is currently commercially available in varieties such as carboxylated chitosan produced by Aladdin reagents, which can be used according to "Synthesis of carboxylated chitosan and its adsorption of cadmium (II), lead (II) and Copper(II) properties” (Synthesis of carboxylated chitosan and its adsorption properties for cadmium(II), lead(II) and copper(II) from aqueous solutions) (KLLv, YLDu, CMWang;.Water Sci Technol 1July 2009; 60 (2): 467–474.doi: https://doi.org/10.2166/wst.2009.369 ) and other documents disclosed method preparation.
- the fermentation cell culture of bacteria and fungi described herein includes the technology in the art that uses fermentation medium as a medium to culture bacteria and fungi cells under appropriate conditions to amplify and produce desired target products.
- the fermentation culture in this paper can be either laboratory scale or industrial production scale.
- bacteria includes but is not limited to Gram-positive bacteria and Gram-negative bacteria, especially bacteria commonly used in fermentation culture, such as Bacillus, Escherichia coli, Lactobacillus, Lactococcus, etc., preferably Escherichia coli, Bacillus subtilis.
- fungus as used herein includes, but is not limited to, eukaryotic spore-forming microorganisms, including but not limited to yeast, preferably baker's yeast, feed yeast, Saccharomyces cerevisiae, Pichia, etc.; Candida; molds, such as Rhizopus , Aspergillus, etc. Saccharomyces cerevisiae and Pichia pastoris are preferred.
- LB Broth Medium (Sangon Biotech, A507002): 10g/L Tryptone, 10g/L NaCl, 5g/L Yeast Extract; Chitosan Solution: Dissolve 1.5g Chitosan (Aladdin Reagent, C105799) to 100mL of 1% acetic acid solution (FISHER, A35-500) to obtain 1.5% chitosan acetic acid solution.
- Carboxylated chitosan solution 1.5 g of carboxylated chitosan (Aladdin reagent, C105800) was dissolved in 100 mL of ultrapure water to obtain a 1.5% carboxylated chitosan aqueous solution.
- Sodium alginate solution 1.0 g of sodium alginate (Aladdin's reagent, S100127) was dissolved in 100 mL of ultrapure water to obtain a 1% sodium alginate aqueous solution.
- Bacterial suspension 50mM glucose (Shanghai test, 10010518), 25mM Tris-HCl (pH8.0) (Shanghai test, 30188336), 10mM EDTA (pH8.0) (Shanghai test, 10009717).
- Lysis solution 0.2mol/L NaOH (Shanghai Test, 10019718), 1% (m/V) SDS (Shanghai Test, 30166428).
- Neutralizing solution 100mL: 60mL 5M potassium acetate (Shanghai Test, 30154518), 11.5mL glacial acetic acid (FISHER, A35-500), 28.5mL distilled water.
- RNase A 10mg/mL (Yisheng Biology, 10405ES03).
- the sequence of the pUC19 plasmid is as follows:
- the top 10 E. coli strains containing the pUC19 plasmid were cultivated in LB medium overnight to obtain E. coli fermentation liquid, and samples were taken to measure OD600.
- control group 3500g centrifugation of bacterial liquid for 1min, remove the fermentation supernatant to obtain the bacterial body
- experimental group use a pipette gun to absorb the supernatant of the fermented liquid after sedimentation, and obtain the bacterium settled at the bottom of the centrifuge tube bacteria.
- Carboxylated chitosan group Add 12ml (added amount 8%) carboxylated chitosan solution to the shake flask, shake it horizontally for 30 seconds manually, then add the same volume of sodium alginate solution (added amount 8%), shake it manually Uniform 30s. Let stand at low temperature for 4 hours to allow the E. coli cells to fully settle, then take the supernatant to measure OD600, and calculate the sedimentation rate.
- Chitosan group add 6ml (addition amount 4%) chitosan solution in the shaking flask, mix 2-3min quickly with the rotating speed of 120-150rpm on the shaker, add the sodium alginate solution of 3ml afterwards (addition Volume 2%), mix slowly on the shaker at 50-60rpm for 2-5min. Let stand at low temperature for 4 hours to allow the E. coli cells to fully settle, then take the supernatant to measure OD600, and calculate the sedimentation rate.
- Carboxylated chitosan and the concentrated bacterial solution obtained after chitosan sedimentation were used for the plasmid DNA extraction test, and the bacterial cells obtained by centrifuging 150 mL of overnight cultured E. coli bacterial liquid at 3500 g for 10 min were used as the control group. Due to the large volume of remaining bacterial liquid after chitosan treatment, an electric pipette was used to carefully remove the culture medium that could not be poured cleanly, so that the volume of the final concentrated bacterial liquid was the same as that of the carboxylated chitosan treatment group (about 10ml).
- agarose gel electrophoresis The experimental steps of agarose gel electrophoresis refer to "Molecular Cloning: A Laboratory Manual (Fourth Edition), Michael R. Green, Joseph Sambrook, Cold Spring Harbor Laboratory Press, 2012."
- Control group Add 12ml of ultrapure water to the shaker flask, shake it horizontally for 30s by hand, let it stand at low temperature, take the supernatant at 4h and 6h to measure OD600, and calculate the sedimentation rate.
- Table 1 The effect of different additions of carboxylated chitosan and sodium alginate on the bacterial body sedimentation in 1mL E. coli fermentation broth
- the above-mentioned obtained carboxylated chitosan and sodium alginate experimental groups 1, 2, and 3 were selected to do a small amount of plasmid preparation test, and the bacteria collected by centrifugation were used as a control (as in method part 2.2), and the results See Table 2.
- the plasmid yield of the control group was 9.4 ⁇ g
- the experimental group 3 obtained the highest plasmid yield of 14.5 ⁇ g by using 8% carboxylated chitosan and sodium alginate to settle the bacteria, which was 1.5 times that of the control group; With the increase of cell sedimentation rate, the plasmid yield showed a linear growth trend.
- the plasmid DNA A260/A280 obtained by the small-scale extraction of the control group and the experimental group was between 1.8 and 2.0, without RNA and protein contamination, and could be used for further research. It is worth noting that the plasmid yield obtained by sedimentation collection of bacteria under small volume conditions is significantly higher than that of centrifugation collection of bacteria, and carboxylated chitosan or sodium alginate may have a certain promotion on the yield of plasmids. effect.
- Plasmid DNA was prepared from the bacteria collected by centrifugation as a control. Due to the large volume of bacterial liquid after sedimentation treatment in the chitosan group, a pipette gun was used to manually remove the remaining medium to reduce the final bacterial liquid volume to about 10 mL. Then add 10 ⁇ L RNase A to the bacterial solution, shake it horizontally to mix well, and then add alkaline lysate to lyse the bacterial cells. Subsequently, the plasmid DNA was extracted according to the method described in 2.4. The yield of the extracted plasmid DNA is shown in Table 4.
- the thalline obtained by carboxylated chitosan sedimentation is not much different from the plasmid yield obtained by the thalline collected by centrifugation when carrying out plasmid extraction, respectively. 330.5 ⁇ g and 324.5 ⁇ g.
- the chitosan sedimentation will significantly reduce the plasmid extraction efficiency in the bacteria, and the plasmid yield is only 37.2 ⁇ g, which is about 11% of the carboxylated chitosan treatment group.
- chitosan and sodium alginate to settle the bacteria is more suitable for the application scenario of removing the bacteria in the fermentation broth and collecting the fermentation supernatant; and carboxylated chitosan and sodium alginate to settle the bacteria can not only be used for fermentation
- the collection of supernatant can also be used for the subsequent processing of the bacteria, such as the extraction of biological products inside the bacteria: plasmid DNA, RNA, intracellular proteins, etc.
- both centrifugation and membrane filtration methods have limited batch processing samples, require more manual operations, are difficult to achieve automation, and need to increase the corresponding Centrifuges or membrane filtration equipment pose challenges such as increased costs.
- the sedimentation method of the present invention only needs to add 1-2 kinds of flocculants, and the separation of bacteria and fermentation medium can be realized by gravity sedimentation under low temperature conditions. for centrifugation and membrane filtration methods.
- the inventors have found unexpectedly that adopting water-soluble carboxylated chitosan and sodium alginate or using carboxylated chitosan alone can effectively realize the thalline settlement in E. coli fermentation broth, avoiding Centrifugal or membrane filtration, easy to operate.
- the fermentation supernatant can be removed by pouring to obtain sedimentation bacteria.
- This process can be automated with a robotic arm.
- the bacterium sedimentation obtained by the sedimentation method of the present invention is relatively dispersed and does not agglomerate to form floc, which is beneficial to the lysis of the bacterium and the subsequent extraction of intracellular plasmid DNA, RNA and protein.
- Embodiment 2 Pichia pastoris thalline sedimentation
- YPD medium 1% yeast extract (Thermo Scientific, LP0021B), 2% peptone (Thermo Scientific, LP0042B), 2% dextran (Sinopharm Reagent, 63005518).
- Carboxylated chitosan solution 1.5 g of carboxylated chitosan (Aladdin reagent, C105800) was dissolved in 100 mL of ultrapure water to obtain a 1.5% carboxylated chitosan aqueous solution.
- Pichia GS115 bacterial strain in 4ml YPD medium obtain Pichia GS115 fermented liquid, take a sample and measure OD600.
- the fermentation broth without carboxylated chitosan was taken as blank control.
- the carboxylated chitosan of different addition amount of table 6 is to the thalline sedimentation effect in Pichia pastoris fermented liquid
- carboxylated chitosan has a good effect on the cell sedimentation of Pichia pastoris, and can be used for collection of Pichia pastoris cells or culture supernatant after sedimentation for subsequent protein purification and other applications.
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Abstract
公开了一种从细菌或真菌发酵培养液中收集菌体的方法,该方法包括步骤:a)混合细菌或真菌发酵培养液和羧化壳聚糖,得到混合物;可任选地,b)在混合物中加入海藻酸钠溶液,混合均匀;和c)沉淀,获得细菌或真菌的菌体沉淀物或培养液上清。
Description
本发明属于生化工程后处理技术领域,具体涉及一种适合自动化操作的从细菌和真菌的发酵液中回收菌体的方法。
2021年全球前30大畅销药中有24款是生物药。生物药研发的速度和成功与否,与能否快速获得相应的蛋白样品以用于蛋白结构,体外和体内活性研究密切相关。目前,哺乳动物细胞瞬时表达工艺是获取研究用蛋白的主要方式。该工艺需要的质粒DNA量一般在毫克至克级别,且根据前期候选分子库的大小,需要生产的蛋白样品个数一般从几十到上千不等,对应的所需要质粒DNA的数量也将在几十到上千数量级。在生物药加速研发的过程中,如何快速获得用于蛋白生产所需的大量质粒DNA是目前急需解决的问题。
以细菌为例,大肠杆菌是现代生物技术中一种重要的基因工程宿主菌,具有遗传背景清晰,生长速度快,遗传操作简单,易于生产工艺放大等优势,广泛用于实验室级别或工业规模的质粒DNA,重组蛋白,酶等生物制品的生产。以大肠杆菌作为宿主细胞生产质粒DNA时,需要从培养基中回收大肠杆菌细胞,然后从大肠杆菌细胞中提取所需的质粒DNA。从发酵液中回收大肠杆菌细胞的常规方法有两种:高速离心法和膜过滤法。高速离心法通常用于实验室微克至毫克级别质粒DNA的制备,批次处理样品的数量有限,且需要人工操作较多,通量和规模难以放大;膜过滤法单次处理样品的规模较大,适合同种样品的大量处理,但存在膜堵塞,破损,污染等风险,耗材成本偏高,且难以实现多样品、高通量处理。此外,无论离心法还是膜过滤法都依赖较多的人工操作,在需要批次处理样品的数量增加时需要相应地倍增设备和人员投入。因此,迫切需要建立一种适用于高通量、自动化处理的大肠杆菌菌体收集方法。
化学絮凝作用可用于从培养基中分离细菌细胞,是一种可替代离心和膜过滤的低成本方法。已公开的中国专利(CN109439584A)表明壳聚糖和海藻酸钠可通过絮凝作用浓缩大肠杆菌发酵液,达到菌体富集效果。然而,该方法仅对于大肠杆菌菌体的回收进行了研究,并未研究沉降效果对于菌体完整性、产物和更重要的后续质粒回收的效果。
壳聚糖是一种线性氨基多糖,由甲壳素在热碱液中脱乙酰制备而成,化学名称为β-(1,4)-2-氨基-2-脱氧-D-葡聚糖,分子式为(C6H11NO4)n,结构式如下:
壳聚糖不溶于水和有机溶剂,可溶于稀酸溶液。壳聚糖不溶于水的特性,使其使用过程复杂,因此在絮凝收集菌体方面的应用尚未广泛实现。
因此,本领域仍然需要开发一种操作简单,对后续质粒生产得率没有影响,且不依赖离心机或膜过滤、可自动化实现的菌体收集方法,这对质粒DNA的高通量自动化制备和后期生物药研发所需蛋白的快速生产意义重大。
(3)发明内容
因此,本发明的发明目的是提供一种改进的回收细菌和真菌细胞发酵培养的菌体,且保持其质粒DNA、RNA完整以及蛋白和核酸产物提取的高效率的方法。
因此,本发明提供了一种从细菌或真菌发酵培养液中收集菌体的方法,其特征在于,该方法包括步骤:
a)混合细菌或真菌发酵培养所得的发酵培养液和羧化壳聚糖,得到混合物;
b)在混合物中加入海藻酸钠溶液,混合均匀;和
c)沉淀,获得菌体沉淀物。
在本发明的另一个方面,上述方法中的b)步骤被省略,因此在该方面提供了一种从细菌或真菌发酵培养液中收集菌体沉淀物或发酵培养液上清的方法,该方法包括步骤:
a)混合细菌或真菌发酵培养所得的发酵培养液和羧化壳聚糖,得到混合物;和c)沉淀,获得菌体沉淀物或发酵培养液上清。
该方面的一个优选实施方式还包括在步骤c)后回收菌体的步骤d)。
在一个具体实施方式中,细菌选自芽孢杆菌、大肠杆菌、乳杆菌、乳球菌,优选为大肠杆菌或枯草芽孢杆菌;所述真菌选自酵母,优选面包酵母、饲料酵母、 酿酒酵母、毕赤酵母;假丝酵母;霉菌,更优选根霉、曲霉,最优选为酿酒酵母和毕赤酵母。
在一个最优选的实施方式中,所述细菌是大肠杆菌,和/或所述真菌为毕赤酵母。
在一个具体实施方式中,步骤a)中混合的羧化壳聚糖量为发酵培养液的1%-32%重量,优选1-16%重量,更优选4%-8%重量,最优选8%重量。
在一个具体实施方式中,步骤b)中加入的海藻酸钠量为发酵培养液的1%-16%重量,优选1%-8%重量,更优选4%-8%重量,最优选8%重量。
在另一个具体实施方式中,在发酵培养液中加入的羧化壳聚糖和海藻酸钠的量分别为发酵培养液的8%重量,或在发酵培养液中加入的羧化壳聚糖的量为发酵培养基的8%重量,且不加入海藻酸钠。在一个更优选的实施方式中,发酵培养液优选自大肠杆菌发酵培养液和毕赤酵母发酵培养液。
在还有一个优选实施方式中,加入的羧化壳聚糖与海藻酸钠的质量比为1:4-4:1,优选1:2-2:1,更优选1:1。
在一个具体实施方式中,羧化壳聚糖选自氨基(-NH
2),或羟基(-OH),或氨基(-NH
2)和羟基(-OH)被羧化基团修饰后的壳聚糖,其中所述羧化基团是含1-3个羧基取代的C1-C6的直链或支链烃基,优选含3个羧基,更优选2个羧基,最优选为1个羧基;所述直链或支链烃基优选具有1-6个碳原子。
在还有一个具体实施方式中,大肠杆菌的菌株包括E.coli TOP10,E.coli JM109,E.coli HB101,E.coli DH5α,E.coli BL21(DE3),以及其它大肠杆菌K菌株和B菌株的衍生菌株。在一个优选实施方式中,酵母的菌株包括Saccharomyces cerevisiae,Pichia pastoris。
在另一个具体实施方式中,步骤d)的回收选自倾倒培养基或自动化机械臂倾倒培养基获得菌体,采用物理或化学方法破碎菌体释放菌体内DNA或RNA,磁珠吸附DNA或RNA,清洗液清洗纯化DNA或RNA,,优选倾倒,更优选手动倾倒培养基或自动化机械臂倾倒培养基。
在该方面的一个具体实施方式中,获得菌体后还可从菌体中提取蛋白质。
在还有一个优选实施方式中,步骤d)的回收培养基上清通过倾倒培养基或自 动化机械臂倾倒培养基,获得培养基上清,进而纯化上清中DNA或蛋白质,或小分子代谢物。
在还有一个方面,大肠杆菌发酵培养基选自LB肉汤培养基,酵母发酵培养基选自YPD培养基。
因此,本发明的回收细菌和真菌发酵培养的菌体的方法的优点如下:
1.采用可溶于水的羧化壳聚糖和海藻酸钠有效实现了发酵液中的菌体沉降,操作简便,成本低廉,避免离心或膜过滤。
2.发酵液经羧化壳聚糖和海藻酸钠处理后,可通过倾倒方法去除发酵上清,得到沉降菌体。该过程可通过机械臂实现自动化。
(3)沉降得到的菌体沉淀较为分散,不结团形成絮状,有利于菌体裂解和后续胞内质粒DNA,RNA和蛋白的提取。
(4)发酵液经羧化壳聚糖和海藻酸钠处理后,也可直接通过倾倒方法分离得到发酵上清,用该上清可进行后续的生化分离过程得到目标产品(包括但不限于蛋白,醇,酸,酯,酮等生物代谢物)。
各种实施方式的其它特征和优势将在下面的说明书中进行部分阐述,并且部分地根据说明书其将是显而易见的,或者可以通过各种实施方式的实践得到了解。各种实施方式的目标和其它优势将通过特别是在说明书和所附权利要求书中所指出的要素及组合得以实现和达到。
除非另外指出,本发明采用的试剂、细胞、以及仪器装置都是普通的市售和公众可得的。
图1显示了离心收集菌体抽提质粒DNA(泳道1)和羧化壳聚糖收集菌体抽提质粒DNA(泳道2)的琼脂糖凝胶电泳图谱。
现在将详细地参考本发明的一些实施方式,其在下文实施例部分中被阐述。 但是应当理解,它们并非意图使本发明限于那些实施方式。相反,本发明意图覆盖可以被所附权利要求书限定的本发明包括在内所有替换、修改和等价物。
本文具体实施例中使用的羧化壳聚糖是选自氨基(-NH
2),或羟基(-OH),或氨基(-NH
2)和羟基(-OH)被羧化基团修饰后的壳聚糖,其中所述羧化基团是含1-3个羧基取代的C1-C6的直链或支链烃基,优选含3个羧基,更优选2个羧基,最优选为1个羧基;所述直链或支链烃基优选具有1-6个碳原子。羧化壳聚糖具有良好的水溶性。羧化壳聚糖目前市售的品种有例如阿拉丁试剂出品的羧化壳聚糖,其可根据“羧化壳聚糖的合成及其从水溶液中吸附镉(II)、铅(II)和铜(II)的性质”(Synthesis of carboxylated chitosan and its adsorption properties for cadmium(II),lead(II)and copper(II)from aqueous solutions)(K.L.Lv,Y.L.Du,C.M.Wang;.Water Sci Technol 1July 2009;60(2):467–474.doi:
https://doi.org/10.2166/wst.2009.369)等文献公开的方法制备。
本文中所描述的细菌和真菌的发酵细胞培养包括本领域中以发酵培养基作为介质,在适当条件下培养细菌和真菌细胞,使其扩增并生产所需目标产物的技术。本文中的发酵培养可以是实验室级别也可以是工业生产级别的规模。
本文中所指的术语“细菌”包括但不限于革兰氏阳性菌和革兰氏阴性菌,特别是常用于发酵培养的细菌,例如芽孢杆菌、大肠杆菌、乳杆菌、乳球菌等,优选为大肠杆菌、枯草芽孢杆菌。本文中所指的术语“真菌”包括但不限于真核产孢的微生物,包括但不限于酵母,优选面包酵母、饲料酵母、酿酒酵母、毕赤酵母等;假丝酵母;霉菌,例如根霉、曲霉等。优选为酿酒酵母和毕赤酵母。
实施例1大肠杆菌菌体的提取和分析
1.试剂
LB肉汤培养基(生工生物,A507002):10g/L胰蛋白胨,10g/L NaCl,5g/L酵母提取物;壳聚糖溶液:将1.5g壳聚糖(阿拉丁试剂,C105799)溶解至100mL1%乙酸溶液(FISHER,A35-500),得到1.5%的壳聚糖醋酸溶液。
羧化壳聚糖溶液:将1.5g羧化壳聚糖(阿拉丁试剂,C105800)溶解至100mL超纯水中,得到1.5%的羧化壳聚糖水溶液。
海藻酸钠溶液:将1.0g海藻酸钠(阿拉丁试剂,S100127)溶解至100mL超纯水中,得到1%的海藻酸钠水溶液。
菌体重悬液:50mM葡萄糖(沪试,10010518),25mM Tris-HCl(pH8.0)(沪 试,30188336),10mM EDTA(pH8.0)(沪试,10009717)。
裂解液:0.2mol/L NaOH(沪试,10019718),1%(m/V)SDS(沪试,30166428)。
中和液(100mL):60mL 5M乙酸钾(沪试,30154518),11.5mL冰醋酸(FISHER,A35-500),28.5mL蒸馏水。
RNase A:10mg/mL(翌圣生物,10405ES03)。
pUC19质粒:Thermo Scientific SD0061
大肠杆菌Top10菌株:TIANGEN CB104
pUC19质粒的序列如下:
1.pUC19 DNA序列
>pUC19(SEQ ID NO:1)
2.方法:
2.1小体积大肠杆菌发酵液的羧化壳聚糖沉降处理
在LB培养基中过夜培养含pUC19质粒的大肠杆菌Top 10菌株,得到大肠杆菌发酵液,取样测OD600。取6份1mL该发酵液用于羧化壳聚糖的沉降实验。向1mL的发酵液中依次加入不同添加量的羧化壳聚糖溶液,漩涡混匀;之后添加对应不同量的海藻酸钠溶液,漩涡混匀后静置30min。另取1ml发酵液做空白对照。检测对照组和羧化壳聚糖处理组的OD600。羧化壳聚糖和海藻酸钠的添加量见表1。根据沉降前后的OD600计算沉降率,沉降率=(原样品OD600-沉降后样 品OD600)/原样品OD600。
2.2羧化壳聚糖沉降处理小体积样品对质粒DNA抽提的影响
采用不同的处理方法对使用2.1的方法制备得到对照组样品和实验组1,2,3样品中的大肠杆菌菌体。具体流程如下:(1)对照组:3500g离心菌液1min,去除发酵上清得到菌体;(2)实验组:用移液枪吸取沉降后的发酵液上清,得到在离心管底部沉降的菌体。用250μL菌体重悬液和1μL RNase A重悬菌体,之后加入250μL裂解液,轻柔颠倒混匀裂解菌体,之后加入350μL中和液,轻柔颠倒混匀。12000g离心后收集上清,加入适量磁珠混合均匀,使磁珠吸附上清中的DNA。之后用磁铁吸附磁珠,去除上清液。之后加入70%乙醇清洗磁珠两次,晾干乙醇,用50μL的超纯水洗脱磁珠得到质粒DNA,用分光光度计检测质粒DNA浓度,计算质粒DNA得率。
2.3大体积大肠杆菌发酵液的羧化壳聚糖和壳聚糖沉降效果比较
在2个250mL锥形摇瓶中分别加入150mL LB培养基中过夜培养含pUC19质粒的大肠杆菌Top 10菌株,得到大肠杆菌发酵液,取样测0D600。向羧化壳聚糖沉降组添加小试优选的絮凝剂使用量:8%羧化壳聚糖和8%的海藻酸钠;向壳聚糖沉降组添加公开专利(CN109439584A)中优选的絮凝剂添加量:4%壳聚糖和2%的海藻酸钠。两组实验的添加过程和操作如下:
羧化壳聚糖组:向摇瓶中添加12ml(添加量8%)羧化壳聚糖溶液,水平手动摇匀30s,之后加入相同体积的海藻酸钠溶液(添加量8%),手动摇匀30s。低温静置4h,使大肠杆菌菌体充分沉降,之后分别取上清测OD600,计算沉降率。
壳聚糖组:向摇瓶中添加6ml(添加量4%)壳聚糖溶液,在振荡器器上以120-150rpm的转速快速混匀2-3min,之后加入3ml的海藻酸钠溶液(添加量2%),在振荡器上以50-60rpm的转速缓慢混匀2-5min。低温静置4h,使大肠杆菌菌体充分沉降,之后分别取上清测OD600,计算沉降率。
采用手工倾倒的方法从摇瓶中缓慢倒出上清液,用量筒量取剩余菌液的体积,计算培养上清去除率。沉降率=(原样品OD600-沉降后样品OD600)/原样品OD600;培养上清去除率=(培养体积-沉降后菌液体积)/培养体积。
2.4羧化壳聚糖和壳聚糖处理大体积样品对质粒DNA抽提的影响
将羧化壳聚糖和壳聚糖沉降后得到的浓缩菌液用于质粒DNA提取测试,以150mL过夜培养的大肠杆菌菌液3500g离心10min收集得到的菌体为对照组。由于壳聚糖处理后剩余菌液的体积较大,使用电动移液器小心去除未能倾倒干净的培养基,使最终浓缩菌液的体积与羧化壳聚糖处理组相同(10ml左右)。向对照组离心得到的菌体中加入10ml的菌体重悬液,使菌体充分重悬没有结块。之后向对照组 和实验组的菌液中加入10μL RNase A,水平摇匀10s,之后加入10mL裂解液,水平摇匀30s裂解菌体,之后加入12μL中和液,水平摇匀30s。将摇瓶中的中和液转移至50ml离心管,3500g离心10min后收集上清,加入适量磁珠混合均匀,使磁珠吸附上清中的DNA。之后用磁铁吸附磁珠,去除上清液。之后加入70%乙醇清洗磁珠两次,并使乙醇充分挥发后,用1000μL的超纯水洗脱磁珠得到质粒DNA。用分光光度计检测质粒DNA浓度,计算质粒DNA得率。用琼脂糖凝胶电泳方法观察质粒条带和超螺旋情况。琼脂糖凝胶电泳的实验步骤参照《分子克隆实验指南第四版》(Molecular Cloning:A Laboratory Manual(Fourth Edition),Michael R.Green,Joseph Sambrook,Cold Spring Harbor Laboratory Press,2012)。
2.5单独使用羧化壳聚糖处理大体积样品对菌体沉降和质粒DNA抽提的影响在2个250mL锥形摇瓶中分别加入150mL LB培养基中过夜培养含pUC19质粒的大肠杆菌Top 10菌株,得到大肠杆菌发酵液,取样测OD600。向实验组添加小试优选的絮凝剂使用量:8%羧化壳聚糖;向对照组添加等体积的超纯水。两组实验的添加过程和操作如下:
实验组:向摇瓶中添加12ml(添加量8%)羧化壳聚糖溶液,水平手动摇匀30s,低温静置,使大肠杆菌菌体充分沉降,在4h和6h时分别取上清测OD600,计算沉降率。
对照组:向摇瓶中添加12ml超纯水,水平手动摇匀30s,低温静置,在4h和6h时分别取上清测OD600,计算沉降率。
采用手工倾倒的方法从摇瓶中缓慢倒出上清液,用量筒量取剩余菌液的体积,计算培养上清去除率。沉降率=(原样品OD600-沉降后样品OD600)/原样品OD600;培养上清去除率=(培养体积-沉降后菌液体积)/培养体积
3.实验结果与分析
3.1羧化壳聚糖对小体积大肠杆菌发酵液的沉降效果
如方法部分2.1所述使用不同的添加量的羧化壳聚糖和海藻酸钠对1mL过夜培养大肠杆菌菌液的菌体沉降效果的影响,结果如表1所示。在一定范围内,随着羧化壳聚糖和海藻酸钠添加量的提高,菌体沉降率呈上升趋势。当羧化壳聚糖和海藻酸钠的添加量分别为8%和8%时,菌体沉降效果最好可达到93.5%。当两种絮凝剂的添加量继续增加时,沉降效果并未进一步提升,反而有下降趋势。因此在后续的研究中采用8%的羧化壳聚糖添加量和8%的海藻酸钠添加量作为沉降条件。
表1不同添加量的羧化壳聚糖和海藻酸钠对1mL大肠杆菌发酵液中的菌体沉降效果
3.2羧化壳聚糖和海藻酸钠沉降菌体对质粒小体积样品提取的影响
选择上述获得的羧化壳聚糖和海藻酸钠实验组1,2,3得到的沉降菌体做质粒小量制备测试,以离心法收集得到的菌体做对照(如方法部分2.2),结果见表2。对照组的质粒得率为9.4μg,实验组3使用8%的羧化壳聚糖和海藻酸钠沉降菌体得到了最高的质粒得率14.5μg,为对照组的1.5倍;且随着菌体沉降率的提高,质粒得率呈线性增长趋势。对照组和实验组小规模提取得到的质粒DNA A260/A280均在1.8-2.0之间,没有RNA和蛋白污染,可用于下一步研究。值得注意的是,在小体积条件下采用沉降收集菌体得到的质粒得率明显高于离心收集菌体的质粒得率,羧化壳聚糖或海藻酸钠对质粒的得率可能有一定促进作用。
表2羧化壳聚糖和海藻酸钠沉降菌体对质粒小规模提取的影响
3.3羧化壳聚糖和壳聚糖对大体积大肠杆菌发酵液的沉降效果
采用本研究中如方法2.1部分的小规模提取得到的适合羧化壳聚糖沉降菌体的最佳条件:8%添加量的羧化壳聚糖和海藻酸钠和已公开专利申请(CN109439584A)中报道的适合壳聚糖和海藻酸钠沉降菌体的最佳条件:4%添加量的壳聚糖和2%添加量的海藻酸钠,对150mL过夜培养的大肠杆菌菌液进行沉降实验,结果如表3所示。从表3可以看出,在原始发酵液的OD600相近的情况下,壳聚糖对菌体的沉降率略高于羧化壳聚糖,分别为98.4%和92.7%。然而通过直接倾倒去除的培养基上清时,羧化壳聚糖组的培养基去除率远高于壳聚糖组,分别为93%和62%。采用直接倾倒的方法去除培养基上清后,羧化壳聚糖处理组的剩余菌液体积仅为10.5ml。沉降后培养基去除率高的特性,使羧化壳聚糖沉降后的样品可以通过自 动化机械臂的方式实现批量自动样品处理,有利于菌体沉降后下游整个自动化操作的***整合。
表3羧化壳聚糖和壳聚糖对大体积大肠杆菌的沉降结果
3.4羧化壳聚糖和壳聚糖处理大体积样品对质粒DNA抽提的影响
考察了3.3中得到的沉降后大肠杆菌菌液通过碱裂解方法制备质粒DNA的得率。以离心收集得到的菌体制备质粒DNA做对照。由于壳聚糖组沉降处理后的菌液体积较大,因此使用移液***动去除剩余的培养基,使最终菌液体积减少至10mL左右。随后向菌液中加入10μL RNase A,水平摇动混合均匀后加入碱裂解液裂解菌体。随后按照2.4中所述方法提取质粒DNA.抽提得到的质粒DNA得率见表4。
表4羧化壳聚糖和壳聚糖沉降大肠杆菌的质粒得率
从表4的抽提结果可以看出,羧化壳聚糖沉降得到的菌体在进行质粒抽提时与离心方法收集得到的菌体进行质粒抽提得到的质粒得率相差不大,分别为330.5μg和324.5μg。而壳聚糖沉降会显著降低菌体中的质粒抽提效率,质粒得率仅37.2μg,约为羧化壳聚糖处理组的11%。在对沉降得到的菌体进行裂解处理时目测观察发现,壳聚糖和海藻酸钠使大肠杆菌形成较大的絮状菌块,难以分散均匀,进一步在碱裂解时会产生裂解不充分的现象。而羧化壳聚糖和海藻酸钠沉降得到的大肠杆菌外观细腻,手动摇晃后可以快速分散均匀,对后期的碱裂解不产生影响(结果未显示)。因此,使用壳聚糖和海藻酸钠沉降菌体更适合于去除发酵液中的菌体,收集发酵上清的应用场景;而羧化壳聚糖和海藻酸钠沉降菌体不仅可用于发酵上清的收集,也可用于菌体的后续处理,例如提取菌体内部的生物制品:质粒DNA,RNA,胞内蛋白等。
根据质粒DNA的A
260/A
280测试结果(表2)和琼脂糖凝胶电泳图中显示的质粒 超螺旋比例结果(图1)也可以看出羧化壳聚糖方法收集菌体对抽提得到的质粒DNA的质量无影响。
3.5单独使用羧化壳聚糖处理大体积样品对菌体沉降和质粒DNA抽提的影响考察了单独使用羧化壳聚糖对大肠杆菌菌体沉降和质粒DNA抽提得率的影响。以不加羧化壳聚糖的样品做沉降效率对照和质粒得率对照。结果见表5.
表5单独使用羧化壳聚糖对大体积菌液沉降和质粒DNA抽提得率的影响
从表5可以看出,沉降4h后实验组的菌体沉降率达到80.5%,低于羧化壳聚糖和海藻酸钠沉降4h的菌体沉降率92.7%(表3)。然而,在沉降6h后单独使用8%的羧化壳聚糖能够达到羧化壳聚糖和海藻酸钠联用时沉降4h时相当的菌体沉降效果(95.2%)。虽然单独使用羧化壳聚糖的沉降时间比使用两种絮凝剂的沉降时间多了2h,但简化了试剂添加流程,且避免了自动化操作过程中由于海藻酸钠粘度过高造成的加液不准和管路堵塞风险,具有更大的应用价值。
此外,当所需要处理的大肠杆菌菌液体积或批次量需要放大时,离心法和膜过滤法均存在批次处理样品的数量有限,需要较多人工操作,难以实现自动化,且需要增加相应的离心机或膜过滤设备造成成本增加等挑战。而本发明的沉降法仅需要添加1-2种絮凝剂,在低温条件下通过重力沉降即可实现菌体和发酵培养基的分离,方法简单,成本较低,容易实现自动化批量放大处理,优于离心和膜过滤方法。
4.结果总结:
如上所述,发明人出乎意料地发现,采用可溶于水的羧化壳聚糖和海藻酸钠或单独使用羧化壳聚糖均可以有效实现大肠杆菌发酵液中的菌体沉降,避免离心或膜过滤,操作简便。大肠杆菌发酵液经絮凝剂处理后,可通过倾倒方法去除发酵上清,得到沉降菌体。该过程可通过机械臂实现自动化。用本发明的沉降方法沉降得到的菌体沉淀较为分散,不结团形成絮状,有利于菌体裂解和后续胞内质粒DNA,RNA和蛋白的提取。
对于本领域普通技术人员来说显而易见的是,对本文描述的各种实施方式可以进行各种更改和改变,而不背离本文教导的精神或范围。因而,拟使各种实施方式将各种实施方式的其它更改和改变覆盖在本教导的范围之内。
实施例2毕赤酵母菌体沉降
1.试剂
YPD培养基:1%酵母提取物(Thermo Scientific,LP0021B),2%蛋白胨(Thermo Scientific,LP0042B),2%葡聚糖(国药试剂,63005518)。
羧化壳聚糖溶液:将1.5g羧化壳聚糖(阿拉丁试剂,C105800)溶解至100mL超纯水中,得到1.5%的羧化壳聚糖水溶液。
2.方法
在4ml YPD培养基中培养毕赤酵母GS115菌株,得到毕赤酵母GS115发酵液,取样测OD600。在发酵液中加入不同添加量的羧化壳聚糖溶液,漩涡混匀后4℃静置。取不添加羧化壳聚糖的发酵液做空白对照。每小时检测对照组和羧化壳聚糖处理组的OD600。根据沉降前后的OD600计算沉降率,沉降率=(原样品OD600-沉降后样品OD600)/原样品OD600。
3.结果
如方法部分所述使用不同的添加量的羧化壳聚糖对4mL毕赤酵母菌液的菌体沉降效果的影响,结果如表6所示。
表6不同添加量的羧化壳聚糖对毕赤酵母发酵液中的菌体沉降效果
在一定范围内,随着羧化壳聚糖添加量的提高,菌体沉降率呈上升趋势。当羧化壳聚糖添加量分别为8%时,菌体沉降效果最好可达到94.2%。进一步增加羧化壳聚糖的添加量,沉降效果未进一步提升。
该实施例说明,羧化壳聚糖对毕赤酵母的细胞沉降有较好作用,可以用于毕赤酵母菌体收集或沉降后的培养基上清收集,用于后续的蛋白提纯等应用。
Claims (11)
- 一种从细菌或真菌发酵培养液中收集菌体的方法,其特征在于,该方法包括步骤:a)混合细菌或真菌发酵培养所得的发酵培养液和羧化壳聚糖,得到混合物;b)在混合物中加入海藻酸钠溶液,混合均匀;和c)沉淀,获得菌体沉淀物或发酵培养液上清。
- 如权利要求1所述的方法,其中b)步骤被省略。
- 如权利要求1或2所述的方法,还包括在步骤c)后回收菌体或回收发酵培养液上清的步骤d)。
- 如权利要求1或2所述的方法,其中所述细菌选自芽孢杆菌、大肠杆菌、乳杆菌、乳球菌,优选为大肠杆菌或枯草芽孢杆菌;所述真菌选自酵母,优选面包酵母、饲料酵母、酿酒酵母、毕赤酵母;假丝酵母;霉菌,更优选根霉、曲霉,最优选为酿酒酵母和毕赤酵母。
- 如权利要求1或2所述的方法,其中加入的羧化壳聚糖量为发酵培养液的1%-32%重量,优选1-16%重量,更优选4%-8%重量,最优选8%重量。
- 如权利要求1所述的方法,其中步骤b)中混合的海藻酸钠量为发酵培养液的0%-16%重量,优选0%-8%重量,更优选0%或8%重量。
- 如权利要求1所述的方法,其中在发酵培养液中加入的羧化壳聚糖海藻酸钠分别为发酵培养液的8%重量,优选发酵培养液是大肠杆菌和毕赤酵母发酵培养液。
- 如权利要求2所述的方法,其中在发酵培养液中加入的羧化壳聚糖是发酵培养液的8%重量,优选发酵培养液是大肠杆菌和毕赤酵母发酵培养液。
- 如权利要求1或2所述的方法,其中所述羧化壳聚糖选自氨基(-NH 2),或羟基(-OH),或氨基(-NH 2)和羟基(-OH)被羧化基团修饰后的壳聚糖,其中所述羧化基团是含1-3个羧基取代的C1-C6的直链或支链烃基,优选含3个羧基,更优选2个羧基,最优选为1个羧基;所述直链或支链烃基优选具有1-6个碳原子。
- 如权利要求4所述的方法,其中大肠杆菌的菌株包括E.coli TOP10,E.coli JM109,E.coli HB101,E.coli DH5α,E.coli BL21(DE3),以及其它大肠杆菌K菌株和B菌株的衍生菌株,包括E.coli DH10B,E.coli JM110,E.coli MC1061,E.coli MG1655,E.coli Stbl2,E.coli Stbl3,E.coli T-Fast,E.coli XL1 Blue,E.coli Rosetta 2(DE3)等,或其中酵母的菌株包括酿酒酵母(Saccharomyces cerevisiae),毕赤酵母(Pichia pastoris).
- 如权利要求3所述的方法,其中所述步骤d)中的回收选自倾倒培养基或自动化机械臂倾倒培养基获得菌体或培养基上清,磁珠吸附DNA/RNA或蛋白质,清洗DNA/RNA或蛋白质,优选倾倒,更优选手动倾倒培养基上清或自动化机械臂倾倒培养基上清。
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