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Construction of Glycine Oxidase Mutant Libraries by Random Mutagenesis, Site Directed Mutagenesis and DNA Shuffling
通过随机突变、点位定向诱变和DNA改组构建甘氨酸氧化酶突变库   

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Abstract

Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate (Pedotti et al., 2009). Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution.

Materials and Reagents

  1. Bacillus cereus HYC-7
  2. Escherichia coli (E.coli) DH5α strain, bacteriophage T7
  3. Glyphosate (Sigma-Aldrich, catalog number: PS1051 )
  4. Tryptone (Difco)
  5. Yeast extract (Difco)
  6. Ampicillin
  7. o-Dianisidine dihydrochloride (Sigma-Aldrich, catalog number: D3252 )
  8. Horseradish peroxidase (Sigma-Aldrich, catalog number: P6782 )
  9. Protein expression vector of pGEX-6P-1 (the plasmid full length 4,984 bp) (GE Healthcare, catalog number: 28-9546-48 ; Genbank accession number: U78872.1)
  10. Recombinant plasmid pGEX-GO contains encoding gene of glycine oxidase from Bacillus cereus HYC-7
    The nucleotide sequence (1,110 bp) was submitted to the NCBI Genbank and gained the accession number (KC203486.1).
  11. Taq DNA polymerase (Takara, catalog number: R500A )
  12. DpnI restriction enzyme (Takara, catalog number: 1235A )
  13. dATP, dTTP, dCTP, dGTP (Takara, catalog numbers: 4026Q , 4029Q , 4028Q , 4027Q )
  14. TransStart® FastPfu DNA polymerase (TransGen Biotech, catalog number: AP221-01 )
  15. High Pure dNTPs (TransGen Biotech, catalog number: AD101-01 )
  16. Luria-Bertani medium (see Recipes)

Equipment

  1. 96 deep-well plates (Axygen, catalog number: P-DW-20-C-S )
  2. Gel purification column (Axygen)
  3. Thermo Multiskan spectrum plate reader (Thermo Scientific, catalog number: 51118600 )
  4. Thermal cyclers (Bio-Rad Laboratories, catalog number: 186-1096 )
  5. Ultrasonic processor (Sigma-Aldrich, catalog number: Z412619-1EA )

Procedure

  1. Random mutagenesis
    1. Prepare the amplification mixture (100 µl) as follows:
      10 µl of 10x Taq buffer (Mg2+ plus)
      5 µl of 10 mM Mn2+
      2 µl of 10 mM dGTP and dCTP
      1 µl of 10 mM dATP and dTTP
      2 µl of 100 nM oligonucleotide primer F
      2 µl of 100 nM oligonucleotide primer R
      1 µl of recombinant plasmid pGEX-GO as template
      2 µl of Taq DNA polymerase
      Add ddH2O to a final volume of 100 μl
    2. The error-prone PCR procedure was performed using the following parameters:

      Segment   
      Cycles   
      Temperature   
      Time
      1   
      1   
      94 °C   
      3 min
      2   
      30   
      94 °C   
      30 sec
      59 °C
      30 sec
      72 °C
      80 sec
      3   
      1   
      72 °C   
      7 min

    3. Check product by electrophoresis of 5 μl of error-prone PCR product on 1% agarose gel.
    4. Error-prone PCR products were purified, digested with BamHI and XhoI, cloned into pGEX-6P-1, and transformed into E.coli DH5α to construct the random mutant library.


      Figure 1. Agarose gel electrophoresis of PCR products by the first round error-prone PCR and recombinant plasmids. A. PCR products. Lane 1: Wide Range DNA Marker (500~12,000 bp); Lane 2-5: Error-prone PCR products; B. Recombinant plasmids. Lane 1: The empty vector pGEX-6P-1; Lane 2-11: Recombinant plasmids pGEX-GOs from colonies.

  2. Site directed mutagenesis
    1. Prepare the amplification mixture (50 µl) as follows:
      10 µl of 10x FastPfu buffer (Mg2+ plus)
      1 µl of 10 mM high pure dNTPs
      1 µl of 100 nM oligonucleotide primer F
      1 µl of 100 nM oligonucleotide primer R
      1 µl of dsDNA template
      1 µl of FastPfu DNA polymerase
      Add ddH2O to a final volume of 50 μl
    2. The site directed mutagenesis PCR procedure was performed as the following parameters:

      Segment   
      Cycles   
      Temperature   
      Time
      1  
      1   
      97 °C   
      2 min
      2   
      20   
      94 °C   
      20 sec
      54 °C
      30 sec
      72 °C
      1 min/kb of plasmid length
      3  
      1   
      72 °C   
      7 min

    3. Check product by electrophoresis of 5 μl of the site directed mutagenesis PCR product on 1% agarose gel.
    4. The site directed mutagenesis PCR products were purified, digested with DpnI, and transformed into E.coli DH5α.


      Figure 2 Agarose gel electrophoresis analysis of cylcled PCR product and mutated plasmid.  A. PCR product. Lane 1: Wide Range DNA Marker (500~12,000 bp); Lane 2: Cycled PCR product. B. Mutated plasmid. Lane 1: The empty vector pGEX-6P-1; Lane 2: Mutated plasmid.

  3. DNA shuffling
    1. Obtaining DNA fragments for shuffling.
      1. Prepare the parental genes by PCR amplification (100 µl) as follows:
        10 µl of 10x Taq buffer (Mg2+ plus)
        2 µl of 10 mM High Pure dNTPs
        2 µl of 100 nM oligonucleotide nested primer F1
        2 µl of 100 nM oligonucleotide nested primer R1
        1 µl of beneficial mutatant as template
        2 µl of Taq DNA polymerase
        Add ddH2O to a final volume of 100 μl
      2. The DNA shuffling PCR procedure was performed as the following parameters:

        Segment   
        Cycles  
        Temperature   
        Time
        1   
        1   
        94 °C   
        3 min
        2   
        30   
        94 °C   
        30 sec
        62 °C
        30 sec
        72 °C
        80 sec
        3   
        1   
        72 °C   
        7 min

      3. Check product by electrophoresis of 5 μl of product on 1% agarose gel.
      4. Mix ~1 μg of each purified PCR products.
      5. Prepared for DNA fragmentation by ultrasonic treatment at 0 °C for 40 min to generate a pool of fragments, then the DNA fragments between 100~200 bp were purified using gel purification column.
      6. Check the DNA fragments by electrophoresis of 10 μl of product on 3% agarose gel.


        Figure 3. Schematic of parental genes with the nested primers in the process of DNA shuffling

    2. Reassembled by primerless PCR.
      1. Prepare the amplification mixture (50 µl) as follows:
        10 µl of 10x Taq buffer (Mg2+ plus)
        2 µl of 10 mM high pure dNTPs
        42 µl of purified fragment DNA
        1 µl of Taq DNA polymerase
        Add ddH2O to a final volume of 50 μl
      2. The primerless PCR procedure was performed as the following parameters:

        Segment   
        Cycles   
        Temperature   
        Time
        1   
        1   
        94 °C   
        3 min
        2  
        60   
        94 °C   
        30 sec
        40 °C
        30 sec
        72 °C
        20 sec + 1 sec per cycle
        3   
        1
        72 °C   
        7 min

      3. Check product by electrophoresis of 5 μl of PCR product on 1% agarose gel. A smear of reassembled product that extends above the molecular weight of the parent gene should be visible.
    3. Amplification of full-length sequences.
      1. Prepare the amplification mixture (50 µl) as follows:
        10 µl of 10x Taq buffer (Mg2+ plus)
        2 µl of 10 mM high pure dNTPs
        1 µl of 100 nM oligonucleotide nested primer F2
        1 µl of 100 nM oligonucleotide nested primer R2
        5 µl of unpurified reassembly reaction mixture as template
        1 µl of Taq DNA polymerase
        Add ddH2O to a final volume of 50 μl
      2. The PCR procedure was performed as the following parameters:

        Segment   
        Cycles   
        Temperature   
        Time
        1   
        1  
         94 °C   
        3 min
        2   
        30   
        94 °C   
        30 sec
        59 °C
        30 sec
        72 °C
        80 sec
        3   
        1   
        72 °C   
        7 min

      3. Check product by electrophoresis of 5 μl of product on 1% agarose gel.
      4. PCR products were purified, digested with BamHI and XhoI, cloned into pGEX-6P-1, and transformed into E.coli DH5α to create the DNA shuffling library.


        Figure 4. The schematic of glyphosate oxidase gene by DNA shuffling. A. Amplification of six variants GO. Lane 1: Wide Range DNA Marker (500~12,000 bp); Lanes 2-7: DNA fragments encoding variants GO. B. DNA fragmentation. Lanes 1-8: DNA fragments were treated by ultrasonic at 0 °C with a time gradient of 5 min from 0 to 35 min; Lane 9: 20 bp DNA Ladder Marker (20~500 bp). C. Purification of DNA fragments. Lane 1: 20 bp DNA Ladder Marker (20~500 bp); Lane 2: DNA fragments of 100~200 bp were purified from an agarose gel. D. Fragments were reassembled without primers. Lane 1: Wide Range DNA Marker (500~12,000 bp); Lane 2: DNA fragments were reassembled into a full-length gene by 60 cycles without primers. E. The reassembled full-length products were amplified by the standard PCR. Lane 1: Wide Range DNA Marker (500~12,000 bp); Lane 2: PCR product with primers.

  4. Screening
    1. The resulting library of BceGO mutants were expressed into 96 deep-well plates (containing 0.6 ml Luria-Bertani medium) and transferred onto Luria-Bertani agar plates as corresponding copies, followed by an overnight growth (37 °C, 300 rpm).
    2. When the cultures grew to saturation, both IPTG (at a final concentration of 0.1 mM) and the bacteriophage T7 (above 100 particles per cell) were added into 96 deep-well plates to synchronize the induction of recombinant mutants with the release of the lysis of the host E.coli DH5α at 37 °C with shaking for 6 h.
    3. The enzyme-coupled colorimetric assay (200 µl) was performed as follows:
      159 µl of lysis cell extracts
      20 µl of 50 mM glyphosate (at a decreasing substrate concentration gradient in sequential rounds of screening system)
      20 µl of 0.32 mg/ml o-dianisidine dihydrochloride
      1 µl of 5 unit/ml horseradish peroxidase
      Then incubated at 25 °C for 8 h
    4. The absorbance change at 450 nm for each well in the microtiter plates was measured and compared with the control (harboring wild-type BceGO or containing the empty vector pGEX-6P-1). Mutants that outperformed the wild-type were chosen for further activity analysis (Figure 1).


      Figure 5. The screening process of glycine oxidase mutant library

Representative data

Table 1. The apparent kinetic parameters on glycine and glyphosate measured for wild-type BceGO and variants obtained by random mutagenesis, site saturation mutagenesis and DNA shuffling


Glycine
Glyphosate
kcat,app (min-1)
Km,app (mM)
kcat,app (min-1)
Km,app (mM)
Wild-type
8.17 ± 0.31
1.04 ± 0.17
5.72 ± 0.42
84.79 ± 4.25
22D11
1.16 ± 0.05
54.6 ± 3.47
2.95 ± 0.21
8.29 ± 0.27
23B1
4.56 ± 0.38
0.99 ± 0.04
7.15 ± 0.62
18.88 ± 2.52
B1R
0.44 ± 0.03
58.5 ± 5. 26
2.78 ± 0.46
2.45 ± 0.15
B2R11
1.86 ± 0.09
105.6 ± 7.31
3.83 ± 0.17
2.77 ± 0.21
B2R14
1.35 ± 0.12
92.5 ± 7. 43
4.16 ± 0.14
2.17 ± 0.37
B2R23
13.02 ± 0.96
101.8 ± 8.29
30.80 ± 1.33
3.80 ± 0.26
B2R81
5.41 ± 0.83
134.4 ± 10.33
7.27 ± 0.75
4.37 ± 0.30
B3S1
5.43 ± 0.79
41.55 ± 3.32
11.67 ± 0.98
0.53 ± 0.03
B3S4
5.68 ± 0.64
80.43 ± 5.01
12.99 ± 1.14
1.37 ± 0.07
B3S6
10.14 ± 1.32
138.1 ± 12.16
13.22 ± 1.78
1.69 ± 0.08
B3S7
2.30 ± 0.31
41.64 ± 2.10
4.63 ± 0.39
0.57 ± 0.02

Especially, B3S1 demonstrated a 160-fold increase in substrate affinity for glyphosate, a 326-fold increase in catalytic efficiency towards glyphosate and a significant enhancement in the specificity constant over the wild-type BceGO, achieving the goal of efficient oxidation of glyphosate by evolution of glycine oxidase.

Notes

  1. The error-rate is controlled by varying the amount of MnCl2 and unblanced dNTPs added to the error-prone PCR reaction.
  2. Design nested primer to amplify the parental genes for the DNA fragmentation and full-length sequences from reassembly products.

Recipes

  1. Luria-Bertani medium
    10 g/L tryptone
    5 g/L yeast extract
    10 g/L NaCl
    Ampicillin (50 mg/L) was added as needed.

Acknowledgments

We thank Drs. Ziduo Liu and Dexin Kong for valuable discussions about this article.

References

  1. GST Gene Fusion System Handbook. (2002). Biosciences, Amersham.
  2. Zhan, T., Zhang, K., Chen, Y., Lin, Y., Wu, G., Zhang, L., Yao, P., Shao, Z. and Liu, Z. (2013). Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution. PLoS One 8(11): e79175.

简介

草甘膦,广泛应用于全世界农业的广谱除草剂在莽草酸途径中抑制5-烯醇丙酮酸莽草酸-3-磷酸合酶,并且已经报道甘氨酸氧化酶(GO)能够催化各种胺的氧化脱氨基, 切割草甘膦中的CN键(Pedotti等人,2009)。 在这里,为了改善从草甘膦降解的蜡状芽孢杆菌菌株(BceGO)克隆的甘氨酸氧化酶的催化活性,我们使用基于噬菌体T7裂解的方法, 通过筛选氧化酶活性并通过定向进化改造编码BceGO的基因。

材料和试剂

  1. <蜡>芽孢杆菌 HYC-7
  2. 大肠杆菌(大肠杆菌)DH5α菌株,噬菌体T7
  3. 草甘膦(Sigma-Aldrich,目录号:PS1051)
  4. 胰蛋白胨(Difco)
  5. 酵母提取物(Difco)
  6. 氨苄西林
  7. 二盐酸二噻吩(Sigma-Aldrich,目录号:D3252)
  8. 辣根过氧化物酶(Sigma-Aldrich,目录号:P6782)
  9. pGEX-6P-1(质粒全长4,984bp)的蛋白质表达载体(GE Healthcare,目录号:28-9546-48; Genbank登录号:U78872.1)
  10. 重组质粒pGEX-GO含有来自蜡状芽孢杆菌 HYC-7的甘氨酸氧化酶的编码基因 核苷酸序列(1,110bp)提交NCBI Genbank并获得登录号(KC203486.1)。
  11. Taq DNA聚合酶(Takara,目录号:R500A)
  12. (Takara,目录号:1235A)。
  13. dATP,dTTP,dCTP,dGTP(Takara,目录号:4026Q,4029Q,4028Q,4027Q)
  14. TransStart FastPfu DNA聚合酶(TransGen Biotech,目录号:AP221-01)
  15. 高纯dNTP(TransGen Biotech,目录号:AD101-01)
  16. Luria-Bertani培养基(见配方)

设备

  1. 96深孔板(Axygen,目录号:P-DW-20-C-S)
  2. 凝胶纯化柱(Axygen)
  3. Thermo Multiskan光谱板读数器(Thermo Scientific,目录号:51118600)
  4. 热循环仪(Bio-Rad Laboratories,目录号:186-1096)
  5. 超声波处理器(Sigma-Aldrich,目录号:Z412619-1EA)

程序

  1. 随机诱变
    1. 如下制备扩增混合物(100μl):
      10μl10×Taq 缓冲液(Mg 2 + 加)
      5μl10mM Mn 2+ 2 +
      2μl10mM dGTP和dCTP 1μl10mM dATP和dTTP
      2μl100nM寡核苷酸引物F
      2μl100nM寡核苷酸引物R
      1μl重组质粒pGEX-GO作为模板 2μl的Taq DNA聚合酶
      将ddH O加到最终体积为100μl的
    2. 使用以下参数进行易错PCR过程:

      区隔   
      周期   
      温度   
      时间
      1   
      1   
      94°C   
      3分钟
      2   
      30   
      94°C   
      30秒
      59°C
      30秒
      72℃
      80秒
      3   
      1   
      72℃   
      7分钟

    3. 通过在1%琼脂糖凝胶上电泳5μl易错PCR产物检查产物
    4. 纯化易错PCR产物,用Bam HI和Xho I I消化, 克隆到pGEX-6P-1中,并转化到大肠杆菌DH5α中以构建   随机突变体库

      图1. PCR的琼脂糖凝胶电泳 产物通过第一轮易错PCR和重组质粒。。   PCR产物。 泳道1:宽范围DNA标记(500〜12,000bp); 泳道2-5: 易错PCR产物; B.重组质粒。 通道1:空 载体pGEX-6P-1; 泳道2-11:重组质粒pGEX-GOs 群落。

  2. 定点诱变
    1. 按如下步骤制备扩增混合物(50μl):
      10μl10x FastPfu 缓冲液(Mg 2 + 加)
      1μl10mM高纯度dNTPs
      1μl100nM寡核苷酸引物F
      1μl100nM寡核苷酸引物R
      1μldsDNA模板
      1μl的FastPfu DNA聚合酶
      将ddH 2 O加至最终体积为50μl
    2. 定点诱变PCR程序如下参数进行:

      区隔   
      周期   
      温度   
      时间
      1  
      1   
      97°C   
      2分钟
      2   
      20   
      94°C   
      20秒
      54°C
      30秒
      72℃
      1分钟/kb的质粒长度
      3  
      1   
      72℃   
      7分钟

    3. 通过在1%琼脂糖凝胶上电泳5μl位点定向诱变PCR产物检查产物
    4. 纯化位点定向诱变PCR产物,用Dpn I消化,并转化到大肠杆菌DH5α中。


      图2圆柱PCR产物的琼脂糖凝胶电泳分析 和突变的质粒。 A. PCR产物。 泳道1:宽范围DNA标记 (500〜12,000bp)。 泳道2:循环PCR产物。 B.突变的质粒。 泳道1:   空载体pGEX-6P-1; 泳道2:突变的质粒。

  3. DNA改组
    1. 获得用于改组的DNA片段。
      1. 通过PCR扩增(100μl)如下制备亲本基因:
        10μl10×Taq 缓冲液(Mg 2 + 加)
        2μl10 mM High Pure dNTPs
        2μl100nM寡核苷酸嵌套引物F1
        2μl100nM寡核苷酸嵌套引物R1 / 1μl有益突变体作为模板
        2μlTaq DNA聚合酶
        将ddH O加到最终体积为100μl的
      2. DNA改组PCR过程如下参数进行:

        区隔   
        周期  
        温度   
        时间
        1   
        1   
        94°C   
        3分钟
        2   
        30   
        94°C   
        30秒
        62°C
        30秒
        72℃
        80秒
        3   
        1   
        72℃   
        7分钟

      3. 通过在1%琼脂糖凝胶上电泳5μl产物检查产物
      4. 混合〜1μg每种纯化的PCR产物
      5. 通过在0℃下超声处理来制备DNA断裂 40分钟以产生片段集合,然后产生DNA片段 在100〜200bp之间使用凝胶纯化柱纯化
      6. 通过在3%琼脂糖凝胶上电泳10μl产物检查DNA片段

        图3.在DNA改组过程中亲本基因与嵌套引物的示意图

    2. 通过无引物PCR重组。
      1. 按如下步骤制备扩增混合物(50μl):
        10μl10×Taq 缓冲液(Mg 2 + 加)
        2μl10mM高纯度dNTPs
        42μl纯化的片段DNA
        1μl的Taq DNA聚合酶
        将ddH 2 O加至最终体积为50μl
      2. 无引物PCR操作如下参数进行:

        区隔   
        周期   
        温度   
        时间
        1   
        1   
        94°C   
        3分钟
        2  
        60   
        94°C   
        30秒
        40℃
        30秒
        72℃
        20秒+ 1秒/周期
        3   
        1
        72℃   
        7分钟

      3. 检查产物通过电泳5μlPCR产物在1% 琼脂糖凝胶。 重新组装的产品的涂片在上面延伸 亲本基因的分子量应该是可见的。
    3. 全长序列的扩增。
      1. 按如下步骤制备扩增混合物(50μl):
        10μl10×Taq缓冲液(Mg 2+ + 加) 2μl10mM高纯度dNTPs
        1μl100nM寡核苷酸嵌套引物F2
        1μl100nM寡核苷酸嵌套引物R2 / 5μl未纯化的重组反应混合物作为模板
        1μlTaq DNA聚合酶
        将ddH 2 O加至最终体积为50μl
      2. PCR过程如下参数进行:

        区隔   
        周期   
        温度   
        时间
        1   
        1  
          94°C   
        3分钟
        2   
        30   
        94°C   
        30秒
        59°C
        30秒
        72℃
        80秒
        3   
        1   
        72℃   
        7分钟

      3. 通过在1%琼脂糖凝胶上电泳5μl产物检查产物
      4. 纯化PCR产物,用克隆的BamHI和XhoI I消化 转化到pGEX-6P-1中,并转化到大肠杆菌DH5α中以产生DNA shuffling库。


        图4.草甘膦氧化酶的示意图 基因通过DNA改组。 A.扩增六个变体GO。 泳道1:宽   范围DNA标记(500〜12,000 bp); 泳道2-7:编码DNA片段 变体GO。 B.DNA断裂。 泳道1-8:处理DNA片段 通过在0℃下用0至35分钟的5分钟的时间梯度进行超声波处理; 泳道9:20bp DNA梯形标志物(20〜500bp)。 C.DNA的纯化 片段。 泳道1:20bp DNA梯形标志物(20〜500bp); 泳道2:DNA 从琼脂糖凝胶纯化100〜200bp的片段。 D.片段 无需引物重组。 泳道1:宽范围DNA标记 (500〜12,000bp)。 泳道2:将DNA片段重新装配成a 全长基因60个循环,无引物。 E.重新组装 通过标准PCR扩增全长产物。 泳道1:宽 范围DNA标记(500〜12,000 bp); 泳道2:具有引物的PCR产物。

  4. 筛选
    1. 将所得的BceGO突变体文库表达到96个深孔中 平板(含有0.6ml Luria-Bertani培养基)中并转移 Luria-Bertani琼脂平板作为相应的拷贝, 过夜生长(37℃,300rpm)。
    2. 当文化成长 饱和,IPTG(终浓度为0.1mM) 将噬菌体T7(每个细胞超过100个颗粒)加入96中 深孔板以同步重组突变体的诱导 随后在37℃下释放宿主大肠杆菌DH5α的裂解 摇动6小时
    3. 酶联色比色测定(200μl)如下进行:
      159μl裂解细胞提取物
      20μl50mM草甘膦(在连续轮筛选系统中底物浓度梯度递减)
      20μl0.32mg/ml阳离子 - 二噻啶二盐酸盐
      1μl5单位/ml辣根过氧化物酶
      然后在25℃下孵育8小时
    4. 在微量滴定板中每个孔在450nm处的吸光度变化 测量板并与对照(含有野生型)比较 BceGO或含有空载体pGEX-6P-1)。 突变体 超越野生型进行进一步的活性分析 (图1)。


      图5.甘氨酸氧化酶突变体文库的筛选过程

代表数据

表1.对野生型BceGO和通过随机诱变,位点饱和诱变和DNA改组获得的变体测量的甘氨酸和草甘膦的表观动力学参数


甘氨酸
草甘膦
k cat,app (min -1
K m,app(mM)
k cat,app (min -1
K m,app(mM)
野生型
8.17±0.31
1.04±0.17
5.72±0.42
84.79±4.25
22D11
1.16±0.05
54.6±3.47
2.95±0.21
8.29±0.27
23B1
4.56±0.38
0.99±0.04
7.15±0.62
18.88±2.52
B1R
0.44±0.03
58.5±5. 26
2.78±0.46
2.45±0.15
B2R11
1.86±0.09
105.6±7.31
3.83±0.17
2.77±0.21
B2R14
1.35±0.12
92.5±7. 43
4.16±0.14
2.17±0.37
B2R23
13.02±0.96
101.8±8.29
30.80±1.33
3.80±0.26
B2R81
5.41±0.83
134.4±10.33
7.27±0.75
4.37±0.30
B3S1
5.43±0.79
41.55±3.32
11.67±0.98
0.53±0.03
B3S4
5.68±0.64
80.43±5.01
12.99±1.14
1.37±0.07
B3S6
10.14±1.32
138.1±12.16
13.22±1.78
1.69±0.08
B3S7
2.30±0.31
41.64±2.10
4.63±0.39
0.57±0.02

特别地,B3S1表现出对草甘膦的底物亲和力增加160倍,对草甘膦的催化效率增加326倍并且特异性常数显着增加超过野生型BceGO,实现通过进化有效氧化草甘膦的目标 的甘氨酸氧化酶。

笔记

  1. 错误率通过改变添加到易错PCR反应中的MnCl 2和未消除的dNTP的量来控制。
  2. 设计嵌套引物以扩增用于DNA断裂的亲本基因和来自重组产物的全长序列

食谱

  1. Luria-Bertani培养基
    10 g/L胰蛋白酶
    5g/L酵母提取物
    10g/L NaCl
    根据需要加入氨苄青霉素(50mg/L)

致谢

我们感谢博士。 Ziduo Liu和Dexin Kong对本文的宝贵讨论。

参考文献

  1. GST基因融合系统手册。 (2002)。 Biosciences,Amersham。
  2. Zhan,T.,Zhang,K.,Chen,Y.,Lin,Y.,Wu,G.,Zhang,L.,Yao,P.,Shao,Z.and Liu,Z.(2013)。 通过定向进化改善蜡样芽孢杆菌的甘氨酸氧化酶的草甘膦氧化活性。 PLoS One 8(11):e79175。
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引用:Zhan, T. (2014). Construction of Glycine Oxidase Mutant Libraries by Random Mutagenesis, Site Directed Mutagenesis and DNA Shuffling. Bio-protocol 4(19): e1252. DOI: 10.21769/BioProtoc.1252.
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