WO2020199612A1 - 一种提高重组染料脱色过氧化物酶酶活的方法 - Google Patents

一种提高重组染料脱色过氧化物酶酶活的方法 Download PDF

Info

Publication number
WO2020199612A1
WO2020199612A1 PCT/CN2019/120475 CN2019120475W WO2020199612A1 WO 2020199612 A1 WO2020199612 A1 WO 2020199612A1 CN 2019120475 W CN2019120475 W CN 2019120475W WO 2020199612 A1 WO2020199612 A1 WO 2020199612A1
Authority
WO
WIPO (PCT)
Prior art keywords
dyp
peroxidase
medium
heme
dye
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2019/120475
Other languages
English (en)
French (fr)
Inventor
唐蕾
朱竹兵
孙亚武
顾鹏帅
沈微
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangnan University
Original Assignee
Jiangnan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangnan University filed Critical Jiangnan University
Publication of WO2020199612A1 publication Critical patent/WO2020199612A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to a method for improving the enzymatic activity of recombined dye decolorization peroxidase, and belongs to the technical field of bioengineering.
  • Dye decoloring peroxidase is a new member of the peroxidase family. It uses heme as a prosthetic group and uses hydrogen peroxide as an electron acceptor to catalyze various organics. It can be used as anthraquinone dyes and lignin model compounds.
  • the substrate has potential application value, which can realize the degradation of dye wastewater by biological method, thereby avoiding the secondary pollution problem of degradation by chemical and physical methods.
  • DyP is mainly derived from microorganisms, but DyP obtained from primitive bacteria is not only low in content, but also difficult to separate from other intracellular proteins. Therefore, DyP is often produced by the method of constructing genetic engineering bacteria, such as heterologous expression of DyP in E. coli , But the content of recombinant protein is low, generally below 10mg/L. Recent studies have found that DyP and modified protein fusion expression, the amount of recombinant protein can be increased to about 200mg/L, but the highly overexpressed fusion DyP has almost no activity.
  • Adding 5-ALA to the culture medium is another way to increase the yield of DyP, but the cost is high and it is not suitable for large-scale promotion and application.
  • An object of the present invention is to provide a method for improving the enzyme activity of recombinant DyP, the method being the following (1) and/or (2):
  • the final concentration of Glu and/or Fe 2+ added is 20-100 ⁇ mol/L.
  • the final concentration of added Glu and/or Fe 2+ is 40-100 ⁇ mol/L.
  • the final concentration of the added Glu and/or Fe 2+ is 60-100 ⁇ mol/L.
  • the amino acid sequence of the DyP is the sequence shown in GenBank numbering AAZ57111.1 and UniProtKB/Swiss-Prot numbering Q47KB1.
  • the recombinant strain uses a pET series vector as a vector.
  • the fermentation conditions are: after seed culture, transfer to LB medium with an inoculum of 1-5% (v/v), 200-220r/min, 35- Cultivate at 38°C until the OD 600 of the bacteria reaches 0.6-0.8, add IPTG to the medium at a final concentration of 0.1-0.5mmol/L, and induce culture at 28-30°C for 12-20 hours.
  • the seed culture is to inoculate the recombinant strain into the LB medium, and cultivate for 10-15 hours at 200-220 r/min at 35-38°C.
  • the formula of LB medium is: tryptone 10g/L, yeast extract 5g/L, and sodium chloride 10g/L.
  • the E. coli is E. coli BL21 (DE3).
  • the co-expression uses pET-28a as an expression vector.
  • the second purpose of the present invention is to provide a genetically engineered bacteria that co-expresses the gene encoding DyP and the key enzyme gene heme in the heme synthesis pathway in Escherichia coli.
  • the amino acid sequence of the DyP is as shown in GenBank numbering AAZ57111.1
  • the sequence shown, the amino acid sequence of the key enzyme of the heme synthesis pathway is the sequence shown in GenBank numbering CAQ31712.1.
  • the third objective of the present invention is to provide a method for producing DyP, which is to first co-express the gene encoding DyP and the key enzyme gene heme synthesis pathway heme in E. coli, and then use the co-expressed strain to ferment to produce DyP.
  • the amino acid sequence of DyP is the sequence shown in GenBank numbering AAZ57111.1
  • the amino acid sequence of the key enzyme in the heme synthesis pathway is the sequence shown in GenBank numbering CAQ31712.1.
  • the fermentation conditions are to activate the co-expressed strain at 35-38°C, 200-220r/min for 8-14h, and then press 1-5% inoculum (V/V ) Transfer to LB medium, culture until the OD 600 value of the bacteria is 0.6-0.8, add IPTG at a final concentration of 0.1-1 mmol/L, and induce 6-10 hours at 35-38°C.
  • the fermentation medium is LB medium.
  • the fourth object of the present invention is to provide a method for decolorizing Reactive Blue 19, which uses Reactive Blue 19 as a substrate and adds the protein expressed by the genetically engineered bacteria.
  • the decolorization reaction system includes 20 mmol/L acetate buffer, 0.1-0.3 mmol/L final concentration of reactive blue 19, and 180-220 ⁇ g of the protein expressed by the genetically engineered bacteria. , H 2 O 2 with a final concentration of 0.02-0.2 mmol/L.
  • Another object of the present invention is to provide the application of the above method in the biological, textile or chemical fields.
  • the invention provides a method for improving the enzyme activity of recombinant DyP.
  • exogenous substances Glu and/or Fe 2+ By adding exogenous substances Glu and/or Fe 2+ to the fermentation medium, the heme saturation in recombinant DyP is increased.
  • the specific enzyme activity can be increased to 1.37 times that of the control; when adding 80 ⁇ mol/L of FeCl 2 to the medium, the enzyme activity can be increased to that of the control 1.45 times.
  • the method is simple and easy to operate, and the added foreign substances such as Glu and FeCl 2 are cheap and economical.
  • the intracellular hemoglobin content is greatly increased: after the recombinant bacteria are induced at 37°C and 0.3mmol/L IPTG, the intracellular hemoglobin test is performed.
  • the heme concentration in the strain pD is 3.4 ⁇ mol/L, and the co-expressing strain pAD
  • the concentration of heme in the medium increased significantly, reaching 9.8 ⁇ mol/L; adding 20 ⁇ mol/L FeCl 2 and 40 ⁇ mol/L Glu to the culture medium of the strain pAD further increased the concentration of heme to 11.3 ⁇ mol/L and 13.5 ⁇ mol/L;
  • DyP binds more heme: the heme and protein content of DyP is expressed by 408nm (Soret peak) and 280nm wavelength. Soret peak is the characteristic absorption peak of heme, which can be used to characterize the level of heme incorporated into DyP. Except for the strains containing empty plasmids, all other strains showed a Soret peak at 408nm; the analysis peak showed that compared with pD strain 0.637, the co-expression strain pAD was 0.794. It can be seen that the DyP gene and the heme synthesis pathway key enzyme gene heMA Co-expression enhances the binding degree of Dyp and cofactor heme. Exogenous addition of 20 ⁇ mol/L FeCl 2 and 40 ⁇ mol/L Glu respectively increases the peak at 408nm to 0.865 and 0.889;
  • the Dyp enzyme activity was measured with 2,2-diazo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) as a substrate: the Dyp enzyme activity of strain pD was 4.5 U /mg, while the pAD enzyme activity of the co-expression strain reached 9.2U/mg, which was 1.1 times higher than the original. Adding 20 ⁇ mol/L FeCl 2 and 40 ⁇ mol/L Glu separately, the enzyme activity increased to 11.8U/mg, 13.2U/mg .
  • ABTS 2,2-diazo-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt
  • Figure 1 The effects of metal ions and amino acids on cell mass and recombinant DyP enzyme activity.
  • Figure 2 The effect of additives on enzyme activity and heme saturation.
  • Figure 4 The effect of various strains and additives on the content of recombinant DyP heme.
  • Figure 5 The effects of various strains and additives on the spectral absorption of recombinant DyP.
  • Figure 6 The effects of strains and additives on the level of recombinant DyP enzyme activity.
  • DyP dye decolorizing peroxidase
  • H 2 O 2 hydrogen peroxide
  • Strain pD a strain co-expressing the dye decolorizing peroxidase gene
  • Strain pAD a strain that co-expresses the dye decoloring peroxidase gene and the key enzyme gene heme in the heme synthesis pathway;
  • Strain pAD FeCl 2
  • Strain pAD (Glu): Co-express the dye decoloring peroxidase gene with hema, a key enzyme gene in the heme synthesis pathway, and add Glu exogenously;
  • OD 600 absorbance value at a wavelength of 600nm
  • CoCl 2 Cobalt chloride
  • MnCl 2 Manganese chloride
  • 5-ALA 5-aminolevulinic acid.
  • the full-length DyP protein sequence (GenBank numbering AAZ57111.1) was obtained using GenBank database, and the target gene encoding the protein was synthesized.
  • plasmid pET-28a(+) as a vector, through restriction digestion and ligation, a recombinant plasmid containing the target gene was synthesized.
  • the recombinant plasmid containing the target gene was transformed into E. coli BL21 for heterologous expression, and a recombinant strain was obtained.
  • the recombinant strain was inoculated into LB medium containing kanamycin, cultured at 200r/min, 37°C for 12h, and transferred to a 500mL shake flask containing 100mL of fresh LB medium at 4% (v/v) inoculum , And cultivate under the same conditions until the OD 600 of the bacteria reaches 0.6-0.8.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • Glu 100 ⁇ mol/L Glu, FeCl 2 , CoCl 2 , MnCl 2 , Glu+FeCl 2 were added to the culture medium, the final concentration of IPTG added was 0.3 mmol/L, and the culture was induced at 30°C for 14 hours.
  • the cells were collected and centrifuged. Suspend, break, centrifuge, collect the supernatant and pass through a 0.22 ⁇ m aqueous membrane to obtain the crude enzyme solution, which is separated and purified by a 1mL His Trap TM HP affinity nickel column, and linearly eluted with 10 column volumes of elution buffer.
  • the target protein performs full-wavelength scanning and hemoglobin saturation calculation.
  • Heme saturation is defined as the ratio of absorbance at 408nm to 280nm, that is, A 408 /A 280 .
  • IPTG IPTG to a final concentration of 0.3mmol/L
  • induce culture at 30°C for 14h collect the bacteria by centrifugation, suspension, breakage, centrifugation, collect the supernatant and pass through 0.22 ⁇ m water filter membrane to obtain the crude enzyme solution, pass 1mL His Trap TM HP affinity nickel column separation and purification, linear elution with 10 column volumes of elution buffer, full wavelength scanning of the target protein and calculation of specific enzyme activity.
  • DyP enzyme activity at 25°C and pH 4.5, the amount of enzyme required to oxidize 1 ⁇ mol of Reactive Blue 19 per minute is 1 U.
  • CoCl 2 has a certain inhibitory effect on the recombinant DyP enzyme activity. Adding His can basically keep the recombinant DyP enzyme activity and bacterial growth unchanged. Exogenous addition of FeCl 2 , MnCl 2 and Glu can all Improve recombinant DyP enzyme activity and bacterial growth.
  • NCBI https://www.ncbi.nlm.nih.gov/nuccore/AM946981.2:1251856..1253112
  • the Dyp gene was cloned into the pET-28a vector to construct the recombinant plasmid pDyP.
  • the hema and Dyp genes were co-expressed on the pET-28a vector to construct the recombinant plasmid phemA-DyP.
  • the plasmid pDyP and phemA-DyP were introduced into E. coli BL21 (DE3) to form recombinant bacteria pD and pAD respectively.
  • the recombinant bacteria were activated and cultured overnight at 37°C and 200 r/min, they were transferred to the LB liquid medium containing 50 ⁇ g/mL Kan antibiotic according to the 4% inoculum.
  • the OD 600 value of the bacteria is 0.6-0.8, add IPTG at a final concentration of 0.3 mmol/L and induce 8 hours at 37°C.
  • IPTG induction intracellular hemoglobin was detected.
  • the heme concentration of pD strain was 3.4 ⁇ mol/L, while the heme concentration of co-expression strain pAD increased significantly to 9.8 ⁇ mol/L ( Figure 4).
  • the cells were suspended in phosphate buffer (20mmol/L, pH 7.4), the cells were disrupted by ultrasound for 20 minutes, and centrifuged at 4°C for 30 minutes at 10,000 r/min.
  • the crude enzyme solution is filtered with a 0.45 ⁇ m filter membrane to remove impurities.
  • the purified protein was analyzed by 10% SDS-PAGE.
  • Fluorescence method is used to detect the heme concentration.
  • the heme is heated in an oxalic acid reagent to remove ferrous ions and reduced to porphyrin with fluorescent characteristics.
  • the fluorescence value is detected by a microplate reader to calculate the hemoglobin concentration.
  • DyP has an obvious characteristic absorption peak of heme at 408nm, so the binding degree of DyP and heme can be analyzed by spectrum. Take 200 ⁇ L of pure enzyme sample, use the multifunctional microplate reader biotek to scan the wavelength at 280-700nm.
  • the heme and protein content of DyP is expressed at 408nm (Soret peak) and 280nm wavelength.
  • Soret peak is the characteristic absorption peak of heme, which can be used to characterize the level of heme incorporated into Dyp. Except for the strains containing empty plasmids, all other strains showed a Soret peak at 408nm. The peak analysis showed that, compared with pD strain 0.637, the co-expression strain pAD was 0.794. It can be seen that co-expression of Dyp gene and heMA gene enhances the binding degree of Dyp and cofactor heme (see Figure 5).
  • the reaction system includes 20mmol/L acetate buffer, 0.2mmol/L ABTS, 100 ⁇ L pure enzyme solution after proper dilution, H 2 O 2 with final concentration 0.2mmol/L to start the reaction, reaction time is 30S, terminator It is 200 ⁇ L, 2% SDS.
  • An enzyme activity unit is defined as the amount of enzyme required to oxidize 1 ⁇ mol ABTS under the conditions of 25°C and pH 4.5.
  • Dyp's enzyme activity detection method two the Dyp enzyme activity was measured with ABTS as a substrate.
  • the Dyp enzyme activity of the overexpression strain pD alone was 4.5 U/mg, while the pAD enzyme activity of the co-expression strain reached 9.2 U/mg, which was 1.1 times higher than the original (see Figure 6).
  • the reaction system includes 20mmol/L acetate buffer, 0.1mmol/L final concentration of Reactive Blue 19, and appropriate dilution of 100 ⁇ L pure enzyme solution (about 200 ⁇ g of protein), final concentration 0.2mmol/L L H 2 O 2 initiates the reaction, and the terminator is 200 ⁇ L, 2% SDS.
  • the light absorption value of the solution at 595nm was detected after 15 minutes of reaction, and it was recorded as A.
  • Decolorization rate (%) of Reactive Blue 19 (A 0 -A) ⁇ 100%/A 0 . Where A 0 is the light absorption value of the control group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种提高重组染料脱色过氧化物酶酶活的方法，属于生物工程技术领域。包括方法（1）和/或（2），（1）先将染料脱色过氧化物酶（DyP）在大肠杆菌中进行异源表达，获得重组菌株，进行发酵，通过在发酵培养基中添加外源物质谷氨酸和/或FeCl2，当向培养基中添加80μmol/L的FeCl2时，可将酶活提高为对照的1.45倍；（2）通过血红素合成途径关键酶基因hemA和DyP基因的共表达，结合外源添加40μmol/L谷氨酸，Dyp的酶活可提高至13.2U/mg，染料脱色率提高到53.3％。

Description

一种提高重组染料脱色过氧化物酶酶活的方法 技术领域
本发明涉及一种提高重组染料脱色过氧化物酶酶活的方法，属于生物工程技术领域。
背景技术
染料脱色过氧化物酶(DyP)是过氧化物酶家族的新成员，以血红素为辅基，利用过氧化氢作为电子受体催化各种有机物，可以蒽醌染料及木质素模型化合物等为底物，具有潜在的应用价值，可以实现生物法对染料污水的降解，从而避免化学、物理等方法降解的二次污染问题。
目前DyP主要来源于微生物，但是从原始菌中获取DyP，不仅含量低，而且不易与胞内其他蛋白相分离，所以常通过构建基因工程菌的方法生产DyP，如在大肠杆菌中异源表达DyP，但重组蛋白的含量偏低，一般在10mg/L以下。最近有研究发现，DyP与修饰蛋白融合表达，重组蛋白量可增至约200mg/L，然而高度过表达的融合DyP几乎没有活性。通过紫外-可见吸收光谱法和高分辨率质谱法对酶的分析表明，大部分高度过表达的酶只包含有铁缺乏的血红素前体原卟啉IX(PPIX)而不是血红素。在大肠杆菌中血红素合成始于谷氨酸(Glu)，经由关键前体5-氨基乙酰丙酸(5-ALA)，和多步生化反应生成原卟啉IX(PPIX)，PPIX螯合铁后形成血红素。改造血红素合成基因提高血红素含量是提高DyP产量的一种方式，但因途径中涉及多个基因且存在反馈控制，提升幅度有限。
在培养基中添加5-ALA是提高DyP产量的另一种方式，但成本较高，不适于大规模推广应用。
因此，提供一种经济、简单、有效的提高DyP产量的方法，对于DyP的进一步应用有重要意义。
发明内容
本发明的一个目的是提供一种提高重组DyP酶活的方法，所述方法为下述(1)和/或(2)：
(1)在大肠杆菌中异源表达DyP，获得重组菌株，应用所述重组菌株进行发酵，在发酵培养基中添加Glu和/或Fe 2+；
(2)在大肠杆菌中共表达编码DyP的基因和血红素合成途径关键酶基因hemA，所述血红素合成途径关键酶的氨基酸序列是如GenBank编号为CAQ31712.1所示的序列。
在本发明的一种实施方式中，添加的Glu和/或Fe 2+的终浓度为20-100μmol/L。
在本发明的一种实施方式中，添加的Glu和/或Fe 2+的终浓度为40-100μmol/L。
在本发明的一种实施方式中，添加的Glu和/或Fe 2+的终浓度为60-100μmol/L。
在本发明的一种实施方式中，所述DyP的氨基酸序列是如GenBank编号为AAZ57111.1，UniProtKB/Swiss-Prot的编号为Q47KB1所示的序列。
在本发明的一种实施方式中，所述重组菌株以pET系列载体为载体。
在本发明的一种实施方式中，所述发酵的条件为：种子培养后，以1-5％(v/v)的接种量转接至LB培养基中，200-220r/min、35-38℃培养至菌体OD 600达到0.6-0.8，向培养基中添加终浓度为0.1-0.5mmol/L IPTG，28-30℃诱导培养12-20h。
在本发明的一种实施方式中，种子培养是将重组菌株接种到LB培养基中，200-220r/min、35-38℃培养10-15h。
在本发明的一种实施方式中，LB培养基的配方为：胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L。
在本发明的一种实施方式中，所述大肠杆菌为大肠杆菌BL21(DE3)。
在本发明的一种实施方式中，所述共表达以pET-28a为表达载体。
本发明的第二个目的是提供一种基因工程菌，是在大肠杆菌中共表达编码DyP的基因和血红素合成途径关键酶基因hemA，所述DyP的氨基酸序列是如GenBank编号为AAZ57111.1所示的序列，所述血红素合成途径关键酶的氨基酸序列是如GenBank编号为CAQ31712.1所示的序列。
本发明的第三个目的是提供一种生产DyP的方法，是先在大肠杆菌中共表达编码DyP的基因和血红素合成途径关键酶基因hemA，再利用共表达的菌株进行发酵生产DyP，所述DyP的氨基酸序列是如GenBank编号为AAZ57111.1所示的序列，所述血红素合成途径关键酶的氨基酸序列是如GenBank编号为CAQ31712.1所示的序列。
在本发明的一种实施方式中，所述发酵的条件是将共表达的菌株于35-38℃，200-220r/min活化培养8-14h后，按1-5％接种量(V/V)转接至LB培养基中，培养至菌体的OD 600值为0.6-0.8时，加入终浓度为0.1-1mmol/L IPTG，35-38℃条件诱导6-10h。
在本发明的一种实施方式中，所述发酵的培养基是LB培养基。
本发明的第四个目的是提供一种对活性蓝19进行脱色的方法，是以活性蓝19为底物，添加上述基因工程菌表达后的蛋白。
在本发明的一种实施方式中，所述脱色的反应体系包括20mmol/L的醋酸缓冲液、终浓度0.1-0.3mmol/L的活性蓝19、180-220μg的上述基因工程菌表达后的蛋白，终浓度0.02-0.2 mmol/L的H 2O 2。
本发明的另一个目的是提供上述方法在生物、纺织或化工领域内的应用。
本发明的有益效果：
本发明提供了一种提高重组DyP酶活的方法。
(1)先将DyP在大肠杆菌中进行异源表达，获得重组菌株，进行发酵，通过在发酵培养基中添加外源物质Glu和/或Fe 2+，提高了重组DyP中血红素饱和度，当向培养基中添加100μmol/L的Glu+FeCl 2时，可将比酶活提高为对照的1.37倍；当向培养基中添加80μmol/L的FeCl 2时，可将酶活提高为对照的1.45倍。该方法简单易操作，添加的外源物质如Glu，FeCl 2价格便宜，经济实用。
(2)通过E.coli内源血红素合成途径关键酶基因hemA和DyP基因的共表达，结合廉价外源血红素合成前体的添加，结果发现：
(a)胞内血红素含量大幅提升：将重组菌在37℃、0.3mmol/L IPTG诱导后，进行胞内血红素检测，菌株pD中血红素浓度为3.4μmol/L，而共表达菌株pAD中血红素浓度明显提高，达到9.8μmol/L；在菌株pAD的培养基中外源添加20μmol/L FeCl 2、40μmol/L Glu，血红素浓度进一步提高到11.3μmol/L、13.5μmol/L；
(b)DyP结合了更多的血红素：DyP的血红素和蛋白质含量通过408nm处(Soret峰值)和280nm波长表示。Soret峰值为血红素的特征吸收峰，可用来表征血红素掺入DyP中的水平。除包含空载质粒的菌株外，其他菌株均在408nm处出现Soret峰值；分析峰值表明，与pD菌株0.637相比，共表达菌株pAD为0.794，可知将DyP基因与血红素合成途径关键酶基因hemA共表达，增强了Dyp与辅因子血红素的结合度，外源分别添加20μmol/L FeCl 2、40μmol/L Glu，408nm处的峰值进一步提高至0.865、0.889；
(c)以2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)为底物测定Dyp酶活性：菌株pD的Dyp酶活为4.5U/mg，而共表达菌株pAD酶活性达到9.2U/mg，比原来提高1.1倍，外源分别添加20μmol/L FeCl 2、40μmol/L Glu，酶活提高到11.8U/mg、13.2U/mg。
(d)Dyp对活性蓝19的脱色作用：选用难降解的蒽醌类染料活性蓝19作为底物，进行Dyp的脱色测定；结果显示，重组菌株都表现出对染料活性蓝19的脱色活性，菌株pD表达的Dyp脱色率为24.4％，共表达菌株pAD的Dyp为37.4％，外源分别添加20μmol/L FeCl 2、40μmol/LGlu，染料脱色效率增强至42.7％、53.3％。
附图说明
图1：金属离子和氨基酸对菌体量和重组DyP酶活性的影响。
图2：添加剂对酶活与血红素饱和度的影响。
图3：FeCl 2浓度对重组DyP酶活性的影响。
图4：各菌株及添加剂对重组DyP血红素含量的影响。
图5：各菌株及添加剂对重组DyP光谱吸收的影响。
图6：各菌株及添加剂对重组DyP酶活水平的影响。
图7：各菌株及添加剂对重组DyP染料脱色效率的影响。
具体实施方式
本发明中涉及的英文缩写与全称对应关系如下：
DyP：染料脱色过氧化物酶；
Glu：谷氨酸；
Fe 2+：亚铁离子；
FeCl 2：氯化亚铁；
H 2O 2：过氧化氢；
菌株pD：共表达染料脱色过氧化物酶基因的菌株；
菌株pAD：共表达染料脱色过氧化物酶基因与血红素合成途径关键酶基因hemA的菌株；
菌株pAD(FeCl 2)：将染料脱色过氧化物酶基因与血红素合成途径关键酶基因hemA共表达，外源添加FeCl 2；
菌株pAD(Glu)：将染料脱色过氧化物酶基因与血红素合成途径关键酶基因hemA共表达，外源添加Glu；
OD 600：波长为600nm时的吸光度值；
CoCl 2：氯化钴；
MnCl 2：氯化锰；
His：组氨酸；
ABTS：2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐；
5-ALA：5-氨基乙酰丙酸。
实施例1
利用GenBank数据库获得DyP全长蛋白序列(GenBank编号为AAZ57111.1)，合成编码该蛋白的目的基因。以质粒pET-28a(+)为载体，通过酶切连接，合成含有目的基因的重组质粒。将含有目的基因的重组质粒转化到E.coli BL21进行异源表达，获得一株重组菌。
将重组菌株接种到含有卡那霉素的LB培养基中，200r/min、37℃培养12h，以4％(v/v) 接种量转接至含有100mL新鲜的LB培养基的500mL摇瓶中，相同条件培养至菌体OD 600达到0.6-0.8时。向培养基中添加终浓度为0.3mmol/L异丙基-β-D-硫代半乳糖吡喃苷(Isopropylβ-D-thiogalactopyranoside,IPTG)，30℃诱导培养14h，8000rpm离心20min得到菌体，用50mmol/L的pH 7.4磷酸钾缓冲溶液重新悬浮细胞，超声破碎30min，10000rpm离心30min收集上清液，过0.22μm水系滤膜得到粗酶液，通过1mL His Trap TM HP亲和镍柱分离纯化，收集目的蛋白，进行SDS-PAGE电泳以鉴定重组DyP纯度。
分别将100μmol/L的Glu、FeCl 2、CoCl 2、MnCl 2、Glu+FeCl 2加入培养基中，添加的IPTG终浓度为0.3mmol/L，在30℃下诱导培养14h，收集菌体离心、悬浮、破碎、离心，收集上清液过0.22μm水系滤膜得到粗酶液，通过1mL His Trap TM HP亲和镍柱分离纯化，用10个柱体积的洗脱缓冲液进行线性洗脱，对目的蛋白进行全波长扫描和血红素饱和度的计算。
血红素饱和度定义为在408nm处的吸光值与280nm处之比，即A 408/A 280。
结果表明，CoCl 2的加入降低了血红素饱和度，添加MnCl 2没有显著影响(表1)，而添加Glu+FeCl 2，显著提高了重组DyP中血红素的饱和度。
表1不同外源添加剂对重组DyP中血红素饱和度的影响
Figure PCTCN2019120475-appb-000001
注：B为不加任何添加剂的对照组。
向培养基中分别添加终浓度为100μmol/L的His、Glu以及FeCl 2、CoCl 2、MnCl 2、Glu+FeCl 2，考察氨基酸和金属离子对重组DyP酶活和菌体生长的影响，其中不添加任何添加剂为对照组(B)。添加的IPTG终浓度为0.3mmol/L，在30℃下诱导培养14h，收集菌体离心、悬浮、破碎、离心，收集上清液过0.22μm水系滤膜得到粗酶液，通过1mL His Trap TM HP亲和镍柱分离纯化，用10个柱体积的洗脱缓冲液进行线性洗脱，对目的蛋白进行全波长扫描和比酶活的计算。
DyP酶活测定方法一：
向反应体系中依次加入100mmol/L醋酸钠缓冲溶液(pH 4.5)，100μmol/L活性蓝19和适量的酶液，最后加入终浓度为0.1mMH 2O 2启动反应，酶促反应10min，然后通过加入200 μL 2％SDS终止反应。在室温下，于波长595nm处检测吸光值变化。蛋白质浓度测定采用BCA法，以牛血清蛋白为标准蛋白。
DyP酶活定义：在25℃，pH 4.5条件下，每分钟氧化1μmol活性蓝19所需的酶量为1U。
比酶活(U/mg)＝酶活(U/mL)×[蛋白浓度(mg/mL)] -1。
使用DyP比酶活测定方法一测定DyP酶活。结果如下：
(1)如图1所示，CoCl 2对重组DyP酶活有一定抑制作用，添加His对重组DyP酶活和菌体的生长基本保持不变，外源添加FeCl 2、MnCl 2以及Glu都能提高重组DyP酶活和菌体生长量。当向培养基中添加100μmol/L的FeCl 2时，重组DyP酶活从22.34U/L提高至26.20U/L，菌体生长量从5.45g/L提高至6.17g/L；当向培养基中添加100μmol/L的Glu时，重组DyP酶活从22.34U/L提高至23.33U/L，菌体生长量从5.45g/L提高至5.78g/L；
(2)如图2所示，当向培养基中添加100μmol/L的Glu+FeCl 2时，比酶活提高为对照(101U/g)的1.37倍；
(3)向培养基中分别添加终浓度为40、60、80、100、120μmol/L的FeCl 2，如图3所示，随着FeCl 2浓度增加，酶活先增后降，当浓度为80μmol/L时，酶活达到31.31U/L，是对照的1.45倍。
实施例2
(1)重组质粒的构建
编码来源于大肠杆菌的谷氨酰胺tRNA还原酶的hemA基因的核苷酸序列参考NCBI(https://www.ncbi.nlm.nih.gov/nuccore/AM946981.2:1251856..1253112)。
编码来源于Thermobifida fusca的DyP的基因的核苷酸序列参考NCBI(https://www.ncbi.nlm.nih.gov/nuccore/CP000088.1:3600559..3601851)。将Dyp基因克隆在pET-28a载体上，构建重组质粒pDyP。将hemA和Dyp基因共表达在pET-28a载体上，构建重组质粒phemA-DyP。将质粒pDyP、phemA-DyP导入大肠杆菌BL21(DE3)中，分别构成重组菌pD、pAD。
(2)重组蛋白的表达与纯化鉴定
重组菌于37℃，200r/min过夜活化培养后，按4％接种量转接至含50μg/mL Kan抗生素的LB液体培养基中。待菌体的OD 600值为0.6-0.8时，加入终浓度0.3mmol/L IPTG，37℃条件诱导8h。IPTG诱导后，进行胞内血红素检测，pD菌株血红素浓度为3.4μmol/L，而共表达菌株pAD血红素浓度明显提高，达到9.8μmol/L(图4)。
用磷酸缓冲液(20mmol/L，pH 7.4)悬浮菌体，超声破碎细胞20min，4℃，10000r/min离心30min。粗酶液用0.45μm过滤膜进行过滤，以除去杂质。用含有20mmol/L咪唑的磷酸缓冲液进行平衡后上样，最后用含有500mmol/L咪唑的磷酸缓冲液进行线性洗脱。根据洗脱图谱，收集对应洗脱峰的酶液。纯化后的蛋白用10％的SDS-PAGE进行分析。
(3)重组菌培养基中添加血红素合成前体
在菌株pAD的培养基中外源添加20μmol/L FeCl 2、40μmol/L Glu。
(4)血红素浓度测定
利用荧光法来检测血红素浓度，血红素在草酸试剂中受热脱去亚铁离子，还原成具有荧光特征的卟啉，通过酶标仪检测其荧光值，推算出血红素浓度。根据公式V＝8/OD取诱导后的菌液，4℃、8000g、离心5min，弃上清。超纯水水洗后将菌体重悬转移至1.5mL琥珀色离心管。向每个离心管中加入500μL，20mM草酸溶液，于4℃暗室放置16h。而后，加入500μL、2mol/L草酸溶液，对照组与实验组分别在室温和95℃水浴处理30min。待样品自然冷却后，12000g、4℃、离心5min，取200μL上清于黑色96孔荧光板，进行荧光值检测(激发波长、发射波长为分别设定为400nm和620nm)。
(5)DyP的光谱分析
DyP在408nm处有一个明显的亚铁血红素酶的特征吸收峰，因此可以通过光谱分析DyP与血红素结合度。取200μL纯酶样品，运用多功能酶标仪biotek在280-700nm对其进行波长扫描。
DyP的血红素和蛋白质含量通过408nm处(Soret峰值)和280nm波长表示。Soret峰值为血红素的特征吸收峰，可用来表征血红素掺入Dyp中的水平。除包含空载质粒的菌株外，其他菌株均在408nm处出现Soret峰值。分析峰值表明，与pD菌株0.637相比，共表达菌株pAD为0.794，可知，将Dyp基因与hemA基因共表达，增强了Dyp与辅因子血红素的结合度(见图5)。
(6)Dyp的酶活性检测方法二
以ABTS为底物，检测DyP的活性。反应体系包括20mmol/L的醋酸缓冲液、终浓度0.2mmol/L的ABTS、适量稀释后的100μL纯酶液，终浓度0.2mmol/L的H 2O 2启动反应，反应时间为30S，终止剂为200μL、2％SDS。使用紫外可见分光光度计检测反应产物在420nm(ε＝36,000M -1cm -1)的光吸收值。
一个酶活力单位定义为：在25℃、pH 4.5条件下，氧化1μmol ABTS所需要的酶量。
比酶活(U·mg -1)＝酶活(U·mL -1)×[蛋白浓度(mg·mL -1)] -1
使用Dyp的酶活性检测方法二，以ABTS为底物测定Dyp酶活性。单独过表达菌株pD的Dyp酶活为4.5U/mg，而共表达菌株pAD酶活性达到9.2U/mg，比原来提高1.1倍(见图6)。
(7)DyP对活性蓝19的脱色作用
以活性蓝19为研究对象，反应体系包括20mmol/L的醋酸缓冲液、终浓度0.1mmol/L的活性蓝19、适量稀释后的100μL纯酶液(蛋白量约200μg)，终浓度0.2mmol/L的H 2O 2启动反应，终止剂为200μL、2％SDS。在25℃、pH 4.5条件下，反应15分钟后检测溶液在595nm的光吸收值，记为A。活性蓝19的脱色率(％)＝(A 0-A)×100％/A 0。其中A 0为对照组光吸收值。
结果显示，重组菌株都表现出对染料活性蓝19的脱色活性。单独表达菌株pD脱色率为24.4％，共表达菌株pAD为37.4％(见图7)。
实施例3
在菌株pAD培养基中外源添加20μmol/L FeCl 2，其他条件同实施例2。血红素浓度进一步提高到11.3μmol/L，408nm处的峰值提高至0.865，Dyp的酶活提高至11.8U/mg(测定方法见Dyp的酶活性检测方法二)，染料脱色率提高到42.7％。
在菌株pAD培养基中外源添加40μmol/L Glu，其他条件同实施例2。血红素浓度提高到13.5μmol/L，408nm处的峰值提高至0.889，Dyp的酶活提高至13.2U/mg(测定方法见Dyp的酶活性检测方法二)，染料脱色率提高到53.3％。
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 (18)

  1. 一种提高重组染料脱色过氧化物酶酶活的方法，其特征在于，所述方法为下述(1)和/或(2)：
    (1)在大肠杆菌中异源表达染料脱色过氧化物酶，获得重组菌株，应用所述重组菌株进行发酵，在发酵培养基中添加Glu和/或Fe 2+；
    (2)在大肠杆菌中共表达编码染料脱色过氧化物酶的基因和血红素合成途径关键酶基因hemA，所述血红素合成途径关键酶的氨基酸序列是如GenBank编号为CAQ31712.1所示的序列。
  2. 如权利要求1所述的方法，其特征在于，添加的Glu和/或Fe 2+的终浓度为20-100μmol/L。
  3. 如权利要求1或2所述的方法，其特征在于，添加的Glu和/或Fe 2+的终浓度为40-100μmol/L。
  4. 如权利要求1所述的方法，其特征在于，所述染料脱色过氧化物酶的氨基酸序列是如GenBank编号为AAZ57111.1所示的序列。
  5. 如权利要求1所述的方法，其特征在于，所述大肠杆菌为大肠杆菌BL21(DE3)。
  6. 如权利要求1或5所述的方法，其特征在于，所述重组菌株以pET系列载体为载体。
  7. 如权利要求1所述的方法，其特征在于，发酵条件为：种子培养后，以1-5％(v/v)的接种量转接至LB培养基中，200-220r/min、35-38℃培养至菌体OD 600达到0.6-0.8，向培养基中添加终浓度为0.1-0.5mmol/L IPTG，28-30℃诱导培养12-20h。
  8. 如权利要求7所述的方法，其特征在于，种子培养是将重组菌株接种到LB培养基中，200-220r/min、35-38℃培养10-15h。
  9. 如权利要求7所述的方法，其特征在于，LB培养基的配方如下:胰蛋白胨10g/L，酵母提取物5g/L，氯化钠10g/L。
  10. 如权利要求1所述的方法，其特征在于，所述共表达以pET-28a为表达载体。
  11. 一种基因工程菌，是在大肠杆菌中共表达编码染料脱色过氧化物酶的基因和血红素合成途径关键酶基因hemA，所述染料脱色过氧化物酶的氨基酸序列是如GenBank编号为AA257111.1所示的序列，所述血红素合成途径关键酶的氨基酸序列是如GenBank编号为CAQ31712.1所示的序列。
  12. 一种生产染料脱色过氧化物酶的方法，是先在大肠杆菌中共表达编码染料脱色过氧化物酶的基因和血红素合成途径关键酶基因hemA，再利用共表达的菌株进行发酵生产染料脱色过氧化物酶；所述染料脱色过氧化物酶的氨基酸序列是如GenBank编号为AA257111.1所 示的序列，所述血红素合成途径关键酶的氨基酸序列是如GenBank编号为CAQ31712.1所示的序列。
  13. 如权利要求12所述的方法，其特征在于，所述发酵的条件是将共表达的菌株于35-38℃，200-220r/min活化培养8-14h后，按1-5％接种量(V/V)转接至LB培养基中，培养至菌体的OD 600值为0.6-0.8时，加入终浓度为0.1-1mmol/L IPTG，35-38℃条件诱导6-10h。
  14. 如权利要求12或13的方法，其特征在于，所述发酵的培养基是LB培养基。
  15. 一种对活性蓝19进行脱色的方法，其特征在于，以活性蓝19为底物，添加权利要求11所述基因工程菌表达后的蛋白。
  16. 如权利要求15所述的方法，其特征在于，所述脱色的反应体系包括20mmol/L的醋酸缓冲液、终浓度0.1-0.3mmol/L的活性蓝19、180-220μg的权利要求11所述基因工程菌表达后的蛋白，终浓度0.02-0.2mmol/L的H 2O 2。
  17. 权利要求1-10任一所述的方法在生物、纺织或化工领域内的应用。
  18. 权利要求12-16任一所述的方法在生物、纺织或化工领域内的应用。
PCT/CN2019/120475 2019-04-02 2019-11-25 一种提高重组染料脱色过氧化物酶酶活的方法 Ceased WO2020199612A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910261137.5A CN109943544A (zh) 2019-04-02 2019-04-02 一种提高重组染料脱色过氧化物酶酶活的方法
CN201910261137.5 2019-04-02

Publications (1)

Publication Number Publication Date
WO2020199612A1 true WO2020199612A1 (zh) 2020-10-08

Family

ID=67013532

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/120475 Ceased WO2020199612A1 (zh) 2019-04-02 2019-11-25 一种提高重组染料脱色过氧化物酶酶活的方法

Country Status (2)

Country Link
CN (1) CN109943544A (zh)
WO (1) WO2020199612A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111593061A (zh) * 2020-07-06 2020-08-28 江南大学 一种提高染料脱色过氧化物酶活性的方法
CN113683813A (zh) * 2021-08-18 2021-11-23 江南大学 染料脱色过氧化物酶在聚苯乙烯降解中的应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109943544A (zh) * 2019-04-02 2019-06-28 江南大学 一种提高重组染料脱色过氧化物酶酶活的方法
CN111849847B (zh) * 2020-07-08 2022-05-06 江南大学 一种提高大肠杆菌胞内血红素含量的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106754803A (zh) * 2017-01-18 2017-05-31 四川爱奇生物科技有限公司 DypB活性蛋白异源高表达的方法及应用
CN107460176A (zh) * 2017-06-28 2017-12-12 江苏大学 一种过氧化物酶DyP35基因及其表达蛋白和应用
CN109943544A (zh) * 2019-04-02 2019-06-28 江南大学 一种提高重组染料脱色过氧化物酶酶活的方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102016205671A1 (de) * 2016-04-06 2017-10-12 Henkel Ag & Co. Kgaa Wasch- oder Reinigungsmittel mit lebenden Mikroorganismen
CN108676783B (zh) * 2018-05-28 2020-12-22 南华大学 一种脱色过氧化物酶及其制备方法与应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106754803A (zh) * 2017-01-18 2017-05-31 四川爱奇生物科技有限公司 DypB活性蛋白异源高表达的方法及应用
CN107460176A (zh) * 2017-06-28 2017-12-12 江苏大学 一种过氧化物酶DyP35基因及其表达蛋白和应用
CN109943544A (zh) * 2019-04-02 2019-06-28 江南大学 一种提高重组染料脱色过氧化物酶酶活的方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHEN DAN-YUAN , SHEN YUN-JIE , YANG YAN , TANG LEI: "Regulation of Heme Synthesis In Escherichia Coli by Overexpression of Genes for The Key Enzymes and Environmental Factors", FOOD AND FERMENTATION INDUSTRIES, vol. 44, no. 11, 31 May 2018 (2018-05-31), pages 7 - 14, XP055739899, DOI: 10.13995/j.cnki.11-1802/ts.017222 *
DATABASE Genbank 27 February 2015 (2015-02-27), ANONYMOUS: "glutamyl-tRNA reductase [Escherichia coli BL21(DE3)", XP055739904, retrieved from NCBI Database accession no. CAQ31712.1 *
DATABASE Genbank 28 January 2014 (2014-01-28), ANONYMOUS: "dyp-type peroxidase [Thermobifida fusca YX], access no. AAZ57111.1.", XP055739906, retrieved from NCBI Database accession no. AAZ57111.1 *
LIQUN WANG,ALAN K.CHANG,WENJIE YUAN, FENGWU BAI: "Recombinant Expression, Purification and Characterization of a Novel Dyp-Type Peroxidase in Escherichia Coli", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 29, no. 6, 25 June 2013 (2013-06-25), pages 772 - 784, XP055739911, DOI: 10.13345/j.cjb.2013.06.005 *
RAMZI AHMAD BAZLI; HYEON JEONG EUN; HAN SUNG OK: "Improved catalytic activities of a dye-decolorizing peroxidase (DyP) by overexpression of ALA and heme biosynthesis genes in Escherichia coli.", PROCESS BIOCHEMISTRY, vol. 50, 18 May 2015 (2015-05-18), pages 1272 - 1276, XP029189758, ISSN: 1359-5113, DOI: 10.1016/j.procbio.2015.05.004 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111593061A (zh) * 2020-07-06 2020-08-28 江南大学 一种提高染料脱色过氧化物酶活性的方法
CN113683813A (zh) * 2021-08-18 2021-11-23 江南大学 染料脱色过氧化物酶在聚苯乙烯降解中的应用

Also Published As

Publication number Publication date
CN109943544A (zh) 2019-06-28

Similar Documents

Publication Publication Date Title
JP5895004B2 (ja) 組み換え大腸菌及び５−アミノレブリン酸の生産におけるその応用
Joseph et al. Increased biomass production and glycogen accumulation in apcE gene deleted Synechocystis sp. PCC 6803
Sygmund et al. Heterologous overexpression of Glomerella cingulata FAD-dependent glucose dehydrogenase in Escherichia coli and Pichia pastoris
Tan et al. Stepwise optimization of genetic RuBisCO-equipped Escherichia coli for low carbon-footprint protein and chemical production
CN111849847B (zh) 一种提高大肠杆菌胞内血红素含量的方法
CN116162583B (zh) 一种提高重组大肠杆菌血红素合成的方法
CN109943544A (zh) 一种提高重组染料脱色过氧化物酶酶活的方法
Poladyan et al. Valorization of whey-based side streams for microbial biomass, molecular hydrogen, and hydrogenase production
Huang et al. Heterologous overproduction of 2 [4Fe4S]-and [2Fe2S]-type clostridial ferredoxins and [2Fe2S]-type agrobacterial ferredoxin
CN102041264B (zh) 一种氧化葡萄糖酸杆菌(G.oxydans)的基因工程菌及其应用
CN102796720A (zh) 拆分消旋体γ-内酰胺活性的（+）γ-内酰胺酶及应用
Bertsova et al. The flavin transferase ApbE flavinylates the ferredoxin: NAD+-oxidoreductase Rnf required for N2 fixation in Azotobacter vinelandii
Li et al. Improved stress tolerance of recombinant Escherichia coli strain expressing nitrile hydratase by combining Global Regulator IrrE
US20250171748A1 (en) Dioxygenase mutants and use thereof in synthesizing (2s,3r)-3-hydroxypipecolic acid
CN112941062B (zh) 腈水合酶赖氨酸突变体hba-k2h1、编码基因及应用
CN115287294A (zh) 改造ala促进枯草芽孢杆菌内血红素生物合成的方法
CN1351665A (zh) 山梨糖醇脱氢酶、其编码基因及它们的用途
CN111593061A (zh) 一种提高染料脱色过氧化物酶活性的方法
CN114891710B (zh) 一种通过重构辅酶再生系统强化重组大肠杆菌催化合成胆绿素的方法
Tong et al. Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli
KR102784639B1 (ko) 일종 원핵 표현 시스템을 이용한 바이러스 유사입자 조제 방법
CN118109428A (zh) 一种细胞色素p450酶突变体及其制备方法和应用
CN116590208A (zh) 一种构建重组大肠杆菌高效合成血红素的方法
Ma et al. Triple fusion of D-amino acid oxidase from Trigonopsis variabilis with polyhistidine and Vitreoscilla hemoglobin
Huston et al. Expression and characterisation of a major c-type cytochrome encoded by gene kustc0563 from Kuenenia stuttgartiensis as a recombinant protein in Escherichia coli

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19922467

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19922467

Country of ref document: EP

Kind code of ref document: A1