A recombinant human copper / zinc superoxide dismutase and its preparation and application

Abstract

The invention discloses a recombinant human copper / zinc superoxide dismutase and its preparation and application. The invention is based on the amino acid sequence of human copper / zinc superoxide dismutase, and with reference to the chloroplast Cu / Zn-SOD amino acid sequence of Withania somnifera and the Cu / Zn-SOD amino acid sequence of other biological species, to obtain a recombinant human copper / zinc superoxide dismutase (rmh Cu / Zn-SOD) having an amino acid sequence as shown in SEQ ID NO.1. Relative to the wild type, rmh Cu / Zn-SOD not only has greatly improved water solubility and enzyme specific activity, but also significantly improves tolerance to acidic environments and tolerance to trypsin, and therefore has more commercial value, and is particularly suitable for preparing oral SOD products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant human copper / zinc superoxide dismutase and a preparation method and application thereof. Background Art

[0002] Superoxide dismutase (SOD) catalyzes the dismutation reaction of superoxide anion free radicals to generate hydrogen peroxide (H2O2) and molecular oxygen (O2), and hydrogen peroxide is then catalyzed by catalase to generate water and molecular oxygen. According to the different metal cofactors in the active center of SOD enzyme, it is mainly divided into the following three categories: 1) Cu / Zn-SOD with Cu / Zn as cofactor; 2) Mn-SOD with Mn as cofactor; 3) Fe-SOD with Fe as cofactor. These three types of SOD can exist in different species, or in different subcellular structures of the same species. Human superoxide dismutase (hSOD) is mainly SOD (Cu / Zn-SOD) with copper and zinc as cofactors. It can quickly decompose superoxide anion free radicals produced by the human body in the normal metabolic process, and can also decompose reactive oxygen species (ROS) produced in the process of clinical diseases or medical clinical treatment, such as: ROS produced in large quantities after ischemia reperfusion therapy and radiotherapy, decomposing them into water molecules and hydrogen peroxide molecules that are harmless to the human body, reducing lipid peroxidation in tissue cells and protecting body cells. Due to the important role of SOD in maintaining human health, it is widely used in biotechnology products such as medicine, health foods, cosmetics, and feed additives.

[0003] As the role of SOD in human health and animal husbandry is increasingly recognized in the field of life sciences, the demand for SOD in modern society is increasing, and the mass production of highly biologically active SOD is a major issue. The E. coli expression system is the most commonly used expression system for large-scale production of recombinant proteins. However, this expression system usually forms recombinant proteins with inclusion body structures. SOD inclusion body proteins have only low or no biological activity. Moreover, human superoxide dismutase is easily destroyed by the acidic environment in gastric juice and trypsin in the intestine (the main digestive enzyme in the gastrointestinal tract of humans and higher animals) during oral administration. Therefore, overcoming the shortcomings of E. coli inclusion body expression, improving the solubility and enzyme activity of recombinant SOD expression, and improving the tolerance of SOD expression products to physical and chemical factors (such as high temperature, extreme pH environment, etc.) and resistance to gastrointestinal digestive enzymes are key technologies that must be solved for large-scale production of recombinant SOD.

[0004] SOD is a homologous protein. Almost all organisms have SOD genes and expression products. In the long process of biological evolution, some amino acids of SOD molecules of different species have remained unchanged, which are called invariant residues. These invariant residues can maintain their basic spatial conformation and active center unchanged to maintain the basic activity or function of SOD; the amino acid residues in other positions have a certain degree of variation between different species, which are called variable residues. Variable residues can be replacements between amino acids with similar functions or between amino acids with different functions between different species. Changes in variable amino acid residues in different species will also lead to differences in SOD traits and activity between species. Withania somnifera, also known as Ashwagandha (Withania somnifera, also known as Indian ginseng, or South African Withania somnifera), belongs to the Solanaceae family. It is a highly adaptable species that grows mostly in wasteland, arid and semi-arid areas and has very good adaptability to high temperatures and drought. It is widely distributed in Africa, the Middle East, and South Asia. A document (R. Madanala, V. Gupta, F. Deeba, S. K. Upadhyay, V. Pandey, P. K. Siingh, R. Tuli., A highly stable Cu / Zn superoxide dismutase from Withania somnifera plant: gene cloning, expression and characterization of the combined protein. Biotechnol Lett (2011) 33: 2057-2063.) reported that Withania somnifera chloroplast Cu / Zn SOD has excellent stability and is particularly resistant to high temperature, acidic environment, and trypsin. Summary of the invention

[0005] After analyzing the amino acid sequence of Withania somnifera chloroplast Cu / Zn-SOD, the inventors found that the number of lysine (K) and arginine (R) in its amino acid composition is much less than that of human Cu / Zn-SOD. In view of this, the present invention aims to obtain a technical scheme for recombinant modification of human Cu / ZnSOD (Recombinant modify human Cu / Zn superoxide dismutase, rmh Cu / Zn-SOD) amino acid sequence based on the amino acid sequence of human copper / zinc superoxide dismutase, with reference to the amino acid sequence of Withania somnifera chloroplast Cu / Zn-SOD and the amino acid sequence of Cu / Zn-SOD of other biological species, and the SOD product obtained according to the scheme has greatly improved water solubility and enzyme specific activity, and has stronger tolerance to acidic environment and trypsin.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a recombinant human copper / zinc superoxide dismutase (rmh Cu / Zn-SOD), the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] After comparing the amino acid sequences of copper / zinc superoxide dismutases of a large number of different organisms (partial representative organisms are shown in Table 1), the inventors determined the variable amino acid residues and invariant amino acid residues of copper / zinc superoxide dismutases. Based on this, an experiment was designed to replace the variable amino acid residues of lysine and arginine in a part of human Cu / Zn-SOD with other amino acids. After exploration, the replacement scheme in Table 2 was finally determined, and the resulting amino acid sequence is shown in SEQ ID NO.1.

[0009] Table 1

[0010]

[0011]

[0012] Table 2

[0013] Mutation number Mutated amino acid residue position* Withania somnifera SOD Human SOD rmh Cu / Zn-SOD Nucleotide code 1 24 -- K Q** CAG 2 31 T K T ACC 3 37 T K T ACT 4 70 L R L CTG 5 71 T K T ACC 6 76 G K G GGC 7 92 A K A GCC 8 123 L K L CTG 9 137 T K T ACC

[0014] Note: *Numbering is based on the amino acid sequence of human Cu / Zn-SOD; **Determined with reference to the corresponding amino acid residues in organisms such as chicken and fruit fly.

[0015] Compared with human Cu / Zn-SOD, the biggest feature of the amino acid sequence of rmh Cu / Zn-SOD provided by the present invention is that the number of lysine (K) and arginine (R) belonging to variable amino acid residues in the expressed protein is reduced. Lysine and arginine are the main alkaline amino acids in the body, and are positively charged under physiological pH (pH=7.3). At the same time, the peptide bond formed by the carboxyl group of these two amino acids and the amino group of other amino acids is the bond-specific hydrolysis site of trypsin, and its bond specificity is very strict. The reduction in the number of lysine and arginine can reduce the isoelectric point of the protein to a certain extent, so that Cu / Zn-SOD has better catalytic activity under lower pH conditions. Moreover, the number of lysine and arginine on the surface of the SOD molecule is reduced, so that the specific enzymatic cleavage site of trypsin is also reduced accordingly, and the modified SOD is more able to tolerate or resist the hydrolysis of trypsin.

[0016] It is understandable that proteins with the same function obtained by connecting a tag sequence or other sequence to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1 still fall within the scope of protection of the present invention.

[0017] The second aspect of the present invention provides some biological materials related to the rmh Cu / Zn-SOD, specifically including the following:

[0018] (i) Nucleic acid molecules (i.e. gene sequences) encoding rmh Cu / Zn-SOD;

[0019] (ii) a recombinant vector, expression cassette or transposon comprising the gene described in (i);

[0020] (iii) A recombinant bacterium comprising (i) the nucleic acid molecule or (ii) the recombinant vector.

[0021] In some embodiments of the present invention, a nucleotide sequence suitable for prokaryotic cell expression is deduced from the amino acid sequence shown in SEQ ID NO. 1, specifically as shown in SEQ ID NO. 2. Further, an Nco I restriction endonuclease site can be added to the 5' end of the sequence shown in SEQ ID NO. 2, and an Xho I restriction endonuclease site can be added to the 3' end to facilitate gene manipulation.

[0022] In some embodiments of the present invention, the recombinant vector is obtained by inserting the rmh Cu / Zn-SOD encoding gene into a pET plasmid, wherein the pET plasmid includes but is not limited to pET28a, pET30a, pET32a, and the like.

[0023] In some embodiments of the present invention, the recombinant bacteria are obtained by transforming the recombinant vector into Escherichia coli, wherein the Escherichia coli includes but is not limited to BL21, DH5α, JM109, etc.

[0024] The third aspect of the present invention provides a method for preparing rmh Cu / Zn-SOD, which specifically comprises: inserting a gene sequence encoding rmh Cu / Zn-SOD into a vector, then transforming the obtained recombinant vector into a receptor cell, inducing expression, and purifying the product.

[0025] In one embodiment of the present invention, the gene sequence shown in SEQ ID NO.2 is used, pET28a is used as a vector, and Escherichia coli BL21 is used as a recipient cell to finally prepare the engineered bacteria E.coli BL21 / pET28a rmhSOD, and α-lactose is used as an inducer to induce the expression of the engineered bacteria E.coli BL21 / pET28a rmhSOD. The test results show that the expression amount of the obtained target protein accounts for about 36.9% of the total protein amount, and more than 90% of the target protein is in the supernatant of the expression product lysis solution, which shows that the rmh Cu / Zn-SOD prepared by this method is a highly soluble protein.

[0026] The properties of the rmh Cu / Zn-SOD prepared by the present invention were further analyzed, and the results showed that compared with recombinant wild-type human SOD (rwhCu / Zn-SOD), the isoelectric point of rmhCu / Zn-SOD moved toward the low pH direction, the water solubility was significantly improved, the specific activity was also significantly increased, and it had better tolerance to temperature, pH, and trypsin.

[0027] It can be seen that the rmh Cu / Zn-SOD provided by the present invention is more suitable for exerting the catalytic effect of SOD enzyme in the gastrointestinal tract of humans and animals and exerting its due function after entering the body, and its original function is not affected in any way. Therefore, the rmh Cu / Zn-SOD provided by the present invention can be applied to at least the following fields:

[0028] 1) Used in the preparation of functional foods, such as SOD health capsules, health drinks, etc. It has the effects of improving immunity, delaying aging, effectively reducing blood lipids, blood pressure, cholesterol, blood viscosity, improving cardiovascular and cerebrovascular conditions, preventing Alzheimer's disease, improving diabetes symptoms, and preventing complications.

[0029] 2) Used for preparing skin care products, such as SOD essence, SOD cream (emulsion), eye cream, etc., which have the functions of whitening, removing spots, removing wrinkles, removing acne, repairing ultraviolet radiation damage, etc.; skin care products are suitable for weakly acidic buffer systems, so the rmhCu / Zn-SOD provided by the present invention is more suitable for use in skin care products; and rmh Cu / Zn-SOD has better solubility and higher activity, which can further improve the skin care function.

[0030] 3) Used in the preparation of medicines, such as drugs for the treatment of acute myocardial infarction, drugs for anti-radiation or repair of radiation damage, etc.

[0031] 4) Used in the preparation of feed additives, which have the functions of anti-oxidation, improving the immunity of farmed animals, and resisting the stress response of farmed animals.

[0032] Compared with the prior art, the beneficial effects of the present invention are:

[0033] The present invention confirms the variable amino acid residues and invariant amino acid residues of copper / zinc superoxide dismutase, takes the amino acid sequence of human copper / zinc superoxide dismutase as a basis, replaces part of the lysine and arginine in its variable residues with other amino acids, thereby improving the activity of SOD and its tolerance to physical and chemical factors (such as high temperature, extreme pH environment, etc.), reducing the action sites of trypsin, and enhancing the tolerance of SOD to trypsin. It can be seen that the present invention provides a new idea for the modification of SOD enzyme.

[0034] Compared with rwh Cu / Zn-SOD, the rmh Cu / Zn-SOD provided by the present invention has the following advantages: ① the water solubility is greatly improved (from about 36% to more than 90%); ② the specific activity of the SOD enzyme is greatly improved; ③ the tolerance to high temperature is significantly improved, and it is more able to tolerate temperature changes during the preparation and processing; ④ the pH range suitable for SOD catalysis is expanded, and it is more suitable for acidic environmental conditions; ⑤ the resistance to trypsin is significantly improved. Therefore, the rmh Cu / Zn-SOD provided by the present invention is more suitable for large-scale production, and is more suitable for the production and preparation of oral SOD products. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The figure is the SDS-PAGE result of the induced expression product of the engineered bacteria E. coli BL21 / pET28a rmhSOD in Example 1, wherein lane 1 is a standard protein molecular weight marker, lane 2 is a whole solution of E. coli BL21 cells (negative control), lane 3 is a whole solution of the engineered bacteria E. coli BL21 / pET28a rmhSOD after expression, and lane 4 is a supernatant of the engineered bacteria E. coli BL21 / pET28a rmhSOD after expression.

[0036] Figure 2 This is a graph showing the purification results of the target protein rmh Cu / Zn-SOD in Example 1, wherein lane 1 is a standard protein molecular weight marker, lane 2 is a hydrophobic chromatography result of rmh Cu / Zn-SOD, and lane 3 is a hydrophobic chromatography + ion exchange chromatography result of rmh Cu / Zn-SOD. DETAILED DESCRIPTION

[0037] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific embodiments and drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0038] Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook and other molecular cloning experimental manuals, or the conditions recommended by the manufacturer's instructions. All reagents and materials used, unless otherwise specified, can be obtained from commercial sources.

[0039] Example 1 Preparation of rmh Cu / Zn-SOD

[0040] (1) Genetic design and construction of engineered bacteria.

[0041] Based on the amino acid sequence of rmh Cu / Zn-SOD (as shown in SEQ ID NO.1), a nucleotide sequence suitable for prokaryotic cell expression was deduced, specifically as shown in SEQ ID NO.2. Then, an Nco I restriction endonuclease site was added to the 5' end of the sequence, and an XhoI restriction endonuclease site was added to the 3' end. Finally, the designed sequence was sent to Shanghai Biotechnology Co., Ltd. (Shanghai Biotechnology) for whole gene synthesis.

[0042] The synthesized gene sequence was inserted into the E. coli expression plasmid pET28a, and then the recombinant plasmid was used to transform E. coli BL21. The resulting engineered bacteria was named: E. coli BL21 / pET28a rmhSOD.

[0043] (2) Induced expression in engineered bacteria and analysis of expression products.

[0044] The constructed engineered bacteria E. coli BL21 / pET28a rmhSOD was induced to express, and the inducer was α-lactose, with a working concentration of 0.03 mol / L. After the expression, the bacteria were broken by ultrasound, and the soluble protein and insoluble protein were separated by centrifugation. The whole bacterial liquid before centrifugation after ultrasonic breaking of the bacteria and the supernatant after centrifugation were subjected to SDS-PAGE electrophoresis detection. The results are as follows: Figure 1 shown.

[0045] from Figure 1 It can be seen that the molecular weight of the target protein subunit is about 17-18kD, and the expression amount accounts for about 36.9% of the total protein. The target protein is basically in the supernatant, indicating that it is a soluble protein. According to the analysis of soluble expression level, more than 90% of the target protein is present in the supernatant, which is a highly soluble protein.

[0046] (3) Purification of rmh Cu / Zn-SOD.

[0047] Take the wet cells of the engineered bacteria expressing rmhCu / Zn-SOD, add PBS to resuspend the cells at a mass volume ratio of 1:10 (W / V), and add 600mM CuSO4-ZnSO4 / L solution to make the Cu content of the resuspended cell liquid + and Zn + The final concentration is 0.1mM / L. The heavy suspension of the bacteria is broken by ultrasound. Then add ammonium sulfate to 50% saturation, precipitate at 2-8℃ overnight, and heat treat in a 70℃ water bath for 30 minutes. After heat treatment, cool quickly and centrifuge to remove the precipitate. The supernatant is filtered with a 0.45μm filter membrane and chromatographic separation is performed step by step. The first step is hydrophobic chromatography, and the filler is Phenyl Sepharose 6Fast Flow, and the collected target protein is desalted. Then the second step of chromatography is carried out, namely DEAE-Sepharose Fast Flow ion exchange chromatography.

[0048] Purity analysis: The protein samples collected by hydrophobic chromatography and DEAE ion exchange chromatography were analyzed by 15% SDS-PAGE. Figure 2 After grayscale scanning purity analysis, the protein purity was 92% and 99% or more respectively.

[0049] Comparative Example 1

[0050] Referring to Example 1, this example prepared recombinant wild-type human SOD (rwhCu / Zn-SOD). The difference between the amino acid sequence of rwhCu / Zn-SOD (i.e., human SOD in Table 1, see GenBank, AY450286 for the specific sequence) and rmh Cu / Zn-SOD is shown in Table 2.

[0051] The results of SDS-PAGE electrophoresis showed that the protein solubility ratio of the wild-type copper / zinc superoxide dismutase without any modification after induced expression was 36.1%, which was much lower than the solubility ratio of the modified copper / zinc superoxide dismutase (more than 90%).

[0052] Example 2: Property detection of rmh Cu / Zn-SOD

[0053] Taking rwhCu / Zn-SOD as control, this example tested the properties of the modified rmh Cu / Zn-SOD, including the following aspects:

[0054] (1) Comparison of isoelectric points (pI) between rmhCu / Zn-SOD and rwhCu / Zn-SOD.

[0055] The isoelectric points of rmhCu / Zn-SOD and rwhCu / Zn-SOD were determined by isoelectric focusing electrophoresis. The results showed that the isoelectric point of rmhCu / Zn-SOD was 5.4, while that of rwhCu / Zn-SOD was 5.7. After the human superoxide dismutase was modified, 9 basic amino acids (8 lysines and 1 arginine) were transformed into threonine (T), leucine (L), glycine (G), and alanine (A) (see Table 2), which moved the isoelectric point (pI) of the protein toward the low pH (acidic) direction.

[0056] In addition, the data of Example 1 and Comparative Example 1 show that after the human superoxide dismutase is modified in the present invention, the water solubility is significantly improved, and the reason may also be that the pI of the protein is reduced after the modification, so that the protein has more negative charges on the surface under physiological pH conditions (pH 7.3), thereby improving the water solubility.

[0057] (2) Comparison of the specific activities of rmh Cu / Zn-SOD and rwh Cu / Zn-SOD.

[0058] Activity determination method: in accordance with the national standard for the determination of superoxide dismutase activity in health foods, number: GB / T5009.171-2003.

[0059] SOD enzyme activity determination: Take 2.35mL substrate buffer and 2mL PB in a 10mL centrifuge tube, add 0.15mL pyrogallol and mix immediately, take 2mL in a quartz cuvette, put it in a UV spectrophotometer to measure its initial absorbance at 325nm and the absorbance after 1min, the difference is the pyrogallol self-oxidation rate ΔA. Replace 2mL PB with 1.8mL PB and 0.20mL sample, and detect the sample's self-oxidation rate ΔA' in the same way. By diluting the sample by different multiples, ΔA' is equal to 1 / 2ΔA, that is, the inhibition ratio of SOD to the pyrogallol self-oxidation rate is 50%. The enzyme activity of SOD is calculated using the formula:

[0060]

[0061] Wherein: ΔA is the self-oxidation rate of pyrogallol, ΔA' is the self-oxidation rate of pyrogallol inhibited by the sample, 4.5 is the total reaction volume (mL), V is the sample volume (mL), and D is the dilution factor.

[0062] After rmhCu / Zn-SOD was purified, the protein content was determined by the BCA method and the SOD activity was determined by the above national testing standards. After the determination, the specific activity of rmhCu / Zn-SOD was calculated to be 7000U / mg. The specific activity of the unmodified rwhCu / Zn-SOD was about 4200U / mg protein. In contrast, the specific activity of the modified SOD was greatly improved. The reason may be related to the improved solubility of the modified protein, the increased solubility ratio of the expressed protein, the better spatial structure of the enzyme, and the significantly improved specific activity of SOD.

[0063] (3) Comparison of temperature tolerance between rmh Cu / Zn-SOD and rwh Cu / Zn-SOD.

[0064] The rmhCu / Zn-SOD and rwhCu / Zn-SOD solutions were placed in a 80°C water bath for 5, 10, 20, 30, 40, 50, and 60 minutes, respectively, and the enzyme activity was determined using the activity determination method in (2), and the enzyme activity without heat treatment was set as 100% residual activity. The SOD activity at different heat treatment times was determined, and the relative enzyme activity of each heat treatment time period was calculated. Each data was measured 3 times, and the average value was taken. The results are shown in Table 3.

[0065] Table 3

[0066] Insulation time (min) Residual activity of rmhCu / Zn-SOD (%) Residual activity of rwhCu / Zn-SOD (%) 5 100.0 100.0 10 100.0 100.0 20 99.1 92.8 30 96.8 85.7 40 94.9 77.8 50 92.2 69.4 60 90.7 60.3

[0067] As can be seen from Table 3, rmhCu / Zn-SOD still retains more than 90% of SOD enzyme activity after being kept at 80°C for 60 minutes. Its tolerance to high temperatures is significantly improved compared to the wild type, and it is more able to tolerate temperature changes during the preparation and processing of the enzyme.

[0068] (4) Comparison of the pH ranges of action of rmhCu / Zn-SOD and rwhCu / Zn-SOD.

[0069] The pH range from pH 2.2 to pH 11.0 was constructed using three buffer systems: ① pH 2.2 to pH 8.0 sodium dihydrogen phosphate-citric acid buffer; ② pH 9.0 to pH 10.0 Tris-HCl buffer; ③ pH 11.0 glycine-sodium hydroxide buffer. Take 1 mL of buffer and add 50 μL of purified SOD enzyme solution. After standing at 32°C for 1 hour, the enzyme activity was determined by the pyrogallol auto-oxidation method. The highest activity value was defined as the relative residual activity of 100, and the relative residual activity of each pH range was calculated. The measurement data was repeated 3 times and the average value was taken. The results are shown in Table 4.

[0070] Table 4

[0071] pH Residual activity of rmhCu / Zn-SOD (%) Residual activity of rwhCu / Zn-SOD (%) 2.2 72.6 35.3 3.0 87.0 60.2 4.0 98.2 88.0 5.0 99.8 95.1 6.0 100.0 100.0 7.0 98.6 98.7 8.0 92.4 93.2 9.0 73.7 75.2 10.0 68.5 78.2 11.0 58.3 67.6

[0072] As can be seen from Table 4, after the transformation, along with the decrease in pI, rmhCu / Zn-SOD obtained a wider enzyme activity range, and was able to reach more than 90% relative residual activity in the pH 4.0-pH 8.0 section, 87% residual activity under pH 3.0 conditions, and 72% residual enzyme activity in the pH 2.2-pH 3.0 range. Compared with rwhCu / Zn-SOD, it has better acid resistance; the activity of the modified rmhCu / ZnSOD did not decrease significantly compared with the wild type under the conditions of pH 8.0-pH 9.0.

[0073] The pH value of the stomach of a normal person after eating or drinking is about 3.5, the pH value of the small intestinal fluid of a normal person is about pH 7.6, and the pH value of the large intestinal fluid is about pH 8.3. It can be seen that the modified rmhCu / Zn SOD can play a good catalytic role in the pH environment of the human stomach and various intestinal sections.

[0074] In addition, although the catalytic activity of rmh Cu / Zn-SOD at pH 10 to pH 11 is slightly lower than that of rwhCu / Zn-SOD, this pH condition does not exist in the gastrointestinal tract of humans and animals.

[0075] (5) Comparison of the trypsin resistance of rmhCu / Zn-SOD and rwhCu / Zn-SOD.

[0076] Bovine trypsin (purchased from aladdin TPCK-trypsin), derived from bovine pancreas, CAS: 9002-07-7. The trypsin concentration was prepared to be 1 mg / ml. The enzyme action conditions were 37°C, pH 7.4, 0.05M Tris-HCl buffer. During the operation, trypsin and SOD were mixed at a ratio of 1:20 (W / W). The SOD activity at different action time periods was measured. The SOD enzyme activity at 0 hours of action was taken as the residual activity 100, and the measurement data was repeated 3 times, and the average value was taken. The results are shown in Table 5.

[0077] As shown in Table 5, rmhCu / Zn-SOD can maintain more than 90% residual activity in the presence of trypsin within 6 hours, while rwhCu / Zn-SOD can only maintain more than 90% residual activity within 2 hours. It can be seen that the modification of the present invention has greatly improved the hydrolysis tolerance of trypsin.

[0078] The pH of normal human small intestinal fluid is about 7.6, which is a weakly alkaline liquid with high trypsin activity. rmhCu / ZnSOD has a stronger tolerance to trypsin than the unmodified rwhCu / Zn-SOD, which makes it easier for it to play a catalytic role in the intestine or be absorbed into the body to play a role.

[0079] Table 5

[0080] Action time (h) Residual activity of rmhCu / Zn-SOD (%) Residual activity of rwhCu / Zn-SOD (%) 0 100.0 100.0 1 100.0 99.1 2 100.0 92.2 3 99.0 88.9 4 98.5 71.4 5 96.2 50.8 6 97.0 38.5 7 89.1 23.9 8 58.8 21.4 9 50.0 20.5

[0081] In summary, the present invention selectively replaces the variable amino acid residues of human copper / zinc superoxide dismutase to obtain efficient and soluble expressed rmh Cu / Zn-SOD; compared with rwh Cu / Zn-SOD, rmh Cu / Zn-SOD not only has greatly improved water solubility and enzyme specific activity, but also significantly improves tolerance to acidic environment and tolerance to trypsin; it can be seen that the rmh Cu / Zn-SOD provided by the present invention is suitable for large-scale production, has more commercial value, and is particularly suitable for the production and preparation of oral SOD products.

[0082] The above implementation cases are only for illustrating the technical solutions and features of the present invention, and their purpose is to enable people familiar with the technology to implement them better. They cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention are within the protection scope of the present invention, and those not described in detail are prior art.

Claims

1. A recombinant human copper / zinc superoxide dismutase, characterized in that: The amino acid sequence of the recombinant human copper / zinc superoxide dismutase is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the recombinant human copper / zinc superoxide dismutase according to claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.

2.

4. An expression cassette or recombinant vector comprising the nucleic acid molecule of claim 2.

5. A recombinant bacterium comprising the nucleic acid molecule according to claim 2 or the recombinant vector according to claim 4.

6. A method for preparing the recombinant human copper / zinc superoxide dismutase according to claim 1, characterized in that: The gene sequence encoding the recombinant human copper / zinc superoxide dismutase according to claim 1 is inserted into a vector, and the obtained recombinant vector is then transformed into a receptor cell, induced to express, and purified to obtain the product.

7. The method according to claim 6, characterized in that The recipient cell is Escherichia coli.

8. Use of the recombinant human copper / zinc superoxide dismutase as claimed in claim 1 in preparing food.

9. Use of the recombinant human copper / zinc superoxide dismutase as claimed in claim 1 in the preparation of a feed additive.

10. Use of the recombinant human copper / zinc superoxide dismutase according to claim 1 in the preparation of skin care products.

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