KR100687884B1 - 1 2 A Method for Mass Production of Recombinant Trypsin by Employing Regulatory Genes sgtR1 and sgtR2 from Streptomyces griseus - Google Patents

Abstract

A method for mass production of recombinant trypsin by using regulatory genes sgtR1 and sgtR2 derived from Streptomyces griseus is provided to improve production yield of trypsin compared to the conventional methods by using a transformant. A trypsin recombinant expression vector for transforming prokaryotes contains sprT gene derived from Streptomyces griseus, and sgtR1 and sgtR2 genes derived from Streptomyces griseus, wherein the trypsin recombinant expression vector is pWHM3-TR1, pWHM3-TR2 or pWHM3-TR1R2. The method for mass production of recombinant trypsin comprises the steps of: constructing the trypsin recombinant expression vector; producing transformed Streptomyces griseus with the trypsin recombinant expression vector; and culturing the transformed Streptomyces griseus and harvesting trypsin from the cultured medium.

Description

A Method for Mass Production of Recombinant Trypsin by Employing Regulatory Genes, sgtR1 and sgtR2 from Streptomyces griseus}

1 shows the genetic maps of recombinant vectors pWHM3-T, pWHM3-TR1, pWHM3-TR2 and pWHM3-TR1R2, respectively.

Figure 2 is an electrophoresis photograph indirectly comparing the amount of mRNA expressed in the transformants of the recombinant vector pWHM3-T and pWHM3-TR1R2.

The present invention is Streptomyces griseus ) and a method for mass production of recombinant trypsin using the regulatory genes sgt R1 and sgt R2. More specifically, the present invention provides a recombinant expression vector of a trypsin comprising a trypsin gene and a regulatory gene derived from Streptomyces griesus , sgt R1 and sgt R2, a transformant transformed with an electric expression vector, and an electric trait. The present invention relates to a method for mass production of recombinant trypsin using a transformant.

Enzymes have been used in various industrial fields, such as beverage production, meat processing, dairy processing, brewing, baking, fats and oils, and enzyme sensors for a long time. Currently, proteolytic enzymes among industrially used enzymes play a commercially important role in various fields such as making foodstuffs such as Cheonggukjang, shrimp sauce, bakery, cheese, and medicines and detergents such as digestive and anti-inflammatory agents.

In particular, trypsin, which exhibits enzymatic reactivity to various substrates among proteolytic enzymes, has been industrially useful. However, because of the property of secreting extracellularly to break down proteinaceous substrates, trypsin genes are present in very low concentrations intracellularly and are rapidly degraded once secreted out of the cell and subjected to enzymatic reactions. Even when using a genetic recombination method, there is a disadvantage that the production yield is relatively lower than other enzymes. In order to overcome this drawback, studies are being actively conducted to improve the activity of the produced trypsin or to maintain the activity of trypsin for a long time.

For example, US Pat. No. 6,177,268 discloses a method for improving the enzyme activity of trypsin comprising adding 3 to 10 mmol / L of calcium ions and manganese ions to an enzyme reaction solution to prevent deterioration of the enzyme activity of trypsin. US Pat. No. 5945328 discloses a method for introducing a tryptic gene into a mycelial fungus, producing a transformed mycelial fungus, and culturing the same to produce an improved trypsin, US Pat. No. 5,804,410. No. discloses a novel protease that exhibits enzymatic activity similar to trypsin and can exhibit enzymatic activity for a long time. International Patent Publication No. WO 02/061064 discloses inoculation of Pichia pastoris strains into pancreatic cells of pigs. And a method for producing trypsin with improved enzymatic activity by culturing.

However, even if the enzyme activity of trypsin is improved by using this method, the amount of trypsin itself does not increase, so it is not a fundamental solution in terms of industrial applications requiring trypsin above a certain level. .

Therefore, there is a need to develop a method for mass production of trypsin.

Accordingly, the present inventors have made intensive studies to develop a method for producing trypsin in large quantities. As a result, the present inventors expressed expressions including sgt R1 and sgt R2, which are regulatory genes derived from Streptomyces griseus together with the trypsin gene. When using a transformant transformed with a vector, it was confirmed that the expression level of recombinant trypsin can be significantly improved, and the present invention was completed.

After all, the main object of the present invention is to provide a trypsin recombinant expression vector comprising sgt R1 and sgt R2, which are regulatory genes derived from Streptomyces griseus with trypsin gene.

Another object of the present invention is to provide a transformant transformed with the trypsin recombinant expression vector.

Still another object of the present invention is to provide a method for mass production of recombinant trypsin, which comprises the step of culturing the transformant and obtaining trypsin therefrom.

The method for mass production of recombinant trypsin of the present invention comprises a trypsin gene and Streptomyces culturing Streptomyces glisus transformed with a trypsin recombinant expression vector comprising a regulatory gene sgtR1 , a regulatory gene sgtR2 or both derived from griseus ) and obtaining trypsin from the culture. In this case, the recombinant expression vector including the electric trypsin gene, the regulatory gene sgtR1 , and the regulatory gene sgtR2 is not particularly limited, and the Streptomyces glisus strain transformed with the electric recombinant expression vector is not particularly limited.

The present inventors came to pay attention to Streptomyces griceus, which is known to produce various proteolytic enzymes while researching to improve the expression level of trypsin. So far as is known, Streptomyces griesus contains trypsin gene ( sprT ) and its base sequence has been reported with the fact that there is an unknown gene downstream of the electric gene ( Ko , B.-J. et al, Journal of Microbiology and Biotechnology, 8 (4): 333-340, 1998; GenBank accession no. AY588948). It was anticipated that genes with unknown electrical function could regulate the expression of trypsin.

Thus, the present inventors, in order to confirm the above-described assumption, as a result of transforming the actinomycetes by introducing the sprT and the regulatory gene together into the expression vector, it was confirmed that the expression level of trypsin increased by 20 times or more by the regulatory gene.

Recombinant trypsin production method of the present invention can significantly improve the productivity of trypsin compared to the conventional method, it will be widely used for economic production of trypsin-based products.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Obtaining Trypsin Gene and Regulatory Gene from Streptomyces gliseus

Streptomyces System to give the trypsin gene (sprT) (SEQ ID NO: 1) and the sgt R1 regulatory gene present downstream thereof (SEQ ID NO: 2) and sgt R2 (SEQ ID NO: 3) from the draw three-house.

First of all, Streptomyces TK24 ( Streptomyces) griseus In order to mass isolate chromosomal DNA of TK24 , KCTC-9080, 100 ml R2YE medium (sucrose 103g / L, 0.25g / L, 10.12g / L, glucose 10g / L, casamino acid 0.1g / L) , yeast extract 5 g / L, (0.5%) 5 ml / L, (3.68%) 40 ml / L, L-proline (20%) 7.5 ml / L, TES (5.73%, pH 7.2) 50 ml / L, trace element 1ml / L: 0.04g / L, 0.2g / L, 0.01g / L, 0.01g / L, 0.01g / L, 0.01g / L) for 36 hours, and lysozyme 2mg / 10 ml of Buffer I (0.3 M sucrose, 25 mM TrisHCl pH 8.0, 25 mM EDTA pH 8.0) containing ㎖ and RNase was added and reacted at 37 ° C. for 1 hour, followed by 10% (w / v). 1,200 μl of SDS solution was added and reacted at 60 ° C. for 30 minutes. Then, 2 ml of 5M NaCl solution was added and stirred slowly for 2 hours, and then 15 ml of chloroform was added and stirred for 2 hours. The reaction was then centrifuged at 3,500 rpm for 20 minutes to give a supernatant, 30 ml of 100% ethanol was added and stirred overnight. Finally, the precipitated chromosomal DNA was obtained, dried, and then dissolved in 10 ml of Al-A-TE buffer to obtain the desired chromosomal DNA.

The 2.2 kb fragment obtained by treating the electrochromosomal DNA with Eco RI and Bgl II and the 0.46 kb fragment obtained by treating with Bgl II and Nco I were inserted into ptrc ΔT (TaKaRa Shuzo Co., Japan), and then again. A fragment obtained by treatment with Bgl II and Hind III was introduced into the Eco RI and Hind III sites of the pWHM3 vector (TaKaRa Shuzo Co., Japan), which is an E. coli-actinomycetes covalent vector, to construct a 9.82 kb pWHM3-T recombinant vector. (See FIG. 1).

On the other hand, the obtained chromosomal DNA was used as a template, and primers sgtR1- F (5'-CCCGCACGCTCTGCAGGCACGTACCG-3 ', pst I) (SEQ ID NO: 4), sgt R1-R (5'-CGCCCTCGACCTGGAGGTCTGCAGAACCG-3', pst I) by performing PCR using a (SEQ ID NO: 5), to give the sgt R1 gene fragment of 727bp size.

In addition, the electrical yield and the chromosome DNA as a template, sgt R2-F (5'- GCCGGCCCGGAAGCTTCGGTCGAAGG-3 ', Hind III) ( SEQ ID NO: 6), sgtR2-R (5' -CGGCCCCCTCAAGCTTCGACCCCTGC -3', Hind III) ( by performing PCR using SEQ ID NO: 7) yielded the sgt R2 gene fragment of 671bp size.

Finally, using the obtained chromosomal DNA as a template, PCR was performed using primers sgtR1 -F and primers sgtR2 -R to obtain gene fragments containing sgtR1 and sgtR2 regions of 1,223bp size at the same time.

Example 2 Construction of Trypsin Recombinant Vector and Transformant Comprising the Same

The three gene fragments obtained in Example 1 were cut into Pst I and Hind III, respectively, and then cloned into pWHM3-T treated with the same restriction enzyme to sgt R1, sgt R2, and sgt R1R2 downstream of the spr T gene. Recombinant vectors introduced (pWHM3-TR1, pWHM3-TR2, pWHM3-TR1R2) were constructed (see Fig. 1). 1 shows the genetic maps of recombinant vectors pWHM3-T, pWHM3-TR1, pWHM3-TR2 and pWHM3-TR1R2, respectively.

On the other hand, Streptomyces glisus was incubated for 2 days in 100ml R2YE liquid medium, and then centrifuged for 5 minutes at 2,800rpm to obtain the cultured cells. Next, P buffer (lyucose 30.9g / L, K ₂ SO ₄ 75mg / L, MgCl ₂ 6H ₂ O 0.606g / L, trace element 0.6ml / L, KH ₂ PO _{4 in} which 20 mg of lysozyme dissolved) (0.5%) 10 mL, 100 mL of CaCl ₂ H ₂ O (3.68%), 100 mL of TES (0.25 M, pH 7.2) was suspended, the cells obtained were reacted at 30 ° C for 1 hour, and filtered. , Protoplasts were obtained. It was then washed several times with P buffer and stored at -70 ° C.

Protoplasts stored at −70 ° C. were dissolved, PEG solution (sucrose (10.3%) 250 mL / l, K ₂ SO ₄ (2.5%) 10ml / l, trace element 2ml / l, 5M CaCl ₂ 0.2 ml, 1 M Tris maleic acid 0.5 ml (pH 8.0)) and 200 µl of each of the recombinant vectors thus obtained were left to stand for 5 minutes, and then plated in a pre-prepared R2YE plate medium and incubated at 30 ° C. Then, after 15 hours, 3 ml of the liquid medium containing antibiotics and 0.4% agar was again applied, and when completely dried, incubated for 4 more days at 30 ° C. to obtain recombinant colonies, and each transformant was It was constructed.

Example 3 Determination of Trypsin Activity Produced by Each Transformant

Each transformant transformed with the recombinant vectors pWHM3-T, pWHM3-TR1, pWHM3-TR2 and pWHM3-TR1R2 prepared in Example 2 were cultured in the R2YE liquid medium for 3 days, 5 days, 7 days, and 5,000 Centrifugation at rpm for 3 minutes yielded a supernatant, which was used as a sample.

10ul of DMSO solution in which trypsin substrate (N-α-benzoyl-DL-arginine-ρ-nitroanilide) was dissolved in 890ul of Tris buffer (pH 8.0) at a concentration of 1mg / ml, and 100ul of each supernatant sample prepared before Were mixed and reacted at 37 ° C. for 1 hour. Subsequently, 30% (v / v) acetic acid was added to terminate the reaction, absorbance was measured at 405 nm, and the measured value was converted to trypsin activity (see Koo, B.-J. et al, Journal of Microbiology and Biotechnology, 8 (4): 333-340, 1998). Comparison was made for each transformant. At this time, the vector pWHM3 into which nothing was introduced was used (see Table 1).

Activity comparison of trypsin expressed in transformants transformed with each recombinant vector (OD ₄₀₅ ) Recombinant vector Culture 3 days 5 days 7 days Control pWHM3-T pWHM3-TR1 pWHM3-TR2 pWHM3-TR1R2 0.029 0.01 0.028 0.097 0.204 0.039 0.015 0.048 0.186 0.392 0.023 0.061 0.102 0.400 0.542

As shown in Table 1, the trypsin expressed in the transformant containing a recombinant vector containing the trypsin gene activity was improved as the culture period elapsed, in the transformant containing a recombinant vector containing only the trypsin gene The activity of trypsin expressed in a transformant comprising a recombinant vector comprising a trypsin gene and a regulatory gene, rather than the expressed trypsin. In particular, the activity of trypsin expressed in a transformant comprising a recombinant vector comprising a trypsin gene and two regulatory genes showed high levels.

Considering that the change in activity includes the same trypsin gene, it was analyzed that the change in the expression level of trypsin was found, and it was confirmed that the recombinant vector containing the regulator gene could increase the expression level of trypsin. .

Example 4 Comparison of Expression of Trypsin Gene by Reverse Polymerase Chain Reaction (RT-PCR)

To determine at what stage the increase in trypsin expression by the regulatory genes sgt R1 and sgt R2 is regulated, the expression levels of the trypsin gene ( sprT ) were compared by mRNA levels: pWHM3-T and pWHM3-TR1R2 Transformants were cultured in R2YE liquid medium for 2 and 6 days to extract total RNA. After removing DNA by DNase treatment on the extracted total RNA, pure RNA was obtained, and reverse polymerase chain reaction was used to obtain respective cDNAs and compared by electrophoresis (see FIG. 2). 2 is an electrophoresis photograph indirectly comparing the amount of mRNA expressed in the transformants of pWHM3-T and pWHM3-TR1R2 recombinant vectors, where M is a marker and lane 1 is pWHM3-T cultured for 2 days Lane 2 is a transformant of pWHM3-TR1R2 cultured for 2 days, lane 3 is a transformant of pWHM3-T cultured for 6 days, lane 4 is cultured for 6 days One pWHM3-TR1R2 is a transformant.

As shown in Figure 2, sprT For pWHM3-TR1R2 a was observed only after 6 days without For pWHM3-T transformant genes only introduced the expression of the second day of the spr T is not observed, control gene sgtR1 and sgtR2 is introduced with the expression of the spr T Significant induction was induced from day 2, and on day 6 it was confirmed that it is significantly overexpressed than in the case of pWHM3-T transformants. These results suggest that co-expression of regulatory genes maintains gene transcription in excess of stable sprT gene expression from early to late cell growth.

As described and demonstrated in detail above, the present invention is transformed with a recombinant expression vector and an electric expression vector of trypsin, including sgt R1 and sgt R2, which are regulatory genes derived from trypsin gene and Streptomyces griseus The present invention provides a method for mass production of recombinant trypsin using the transformants and the electric transformants. Recombinant trypsin production method of the present invention can significantly improve the productivity of trypsin compared to the conventional method, it will be widely used for economic production of trypsin-based products.

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Claims (4)

Streptomyces cis draw and aged mouse (Streptomyces griseus), including both the trypsin gene (sprT) derived, Streptomyces cis draw three-house (Streptomyces griseus) derived from the control gene sgtR1, regulatory genes sgtR2 thereof, transforming a prokaryotic Trypsin recombinant expression vector. The method of claim 1, PWHM3-TR1, pWHM3-TR2 or pWHM3-TR1R2 disclosed in FIG. Trypsin recombinant expression vector. Streptomyces griseus , transformed with the recombinant expression vector of claim 1. A method of mass producing recombinant trypsin comprising culturing the transformed Streptomyces griseus of claim 3 and obtaining trypsin from the culture.

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