NOVEL SERINE PROTEASE VARIANT

Abstract

The present disclosure provides a novel serine protease variant.

Claims

1. A serine protease variant having a sequence homology of at least 80% and less than 100% with an amino acid sequence of SEQ ID NO: 31, wherein at least one amino acid selected from an amino acid corresponding to a 12.sup.th position and an amino acid corresponding to a 116.sup.th position of the amino acid sequence of SEQ ID NO: 31 is substituted with a different amino acid.

2. The serine protease variant according to claim 1, wherein the amino acid corresponding to the 12.sup.th position is substituted with tyrosine (Y), alanine (A), serine (S), or arginine (R).

3. The serine protease variant according to claim 1, wherein the amino acid corresponding to the 116.sup.th position is substituted with aspartate (D), serine (S), threonine (T), or glycine (G).

4. The serine protease variant according to claim 1, wherein the serine protein variant has a homology to SEQ ID NO: 2 of at least 80% and less than 100%.

5. The serine protease variant according to claim 1, wherein the serine protease variant comprises an amino acid sequence as set forth in any one selected from the group consisting of SEQ ID NOS: 3 to 10 and SEQ ID NOS: 32 to 39.

6. A polynucleotide encoding the serine protease variant of claim 1 .

7. (canceled)

8. A microorganism expressing the serine protease variant of claim 1.

9. The microorganism according to claim 8, wherein the microorganism belongs to the genus Bacillus.

10. The microorganism according to claim 9, wherein the microorganism is Bacillus subtilis.

11. A feed composition comprising at least one between the serine protease variant of claim 1 and a microorganism expressing the same.

12. The polynucleotide according to claim 6, wherein the amino acid corresponding to the 12.sup.th position of SEQ ID NO: 31 is substituted with tyrosine (Y), alanine (A), serine (S), or arginine (R).

13. The polynucleotide according to claim 6, wherein the amino acid corresponding to the 116.sup.th position of SEQ ID NO: 31 is substituted with aspartate (D), serine (S), threonine (T), or glycine (G).

14. The polynucleotide of claim 6, wherein the serine protein variant has a homology to SEQ ID NO: 2 of at least 80% and less than 100%.

15. The polynucleotide according to claim 6, wherein the serine protease variant comprises an amino acid sequence as set forth in any one selected from the group consisting of SEQ ID NOS: 3 to 10 and SEQ ID NOS: 32 to 39.

16. The microorganism according to claim 8, wherein the amino acid corresponding to the 12.sup.th position is substituted with tyrosine (Y), alanine (A), serine (S), or arginine (R).

17. The microorganism according to claim 8, wherein the amino acid corresponding to the 116.sup.th position is substituted with aspartate (D), serine (S), threonine (T), or glycine (G).

18. The microorganism according to claim 8, wherein the serine protease variant has a homology to SEQ ID NO: 2 of at least 80% and less than 100%.

19. The microorganism according to claim 8, wherein the serine protease variant comprises an amino acid sequence as set forth in any one selected from the group consisting of SEQ ID NOS: 3 to 10 and SEQ ID NOS: 32 to 39.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0093] The serine protease variant of the present disclosure may be efficiently used in various industrial fields due to its enhanced activity compared to conventional serine proteases and an activity at high temperature and heat resistance.

BEST MODE

[0094] Hereinafter, the present disclosure will be described in more detail with reference to the following examples and experimental examples. However, these examples and experimental examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1. Selection of Serine Protease Variant Derived from Thermobifida fusca

Example 1-1: Preparation of Thermobifida fusca-Derived Serine Protease Library

[0095] Random mutation was introduced into a gene encoding an amino acid (SEQ ID NO: 31) corresponding to a mature region of a Thermobifida fusca-derived serine protease by error-prone PCR. The Error prone PCR was performed using a Diversify™ PCR Random Mutagenesis Kit (Clontech, Cat No. 630703), and the PCR conditions used are as described in Table 1 below. It was confirmed that the mutation was introduced at a frequency of 6.2 mutations/kb.

TABLE-US-00001 TABLE 1 Amount Remarks Composition Template DNA 0.5 ng SEQ ID NO: 1 Primer_forward 5 μM SEQ ID NO: 11 Primer_reverse 5 μM SEQ ID NO: 12 10X Titanium Taq buffer 5 μL MnSO.sub.4 640 μM dGTP 40 μM 50X Diversify dNTP mix 1 μL 50X dNTP mix 1 μL Titanium Taq Polymerase 1 μL Total 50 μL Adjust to 50 μL with DW PCR Conditions I. 94° C. 30 sec Repeat 50 cycles of II II. 94° C. 30 sec and III III. 68° C. 1 min IV. 68° C. 1 min V. 4° C. ∞

[0096] PCR fragments obtained in the above process were each ligated to a vector amplified using primers shown in Table 2 below using an In-FusionR HD cloning kit (Clontech) and transformed into DH5∝ cells to obtain colonies. The plasmids in the obtained colonies were purified to obtain a library having a size of about 5×10.sup.4.

TABLE-US-00002 TABLE 2 Template DNA (pBE-S-TAP) SEQ ID NO: 1 Primer_forward SEQ ID NO: 13 Primer_reverse SEQ ID NO: 14

Example 1-2: Screening of Thermobifida fusca-Derived Serine Protease Library

[0097] A Bacillus subtilis LB700 strain, which easily releases proteins, was transformed with the protease library prepared in Example 1-1 and screened. The screening was performed by a two-stage process. In a first stage, the Bacillus subtilis strain transformed with the library was plated on a 2% skim milk plate, and the desired colonies were selected based on the halo size. The transformation of Bacillus subtilis was performed according to the Groningen method and compositions of the skim milk used in the screening are as shown in Table 3 below.

TABLE-US-00003 TABLE 3 Composition Amount Remarks M9 minimal salts 11.28 g BD, Cat. No. 248510 Agar 20 g Skim milk 20 g BD, Cat. No. 232100 50% Glucose 20 g 1M MgSO.sub.4 2 mM 1M CaCl.sub.2 0.1 mM Kanamycin 50 μg/mL

[0098] (per 1 L)

[0099] A second stage relates to a method of re-selecting the colonies, which were selected in the first stage, by azocasein color development. A Brain Heart Infusion (BHI, bd, Cat. No. 53286) liquid medium containing a kanamycin antibiotic was added to a 96 deep well plate, and the colonies selected in the first stage were inoculated thereinto, followed by incubation at 37° C. for 20 to 24 hours. After the incubation, a supernatant including an enzyme was obtained by centrifugation, and the supernatant was mixed with an equal amount of 2% (w/v) azocasein, as a substrate, followed by a reaction at 37° C. for 1 hour. The reaction was terminated by adding a 3-fold volume of 10% trichloro acetic acid (TCA) to the enzyme reaction solution and coagulated proteins were removed by centrifugation. The color development reaction was performed by mixing the resultant with an equal amount of NaOH, and then the absorbance was measured at 440 nm to compare the degree of color development. Through this process, colonies having an increase in absorbance by 150% or more compared to the wild-type serine protease were selected.

Example 2. Preparation of Selected Variant and Evaluation of Activity

Example 2-1: Preparation of Variant

[0100] As a result of analyzing the sequences of variants selected from screening, it was confirmed that the 12.sup.th amino acid (phenylalanine, Phe) and the 116.sup.th amino acid (asparagine, Asn) of SEQ ID NO: 31 were substituted with tyrosine (Tyr) and aspartate (Asp), respectively. In FIG. 1, the positions of the mutations are indicated. After the two selected variants (F12 and N116) were re-introduced into pBE-S-TAP plasmid in the form of single mutation by site directed mutagenesis, the activities of the strains with a double mutation and a single mutation, respectively, were compared with the activity of the wild-type strain. Primers used to prepare the variants are as shown in Table 4 below.

TABLE-US-00004 TABLE 4 TAP_F12Y_F SEQ ID NO: 15 TAP_F12Y_R SEQ ID NO: 16 TAP_N116D_F SEQ ID NO: 17 TAP_N116D_R SEQ ID NO: 18

Example 2-2: Evaluation of Activity

[0101] After the Bacillus subtilis LB700 strain was transformed with the prepared plasmid, the activity evaluation of the transformant was performed using a N-SUCCINYL-ALA-ALA-PRO-PHE-P-NITROANILIDE (Sigma, cat #57388, hereinafter, referred to as SUC-AAPF-pNA) peptide as a substrate. The transformed Bacillus subtilis strain was inoculated into the Brain Heart Infusion (BHI, bd, cat #53286) liquid medium containing a kanamycin antibiotic and cultured at 37° C. for 20 to 24 hours, and a portion of the culture solution except for the cells was mixed with a 25 mM Tris-HCI (pH7.5) buffer and a 1 mM Suc-AAPF-pNA, followed by reaction at 37° C. for 30 minutes. The absorbance of the reaction solution was measured at 410 nm. An extinction coefficient of para-nitroaniline produced by the enzyme known in a literature was 8,800M.sup.−1 cm.sup.−1 at 410 nm, and the unit of the enzyme was calculated based thereon (Barrett, A. J., Cathepsin G. Methods Enzymol., 80, Pt. C, 561-565, (1981)). The measured activities are shown in Table 5 below.

TABLE-US-00005 TABLE 5 Enzyme activity (unit/ml) Wild-type 16.3 F12Y 34.0 F12YN116D 63.8

[0102] As a result of the measurement, it was confirmed that the activities of the F12Y and F12YN116D variants were increased in conditions at pH 7.5 and 37° C. by about 2.1- and 3.9-fold, respectively.

Example 2-3: Evaluation of Thermal Stability

[0103] Since thermal stability is a very important property of the enzyme, an experiment described below was performed to confirm the influence of introducing a variant on thermal stability.

[0104] Specifically, the enzyme activities of the samples used in Example 2-2 for the activity evaluation were measured after placing the samples at room temperature, at 70° C., at 80° C., and at 90° C. for 5 minutes, respectively. The measured activities are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Heat Enzyme activity (unit/mL) treatment RT, 70° C., 80° C., 90° C., conditions 5 min 5 min 5 min 5 min Wild-type 16.3 15.9 10.6 0.3 F12Y 34.0 34.7 22.8 0.1 F12YN116D 63.8 63.3 41.7 0.1

[0105] As a result of the measurement, it was confirmed that the F12Y and F12YN116D mutation exhibited about 2-and 4-fold higher enzyme activities than the wild-type strain even at 80° C., respectively. Since it was confirmed that a high activity of the serine protease variant of the present disclosure was maintained even at high temperature, the serine protease variant can be effectively used in the industry.

Example 3. Preparation and Selection of Saturation Mutagenesis Library

Example 3-1. Preparation of Saturation Mutagenesis Library of F12 and N116 Residues

[0106] In order to identify the effects of substitution of each of the residues of F12 and N116 (i.e., previously selected variants) with a residue other than tyrosine and aspartate on their activities, saturation mutagenesis libraries were prepared with respect to the two residues.

[0107] Two PCR fragments were obtained using the pBE-S-TAP plasmid as a template and the primer pairs of SEQ ID NOS: 11 and 12 and SEQ ID NOS: 13 and 14, respectively. The fragments were each ligated using the In-Fusion HD cloning kit and then transformed into DH5α cells, thereby obtaining colonies. The plasmids in the obtained colonies were purified to obtain a library having a size of about 4×10.sup.3.

TABLE-US-00007 TABLE 7 Template DNA (pBE-S-TAP) SEQ ID NO: 1 Saturation mutagenesis_F_1 SEQ ID NO: 19 Saturation mutagenesis_R_1 SEQ ID NO: 20 Saturation mutagenesis_F_2 SEQ ID NO: 21 Saturation mutagenesis_R_2 SEQ ID NO: 22

Example 3-2: Screening of Saturation Mutagenesis Library and Activity Evaluation

[0108] The saturation mutagenesis libraries prepared in Example 3-1 were subjected to screening in the same manner as in Example 1-2. Variants having the same or increased activity compared to the F12YN116D variant were selected by screening, and activity evaluation was performed on these variants by sequence analysis using Suc-AAPF-pNA as a substrate.

TABLE-US-00008 TABLE 8 Enzyme activity (unit/mL) Wild-type 23.52 F12YN116D 65 F12YN116S 77.35 F12SN116D 61.75 F12SN116T 117.65 F12AN116G 94.9 F12A 94.25 F12R 58.5

[0109] As a result, it was found that the F12 and N116 variants increased their activities even when their residues were each substituted with a different amino acid (e.g., F12S, F12A, F12R, N116S, N116T, N116G, etc.) in addition to tyrosine and aspartate confirmed in Example 2.

[0110] From the above results, it was confirmed that the F12 and N116 residues are important residues for exhibiting the activity of the serine protease, and the enzyme activity of the serine protease can be increased by substituting each of these residues with a different amino acid. Therefore, the serine protease variant of the present disclosure with an increased enzyme activity can be effectively used in the industry.

[0111] From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.