CN108315314A - A kind of polygalacturonase mutant TePG28b_ △ S88 and its gene and application - Google Patents

Abstract

The invention relates to the field of agricultural biotechnology, in particular, the invention relates to a polygalacturonase mutant TePG28b_ΔS88 and its gene and application. The mutant is obtained by removing the serine at position 88 of the polygalacturonase whose amino acid sequence is shown in SEQ ID NO.1. The polygalacturonase mutant provided by the present invention has high catalytic efficiency. Under the modified conditions, the K cat /K m of the mutant to polygalacturonic acid is higher than that of the wild type, and the K _cat /K _m of the mutant _ΔS88 is higher than that of the wild type. K _m increased by 1.17 times, and the optimum temperature of the enzymatic reaction remained the same as that of the wild type, but the optimum pH of the mutant N85E/S86W shifted to the right by one unit, and the pH of the other mutants remained unchanged.

Description

A polygalacturonase mutant TePG28b_△S88 and its gene and application

technical field

The invention relates to the field of agricultural biotechnology, in particular, the invention relates to a polygalacturonase mutant TePG28b_ΔS88 and its gene and application.

Background technique

Pectin is a methylated polygalacturonic acid substance, and about 75% of the carboxyl groups in the molecule are methylated. Polygalacturonase, a hydrolytic enzyme, is currently the most researched and most widely used commercial pectinase in the market.

The degradation of pectin by pectinases is of great commercial importance for various industrial applications, including food, feed, paper, textiles, fuels, and chemicals, and, pectinases can be used to produce pectin oligosaccharides, which contribute to the reduction of Damage caused by heavy metals and anti-obesity and antioxidant effects play an important role. Therefore, it is of great economic significance to develop, excavate and improve to obtain better pectinase resources.

Contents of the invention

The invention provides a polygalacturonase mutant with high catalytic efficiency, which is a mutant in which the 88th serine in the wild-type T3 loop region is cut off.

Another object of the present invention is to provide a gene encoding the above-mentioned mutant.

Another object of the present invention is to provide a recombinant vector comprising the above-mentioned mutant gene.

Another object of the present invention is to provide a recombinant strain comprising the above-mentioned mutant gene.

According to the high catalytic efficiency polygalacturonase mutant of the present invention, the amino acid sequence of its parental polygalacturonase is shown in SEQ ID NO.1, and the 88th serine on the wild-type T3loop region The amino acid sequence of the truncated mutant is shown in SEQ ID NO.2.

SEQ ID NO.1:

1 MRSFTQVLSF LLPAVSAAVA GKKGPGDNSE CVATEYSQVP TLAACTNVV

51 LRDIAVPSNS ALDLTSAKDN SVITFEGTTT FGFTNSSSFN PILLSGNNIT

101 ITGAPGSVID GNGQLYWDGL GSNGGVPKPD HFVYIKKLNK GSVIENLHIR

151 NWPVHCFSIN SCSDLTIRNL FLDNSAGNAP NNRSNGLAAA HNSDGFDIST

201 STNVVVKDTT VINQDDCVAV TSGDQITATG LTCIGGHGLS IGSVGGKSAN

251 NVTNVIFSKS AVIDSQNGAR IKTNYGTTGF VANITYEDIL LHNISIYGLD

301 VQQDYLNGGP TGTPTNGVII ENLLFKNLVG TMAKNSNARDYYILCGNGSC

351 SNFVFENVHI VGGESASSCN YPASGCP*

SEQ ID NO.2:

1 MRSFTQVLSF LLPAVSAAVA GKKGPGDNSE CVATEYSQVV PTLAACTNVV

51 LRDIAVPSNS ALDLTSAKDN SVITFEGTTT FGFTNSSFNP ILLSGNNITI

101 TGAPGSVIDG NGQLYWDGLG SNGGVPKPDH FVYIKKLNKG SVIENLHIRN

151 WPVHCFSINS CSDLTIRNLF LDNSAGNAPN NRSNGLAAAH NSDGFDISTS

201 TNVVVKDTTV INQDDCVAVT SGDQITATGL TCIGGHGLSI GSVGGKSANN

251 VTNVIFSKSA VIDSQNGARI KTNYGTTGFV ANITYEDILL HNISIYGLDV

301 QQDYLNGGPT GTPTNGVIIE NLLFKNLVGT MAKNSNARDY YILCGNGSCS

351 NFVFENVHIV GGESASSCNY PASGCP*

The present invention also provides genes encoding the above mutants.

The present invention also provides a recombinant vector comprising the above-mentioned mutant gene.

The present invention also provides a recombinant plasmid comprising the above-mentioned mutant gene.

The present invention also provides a method for preparing polygalacturonase mutants with high catalytic efficiency, comprising the following steps:

1) Transforming host cells with the above-mentioned recombinant vectors to obtain recombinant strains;

2) Cultivate the recombinant strain to induce the expression of recombinant polygalacturonase;

3) recovering and purifying the expressed polygalacturonase ΔS88A with high catalytic efficiency;

The polygalacturonase mutant provided by the present invention has high catalytic efficiency. Under the modified conditions, the K cat /K m of the mutant to polygalacturonic acid is higher than that of the wild type, and the K _cat /K _m of the mutant _ΔS88 is higher than that of the wild type. K _m increased by 1.17 times, and the optimum temperature of the enzymatic reaction remained the same as that of the wild type, but the optimum pH of the mutant N85E/S86W shifted to the right by one unit, and the pH of the other mutants remained unchanged.

The present invention also provides the application of the above polygalacturonase mutant with high catalytic efficiency. The polygalacturonase is industrially produced by means of genetic engineering, which can be used in feed, food, textile and other industries.

Description of drawings

Figure 1 shows the optimum temperature of the high catalytic efficiency polygalacturonase mutant and the parental wild type.

Figure 2 shows the optimum pH of the high catalytic efficiency polygalacturonase mutant and the parental wild type.

Detailed ways

Test materials and reagents

1. Strain: Pichia pastoris GS11, vector: pPIC9r.

2 Enzyme production medium: 30g/L wheat bran, 30g/L corn cob powder, 30g/L soybean meal, 5g/L (NH ₄ )SO ₄ , 1g/L KH ₂ PO ₄ , 0.5g/L MgSO ₄ 7H ₂ O, 0.01g/L FeSO ₄ 7H ₂ O, 0.2g/L CaCl ₂ in 1L deionized water, sterilized at 121°C for 20min

(1) Escherichia coli medium LB (1% peptone, 0.5% yeast extract, 1% NaCI, pH 7.0).

(2) YPD medium: 1% yeast extract, 2% peptone, 2% glucose

(3) MD solid medium: 2% glucose, 1.5% agarose, 1.34% YNB, 0.00004% Biotin

(4) BMGY medium; 1% yeast extract, 2% peptone, 1.34% YNB, 0.000049<Biotin, 1% glycerol (v/v).

(5) BMMY medium: replace glycerol with 0.5% methanol, and the rest of the ingredients are the same as BMGY, pH 4.0.

Example 1 Cloning of High Catalytic Efficiency Polygalacturonase Mutant Encoding Gene ΔS88

Using the recombinant plasmid pPIC9r-TePG28b of the gene TePG28b cloned from T. leycettanus JCM12802 as a template, design primers and then amplify

Table 1. Specific primers used for high catalytic efficiency polygalacturonase mutants

Example 2 Preparation of polygalacturonase mutants with high catalytic efficiency.

The recombinant plasmid pPIC9r-amy6 was amplified by specific point mutation to obtain the high catalytic efficiency polygalacturonase mutant plasmid pPIC9r-ΔS88, which was transformed into Pichia pastoris GS115 to obtain the recombinant yeast strain GS115-ΔS88.

Take the GS115 strain containing the recombinant plasmid, inoculate it in a 1L Erlenmeyer flask with 300mL of BMGY medium, place it at 30°C, and culture it on a shaker at 220rpm for 48h; then centrifuge the culture solution at 3000g for 5min, discard the supernatant, and use 100mL of 0.5% methanol for precipitation. The BMMY medium was resuspended, and placed again at 30°C, 220rpm to induce culture. Add 0.5 mL of methanol every 12 hours to keep the concentration of methanol in the bacterial solution at 0.5%, and take the supernatant for enzyme activity detection.

The optimal temperature of the recombinant polygalacturonase mutant with high catalytic efficiency is 70°C, which has not changed compared with the wild type, and the optimal pH is 3.5, which is consistent with the wild type. The K _cat /K _m of the mutants to polygalacturonic acid was increased compared with the wild type, and the K _cat /K _m of ΔS88 was increased by 1.17 times.

Example 3 Activity Analysis of Recombinant High Catalytic Efficiency Polygalacturonase Mutant and Female Parent Wild Type

1. The activity analysis of the polygalacturonase of the invention is carried out by DNS method. The specific method is as follows: under the given pH and temperature conditions, 1 mL of reaction system includes 100 μL of appropriate diluted enzyme solution, 900 μL of substrate, react for 30 minutes, add 1.5 mL of DNS to terminate the reaction, and boil for 5 minutes. After cooling, the OD value was measured at 540 nm. Definition of amylase activity unit: under the conditions of 70°C and pH 3.5, the amount of enzyme required to catalyze the hydrolysis of the substrate to release 1 μmol of reducing sugar per minute is an enzyme activity unit.

2. Determination of the properties of recombinant high catalytic efficiency polygalacturonase mutant and maternal wild type

1. The optimal temperature determination method of the recombinant high catalytic efficiency polygalacturonase mutant and the female parent wild type is as follows:

The determination of the optimal temperature of the recombinant high catalytic efficiency polygalacturonase mutant and the parental wild type is in the buffer system of 0.1mol/L citric acid-disodium hydrogen phosphate (pH 3.5) and different temperatures Perform enzymatic reactions. The optimum temperature determination results of the enzyme reaction (Figure 1) showed that the optimum temperature of the recombinant polygalacturonase mutant (70°C) with high catalytic efficiency was consistent with that of the wild type (70°C).

2. The pH assay method of the recombinant high catalytic efficiency polygalacturonase mutant and the female parent's wild type is as follows:

The recombinant polygalacturonase mutant with high catalytic efficiency purified in Example 2 and the parental wild type were subjected to enzymatic reactions at different pHs to determine their optimum pH. The substrate polygalacturonic acid was tested for polygalacturonase enzyme activity in 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution with different pH at 70°C. The results ( FIG. 2 ) showed that the optimal pH of the mutant ΔS88 was 3.5, which was consistent with that of the wild type of the mother.

3. The method for determining the specific activity of the polygalacturonase mutant with high catalytic efficiency and the wild type of the mother is as follows:

Using 0.66% polygalacturonic acid as a substrate, reacted for 10min under the optimal conditions (PH 3.5, 70°C) to measure the activity. The recombinant polygalacturonase mutant with high catalytic efficiency was improved compared with the wild type.

4, the kinetic parameter determination method of group high catalytic efficiency polygalacturonase mutant and female parent's wild type is as follows:

The reaction time for determining the kinetic constants of TePG28b and mutants is 5 min, with polygalacturonic acid in the concentration range of 0.4-5 mg/mL as the substrate, pH 3.5, and the temperature of 70 °C to measure the enzyme activity, using the software GraphPad Prism 5 K _m and V _max were calculated by hyperbolic fitting of enzyme kinetics, and K _m and V _max were calculated by using Excel software. Table 2 shows the K _m , V _max , k _cat , and k _cat /K _m values of the polygalacturonase mutant with high catalytic efficiency and the parental wild type under optimal conditions, respectively.

Table 2 Kinetic parameters of recombinant high catalytic efficiency polygalacturonase mutant and maternal wild type

sequence listing

<110> Institute of Feed, Chinese Academy of Agricultural Sciences

<120> A polygalacturonase mutant TePG28b_△S88 and its gene and application

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Claims (6)

1. A high temperature and high specific activity polygalacturonase mutant TePG28b_△S88, characterized in that, by removing the amino acid sequence such as the 88th position of polygalacturonase shown in SEQ ID NO.1 The mutant was obtained from serine. 2. The polygalacturonase mutant gene with high temperature and high specific activity, characterized in that it encodes the polygalacturonase mutant TePG28b_ΔS88 with high temperature and high specific activity according to claim 1. 3. A recombinant vector comprising the polygalacturonase mutant gene with high temperature and high specific activity according to claim 2. 4. A recombinant bacterial strain comprising the high temperature and high specific activity polygalacturonase mutant gene of claim 2. 5. A method for preparing the high temperature and high specific activity polygalacturonase mutant TePG28b_△S88 of claim 1, characterized in that the method comprises the following steps: 1) Constructing a recombinant vector comprising the gene encoding the high temperature and high specific activity polygalacturonase mutant TePG28b_ΔS88. 2) Transforming host cells with the recombinant vector. 3) fermenting the host cell, and isolating the polygalacturonase mutant TePG28b_ΔS88 with high temperature and high specific activity. 6. The application of the high temperature and high specific activity polygalacturonase mutant TePG28b_ΔS88 described in claim 1.