Thermally stable glucoamylase TaGA58040, encoding gene and application thereof

Abstract

The present application relates to the field of agricultural biotechnology, in particular to a heat-stable glucoamylase TaGA58040 and a coding gene and application thereof.The amino acid sequence of the glucoamylase TaGA580400 of the present application is shown in SEQ ID NO:1 or SEQ ID NO:2.Belonging to the 15th family of glycoside hydrolase, the optimal pH of the enzyme is 5.0, and in the pH range of 3.0-6.0, the enzyme can maintain more than 95% of its enzyme activity; the optimal temperature of the enzyme is 60 DEG C, and after being treated at 60 DEG C for 60 min, the enzyme activity can still maintain more than 90%, and the enzyme has good temperature stability.According to the technical scheme of the present application, a glucoamylase with excellent properties and suitable for industrial application can be produced by genetic engineering means.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, in particular to a thermostable glucoamylase TaGA58040, and an encoding gene and application thereof. BACKGROUND

[0002] Glucoamylase is the largest production and most widely used enzyme in China, which has important application value in light industry, food, medicine, feed, fermentation and other industries. However, the industrial glucoamylase has the problems of low optimum reaction temperature and poor thermal stability, which leads to the need to reduce the reaction temperature to adapt to the catalytic performance of glucoamylase in the production and application process. Specifically, in the starch sugar production process, first, high-temperature alpha-amylase is used to liquefy starch into dextrin, maltose and smaller oligosaccharides under the conditions of 95-105℃ and pH 5.5-6.5; then, the temperature is cooled to 60-65℃, the pH is adjusted to 4.0-4.5, and then glucoamylase is added to further decompose the starch liquefaction product into glucose.

[0003] At present, the industrial application of glucoamylase is mainly derived from Aspergillus niger, Rhizopus oryzae and Aspergillus oryzae. The optimum temperature of these enzymes is generally 55-60℃, and the optimum pH is 3.5-5.0. Therefore, in order to ensure the maximum activity of glucoamylase, additional cooling equipment is needed to reduce the temperature from 95℃ to 60℃ when switching from the starch liquefaction process to the saccharification process, which leads to high energy consumption and high cost of the starch sugar industry. Therefore, exploring new types of glucoamylase has great application prospect and practicality for the development of the starch sugar industry. SUMMARY

[0004] In order to solve the problem of low optimum temperature of glucoamylase in the prior art, the present application is proposed and completed.

[0005] The purpose of the present application is to provide a glucoamylase TaGA58040.

[0006] Another purpose of the present application is to provide a glucoamylase gene Taga58040.

[0007] Another purpose of the present application is to provide a recombinant expression vector containing the glucoamylase gene Taga58040.

[0008] Another purpose of the present application is to provide a recombinant strain containing the glucoamylase gene Taga58040.

[0009] Another purpose of the present application is to provide a preparation method of the glucoamylase TaGA58040.

[0010] According to the embodiment of the present application, the glucoamylase TaGA58040 has an amino acid sequence as shown in SEQ ID NO: 1.

[0011] SEQ ID NO: 1

[0012] MVKSFLTLSLRTLGLVQAAIAAPSAPVLPRASGSLDQWLATETPYALQGVLDNIGADGAKAAGAKSGVVIASPSKSNPDYFYTWTRDAALTIKCLVDAFIAGNTDLESEIRNYISSQAYLQTVSNPSGGLSTGGLGEPKFNVDLTQFTGAWGRPQRDGPALRATAMIAYAKWLIANGQSDLADSIVWPIVQNDLSYVTQYWNSTGFDLWEEVQGSSFFTTAVQHRALVEGNALAQQLGHSCSNCVSQAPQVLCFLQSYWTGSYILANFGGGRTGKDTNTILGSIHTFDPEAGCDDATFQPCSARALANHKAVTDSFRSIYSINSGIAQGQAVAVGRYAEDVYQGGNPWYLCTLAAAEQLYDALYQWDRAGQLTITDVSLPFFRDVYPSAAVGTYASSSSTYQDIVAAVKAYADGYMSVAQKYTPSSGALAEQFSRNDGTPLSAADLTWSYAALLTAAARRSAVVPASWDAPSADSVPAVCSATSATGPYSTATNTVWPGEATTTATATATSTPCTAAPTAVAVTFNERVTTVWGENVFLVGSISALGSWDTNSAVALSADRYTSSDPLWYVTVSLPAGTSFEYKYIKKETDGSVVWESDPNRSYTVPKACGVTTATVNDSWR*

[0013] The enzyme has a full length of 622 amino acids, and the N-terminal 30 amino acids are a signal peptide sequence "MVKSFLTLSLRTLGLVQAAIAAPSAPVLPR". Therefore, the theoretical molecular weight of the mature glucoamylase TaGA58040 is 62.85 kDa, and the amino acid sequence is as shown in SEQ ID NO: 2.

[0014] As shown in SEQ ID NO: 2

[0015] ASGSLDQWLATETPYALQGVLDNIGADGAKAAGAKSGVVIASPSKSNPDYFYTWTRDAALTIKCLVDAFIAGNTDLESEIRNYISSQAYLQTVSNPSGGLSTGGLGEPKFNVDLTQFTGAWGRPQRDGPALRATAMIAYAKWLIANGQSDLADSIVWPIVQNDLSYVTQYWNSTGFDLWEEVQGSSFFTTAVQHRALVEGNALAQQLGHSCSNCVSQAPQVLCFLQSYWTGSYILANFGGGRTGKDTNTILGSIHTFDPEAGCDDATFQPCSARALANHKAVTDSFRSIYSINSGIAQGQAVAVGRYAEDVYQGGNPWYLCTLAAAEQLYDALYQWDRAGQLTITDVSLPFFRDVYPSAAVGTYASSSSTYQDIVAAVKAYADGYMSVAQKYTPSSGALAEQFSRNDGTPLSAADLTWSYAALLTAAARRSAVVPASWDAPSADSVPAVCSATSATGPYSTATNTVWPGEATTTATATATSTPCTAAPTAVAVTFNERVTTVWGENVFLVGSISALGSWDTNSAVALSADRYTSSDPLWYVTVSLPAGTSFEYKYIKKETDGSVVWESDPNRSYTVPKACGVTTATVNDSWR*

[0016] The present application also provides a gene encoding the above-mentioned glucoamylase Taga58040, and the nucleotide sequence of the gene is shown in SEQ ID NO: 3.

[0017] SEQ ID NO: 3

[0018]

[0019] The full length of the glucoamylase gene Taga58040 structural gene of the present application is 2177 bp, containing 4 introns, +239-333 bp, +618-676 bp, +774-832, +1471-1565 being intron sequences, the cDNA is 1869 bp, and the cDNA sequence is shown as SEQ ID NO: 4.

[0020] SEQ ID NO: 4

[0021]

[0022] wherein the base sequence of the signal peptide is: "ATGGTGAAAAGTTTCCTGACTCTCTCCCTTCGCACACTTGGCCTCGTCCAAGCAGCAATCGCTGCTCCCTCTGCCCCGGTTCTCCCGCGG". Thus, the coding sequence of the mature gene is shown in SEQ ID NO: 5.

[0023] SEQ ID NO: 5

[0024]

[0025] The present application also provides a recombinant vector comprising the above-mentioned glucoamylase gene Taga58040, preferably SESA-Taga58040. The glucoamylase gene Taga58040 of the present application is inserted downstream of the promoter of the expression vector, so that its nucleotide sequence is operably linked to the expression control sequence. As a most preferred embodiment of the present application, the glucoamylase gene is inserted into the plasmid SESA, so that the nucleotide sequence is downstream of and controlled by the promoter An_201cp, to obtain the recombinant filamentous fungal expression plasmid SESA-Taga58040.

[0026] The present application also provides a recombinant strain comprising the above-mentioned glucoamylase gene, preferably the recombinant strain Δalp1 / TaGA58040.

[0027] The method for preparing the glucoamylase TaGA58040 according to the present application comprises the following steps:

[0028] The host cell is transformed with the above-mentioned recombinant vector to obtain a recombinant strain;

[0029] The recombinant strain is cultured to express the recombinant glucoamylase TaGA58040 constitutively;

[0030] The expressed glucoamylase is recovered and purified.

[0031] The host cell is preferably a Myceliopthora thermophila cell or a Myceliopthora heterothallica cell, and the recombinant filamentous fungal expression plasmid is preferably transformed into the Myceliopthora thermophila cell Δalp1 to obtain the recombinant strain Δalp1 / TaGA58040. The present application provides the application of the above-mentioned glucoamylase TaGA58040, which is produced by using genetic engineering means for industrial production, and can be used in the feed, food, medicine and other industries.

[0032] The glucoamylase TaGA58040 of the present application belongs to the 15th family of glycoside hydrolase, and has a highest consistency of 79% with the existing published glucoamylase sequence. Its optimum pH is 5.0, and it can maintain more than 95% of its enzyme activity in the pH range of 3.0-6.0. Its optimum temperature is 60°C, and it can maintain more than 90% of its enzyme activity after being treated at 60°C for 60 min. Compared with the existing glucoamylase with high sequence consistency, the glucoamylase of the present application has better temperature stability. According to the technical solution of the present application, the glucoamylase with excellent properties and suitable for industrial application can be produced by using genetic engineering means. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1SDS-PAGE results of glucose amylase TaGA58040 after separation and purification are shown;

[0034] Figure 2 Optimum pH of glucose amylase TaGA58040 is shown;

[0035] Figure 3 pH stability of glucose amylase TaGA58040 is shown;

[0036] Figure 4 Optimum temperature of glucose amylase TaGA58040 is shown;

[0037] Figure 5 Temperature stability of glucose amylase TaGA58040 is shown. DETAILED DESCRIPTION

[0038] The materials, reagents, instruments and methods used in the following examples, without special instructions, are conventional materials, reagents, instruments and methods in the art, which can be obtained through commercial channels.

[0039] In the present application, high-fidelity DNA polymerase 2xPhantaMax Master Mix, Trans1-T1 cloning competent cells, pEASY-Blunt Cloning Kit and pEASY-Uni Seamless Cloning and Assembly Kit were used for constructing gene overexpression vectors. Thermomyces lanuginosus ATCC 42464 was purchased from the American Type Culture Collection.

[0040] STC solution: 50 mM calcium chloride, 1 M sorbitol, 10 mM Tris-HCl pH 7.5;

[0041] PEG solution: 25% PEG 6000, 50 mM calcium chloride, 10 mM Tris-HCl pH 7.5;

[0042] Upper medium: 2% sucrose, 20 mL / L 50x Vogel's salt solution, 1 M sorbitol, 0.75% agarose;

[0043] Lower medium: 2% sucrose, 20 mL / L 50x Vogel's salt solution, 1 M sorbitol, 1.5% agarose;

[0044] Mycelium lysis solution: 0.05 M sodium hydroxide, 1 mM ethylenediaminetetraacetic acid, 1% triton X-100;

[0045] Fermentation medium: 40 g / L glucose, 10 g / L yeast extract, 0.15 g / L potassium dihydrogen phosphate, 0.15 g / L dipotassium hydrogen phosphate, 0.10 g / L magnesium sulfate heptahydrate, 0.10 g / L calcium chloride, 0.1 mg / L biotin, 1 mL / L 50x Vogel's salt solution.

[0046] Example 1 Cloning of the glucoamylase encoding gene Taga58040

[0047] The cDNA of Talaromyces sp. GY1 was used as a template to design primers SESA-Taga58040-F (SEQ ID NO: 6) and SESA-Taga58040-R (SEQ ID NO: 7) for PCR amplification. The PCR reaction parameters were as follows: 98°C for 5 min; 95°C for 30 sec, 50°C for 30 sec, 72°C for 2 min, 30 cycles; 72°C for 10 min. A gene fragment Taga58040 of about 1800 bp was obtained. The fragment was recovered and sequenced. The nucleotide sequence is shown as SEQ ID NO: 5.

[0048] SEQ ID NO: 6

[0049] 5'-TCGCCACGGGCGCCGTGGCTGCGAGTGGCTCTCTGGATCAATG-3';

[0050] SEQ ID NO: 7

[0051] 5'-ATAAGAAATTCGGATCCTCATTAGTGGTGGTGGTGGTGGTGTCTCCAGCTATCGTTCACCGTC-3'.

[0052] Example 2 Construction of the glucoamylase TaGA58040 engineering strain

[0053] 2.1 Construction of the expression vector

[0054] The primers Vec-F (SEQ ID NO: 8) and Vec-R (SEQ ID NO: 9) were designed, and the plasmid pEASY-Blunt-SESA was used as a template for PCR amplification with high-fidelity DNA polymerase. The amplification product Vec was recovered by 1% agarose gel electrophoresis detection. Then, the pEASY-Uni Seamless Cloning and Assembly Kit was used to splice the amplification products Taga58040 and Vec, and the splicing product was transformed into E. coli cloning competent cells Trans1-T1. Positive clones were selected for sequencing verification, and the recombinant expression plasmid SESA-Taga58040 of Thielavia sp. was obtained.

[0055] SEQ ID NO: 8

[0056] 5'-TGAGGATCCGAATTTCTTATGATTTATG-3';

[0057] SEQ ID NO: 9

[0058] 5'-AGCCACGGCGCCCGTGGCGAAGAC-3'.

[0059] 2.2 Transformation of expression vector

[0060] The spore suspension of Thielavia sp. was spread on a potato dextrose agar plate covered with glass paper, and the mycelium was collected after 15 hours of culture at 37°C. Then, the mycelium cell wall was digested with 0.5% lywallzyme, and the protoplasts were collected by double-layered filter paper filtration and centrifugation after 2 hours of digestion at 30°C. The protoplasts were washed and resuspended with pre-cooled STC solution, and the concentration of the protoplasts was adjusted to 5x10 7 μL of the protoplast suspension was added to 10 μL of the overexpression vector SESA-Taga58040 and 50 μL of PEG solution, and gently mixed. Then, the mixture was placed in an ice bath for 20 minutes. Then, 2 mL of PEG solution was added, and the mixture was placed at room temperature for 5 minutes. Then, 4 mL of STC solution was added, and the mixture was mixed with the upper medium containing 100 μg / mL geneticin. Then, the mixture was poured into the lower medium containing 100 μg / mL geneticin, and the Thielavia sp. transformants were grown after 3 days of culture at 37°C.

[0061] 2.3 Screening of high-expression transformants

[0062] The transformants with geneticin resistance were transferred from the flat plate to PDA plates, and cultured in a thermostat incubator at 45°C for 3 days. The mycelium lysate was used to extract the genome, and PCR verification was performed. Some transformants amplified a band of about 2.0 kb in size, and the wild-type strain had no corresponding band, indicating that the recombinant Myceliopthora thermophila expression strain containing the Taga58040 gene expression cassette was successfully obtained. Randomly selected recombinant Myceliopthora thermophila expression strains were inoculated into fermentation medium at a concentration of 10 6 spores / mL, and the supernatant protein of the fermentation broth was analyzed by SDS-PAGE. Compared with the wild-type strain, the recombinant TaGA58040 Myceliopthora thermophila expression strain detected the target band, and then the transformant with high expression of glucose amylase was screened.

[0063] Example 3 Analysis of the properties of glucose amylase TaGA58040

[0064] 3.1 Preparation of recombinant glucose amylase TaGA58040

[0065] The transformant with high expression of glucose amylase was inoculated into fermentation medium at a concentration of 10 6 spores / mL, and the supernatant of recombinant glucose amylase TaGA58040 expressed in the shake flask on the 3rd day was collected. The supernatant was first concentrated by a 10 kDa membrane bag, and the medium was replaced with a buffer (20 mM pH 7.4 Na2HPO4-NaH2PO4, 500 mM NaCl). Then, the supernatant was purified by a nickel affinity chromatography column, and the electrophoretically pure elution fraction was collected and dialyzed into a protein storage solution. Figure 1 The SDS-PAGE results of glucose amylase TaGA58040 after isolation and purification are shown.

[0066] 3.2 Analysis of the properties of recombinant glucose amylase TaGA58040

[0067] The activity of glucose amylase TaGA58040 of the present application was analyzed by the DNS method. The specific method is as follows: under the conditions of pH 5.0 and 60°C, 200 μL of the reaction system includes 100 μL of appropriately diluted enzyme solution, 100 μL of soluble starch, reaction for 30 min, addition of 300 μL of DNS to terminate the reaction, boiling in water for 5 min. After cooling, the OD value was measured at 540 nm. The definition of glucose amylase activity unit is: the amount of enzyme required to catalyze the hydrolysis of substrate to release 1 μmol of reducing sugar per minute under the conditions of 60°C and pH 5.0 is one enzyme activity unit (U).

[0068] (1) Optimum pH and pH stability of glucose amylase TaGA58040

[0069] The buffer used was a citric acid-disodium hydrogen phosphate series buffer with a pH of 3.0-8.0. The purified glucoamylase TaGA58040 was subjected to enzymatic reaction in the above buffer at different pH values to determine the optimum pH.

[0070] As shown in Figure 2 , the optimum pH of the glucoamylase TaGA58040 was 5.0 at a temperature of 30°C, and the enzyme could maintain more than 50% of its enzyme activity in the pH range of 3.0-6.0.

[0071] The glucoamylase TaGA58040 enzyme solution was mixed with buffers at different pH values, and then treated at 30°C for 60 min, and the enzyme activity was determined to study the pH stability of the enzyme.

[0072] As shown in Figure 3 , the glucoamylase TaGA58040 could maintain more than 95% of its enzyme activity between pH 3.0-6.0, indicating that the enzyme had excellent pH stability.

[0073] (2) Optimum temperature and temperature stability of the glucoamylase TaGA58040

[0074] The enzyme activity of the purified glucoamylase TaGA58040 was determined at different temperatures (30-80°C) at pH 5.0.

[0075] As shown in Figure 4 , the optimum reaction temperature of the glucoamylase TaGA58040 was 60°C, and it still had 60% of its enzyme activity at 70°C.

[0076] The enzyme activity was determined at 60°C after the enzyme was treated at different temperatures for different times to determine the thermal stability of the glucoamylase TaGA58040.

[0077] As shown in Figure 5 , the enzyme activity of the glucoamylase TaGA58040 could still maintain more than 90% after being treated at 60°C for 60 min, and it had good temperature stability.

[0078] (3) Specific activity of the glucoamylase TaGA58040

[0079] The specific activity of the glucoamylase TaGA58040 was determined by measuring the enzyme activity at 60°C in a 0.1 mol / L citric acid-disodium hydrogen phosphate buffer system at pH 5.0 with 0.5% starch as the substrate, and the reaction time was 30 minutes. The specific activity of the glucoamylase TaGA58040 was determined to be 811 U / mg.

[0080] The above examples are only for understanding the technical solutions of the present application and do not limit the protection scope of the present application.

Claims

1. Glucoamylase Ta GA58040 characterized in that, The glucoamylase Ta The amino acid sequence of GA58040 is set forth in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A glucoamylase gene characterized in that, The glucoamylase gene encodes the glucoamylase of claim 1 Ta GA58040.

3. The glucoamylase gene according to claim 2, characterized in that, The nucleotide sequence of the glucoamylase gene is shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

4. A recombinant expression vector comprising the glucoamylase gene of claim 2.

5. A recombinant strain comprising the glucoamylase gene of claim 2.

6. The glucoamylase of claim 1 Ta Use of GA58040 for hydrolyzing starch.

7. A method of preparing a thermostable glucoamylase, characterized in that, The method comprises the following steps: transforming a host with the recombinant expression vector of claim 4 to obtain a recombinant strain; culturing the recombinant strain to express recombinant glucoamylase constitutively; recovering and purifying the expressed glucoamylase.

8. The method of producing heat-stable glucoamylase according to claim 7, characterized in that, The host is a Myceliopthora thermophile cell.

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