Acidic thermophilic polygalacturonase TEPG28A, and encoding gene and application thereof

Abstract

Provided are acidic thermophilic polygalacturonase TePG28A, encoding gene and application thereof. The amino acid sequence thereof is as shown in SEQ ID NO. 1 or SEQ ID NO. 2. The expressed acidic thermophilic polygalacturonase by means of cloning has advantages such as high enzyme activity and high stability; can adapt to the high temperature environment in the industrial production; has better application prospect; and can effectively degrade pectic substances such as polygalacturonic acid and pectin; and can be effectively applied to the industrial field of feed, food, and textile, etc.

Claims

1. A method of producing a polygalacturonase having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, comprising the steps of: (1) transforming a prokaryotic or eukaryotic host cell in culture with a polynucleotide comprising a nucleotide sequence encoding said polygalacturonase to obtain a recombinant host cell; (2) cultivating the recombinant host cell to induce expression of said polygalacturonase; and (3) isolating and recovering said polygalacturonase.

2. A method of degrading polygalacturonic acid of a water-soluble pectin, comprising contacting said water-soluble pectin with an isolated or purified polygalacturonase having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, thereby degrading the polygalacturonic acid of said water-soluble pectin.

3. The method of claim 2, wherein the degrading of polygalacturonic acid of a water-soluble pectin is during production of a feed, food or textile.

4. The method of claim 2, wherein said polygalacturonase has an optimal pH of 4.5, an optimal temperature of 70 C., pH stability within pH 2.0 to pH 7.0 and[H] maintains 90% polygalacturonase activity at 60 C. for 1 hour.

5. The method of claim 2, wherein said polygalacturonase is produced by a recombinant host cell comprising a heterologous polynucleotide encoding said polygalacturonase.

6. The method of claim 5, wherein said heterologous polynucleotide has the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

(1) FIG. 1 shows the expression and deglycosylation of the recombinant polygalacturonase, wherein 1: purified and deglycosylated TePG28A protein, and 2: only purified TePG28A protein.

(2) FIG. 2 shows optimum pH values for the recombinant polygalacturonase TePG28A.

(3) FIG. 3 shows pH stabilities for the recombinant polygalacturonase TePG28A.

(4) FIG. 4 shows optimum temperature values for the recombinant polygalacturonase TePG28A.

(5) FIG. 5 shows heat stability for the recombinant polygalacturonase TePG28A.

EXAMPLES

(6) The present invention is further illustrated with reference to the following Examples and the appended drawings, which should by no means be construed as limitations of the present invention.

(7) Test Materials and Reagents

(8) 1. Strains and vectors: Talaromyces emersonii 12802; Pichia pastoris strain GS115 (Invitrogen); and vetor pPIC9 (Invitrogen, San Diego, Calif.).

(9) 2. Enzymes and other biochemical reagents: restriction endonucleases (TaKaRa); ligase (Invitrogen); and birch xylan (Sigma)

(10) 3. Medium: (1) taking potato dextrose medium as Talaromyces emersonii 12802 Medium, including 1000 mL of potato juice, 10 g of dextrose, and 25 g of arga, natural pH. (2) E. coli. LB medium: 1% of peptone, 0.5% of yeast extract, and 1% of NaCl, natural pH. (3) BMGY medium: 1% of yeast extract; 2% of peptone; 1.34% of YNB, 0.00004% of Biotin; and 1% of glycerol (V/V). (4) BMMY medium: 1% of yeast extract; 2% of peptone; 1.34% of YNB, 0.00004% of Biotin; and 0.5% of methanol (V/V).

(11) Suitable biology laboratory methods not particularly mentioned in the examples as below can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other kit laboratory manuals.

Example 1 Cloning Polygalacturonase Gene TePG28A from Talaromyces emersonii 12802

(12) Total RNA is isolated from Talaromyces emersonii 12802 having been induced for 3 days. It was possible to design a pair of degenerate primers to amplify part fragment of the polygalacturonase gene based on the conserved fragment of the family 3 of polygalacturonase from the Talaromyces emersonii 12802 DNA by RT-PCR.

(13) TABLE-US-00007 P1: (SEQIDNO.7) 5-GACTACGTAGCCCCAACTGCTGTCGAGAAGCGTG-3; P2: (SEQIDNO.8) 5-GTCGAATTCCTAGCAAGAAACTCCAGAAGGCACATTCTCAC-3.

(14) PCR amplification was performed by optimizing PCR parameters as follows: degenerating at 95 C. for 5 minutes, followed by 30 cycles at: degenerating at 94 C. for 30 seconds/annealing temperature at 60 C. for 30 seconds/extending at 72 C. for 1 minute, and a final extension of 10 minutes at 72 C. PCR product comprising 1000 bp was obtained and linked to vector pEASY-T3 for sequencing. Two flanking sequences were obtained, and assembled into polygalacturonase gene with 1095 bp in full length coding 365 amino acids including a terminal. The mature protein encoded by this gene has molecular weight of 35.2 kDa.

Example 2 Producing Recombinant Polygalacturonase

(15) The coding region of mature protein was amplified. The DNA purified was inserted into pPIC9 at the EcoRI and SnaB sites, as described by the manufacturer instruction to obtain DNA construct pPIC-PG5804. The construct was transformed into Pichia pastoris strain GS115 to obtain the recombinant cell GS115/TePG28A.

(16) The expression vector comprising the full-length gene enconding polygalacturonase was constructed and transformed to Pichia pastoris strain GS115 by the same method as above.

(17) The transformed Pichia pastoris strain GS115 (Invitrogen) were incubated in 300 mL of BMGY for 48 h at 30 C. and 250 rpm, and then the cells were spun down and suspended in 150 mL of BMMY to induce the polygalacturonase gene expression. 72 hours after induction, the supernatant was recovered by spinning to test the activity of the polygalacturonase. The enzyme activity of the purified recombinant polygalacturonase was 41,786 U/mg.

Example 4 Measuring the Properties of the Recombinant Polygalacturonase

(18) 900 l of substrate solution of polygalacturonic acid in concentration of 0.33% was added to 100 L of diluted enzyme solution, which was reacted at 70 C. and pH 3.5 for 10 minutes. Then, 1.5 mL of DNS was added to stop the reaction. OD540 was measured.

(19) 1 unit of polygalacturonase activity was determined to be the enzyme amount releasing 1 mol of reducing sugar by decomposing substrate for 1 minute.

Example 5 Measuring the Properties of the Recombinant Polygalacturonase TePG28A

(20) 1. Optimum pH Values and pH Stability

(21) The polygalacturonase purified in example 4 was reacted in the different pH to determine optimum pH. The activity of polygalacturonase was measured using polygalacturonic acid as substrate in 0.1 mol/L citric acid-sodium dimetallic phosphate buffer with different pH at 70 C. As is shown in FIG. 2, the activity of the recombinant polygalacturonase varied with pH. The highest activity was observed at pH 3.5. The recombinant polygalacturonase was maintained at 37 C. at different pH for 60 min followed by measuring the activity in buffer with pH3.5 at 70 C. FIG. 3 showed the enzyme was stable at pH 1.0 to 7.0 and maintained 90% of activity after being treated for 60 min at pH 1.0 to 7.0.

(22) 2. Optimum Temperature and Heat Stability

(23) The polygalacturonase was reacted in the different temperatures to determine optimum temperature. The activity of polygalacturonase was measured using polygalacturonic acid as substrate in citric acid-sodium dimetallic phosphate buffer (pH 4.0) at different temperatures. As shown in FIG. 4, the activity of polygalacturonase varied with temperatures. The highest activity was observed at 70 C. FIG. 4 showed the enzyme activity was thermalstable at 70 C., more than 90% of the enzyme activity was still maintained when the enzyme was maintained at 60 C. for 1 h.