High-temperature pullulanase as well as preparation method and application thereof

Abstract

The invention discloses high-temperature pullulanase and a preparation method and application thereof.The amino acid sequence of the high-temperature pullulanase is shown as SEQ ID NO.2. The preparation method comprises the steps that a fermentation tank containing a BSM culture medium is inoculated with a secondary seed solution of engineering bacteria converting and carrying coding genes of the high-temperature pullulanase for culture; the high-temperature pullulanase is obtained by sequentially feeding glycerol and methanol, inducing fermentation for 132-144 hours, stopping fermentation and collecting supernate. According to the invention, high-efficiency secretory expression of the high-temperature pullulanase in a pichia pastoris expression system is realized, and the problem that the high-temperature pullulanase is easy to degrade in the pichia pastoris expression process is solved at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering. More specifically, the present invention relates to a thermophilic pullulanase, a preparation method thereof, and an application thereof. Background Art

[0002] Pullulanase (EC 3.2.1.41) is a kind of starch debranching enzyme, which is named because it can specifically hydrolyze pullulan (a polymer formed by maltotriose linked by α-1,6 glycosidic bonds). It belongs to the amylase category and can specifically cleave the α-1,6 glycosidic bond at the branch point of amylopectin, cutting off the entire branch structure to form amylose.

[0003] In the enzymatic industrial production of sugar, due to the temperature difference change in the liquefaction and saccharification steps, and the use of various hydrolases in the saccharification process, these factors increase the production cost of starch sugar production and reduce the production efficiency at the same time. Since thermophilic pullulanase can carry out saccharification under high-temperature liquefaction conditions, the saccharification and liquefaction processes of starch sugar production can be combined into one, greatly reducing the production cost and improving the production efficiency.

[0004] The Pichia pastoris (Komagataella phaffii) expression system is a new type of heterologous protein expression system developed in recent years. It not only has the advantages of simple operation, easy cultivation, and high expression level of the prokaryotic expression system, but also has the characteristics of post-translational modification of heterologous proteins that the prokaryotic expression system does not have. At the same time, it avoids the defects of poor secretion efficiency, unstable expression strains, and easy loss of expression plasmids in the Saccharomyces cerevisiae expression system. Therefore, this expression system has become one of the most excellent and widely used heterologous gene expression systems at present.

[0005] However, when using the Pichia pastoris expression system to express thermophilic pullulanase, the expression level of thermophilic pullulanase is at a relatively low level and is easily degraded during the expression process. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a method for efficiently and stably expressing thermophilic pullulanase in the Pichia pastoris expression system.

[0007] The specific technical solutions for achieving the above invention purpose are as follows.

[0008] In the first aspect of the present invention, there is provided a thermophilic pullulanase, the amino acid sequence of which is as shown in SEQ ID NO: 2.

[0009] In the second aspect of the present invention, there is provided a coding gene for a thermophilic pullulanase, the nucleotide sequence of which is as shown in SEQ ID NO: 1.

[0010] In the third aspect of the present invention, a recombinant plasmid carrying the coding gene of thermophilic pullulanase is provided, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO: 1.

[0011] In the fourth aspect of the present invention, an engineered bacterium transformed with the above recombinant plasmid is provided.

[0012] In the fifth aspect of the present invention, a method for preparing thermophilic pullulanase is provided, comprising the following steps:

[0013] (1) Inoculate the secondary seed liquid of the above engineered bacterium into a fermenter filled with BSM medium, and culture at 28°C - 32°C, pH 5 - 5.2, controlling the DO concentration to be 28% - 32%.

[0014] (2) When the DO concentration starts to increase, feed glycerol, and continue to culture until the wet cell weight reaches 170 g / L - 190 g / L, then stop feeding glycerol.

[0015] (3) When the DO concentration is 95% - 100% and remains unchanged, feed methanol, and continue to culture at 27°C - 29°C, pH 6 - 6.2 for 132 h - 144 h, then stop fermentation and collect the supernatant.

[0016] In the present invention, the inventors first constructed a recombinant expression vector of thermophilic pullulanase HJΔ782. On this basis, a recombinant expression vector without the original signal peptide of thermophilic pullulanase HJΔ782 was constructed. Then, a recombinant expression vector without the original signal peptide of thermophilic pullulanase HJΔ782 and with all five kex2 protease cleavage sites inside thermophilic pullulanase HJΔ782 mutated was constructed. This modified recombinant expression vector was transformed into Pichia pastoris, and using this recombinant Pichia pastoris strain, the high-efficiency secretory expression of thermophilic pullulanase in the Pichia pastoris expression system was achieved, and at the same time, the problem that thermophilic pullulanase is easily degraded during the expression process in Pichia pastoris was solved.

[0017] The present invention further explored the process conditions for the production of thermophilic pullulanase by recombinant Pichia pastoris. When fermenting in a 30 L fermenter, when the induction temperature is 28°C and the induction pH is 6.0, the enzyme activity in the supernatant of the fermentation broth reaches 400 U / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a graph showing the changes in OD 600 , wet cell weight, protein concentration and enzyme activity during the fermentation of the recombinant strain X33 / pPICZαA-HJΔ782Δsp-Kex2 in Example 2 of the present invention.

[0019] Figure 2SDS-PAGE analysis chart of the fermentation supernatant during the fermentation process of the recombinant strain X33 / pPICZαA-HJΔ782Δsp-Kex2 in Example 2 of the present invention. Among them, M is the standard protein Marker, and lanes 1-12 are the fermentation supernatants at 0-132 h respectively.

[0020] Figure 3 SDS-PAGE analysis chart of the fermentation supernatant during the fermentation process of the recombinant strain X33 / pPICZαA-HJΔ782Δsp in Example 2 of the present invention. Among them, M is the standard protein Marker, and lanes 1-12 are the fermentation supernatants at 0-132 h respectively.

[0021] Figure 4 Effect results of pH on OD 600 (A), wet cell weight (B), protein content (C) and enzyme activity (D) of the recombinant strain expressing pullulanase HJΔ782 in Example 3 of the present invention.

[0022] Figure 5 Effect results of temperature on OD 600 (A), wet cell weight (B), protein content (C) and enzyme activity (D) of the recombinant strain expressing pullulanase HJΔ782 in Example 3 of the present invention.

[0023] Figure 6 SDS-PAGE analysis chart of each high-temperature pullulanase in Example 4 of the present invention. Among them, M is the standard protein Marker, and lanes 1-6 are the cell lysate of HJΔ782, the purified enzyme solution of HJΔ782, the fermentation supernatant of HJΔ782Δsp, the purified enzyme solution of HJΔ782Δsp, the fermentation supernatant of HJΔ782Δsp-Kex2, and the purified enzyme solution of HJΔ782Δsp-Kex2 respectively.

[0024] Figure 7 Optimum temperature (A) and thermal stability (B) results of each high-temperature pullulanase in Example 4 of the present invention.

[0025] Figure 8 Optimum pH (A) and pH stability (B) results of each high-temperature pullulanase in Example 4 of the present invention.

[0026] Figure 9 OD during the fermentation process of the recombinant strain X33 / pPICZαA-HJΔ782 in Comparative Example 1 of the present invention 600 , changes in wet cell weight, protein concentration and enzyme activity.

[0027] Figure 10SDS-PAGE results of the fermentation supernatant of the recombinant strain X33 / pPICZαA-HJΔ782 in Comparative Example 1 of the present invention during the fermentation process, where M is the standard protein Marker.

[0028] Figure 11 SDS-PAGE results of the fermentation supernatant, intracellular supernatant, and intracellular precipitate of the recombinant strain X33 / pPICZαA-HJΔ782 in Comparative Example 1 of the present invention at 132 h of fermentation, where M is the standard protein Marker, and lanes 1-3 are the fermentation supernatant, intracellular supernatant, and intracellular precipitate, respectively.

[0029] Figure 12 Pullulanase enzyme activities of the fermentation supernatant and cell lysate of the recombinant strain X33 / pPICZαA-HJΔ782 in Comparative Example 1 of the present invention at 132 h of fermentation.

[0030] Figure 13 Prediction result diagram of the Signal P signal peptide in Comparative Example 1 of the present invention.

[0031] Figure 14 OD during the fermentation process of the recombinant strain X33 / pPICZαA-HJΔ782Δsp in Comparative Example 2 of the present invention 600 , results of changes in wet cell weight, protein concentration, and enzyme activity.

[0032] Figure 15 SDS-PAGE results of the fermentation supernatant of the recombinant strain X33 / pPICZαA-HJΔ782Δsp in Comparative Example 2 of the present invention at 132 h of fermentation, where M is the standard protein Marker, and lanes 1-12 are the fermentation supernatants from 0 to 132 h.

[0033] Figure 16 Cleavage site diagram of Kex2 protease in the amino acid sequence of HJΔ782Δsp in Comparative Example 2 of the present invention.

[0034] Figure 17 Structure diagram of HJΔ782Δsp in Comparative Example 2 of the present invention, where the five amino acids K521, K341, K75, K416, and R268 are marked in red, and the two amino acids E291 and E394 are marked in blue.

[0035] Figure 18 SDS-PAGE analysis diagram of the expression products of 5 single-point mutant recombinant strains in Comparative Example 3 of the present invention, where lanes 1-5 are single-point mutants K75G, R268G, K341G, K416G, and K521G, respectively. Detailed implementation manners

[0036] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0037] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The vectors pPICZαA, the expression host Pichia pastoris X33, and the plasmid pET-29a(+) used were all obtained commercially and stored in the applicant's laboratory. Other materials, reagents, etc., unless otherwise specified, can be purchased from commercial channels.

[0039] In some embodiments of the present invention, a thermophilic pullulanase is disclosed, and its amino acid sequence is as shown in SEQ ID NO:2.

[0040] In other embodiments of the present invention, a coding gene of a thermophilic pullulanase is disclosed, and its nucleotide sequence is as shown in SEQ ID NO:1.

[0041] In other embodiments of the present invention, a recombinant plasmid carrying the coding gene of a thermophilic pullulanase is disclosed, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO:1.

[0042] In other embodiments of the present invention, an engineered bacterium transformed with the above recombinant plasmid is disclosed.

[0043] In some of these embodiments, the starting strain of the engineered bacterium is Pichia pastoris.

[0044] In some of these embodiments, the Pichia pastoris is Pichia pastoris X33.

[0045] In other embodiments of the present invention, the application of the above recombinant plasmid or engineered bacterium in the preparation of thermophilic pullulanase is disclosed.

[0046] In other embodiments of the present invention, a method for preparing a thermophilic pullulanase is disclosed, including the following steps:

[0047] (1) Inoculate the secondary seed liquid of the engineered bacterium into a fermenter containing BSM medium, at 28°C to

[0048] Cultivate at 32°C, pH 5 - 5.2, and control the DO concentration at 28% - 32%.

[0049] (2) When the DO concentration starts to increase, add glycerol dropwise and continue culturing until the wet cell weight reaches 170 g / L - 190 g / L, then stop adding glycerol dropwise.

[0050] (3) When the DO concentration is 95% - 100% and remains constant, add methanol, and continue culturing at 27°C - 29°C, pH 6 - 6.2

[0051] Continue culturing for 132 h - 144 h, then stop fermentation and collect the supernatant.

[0052] In some of these embodiments, the inoculation amount in step (1) is 8% - 12%.

[0053] In some of these embodiments, the feeding rate of glycerol in step (2) is 12 mL / (L·h) - 15 mL / (L·h).

[0054] In some of these embodiments, the feeding rate of methanol in step (3) is 4 mL / h - 6 mL / h.

[0055] In some of these embodiments, when the wet cell weight in step (2) reaches 175 g / L - 185 g / L, stop adding glycerol dropwise.

[0056] In some other embodiments of the present invention, the application of the thermophilic pullulanase prepared by the above preparation method in industrial sugar production is disclosed.

[0057] In the following examples, the media used are as follows:

[0058] 1. Activation medium: 2% glucose, 2% peptone, 1% yeast extract, 2% agar powder, sterilize at 115°C for 20 min, and add Zeocin with a final concentration of 100 μg / mL before pouring the plate.

[0059] 2. Primary seed medium YPD (L -1 ): 2% glucose, 2% peptone, 1% yeast extract, the liquid loading volume in a 500 mL Erlenmeyer flask is 100 mL, sterilize at 115°C for 20 min.

[0060] 3. Secondary seed medium YPD (L -1 ): 2% glucose, 2% peptone, 1% yeast extract, sterilize at 115°C for 20 min. Prepare 1.05 L, the liquid loading volume in a 2 L Erlenmeyer flask is 350 mL, and divide it into 3 bottles.

[0061] 4. PTM1 trace elements (L -1):6.0 g CuSO4·5H2O, 0.08 g NaI, 3.0 g MnSO4·H2O, 0.2 g Na2MoO4·2H2O, 0.02 g H3BO3, 0.5 g CoCl2, 20.0 g ZnCl2, 65.0 g FeSO4·7H2O, 5.0 ml concentrated H2SO4, sterilized at 115 °C for 20 min, and 0.2 g Biotin added in a laminar flow hood.

[0062] 5. BSM medium (L -1 ):26.7 mL H3PO4, 1.176 g CaSO4, 18.2 g K2SO4, 14.9 g MgSO4·7H2O, 4.347 g KOH, 40 g glycerol, 4.35 mL PTM1, 26.7 mL H3PO4, adjusted to pH 5.0 with ammonia water, sterilized at 121 °C for 30 min (prepared according to the formula for 3.5 L, but finally only made up to 3 L, prepared in 3 portions).

[0063] 6. Fed-batch growth medium (L -1 ):700 g glycerol, 12 mL PTM1. Prepared 900 mL, sterilized at 121 °C for 30 min.

[0064] 7. Fed-batch induction medium (L -1 ):100% methanol, 12 mL PTM1.

[0065] In the following examples, the detection methods involved are as follows:

[0066] 1. Wet weight determination: Take 3 2-mL centrifuge tubes, accurately weigh their total weight M, respectively and precisely pipette 2 mL of the fermentation broth into the 2-mL centrifuge tubes, centrifuge at 12000 rpm for 3 min, carefully pour out the supernatant, accurately weigh their total weight M', and calculate the cell wet weight (g / L) = (M' - M) / 6 × 1000

[0067] 2. OD 600 Determination: Using pure water as the blank, dilute the fermentation broth with pure water by a certain multiple so that the OD value measured at a wavelength of 600 nm is between 0.2 and 0.8, and then multiply this value by the dilution multiple to obtain the cell OD value in the fermentation broth.

[0068] 3. Determination of protein content in the fermentation supernatant

[0069] Use the Coomassie Brilliant Blue (Bradford) method protein concentration kit to determine the protein concentration of the fermentation supernatant.

[0070] 4. Enzyme activity determination

[0071] (1) Definition of enzyme activity: One unit of enzyme activity (U) is defined as the amount of enzyme required to release reducing sugar by enzymatic hydrolysis of pullulan within 1 min under certain reaction conditions, and the reducing power of the released reducing sugar is equivalent to that of 1 μmol of glucose, denoted as 1 U.

[0072] (2) Principle of enzyme activity determination: Pullulanase catalyzes the hydrolysis of pullulan to produce reducing sugar, which further reacts with 3,5-dinitrosalicylic acid to form a brownish-red amino compound. After spectral scanning, it has a characteristic light absorption at 540 nm. Within a certain range, the light absorption value at 540 nm is proportional to the amount of reducing sugar produced. The activity of pullulanase is calculated based on the rate of increase in light absorption.

[0073] (3) Method for enzyme activity determination

[0074] Experimental group: Take 100 μL of appropriately diluted crude enzyme solution and add it to a 5 mL centrifuge tube containing 300 μL of 1% pullulan solution. React in an oil bath at 110 °C for 15 min. Add 400 μL of DNS reagent and react in an oil bath at 100 °C for 5 min, then cool in ice water. Add 3 mL of ultrapure water to the above reaction system, mix well, and measure its absorbance at 540 nm.

[0075] Control group: Add 300 μL of 1% pullulan solution to a 5 mL centrifuge tube and react in an oil bath at 110 °C for 15 min. Add 400 μL of DNS reagent, then add 100 μL of appropriately diluted crude enzyme solution and react in an oil bath at 100 °C for 5 min, then cool in ice water. Add 3 mL of ultrapure water to the above reaction system, mix well, and measure its absorbance at 540 nm.

[0076] The present invention will be described in detail below with reference to the accompanying drawings and specific examples.

[0077] Example 1 Construction of recombinant plasmid pPICZαA-HJΔ782Δsp-Kex2

[0078] In this example, the high-temperature pullulanase gene (designated as the HJΔ782 gene in this article, with the nucleotide sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 4) was first cloned, and the recombinant plasmid pPICZαA-HJΔ782 was constructed. Then, its original signal peptide was removed to obtain the recombinant plasmid pPICZαA-HJΔ782Δsp (the nucleotide sequence of the HJΔ782Δsp gene is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6). Subsequently, five Kex2 protease cleavage sites within HJΔ782Δsp were simultaneously mutated (K75G, R268G, K341G, K416G, K521G) to obtain the recombinant plasmid pPICZαA-HJΔ782Δsp-Kex2 (the nucleotide sequence of the HJΔ782Δsp-Kex2 gene is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2).

[0079] Specifically, it includes the following steps:

[0080] 1. Design the plasmid on SnapGene: Insert the HJΔ782 gene between the KpnI and SalI cleavage sites of the pPICZαA vector to obtain the recombinant plasmid pPICZαA-HJΔ782.

[0081] 2. Using the plasmids pET-29a(+)-HJΔ782 and pPICZαA as templates, and pPICZαA-HJΔ782 homologous arm-F / R (SEQ ID NO: 7 and SEQ ID NO: 8), pPICZαA vector linearization-F / R (SEQ ID NO: 9 and SEQ ID NO: 10) as primers, perform PCR amplification (the amplification conditions are according to the Takara Max DNA Polymerase instruction manual). After purifying and recovering the PCR products respectively, perform recombination through seamless cloning experiments.

[0082] SEQ ID NO: 7: CGGCCGTCTCGGATCGGTACCAGGCGGGTGGTTGCCCTA

[0083] SEQ ID NO: 8: GATGATGATGATGGTCGACGGCCTTCAGCTCGACGGGGGT

[0084] SEQ ID NO: 9: GCCGTCGACCATCATCA

[0085] SEQ ID NO: 10: GGTACCGATCCGAGACG

[0086] 3. After the recombination reaction, mix 100 μL of Escherichia coli TOP10 competent cells with 10 μL of the ligation product, quickly place it in an ice box and incubate for 30 min. Then, perform heat shock at 42 °C for 90 s, and after that, ice-bath for another 2 min. Add 800 μL of LB liquid medium, incubate at 37 °C for 45 min, and then spread 200 μL of the bacterial solution on a low-salt LB plate containing bleomycin resistance (25 μg / mL), and culture it in a 37 °C incubator for 12 - 16 h.

[0087] 4. Pick a single colony and culture it in a low-salt LB liquid medium containing bleomycin resistance (25 μg / mL) for 5 h (37 °C, 180 rpm). Then, send the sample for sequencing, screen out the positive transformants, and extract the recombinant plasmid, which is the recombinant plasmid pPICZαA-HJΔ782.

[0088] 5. Using the plasmid pPICZαA-HJΔ782 as a template and the signal peptide-removed -F / R as primers (SEQ ID NO:11 and SEQ ID NO:12), perform whole-plasmid PCR amplification (the amplification conditions are according to the Takara Max DNA Polymerase instruction manual). After purifying and recovering the PCR products respectively, transform the products into the cloning strain Escherichia coli TOP10, verify the transformants by sequencing, screen out the positive transformants, and extract the recombinant plasmid, which is the recombinant plasmid pPICZαA-HJΔ782Δsp.

[0089] SEQ ID NO:11: TCGGTACCGCCGAACCGAAGCCGCTC

[0090] SEQ ID NO:12: GTTCGGCGGTACCGATCCGAGACGGC

[0091] 6. Using the plasmid pPICZαA-HJΔ782Δsp as a template and the K75G-F / R as primers, perform whole-plasmid PCR amplification (the amplification conditions are according to the Takara Max DNA Polymerase instruction manual). After purifying and recovering the PCR products respectively, transform the products into the cloning strain Escherichia coli TOP10, verify the transformants by sequencing, screen out the positive transformants, and repeat the above steps for the second point mutation. Until the fifth point mutation is completed, verify the transformants by sequencing, screen out the positive transformants, and extract the recombinant plasmid, which is the recombinant plasmid pPICZαA-HJΔ782Δsp-Kex2. The specific primer sequences used in this step are shown in Table 1.

[0092] Table 1

[0093] Primer Name Primer Sequence (5' to 3') K75G-F CGGCAAGGGGGACACATACCAGATAATCACAGAG(SEQ ID NO:13) K75G-R GTGTCCCCCTTGCCGTTCATGTAGTCCG(SEQ ID NO:14) R268G-F GGTGAAGGGGGCGGACGAGCTTTACAAG(SEQ ID NO:15) R268G-R TCCGCCCCCTTCACCTGGTCGTTGAAGTC(SEQ ID NO:16) K341G-F ACGGAAAGGGGATCTACCTCTTCCCGCGCGAC(SEQ ID NO:17) K341G-R GTAGATCCCCTTTCCGTTGAACTCGGCCACCC(SEQ ID NO:18) K416G-F ACTCTACAAGGGGCTGACCGAACTCCAGGAGC(SEQ ID NO:19) K416G-R TCGGTCAGCCCCTTGTAGAGTTCCGTCAGGAAGAT(SEQ ID NO:20) K521G-F GAGAAGGGGGACGAGATGAGCCAGGAGGACTG(SEQ ID NO:21) K521R-R ATCTCGTCCCCCTTCTCCATCAGGGTCTTCCTCG(SEQ ID NO:22)

[0094] 7. Inoculate the positive transformant into a low-salt LB liquid medium containing bleomycin (25 μg / mL), and culture it in the dark at 37°C and 180 rpm for 12 h. Extract the recombinant plasmid using a column-type DNA microextraction kit. Use the high-fidelity restriction endonuclease PmeI from NEB and perform the linearization step of the recombinant plasmid according to the instruction manual. After digestion, purify the digested product using a PCR product purification kit and store it at -20°C for later use.

[0095] 8. After purification, mix the linearized product with the Pichia pastoris X33 competent cells and incubate on ice for 5 min. Then, perform electroporation using an electroporator. Add 1 mL of 1 M sorbitol and let it stand at 30°C for 2 h. Spread 200 μL of the bacterial solution on a YPD plate containing bleomycin resistance and culture it at 30°C for 3 days. Select 6 - 10 yeast single colonies with good growth and transfer them respectively to 5 mL of YPD liquid medium (containing 100 μg / mL Zeocin), and culture them with shaking at 30°C and 250 rpm for 16 - 18 h. Extract the genomic DNA of the recombinant Pichia pastoris according to the instruction manual of the yeast genomic DNA extraction kit. Use the specific primers 830-F (SEQ ID NO:23) and 830-R (SEQ ID NO:24) of the HJΔ782 gene to perform PCR identification with the genomic DNA of the recombinant Pichia pastoris and the genomic DNA of the blank group as templates, and construct the recombinant strain X33 / pPICZαA-HJΔ782Δsp-Kex2, which is stored in glycerol.

[0096] SEQ ID NO:23:GATAAAATGATGGCCGCAAA

[0097] SEQ ID NO:24:TAGAGTTCCGTCAGGAAGAT

[0098] The nucleotide sequences and amino acid sequences of the HJΔ782Δsp-Kex2 gene, HJΔ782 gene, and HJΔ782Δsp gene are as follows:

[0099] SEQ ID NO:1:

[0100]

[0101] SEQ ID NO:2:

[0102] AEPKPLNVIIVWHQHQPYYYDPIQDIYTRPWVRLHAANNYWKMAYYLSQYPEVHATI DLSGSLIAQLADYMNGKGDTYQIITEKIANGEPLTVEEKWFMLQAPGGFFDHTIPWNGEPITDPSGNPIRDFWDRYTELKDKMMAAKAKYANLPLEEQKVAVTNEFTEQDYIDLAVLFNLAWIDYNYIMTHPELKALYDKVDEGGYTRDDVKTVLDAQLWLLNHTFEEHEKVNLLLGNGNVEVTVVPYAHPIGPILNDFGWEGDFNDQVKGADELYKQYLGNGTAVPVGGWAAESALNDKTLEILANNGWTWVMTDQLVLDRLGVENTVENYYKPWVAEFNGKGIYLFPRDHALSDRVGFTYAGMNQYQAVDDFVNELLKIQKENYDGSLVYVVTLDGENPWEHYPYDGKIFLTELYKGLTELQEQGLIRTLTPSEYIQLYGDQANKLTPKMMERLDLTGDNVQALLKAQSLGDLYDMVGVKEEMQWPESSWIDGTLSTWIGEPQENYGWYWLYLARKTLMEKGDEMSQEDWEKAHEYLLRAEASDWFWWYGSDQNSGQDFTFDRYLKTYLYEMYRLAGVEPPSYLFGNYFPDGEPYVTRALDGLKEGEMKNYSSMSPLAEGVSVYFDGEGLHFIVRGNLSQFEVSIWEKDERVGNTFTLLQGRPGELRYSMFPFSADSVGLMITKHLVYHDGKAEVYKATDYENSEKLGEATVRETSEGIEVVVPFEYIENPADFYFAVSTVKDGRLEVISTPVELK

[0103] SEQ ID NO:3:

[0104]

[0105] SEQ ID NO:4:

[0106] RRVVALLLAVLMVGSLIGANVKTVGAAEPKPLNVIIVWHQHQPYYYDPIQDIYTRPWVRLHAANNYWKMAYYLSQYPEVHATIDLSGSLIAQLADYMNGKKDTYQIITEKIANGEPLTVEEKWFMLQAPGGFFDHTIPWNGEPITDPSGNPIRDFWDRYTELKDKMMAAKAKYANLPLEEQKVAVTNEFTEQDYIDLAVLFNLAWIDYNYIMTHPELKALYDKVDEGGYTRDDVKTVLDAQLWLLNHTFEEHEKVNLLLGNGNVEVTVVPYAHPIGPILNDFGWEGDFNDQVKRADELYKQYLGNGTAVPVGGWAAESALNDKTLEILANNGWTWVMTDQLVLDRLGVENTVENYYKPWVAEFNGKKIYLFPRDHALSDRVGFTYAGMNQYQAVDDFVNELLKIQKENYDGSLVYVVTLDGENPWEHYPYDGKIFLTELYKKLTELQEQGLIRTLTPSEYIQLYGDQANKLTPKMMERLDLTGDNVQALLKAQSLGDLYDMVGVKEEMQWPESSWIDGTLSTWIGEPQENYGWYWLYLARKTLMEKKDEMSQEDWEKAHEYLLRAEASDWFWWYGSDQNSGQDFTFDRYLKTYLYEMYRLAGVEPPSYLFGNYFPDGEPYVTRALDGLKEGEMKNYSSMSPLAEGVSVYFDGEGLHFIVRGNLSQFEVSIWEKDERVGNTFTLLQGRPGELRYSMFPFSADSVGLMITKHLVYHDGKAEVYKATDYENSEKLGEATVRETSEGIEVVVPFEYIENPADFYFAVSTVKDGRLEVISTPVELK

[0107] SEQ ID NO:5:

[0108]

[0109] SEQ ID NO:6:

[0110] AEPKPLNVIIVWHQHQPYYYDPIQDIYTRPWVRLHAANNYWKMAYYLSQYPEVHATIDLSGSLIAQLADYMNGKKDTYQIITEKIANGEPLTVEEKWFMLQAPGGFFDHTIPWNGEPITDPSGNPIRDFWDRYTELKDKMMAAKAKYANLPLEEQKVAVTNEFTEQDYIDLAVLFNLAWIDYNYIMTHPELKALYDKVDEGGYTRDDVKTVLDAQLWLLNHTFEEHEKVNLLLGNGNVEVTVVPYAHPIGPILNDFGWEGDFNDQVKRADELYKQYLGNGTAVPVGGWAAESALNDKTLEILANNGWTWVMTDQLVLDRLGVENTVENYYKPWVAEFNGKKIYLFPRDHALSDRVGFTYAGMNQYQAVDDFVNELLKIQKENYDGSLVYVVTLDGENPWEHYPYDGKIFLTELYKKLTELQEQGLIRTLTPSEYIQLYGDQANKLTPKMMERLDLTGDNVQALLKAQSLGDLYDMVGVKEEMQWPESSWIDGTLSTWIGEPQENYGWYWLYLARKTLMEKKDEMSQEDWEKAHEYLLRAEASDWFWWYGSDQNSGQDFTFDRYLKTYLYEMYRLAGVEPPSYLFGNYFPDGEPYVTRALDGLKEGEMKNYSSMSPLAEGVSVYFDGEGLHFIVRGNLSQFEVSIWEKDERVGNTFTLLQGRPGELRYSMFPFSADSVGLMITKHLVYHDGKAEVYKATDYENSEKLGEATVRETSEGIEVVVPFEYIENPADFYFAVSTVKDGRLEVISTPVELK

[0111] Fermentation culture of recombinant strain X33 / pPICZαA-HJΔ782Δsp-Kex2 in Example 2

[0112] Comprising the following steps:

[0113] (1) Strain activation: Pick and streak the recombinant yeast X33 / pPICZαA-HJΔ782Δsp-Kex2 preserved in glycerol in Example 1 onto a solid plate medium, and culture it in the incubator at 30°C in the dark for 3 days.

[0114] (2) Preparation of primary seed liquid: Pick a single colony from the cultured plate, inoculate it into the primary seed medium, and continuously shake-culture it at 30°C at a speed of 240 rpm for 24 hours.

[0115] (3) Preparation of secondary seed liquid: Inoculate the primary seed liquid into the secondary seed medium at an inoculation amount of 3%, and continuously shake-culture it at 30°C at a speed of 240 rpm until the OD 600 value of the culture solution exceeds 10.

[0116] (4) Preparation of fermenter: Accurately measure the liquid delivery flow rate of the peristaltic pump in the fermenter. Calibrate and verify the pH electrode using standard solutions with pH values of 6.86 and 4.00 respectively. Generally, the dissolved oxygen electrode does not need to be calibrated for zero point, and only the slope needs to be calibrated before inoculation. Prepare 2.7 L of BSM medium (glycerol batch medium) and add it to the 7 L fermenter. After sterilization at 121°C for 30 min, control the temperature at 30°C through the automatic control system, and automatically add ammonia water to adjust the pH of the medium to 5.0.

[0117] (5) Glycerol batch culture: Inoculate 300 mL of secondary seed liquid into the fermenter, and automatically control the stirring rate through the set program to keep the DO concentration above 30% during the glycerol batch culture stage. When the DO concentration in the fermenter continuously increases, it indicates that the carbon source in the medium is exhausted, and then start to feed glycerol.

[0118] (6) Glycerol fed-batch culture: To ensure that the DO concentration is always maintained above 20% during this stage, adjust the stirring rate and ventilation volume to the maximum limit of the equipment, and then feed glycerol at a speed of 12 - 15 mL / (L·h). At the same time, measure the wet cell weight every 1 h. When the wet cell weight reaches 180 g / L, immediately stop feeding until the dissolved oxygen rises to nearly 100% and remains unchanged, and then start induction.

[0119] (7) Induction culture: When starting induction, set the methanol flow rate to 5 mL / h. In the initial stage of induction, increase the flow rate by 0.6 mL / h every 3 hours. In the later stage of induction, judge whether the dissolved oxygen (DO) in the fermenter is appropriate according to the protein expression and yeast growth conditions (dynamically adjusted according to the well-known techniques in the art).

[0120] Take samples every 12 h during the induction culture stage to measure OD 600, related data such as cell wet weight, protein concentration, and enzyme activity of the fermentation supernatant were measured, and the fermentation supernatant at each time point was collected for SDS-PAGE electrophoresis analysis.

[0121] The results are shown in Figure 1 As can be seen from Figure 1 , during the entire induction process, the cell growth level was normal. The enzyme activity of the supernatant reached 260 U / mL at 144 h of induction, and the protein content tended to be stable at 1.9 g / L. The electrophoresis bands of the fermentation supernatant during the fermentation process were relatively single. The band between 66.2 kDa and 116 kDa was the target protein band, and there were no obvious impurity bands between 44.3 kDa and 66.4 kDa ( Figure 2 ).

[0122] There were obvious impurity bands between 44.3 kDa and 66.4 kDa in the fermentation supernatant of the recombinant strain X33 / pPICZαA-HJΔ782Δsp (without mutating the 5 Kex2 protease cleavage sites), and the band of the target protein was very faint ( Figure 3 ). The results indicate that the production of impurity proteins is related to the cleavage of Kex2 protease.

[0123] By removing the original signal peptide of the HJΔ782 gene and mutating all five Kex2 protease cleavage sites, stable and efficient expression of pullulanase HJΔ782 in Pichia pastoris was achieved.

[0124] Example 3 Optimization of the Fermentation Culture Process of the Recombinant Strain X33 / pPICZαA-HJΔ782Δsp-Kex2

[0125] In this example, the fermentation culture process of the recombinant strain X33 / pPICZαA-HJΔ782Δsp-Kex2 was further explored.

[0126] 1. Induction pH

[0127] The pH value of the medium during fermentation has a great influence on the growth of microorganisms and protein expression. Pichia pastoris can grow and reproduce normally at pH 3.0 - 7.0 and has strong tolerance to acid-base conditions. However, for the expression of different heterologous proteins, the effect of induction pH on their expression levels is also different. Therefore, selecting an appropriate induction pH is crucial for ensuring the high-efficiency expression of pullulanase HJΔ782Δsp-Kex2 in Pichia pastoris. By real-time monitoring the culture pH and setting automatic control of ammonia flow addition, the induction pH was maintained at 5.0, 6.0, and 7.0 respectively. The effects of different induction pH values on the expression of pullulanase HJΔ782Δsp-Kex2 in Pichia pastoris were studied.

[0128] The results are shown in Figure 4As shown. The results showed that Pichia pastoris could grow normally in the environment with pH ranging from 5.0 to 7.0, and pH 5.0 was the optimal growth pH for this expression strain. At the end of fermentation, OD 600 reached the highest 550 in each experimental group ( Figure 4 A in Figure 4 ). The protein expression level was greatly affected by the induction pH. There was a large difference in the total protein content among different pH experimental groups. The protein content was the highest when the induction pH was 6.0, reaching 2.25 mg / mL, which was about 1.8 times the protein content at pH 7.0 ( Figure 4 C in

[0129] 2. Induction temperature

[0130] The induction temperature is one of the key factors for the expression of the target protein by recombinant Pichia pastoris. If the induction temperature is too low, the growth rate of the recombinant bacteria is low, which affects the expression of the target protein. If the induction temperature is too high, the metabolic level of the recombinant bacteria is high, and the growth rate is fast, resulting in a decrease in the expression of recombinant protein per unit cell, thus leading to a decrease in the protein expression level. The effect of induction temperature on the high-density fermentation of Pichia pastoris to produce pullulanase HJΔ782Δsp-Kex2 was investigated in a 30 L bioreactor. When cultured at 30 °C and pH 5.0 until the wet cell weight was about 180 g / L, the induction temperatures were controlled at 20 °C, 24 °C, 28 °C and 30 °C respectively, and the induction pH was 6.0 to start inducing enzyme production.

[0131] The results showed that there were obvious differences in the growth rate of bacteria among different temperature experimental groups. The temperature condition with the most vigorous cell growth was 28 °C. At 28 °C, OD 600 and the wet cell weight were 589 g / L and 455 g / L respectively ( Figure 5 A and B in Figure 5 ). The temperature of 28 °C was the most suitable for the engineering bacteria to express pullulanase HJΔ782Δsp-Kex2, and the enzyme activity of the fermentation broth reached 400 U / mL (

[0132] Example 4 Characterization of the Enzymatic Properties of Thermophilic Pullulanase HJΔ782Δsp-Kex2

[0133] Using the method of Example 2, for the recombinant strain X33 / pPICZαA-HJΔ782, the recombinant strain

[0134] The recombinant strains X33 / pPICZαA-HJΔ782Δsp and X33 / pPICZαA-HJΔ782Δsp-Kex2 were fermented and cultured. After 144 h of fermentation, the fermentation broths of the recombinant strains X33 / pPICZαA-HJΔ782Δsp and X33 / pPICZαA-HJΔ782Δsp-Kex2 were centrifuged, and the supernatants were collected. The fermentation broth of the recombinant strain X33 / pPICZαA-HJΔ782 was centrifuged, and the cell pellet was resuspended in phosphate buffer (pH 6.0). Then the resuspended solution was disrupted in a low-temperature ultra-high pressure cell disruptor, and the supernatant was collected by centrifugation. A 10 kDa Vivaflow 200 membrane package ultrafiltration system was used to perform rough separation on each supernatant - first, 500 mL of the supernatant was concentrated to 100 mL, then 500 mL of buffer (phosphate buffer at pH 6.0) was added and concentrated again. This was repeated three times to displace the enzyme protein from the culture medium into the buffer, obtaining crude enzyme solutions of pullulanase HJΔ782, pullulanase HJΔ782Δsp, and pullulanase HJΔ782Δsp-Kex2.

[0135] The crude enzyme solutions were purified using an affinity chromatography column. First, it was equilibrated with phosphate buffer (pH 6.0) at a flow rate of 5 mL / min. After equilibration, the crude enzyme solution was then bound to the chromatography column. Next, the impurity proteins were eluted with phosphate buffer containing 20 mM imidazole (pH 6.0), and then the target protein was eluted with phosphate buffer containing 100 mM imidazole (pH 6.0). The eluted target protein was further desalted with a G-25 desalting column to remove imidazole, and finally, the enzyme purity was detected by reducing SDS-PAGE vertical electrophoresis. The results are shown as Figure 6 shown. The results indicate that a relatively single band can be obtained after purification, indicating that the target protein can be effectively purified using affinity chromatography.

[0136] 1. Optimal temperature and temperature stability of pullulanase HJΔ782Δsp-Kex2

[0137] Using pullulan as the substrate and 50 mM PB at pH 6.0 as the buffer, the enzyme activity of pullulanase was measured at 90 °C to 120 °C to determine the optimal reaction temperature. To study the thermal stability of pullulanase, the purified enzyme was incubated at 90 °C, 100 °C, and 110 °C respectively. At regular intervals, a portion of the enzyme solution was taken out, quickly cooled, and the residual enzyme activity was measured. The results are shown as Figure 7 shown. The results show that there are no significant differences in the optimal reaction temperature and residual enzyme activity at different temperatures among HJΔ782Δsp-Kex2, HJΔ782Δsp, and HJΔ782.

[0138] 2. Optimal pH and pH stability of pullulanase HJΔ782Δsp-Kex2

[0139] Prepare 50 mM phosphate buffer solutions with different pH values (pH 4.0 - 8.0), measure the activity of pullulanase at the optimal temperature of 110 °C, and investigate the optimal pH of pullulanase. To investigate the pH tolerance of pullulanase, mix 50 mM phosphate buffer solutions with different pH values (pH 4.0 - 8.0) with pullulanase, keep them at 4 °C for 24 h, and measure the residual enzyme activity of pullulanase. The results are as Figure 8 shown. The results show that there are no significant differences in the optimal reaction pH and residual enzyme activities at different pH values of HJΔ782Δsp-Kex2, HJΔ782Δsp, and HJΔ782.

[0140] Construction and fermentation of the recombinant strain X33 / pPICZαA-HJΔ782 in Comparative Example 1

[0141] Referring to Example 1, after constructing the pPICZαA-HJΔ782 recombinant plasmid, construct the recombinant strain X33 / pPICZαA-HJΔ782. And use the method of Example 2 for the fermentation culture of the recombinant strain.

[0142] During the induction culture stage, samples were taken every 12 h to measure OD 600 , cell wet weight, protein concentration, and enzyme activity of the fermentation supernatant and other related data, and collect the fermentation supernatant at each time point for SDS-PAGE electrophoresis analysis.

[0143] The results are as Figure 9 and Figure 10 shown. During the whole induction process, the cell growth level is normal, while the protein content and pullulanase enzyme activity are at relatively low levels ( Figure 9 ), and there are no obvious bands in the expression product (protein molecular weight is 90 kDa) ( Figure 10 ). Observe the expression of the target protein in the cells by cell disruption, collect the intracellular supernatant and intracellular precipitate for SDS-PAGE electrophoresis analysis, and detect the pullulanase enzyme activity of the intracellular supernatant. The results are as Figure 11 shown. As can be seen from Figure 11 , there are obvious bands in the intracellular supernatant and intracellular precipitate lanes of the expressed protein. Detect the pullulanase enzyme activity of the cell lysate. The results are as Figure 12 shown. The pullulanase enzyme activity of the cell lysate reaches 90 U / mL. It can be seen that there is a problem with the transport of the target protein in the recombinant strain X33 / pPICZαA-HJΔ782, and the target protein cannot be expressed by extracellular secretion.

[0144] Analyze the protein sequence of the thermophilic pullulanase HJΔ782 using SignalP-6.0. The results are as Figure 13As shown, there is an original signal peptide consisting of 26 amino acids at the N-terminus of the thermophilic pullulanase HJΔ782. It is speculated that the presence of the original signal peptide affects the extracellular secretion expression of the protein.

[0145] Construction and fermentation of the recombinant strain X33 / pPICZαA-HJΔ782Δsp in Comparative Example 2

[0146] Referring to Example 1, after constructing the recombinant plasmid pPICZαA-HJΔ782Δsp, the recombinant strain

[0147] X33 / pPICZαA-HJΔ782Δsp was constructed and fermented by the method of Example 2.

[0148] Samples were taken every 12 h during the induction culture stage to measure OD 600 , cell wet weight, protein concentration, enzyme activity in the fermentation supernatant and other relevant data, and the fermentation supernatant at each time point was collected for SDS-PAGE electrophoresis analysis. The results are as Figure 14 and Figure 15 shown. During the whole induction process, the cell growth level was normal. At 144 h of induction, the enzyme activity in the supernatant reached 97 U / mL, and the protein content tended to be stable at 1.4 g / L ( Figure 14 ). The band between 66.4 kDa and 97.2 kDa corresponded to the molecular weight of the target protein (87 kDa), but the band of the target protein was very light and the expression level was low. There were also three bands between 44.3 kDa and 66.4 kDa, and these three bands gradually became thicker as the fermentation time increased. It was speculated that the low expression level of the target protein was due to the production of heteroproteins, and the amount of heteroproteins also increased as the fermentation time increased ( Figure 15 ).

[0149] The amino acid sequence of the thermophilic pullulanase HJΔ782Δsp was analyzed and compared with the cleavage sites of various endogenous Pichia pastoris enzymes that act on proteins. It was found that there were five cleavage sites of Kex2 protease in the thermophilic pullulanase HJΔ782Δsp ( Figure 16 ). Homology modeling of HJΔ782Δsp was performed using AlphaFold3, and the structure diagram was made using PyMOL. As Figure 17 shown, among them, E291 and E394 are two key catalytic residues, and the yellow-labeled structure is the amino acid residues within the distance of the two key catalytic residues ; K521, K341, K75, K416, and R268 are the amino acids in the five cleavage sites of Kex2 protease respectively. It can be seen from the figure that these 5 sites are all located on the surface of the protein structure and far from the catalytic active center.

[0150] Combining the results of Example 2 and this comparative example, it can be seen that the generation of heteroproteins is related to the cleavage effect of Kex2 protease. When Kex2 protease cannot exert its cleavage effect, no heteroproteins will be generated, and the expression level of the target protein is high.

[0151] Fermentation culture of the recombinant strain X33 / pPICZαA-HJΔ782Δsp-Kex2 with single point mutations in Comparative Example 3

[0152] Referring to Example 1, five recombinant strains X33 / pPICZαA-HJΔ782Δsp-Kex2 with single point mutations (K75G, R268G, K341G, K416G, K521G) were constructed, and the fermentation culture of the recombinant strains was carried out by the method of Example 2. SDS-PAGE electrophoresis analysis was performed on the fermentation supernatant. The results are as Figure 18 shown. It can be seen from Figure 18 that the target protein bands of the expression products of the five recombinant strains X33 / pPICZαA-HJΔ782Δsp-Kex2 with single point mutations are different. The target protein bands between molecular weights of 66.2 kDa and 116 kDa of K416G and K521G are significantly more than the other three, but there are still obvious impurity bands.

[0153] Combining the results of Example 2 and this comparative example, it can be seen that only the recombinant strain X33 / pPICZαA-HJΔ782Δsp-Kex2 with simultaneous mutations at five sites (K75G, R268G, K341G, K416G, K521G) can efficiently and stably produce the target protein.

[0154] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0155] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A high-temperature pullulanase, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. A coding gene for a thermophilic pullulanase, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

3. A recombinant plasmid carrying a coding gene for thermophilic pullulanase, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

1.

4. An engineered bacterium transformed with the recombinant plasmid according to claim 3.

5. The engineered bacterium according to claim 4, wherein Its starting strain is Pichia pastoris; preferably Pichia pastoris X-33.

6. Use of the recombinant plasmid according to claim 3, the engineered bacterium according to claim 4 or 5 in the preparation of thermophilic pullulanase.

7. A method for preparing a thermophilic pullulanase, characterized in that, Comprising the following steps: (1) Inoculate the secondary seed solution of the engineered bacterium according to claim 4 into a fermenter containing BSM medium, and culture at 28°C to 32°C, pH 5 to 5.2, and control the DO concentration to be 28% to 32%. (2) When the DO concentration starts to increase, feed glycerol, and continue to culture until the wet cell weight reaches 170 g / L to 190 g / L, then stop feeding glycerol. (3) When the DO concentration is 95% to 100% and remains unchanged, feed methanol, and continue to culture at 27°C to 29°C, pH 6 to 6.2 for 132 h to 144 h, then stop fermentation and collect the supernatant.

8. The preparation method of the thermophilic pullulanase according to claim 7, wherein, In step (2), the feeding rate of the glycerol is 12 mL / (L·h) to 15 mL / (L·h); and / or, in step (3), the feeding rate of the methanol is 4 mL / h to 6 mL / h.

9. The preparation method of the thermophilic pullulanase according to claim 7, wherein In step (2), stop feeding glycerol when the wet cell weight reaches 175 g / L to 185 g / L; and / or, in step (1), the inoculation amount is 8% to 12%.

10. Use of the thermophilic pullulanase prepared by the preparation method according to any one of claims 7 to 9 in industrial sugar production.

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