Raw starch hydrolyzed glucamylase mutant with improved specific activity and application thereof

Abstract

The invention discloses a raw starch hydrolyzed glucamylase mutant with improved specific activity and application thereof. According to the invention, raw starch hydrolysis glucamylase SeGA is taken as a starting enzyme, on the basis, the catalytic activity of glucamylase is improved by using an online website FuncLib aided design, and heterologous expression is carried out in pichia pastoris, so that the raw starch hydrolysis glucamylase SeGA-21 with improved specific activity is obtained. When raw corn starch is used as a substrate, the specific enzyme activity of the mutant SeGA-21 to the raw corn starch is 1.21 times that of a starting enzyme, the stability is slightly improved compared with that of the starting enzyme, and the half-life period is 1.25 times that of the starting enzyme under the conditions that the temperature is 40 DEG C and the pH value is 5.5. The mutant has potential application value in starch sugar production with corn raw starch as a substrate.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a mutant of raw starch hydrolysate glucoamylase with enhanced specific activity and its application. Background Technology

[0002] Glucoamylase (EC 3.2.1.3), also known as γ-amylase or starch glucoside enzyme, or simply saccharifying enzyme, belongs to the GH15 and GH97 families. It is an exoamylase that primarily acts on the α-1,4 and α-1,6 glycosidic bonds at the non-reducing ends of oligosaccharides and starch, removing a glucose unit from the non-reducing end of the substrate through conformational inversion. Furthermore, glucoamylase can also hydrolyze α-1,6 and α-1,3 glycosidic bonds through prolonged incubation, thus completely hydrolyzing the substrate into glucose. Glucoamylase can be used to hydrolyze corn to produce corn syrup; raw starch hydrolytic glucoamylase (RSGA) can hydrolyze raw starch granules at gelatinization temperatures. For example, a raw starch hydrolytic glucoamylase from *Aspergillus fumigatus* was successfully heterologously expressed in *Pichia pastoris*, yielding the recombinant enzyme pRSGA. At 40 °C, pRSGA, in synergy with α-amylase and pullulanase, hydrolyzed 200 g / L of raw corn starch, achieving a hydrolysis rate of 92.2% after 36 h. Qiang-Sheng X et al. isolated a novel raw starch hydrolytic glucoamylase, PoGA15A, from *Penicillium oxalicum* GXU20, which can efficiently hydrolyze raw starch from different sources such as corn, wheat, and cassava.

[0003] The catalytic activity of glucoamylase for hydrolyzing raw starch is one of the core technologies that restricts the process of starch sugar production at room temperature. However, the existing glucoamylase for hydrolyzing raw starch has low catalytic activity and low hydrolysis efficiency, which makes it difficult to meet the high efficiency requirements of industrial applications. Therefore, using protein engineering technology to obtain glucoamylase for hydrolyzing raw starch with higher specific enzyme activity and better thermal stability plays an important role in the application of hydrolyzing high-concentration corn raw starch. Summary of the Invention

[0004] This invention addresses the shortcomings of existing technologies by providing a mutant of raw starch-hydrolyzing glucoamylase with enhanced specific activity and its applications. Based on the raw starch-hydrolyzing glucoamylase SeGA, this invention utilizes computer-aided design to obtain a mutant with improved specific activity and stability. Using corn raw starch as a substrate, the mutant's specific activity is 1.21 times that of the original enzyme, and its stability is significantly improved, reaching 1.25 times that of the original enzyme at 40℃ and pH 5.5. In experiments hydrolyzing high-concentration corn raw starch, at 40℃, the mutant alone achieved a hydrolysis rate of 34.86% after 15 hours of treatment. This mutant enzyme has potential value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

[0005] This invention relates to a mutant of raw starch hydrolysis glucoamylase, the amino acid sequence of which is shown in SEQ ID NO: 1. Specifically, the amino acid glutamine at position 339 of the SeGA amino acid sequence is mutated to asparagine, the amino acid alanine at position 426 is mutated to histidine, the amino acid threonine at position 438 is mutated to glycine, and the amino acid alanine at position 440 is changed to glutamic acid.

[0006] The encoding gene of the starch hydrolysis glucoamylase mutant of the present invention has the nucleotide sequence shown in SEQ ID NO: 2.

[0007] The expression strain of the raw starch hydrolysate glucoamylase mutant of this invention, classified and named Komagataellaphaffii GS115 pPIC9K-SeGA-21, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025724, deposit date April 8, 2025, deposit address: Wuhan University, Wuhan, China.

[0008] The method for constructing a mutant expression strain of raw starch hydrolysis glucoamylase of the present invention includes the following steps:

[0009] The amino acid sequence of the starch hydrolase SeGA from Septoria linicola was submitted to the online website FuncLib. FuncLib calculated the site mutation space, selected amino acids within the 5 Å range of SeGA catalytic residues at 206E and 427E, removed highly conserved amino acids, and set non-conserved sites as random mutation sites. Each mutant had no more than 5 mutation sites. The top 50 mutant design structures (the lower the free energy, the higher the ranking, and the more stable the protein) were selected for molecular docking to check the changes in substrate affinity and the rationality of the action space. Five mutants were selected for heterologous expression in Pichia pastoris for verification.

[0010] The amino acid sequences of the five designed mutants were translated into nucleotide sequences, and codons were optimized in Pichia pastoris. EcoRI and NotI restriction sites were selected, and the nucleotide sequences of the mutants were constructed into the vector pPIC9K. The plasmid was then preserved in E. coli strain.

[0011] The expression plasmid vector described in the above construction method includes pPIC9K.

[0012] The host bacteria mentioned in the above construction method include E. coli, Komagataella phaffii, etc.

[0013] The raw starch hydrolysis glucoamylase mutant of the present invention can be obtained by fermentation of the expressed strain.

[0014] The present invention relates to the application of a raw starch hydrolysis glucoamylase mutant in the hydrolysis of raw starch.

[0015] Specifically, the described raw starch hydrolytic glucoamylase mutant can be applied to the hydrolysis of 30% corn raw starch emulsion. Using corn raw starch as a substrate, at 40℃ and pH 5.5, the specific activity of the mutant enzyme is 78.2 ± 6.1 U / mg, which is 1.21 times that of the wild type. At 40℃, its stability is 1.25 times that of the original enzyme. At 40℃ and pH 5.5-5.7, after 15 hours of treatment alone, the mutant can achieve a hydrolysis rate of 34.86% for high-concentration corn raw starch (30%). This mutant enzyme has potential value in industrial applications based on the hydrolysis of high-concentration corn raw starch.

[0016] This invention measured and compared the specific enzyme activity, optimal temperature, optimal pH, and stability of the mutant protein and the original starting enzyme. The results showed that, using corn starch as a substrate, this invention, while maintaining improved specific activity, exhibited 1.25 times the stability of the starting enzyme at 40°C. Attached Figure Description

[0017] Figure 1 This is an electrophoresis image of nucleic acid digested with a single enzyme in the mutant.

[0018] Figure 2 SDS-PAGE images of the purified mutant protein and the starting enzyme SeGA are shown. Where: M is the marker; 1 is purified SeGA; 2 is purified SeGA-21.

[0019] Figure 3 The results show the optimal temperature determination results for SeGA and SeGA-21.

[0020] Figure 4 The results show the optimal pH values ​​for SeGA and SeGA-21.

[0021] Figure 5 The stability of SeGA and SeGA-21 at 40℃ and pH 5.5 was determined.

[0022] Figure 6 Hydrolysis rates of high-concentration corn starch hydrolyzed separately by SeGA and SeGA-21. Detailed Implementation

[0023] Unless otherwise specified, the implementation methods in the following embodiments are all conventional methods.

[0024] (i) Construction of expression strains containing the mutant gene for starch hydrolysis glucoamylase of the present invention

[0025] 1. Selection of mutation sites in the glucoamylase gene for raw starch hydrolysis

[0026] Seven amino acids within a 5 Å range of SeGA catalytic residues were selected as mutation sites. Homology modeling and molecular docking were performed on the top 50 mutant sequences designed and output by FuncLib. Five mutant enzymes with better docking results than wild-type were selected. The amino acid sequences of the mutant enzymes were converted into amino acid sequences, and the nucleotide sequences were carried on the pPIC9K vector for heterologous expression in Pichia pastoris.

[0027] Based on the simulated structure and multiple sequence alignment, the site of the mutation was determined: amino acid glutamine at position 339 was mutated to asparagine, amino acid alanine at position 426 was mutated to histidine, amino acid threonine at position 438 was mutated to glycine, and amino acid alanine at position 440 was changed to glutamic acid.

[0028] 2. Construction of mutant strains of raw starch hydrolysis glucoamylase

[0029] Construction, linearization enzyme digestion, and transformation.

[0030] Based on the gene sequence of the starch hydrolysate glucoamylase SeGA, and the selected mutation sites 339Q, 426A, 438S and 440A.

[0031] The amino acid sequence of glucosylase with the signal peptide sequence removed was converted into a nucleotide sequence. Codons were optimized in Komagataella phaffii, and EcoRI and NotI polyclonal restriction sites were selected. The codon-optimized glucosylase nucleotide sequence was constructed into the vector pPIC9K. The plasmid was preserved by the Top 10 host bacteria and sent to a biotechnology company for full genome synthesis.

[0032] The glycerol bacteria constructed by the biotechnology company were inoculated into LB medium and cultured at 37 °C for 10 h. Plasmids were extracted using a plasmid miniprep kit, and their concentration was determined. The recombinant plasmids were linearized by digestion with Sac I restriction endonuclease.

[0033] After enzyme digestion, the digestion products were purified using a gel extraction kit. A 1% agarose gel was prepared to detect the gel-extracted products. The fully digested plasmid was added to competent cells and incubated on ice for 10 min to ensure sufficient contact between the plasmid and the competent cells. The cells were then transferred to an electroporation cuvette, pulsed, and immediately after the pulsed reaction, 700 μL of pre-chilled sterile sorbitol solution was added. The cells were then incubated at 28 °C and 200 rpm for 1 h to recover. After recovery, the cells were centrifuged at 4000 rpm, and 150 μL of supernatant was retained. The cells were resuspended and plated on MD (Medium Deposition Instrument), and incubated at 28 °C for 3 days.

[0034] Transformants with the correct sequence were selected to obtain the engineered strain Komagataella phaffiiGS115pPIC9K-SeGA-21, which contains the mutant gene of this invention.

[0035] The expression strain of the raw starch hydrolysate glucoamylase mutant of this invention, classified and named Komagataellaphaffii GS115 pPIC9K-SeGA-21, has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025724, deposit date April 8, 2025, deposit address: Wuhan University, Wuhan, China.

[0036] (II) Expression and protein purification of engineered bacteria containing the mutant gene for starch hydrolysis glucose amylase of the present invention

[0037] The selected positive strains were inoculated into test tubes containing 5 mL of BMGY liquid medium at an inoculation rate of 1%, and cultured at 28°C and 200 rpm for 24 h. 1 mL of the bacterial culture was then transferred to a 50 mL BMGY Erlenmeyer flask and cultured at 28°C and 200 rpm for 24 h. The cells were centrifuged and resuspended in BMMY liquid medium until OD200 was reached. 600 Induction was performed using a concentration of 1.0 μg / mL and 0.5% methanol (methanol was added every 24 hours to maintain the concentration at 0.5%), and samples were taken to measure the biomass OD. 600 and enzyme activity OD 540 .

[0038] The crude enzyme solution was concentrated using a peristaltic pump and dialyzed for 12 hours using dialysis buffer (20 mM Tris-HCl, 50 mM NaCl, pH=6.5). The resulting protein was tested and found to meet SDS-PAGE purity.

[0039] When using raw corn starch as a substrate, the optimal temperature for the mutant enzyme is 60℃, the optimal pH is 5.5, and it exhibits over 70% catalytic activity within the pH range of 5-6.0.

[0040] (III) Detection of enzyme activity of the starch hydrolysis glucoamylase mutant of the present invention (DNS method)

[0041] 1. Definition of enzyme activity

[0042] 1 U is the amount of protein required to generate 1 µM of maltose per minute.

[0043] 2. Enzyme activity assay

[0044] The reaction system consisted of 600 μL. 270 μL of 50 mM Na₂HPO₄-KH₂PO₄ buffer was transferred to a 2 mL EP tube. 300 μL of a 1% corn starch solution was added, and the mixture was incubated at 45°C for 10 min. 30 μL of enzyme solution was then transferred to an EP tube. 30 μL of buffer was added to the control group, and the mixture was reacted with the substrate for 10 min. 300 μL of DNS was added, and the reaction was terminated by boiling in water for 15 min. The sample was cooled to room temperature on ice, centrifuged at 12,000 g for 2 min, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance was read at A540 nm. The reducing sugar content was calculated based on the DNS standard curve, and the enzyme activity was calculated according to the definition of enzyme activity.

[0045] The test results showed that, when using corn starch as a substrate, the specific activity of the mutant enzyme obtained in this invention was 1.23 times that of the starting enzyme.

[0046] (iv) Detection of the stability of the starch hydrolysate glucoamylase contained in this invention

[0047] Under conditions of 40℃ and pH 5.5, the starting enzyme SeGA and the mutant enzyme were heat-treated. Samples were taken every 3 hours. With the initial enzyme activity as 100%, the enzyme activity remaining rate after a certain period of heat treatment was calculated. The formula is as follows: Enzyme activity remaining rate = Enzyme activity after heat treatment / Enzyme activity before heat treatment × 100%.

[0048] The results showed that at 40℃, the half-life of the mutant enzyme was 240 h, which was 1.25 times that of the starting enzyme.

[0049] (v) Application of the starch hydrolysis glucoamylase mutant of the present invention in the hydrolysis of high-concentration corn starch

[0050] The hydrolysis system consisted of Na₂HPO₄-KH₂PO₄ buffer (50 mM, pH 7.0) with 1 mM CaCl₂ added, followed by raw starch and 2 mg / g of the raw starch-hydrolyzing glucoamylase mutant. The hydrolysis reaction was carried out in a shaker at 35°C and 200 rpm. Samples were taken at appropriate time intervals, and the reducing sugar content in the hydrolysis system was determined using the DNS method. A control group was prepared by adding the same amount of the starting enzyme SeGA.

[0051] In the experiment of hydrolyzing 30% corn starch, the results showed that the mutant enzyme reached a plateau in hydrolysis after 15 hours, with a hydrolysis rate of 34.86% for corn. Under the same conditions, the starting enzyme had a hydrolysis rate of only 30.29% for high-concentration corn starch. The mutant enzyme of this invention has great application potential in the hydrolysis of high-concentration corn starch to produce starch sugars.

Claims

1. A mutant of raw starch hydrolysate glucoamylase with enhanced specific activity, characterized in that... Its amino acid sequence is shown in SEQ ID NO:

1.

2. The encoding gene of the starch hydrolysis glucoamylase mutant according to claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

2.

3. The expression strain of the starch hydrolysis glucoamylase mutant according to claim 1, characterized in that: The expressed strain is classified as Komagataella phaffii GS115 pPIC9K-SeGA-21 and has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025724, deposited on April 8, 2025, at Wuhan University, Wuhan, China.

4. The application of the raw starch hydrolysis glucoamylase mutant of claim 1 in the hydrolysis of raw starch.

5. The application according to claim 4, characterized in that: The raw starch hydrolysis glucoamylase mutant was used to hydrolyze 30% corn raw starch emulsion.

6. The application according to claim 4, characterized in that: The hydrolysis system was operated at a temperature of 40℃ and a pH value of 5.5-5.7.

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