A method for increasing maltotetraose production

Abstract

The application discloses a method for improving maltotetraose yield, and belongs to the field of genetic engineering and biotechnology. PS The CBM enzyme activity is 310 U / mL, the specific enzyme activity is 1802 U / mg, and compared with wild type (230 U / mL, 1200 U / mg), the CBM enzyme activity and the specific enzyme activity are respectively increased by 34.8% and 50.2%. The application mutates the maltulose-forming enzyme MFA from Pseudomonas saccharophila to obtain the maltulose-forming enzyme MFA with increased maltotetraose proportion in product PS The maltotetraose proportion in product catalyzed by the CBM is 73.1%, the CBM has good process adaptability, and has wide application prospect in the field of biology.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for improving the yield of maltotetraose, belonging to the technical field of genetic engineering and enzyme engineering. BACKGROUND

[0002] Linear malt-oligosaccharides are defined as polymers with 3-10 glucose units linked by α-1,4 glycosidic bonds, which have broad application prospects. First, they have excellent food processing performance: when added to baked or puffed foods, they can act as water regulators due to their good water retention properties; when added to beverages, they can act as thickening agents due to their high viscosity. Second, malt-oligosaccharides also have good physiological effects: after being ingested by humans, they are rapidly used to synthesize glycogen after being hydrolyzed by enzymes in the body, so they are less likely to cause blood sugar to rise; they do not cause an increase in osmotic pressure after entering the blood, so they can be used as an additive in sports drinks to improve athletes' endurance; they are difficult for bacteria such as yeast to utilize, which can slow down their growth rate to some extent and thus effectively prevent tooth decay.

[0003] Among linear malt-oligosaccharides, maltotetraose can reduce the adhesion of vascular smooth muscle cells to monocytes and effectively inhibit the occurrence of atherosclerosis, thus having potential application value in the medical field. In addition, it can inhibit the growth of E. coli and has significant skin moisturizing properties and anti-inflammatory properties.

[0004] Currently, the preparation methods of linear malt-oligosaccharides include acid preparation, acid-enzyme preparation, and enzyme catalysis. Among them, enzyme catalysis is widely used because of its high hydrolysis efficiency, environmentally friendly hydrolysis process, and less damage to production equipment. However, the malt-oligosaccharides generated by the industrial use of malt-oligosaccharide generating enzyme catalytic substrates are usually a mixture of multiple oligosaccharides, with a low maltotetraose content. A low maltotetraose content in the product makes it difficult to separate and produces high costs, making industrial production difficult.

[0005] Currently, most research on improving the application prospects of malt-oligosaccharide generating enzymes focuses on improving their thermal stability, and there is less research on improving their product specificity. Therefore, it is necessary to study mutants of linear maltotetraose generating enzymes with improved product specificity to better meet the needs of industrialization SUMMARY

[0006] To solve the above technical problems, the present application mutates linear malt-oligosaccharide generating enzymes derived from Pseudomonas saccharophila to obtain linear malt-oligosaccharide generating enzymes MFA PS -ΔCBM, which catalyzes the product with a maltotetraose content of 73.1%.

[0007] The present application provides a method for preparing maltotetraose, which comprises using linear malt-oligosaccharide forming enzyme MFA PS -ΔCBM is a catalyst, or using linear malt-oligosaccharide forming enzyme MFA PS -ΔCBM is a catalyst, or using linear malt-oligosaccharide forming enzyme MFA PS -ΔCBM is a catalyst, or using linear malt-oligosaccharide forming enzyme MFA

[0008] In an embodiment of the present application, the linear malt-oligosaccharide forming enzyme MFA PS -ΔCBM is linear malt-oligosaccharide forming enzyme MFA PS obtained after truncation (CBM sequence is removed).

[0009] In an embodiment of the present application, the nucleotide sequence encoding the parent enzyme linear malt-oligosaccharide forming enzyme MFA PS is shown as SEQ ID NO. 4.

[0010] In an embodiment of the present application, the nucleotide sequence encoding the linear malt-oligosaccharide forming enzyme MFA PS -ΔCBM is shown as SEQ ID NO. 2.

[0011] In an embodiment of the present application, the recombinant Bacillus subtilis uses Bacillus subtilis WB600 as an expression host.

[0012] In an embodiment of the present application, the recombinant Bacillus subtilis uses pP43nmk plasmid as an expression vector.

[0013] In an embodiment of the present application, the starch includes but is not limited to corn starch, waxy corn starch, cassava starch, potato starch, rice starch and wheat starch.

[0014] In an embodiment of the present application, the linear malt-oligosaccharide forming enzyme MFA PS -ΔCBM is added in an amount of at least 10 U / g.

[0015] In an embodiment of the present application, the reaction condition is that the reaction temperature is 50°C.

[0016] In an embodiment of the present application, the preparation method of the recombinant enzyme is as follows: after the recombinant Bacillus subtilis is inoculated into a LB culture medium containing Canna resistance for activation, the activated liquid is inoculated into a TB fermentation culture medium containing Canna resistance for fermentation culture, and the fermentation liquid is centrifuged to obtain the recombinant enzyme.

[0017] In an embodiment of the present application, the method is MFA PS -ΔCBM is used as a catalyst, and maltodextrin is used as a substrate, wherein the DE value of the substrate maltodextrin is 7-9, and the addition amount of the substrate maltodextrin is at least 10 U / g.

[0018] The present application also provides the linear malt-oligosaccharide generating enzyme MFA PS -ΔCBM or the linear malt-oligosaccharide generating enzyme MFA PS -ΔCBM of the recombinant Bacillus subtilis in improving the yield of maltotetraose.

[0019] In an embodiment of the present application, the recombinant Bacillus subtilis uses Bacillus subtilis WB600 as an expression host.

[0020] In an embodiment of the present application, the recombinant Bacillus subtilis uses pP43nmk plasmid as an expression vector. In an embodiment of the present application, the nucleotide sequence encoding the linear malt-oligosaccharide generating enzyme MFA PS -ΔCBM is shown as SEQ ID NO. 2.

[0021] The present application also provides the linear malt-oligosaccharide generating enzyme MFA PS -ΔCBM or the linear malt-oligosaccharide generating enzyme MFA PS -ΔCBM of the recombinant Bacillus subtilis in improving the yield of maltotetraose.

[0022] Advantages

[0023] (1) 20% maltodextrin (DE 7-9) is used as a substrate, the enzyme amount is 20 U / g, and the linear malt-oligosaccharide generating enzyme MFA PS -ΔCBM of the present application is used for reaction, and the proportion of maltotetraose in the product is 73.1%, which is higher than that of the wild-type linear malt-oligosaccharide generating enzyme MFA PS(64.6%) was increased by 11.6%, and the content of maltotetraose in the product of the mutant was 161.9 g / L, which was increased by 10.4% compared with the wild type (146.6 g / L).

[0024] (2) The linear malt low oligosaccharide generating enzyme MFA provided by the application PS The enzyme activity of the CBM deletion mutant was 310 U / mL, and the specific enzyme activity was 1802 U / mg, which was increased by 34.8% and 50.2% compared with the wild type (230 U / mL, 1200 U / mg), respectively.

[0025] (3) The method for producing linear malt low oligosaccharides provided by the application: after the substrate is adjusted to a slurry, the slurry is cooled to 50°C, and then the linear malt low oligosaccharide generating enzyme is added, without adding other additives such as pullulanase, calcium ions, isoamylase, etc. The process is simple and convenient, the production cost is lower, and it has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 : SDS-PAGE analysis of the purified linear malt low oligosaccharide generating enzyme MFA PS -ΔCBM and wild type MFA PS . Among them, M: protein standard molecular weight; 1: MFA PS ; 2: MFA PS -ΔCBM.

[0027] Figure 2 : Optimum temperature of the linear malt low oligosaccharide generating enzyme MFA PS -ΔCBM and wild type MFA PS

[0028] Figure 3 : Thermal stability of the linear malt low oligosaccharide generating enzyme MFA PS -ΔCBM and wild type MFA PS

[0029] Figure 4 : Optimum pH of the linear malt low oligosaccharide generating enzyme MFA PS -ΔCBM and wild type MFA PS

[0030] Figure 5 : Optimization results of different signal peptides. DETAILED DESCRIPTION

[0031] The culture medium involved in the following examples is as follows:

[0032] LB liquid medium: 1% (w / v) tryptone, 0.5% (w / v) yeast extract, 1% (w / v) sodium chloride, pH 7.0. ​​​

[0033] LB solid medium: 1.5 (w / v) agar was added to LB liquid medium.

[0034] TB liquid medium: 1.2% (w / v) tryptone, 2.4% (w / v) yeast extract, 0.4% (w / v) glycerol, 17 mM KH2PO4, 72 mM K2HPO4, pH 6.0.

[0035] The detection method involved in the following examples is as follows:

[0036] Linear malt low oligosaccharide generating enzyme enzyme activity determination method: the hydrolysis activity of the enzyme is characterized by the change of reducing sugar content by 3, 5-dinitrosalicylic acid (DNS) method. A 1% (w / v) completely gelatinized soluble starch solution was prepared with deionized water as the substrate, 100 μL of enzyme solution was diluted 200 times and added to 900 μL of substrate, and after 50°C reaction for 15 minutes, 1 mL of DNS solution was added to terminate the reaction, heated in a boiling water bath for 5 min, immediately placed in an ice water bath, cooled to room temperature, and then the absorbance value was determined at 540 nm. Compared with the glucose standard curve, 1 unit of crude enzyme activity (U) was required to produce 1 μmol of reducing sugar (calculated as glucose) per minute.

[0037] Specific enzyme activity represents the enzyme activity per milligram of enzyme protein. Specific enzyme activity (U / mg) = enzyme activity (U / mL) / pure enzyme concentration (mg / mL).

[0038] Detection method of polymerization degree: the supernatant after reaction was diluted and passed through a 0.22 μm water system filter membrane, and high performance anion exchange chromatography (HPAEC-PAD) was used to determine the polymerization degree distribution of the product after linear malt low oligosaccharide generating enzyme treatment. The mobile phase consists of 250 mM (w / v) sodium hydroxide and 1 M (w / v) sodium acetate, the flow rate is 0.5 mL / min, and the column temperature is kept at 35°C. The substrate conversion rate and the proportion of each monosaccharide component in G1-G7 are calculated as follows:

[0039] Monosaccharide proportion = (monosaccharide mass / G1-G7 mass) x 100%.

[0040] Substrate conversion rate = (G1-G7 / substrate dry basis mass) x 100%.

[0041] The pP43nmk vector involved in the following examples was purchased from Fenghui Biology.

[0042] The primers involved in the following examples were synthesized by Jinweizhi Biology Technology Co., Ltd., and the homologous recombination was carried out according to the Mut Express II Fast Mutagenesis Kit V2 kit manual of Vazyme company.

[0043] Example 1: Selection of Recombinant Bacillus subtilis Secretion Expression System

[0044] The specific steps are as follows:

[0045] 1. Recombinant vector pP43nmk-MFA containing different signal peptides PS Construction

[0046] (1) Recombinant vector pP43nmk-SP STP01 -MFA PS Construction

[0047] Linked with signal peptide SP STP01 MFA PS The mfa constructed based on Zhang Ziqian's master's thesis "Study on the secretory expression, structure and properties of maltodextrose synthase derived from Pseudomonassaccharophila" ps Using pST as a template, PCR was performed to replicate fragment 1 using primers F1 and R1;

[0048] The pP43nmk vector fragment 2 was replicated by PCR using primers F2 and R2. The two fragments were then ligated via homologous recombination. The ligated fragment was sent to Genewiz Biotechnology Co., Ltd. for sequencing, and a sample containing the signal peptide SP was prepared. STP01 The recombinant vector pP43nmk-SP STP01 -MFA PS .

[0049] (2) SPs with nucleotide sequences as shown in SEQ ID NO.5 to SEQ ID NO.12 were used respectively. Apre SP Ggt SP Amye SP Npre SP YojL SP NprB SP Bgls SP epr Signal peptide replacement signal peptide SP STP01 The signal peptide SP STP01 The sequence is shown in SEQ ID NO.13. Signal peptide ligation: The signal peptide fragment and the pP43nmk-MFA without the signal peptide were replicated by PCR using the primers listed in Table 1 below. PS Fragments were then linked using homologous recombination to prepare recombinant vectors pP43nmk-MFA containing different signal peptides. PS .

[0050] The primer sequences mentioned are shown in Table 1.

[0051] Table 1: Primer sequences

[0052]

[0053] 2. Construction of different recombinant bacteria

[0054] The recombinant vectors prepared in step 1 were transformed into Bacillus subtilis WB600 to obtain recombinant genetically engineered bacteria containing different signal peptides.

[0055] 3. Enzyme expression

[0056] The recombinant genetically engineered bacteria prepared in step 2 were streaked onto LB medium supplemented with kanamycin and cultured at 37°C for 12 h. A single colony was then inoculated into 50 mL of LB medium, and kanamycin was added. After culturing at 37°C for 8 h, 2 mL of the bacterial culture was added to 50 mL of TB medium, and kanamycin was added again. Finally, the culture was fermented at 37°C on a shaker at 200 rpm for 60-72 h to obtain the fermentation broth. The level of linear maltodextrin-producing enzyme (MFA) in the fermentation broth was then measured. PS The enzyme activity, the results are as follows Figure 5 As shown.

[0057] The results showed that SP was included STB01 Signal peptide replaced with SP Bgls The recombinant vector pP43NMK-SP Bgls -MFA ps The recombinant genetically engineered bacteria express the linear maltodextrin-producing enzyme MFA. PS The enzyme activity is the highest, and its enzyme activity is that of the unreplaced signal peptide (i.e., using SP). STB01 The enzyme activity expressed by the recombinant genetically engineered bacteria (signal peptide) was 1.27 times that of the enzyme.

[0058] Therefore, SP is adopted STB01 Signal peptide replaced with SP Bgls The recombinant vector pP43NMK-SP Bgls Bacillus subtilis WB600 was used as an expression vector and as an expression host for subsequent studies; simultaneously, Bacillus subtilis WB600 / pP43NMK-SP was prepared according to the above method. Bgls -MFA PS .

[0059] Example 2: Construction of a recombinant Bacillus subtilis secretory expression system

[0060] The specific steps are as follows:

[0061] Using the pP43NMK-SP constructed in Example 1 BglsMFA PS As a template, a gene fragment (nucleotide sequence as shown in SEQ ID NO. 2) with CBM removed was amplified by PCR, and then the vector was homologously recombined with the gene fragment of interest.

[0062] The primer sequences involved are shown in Table 2:

[0063] Table 2: Primers

[0064]

[0065] The PCR amplification program involved is as follows: Taq Buffer (Mg 2+ Plus) 10 μL, dNTP Mixture (2.5 mM each) 4 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template DNA 1 μL, Taq DNA Polymerase (1.25 U / μL) 1 μL, and sterile water added to 50 μL. The PCR amplification conditions are as follows: 98°C pre-denaturation for 3 min; then 30 cycles of 98°C for 10 s, 60°C for 15 s, and 68°C for 2 min; and finally 68°C for 10 min.

[0066] The linear maltooligosaccharide-producing enzyme MFA PS The ΔCBM enzyme gene was connected to the pP43nmk-SP Bgls vector by homologous recombination, and after completion, it was transformed into E. coli JM109, spread on an LB plate with ampicillin, and transformants were picked for sequencing and colony PCR verification. The recombinant plasmid containing the linear maltooligosaccharide-producing enzyme MFA PS ΔCBM enzyme gene: pP43nmk-SP Bgls MFA PS ΔCBM.

[0067] The recombinant plasmid pP43nmk-SP Bgls MFA PS ΔCBM was transformed into Bacillus subtilis WB600 to obtain a recombinant genetically engineered bacterium: Bacillus subtilis WB600 / pP43nmk-SP Bgls MFA PS ΔCBM.

[0068] Example 3: Expression and purification of the linear maltooligosaccharide-producing enzyme MFA PS ΔCBM

[0069] The specific steps are as follows:

[0070] (1) Linear maltooligosaccharide-forming enzyme MFA PS - Expression of ΔCBM

[0071] The recombinant Bacillus subtilis WB600 / pP43nmk-SP prepared in Example 1 and Example 2 were respectively inoculated into 50 mL of LB medium containing kanamycin and cultured at 37°C for 12 h. Then, 2 mL of the bacterial solution was added to 50 mL of TB medium containing kanamycin and cultured at 37°C for 8 h. Finally, the solution was subjected to fermentation at 37°C for 60-72 h at 200 rpm to prepare a fermentation solution. The enzyme activity of the original enzyme, linear maltooligosaccharide-forming enzyme MFA Bgls - MFA PS - ΔCBM, Bacillus subtilis WB600 / pP43NMK-SP Bgls - MFA PS The fermentation solution was prepared by inoculating a single colony on LB medium containing kanamycin and culturing at 37°C for 12 h, inoculating 2 mL of the bacterial solution into 50 mL of TB medium containing kanamycin and culturing at 37°C for 8 h, and finally subjecting the solution to fermentation at 37°C for 60-72 h at 200 rpm. The enzyme activity of the original enzyme, linear maltooligosaccharide-forming enzyme MFA PS , linear maltooligosaccharide-forming enzyme MFA PS - ΔCBM was measured, and the results are shown in Table 1.

[0072] (2) Linear maltooligosaccharide-forming enzyme MFA PS - Purification of ΔCBM

[0073] Buffer A1 (10 mmol / L Tris-HCl, 500 mmol / L NaCl, pH 7.5) and buffer B1 (10 mmol / L Tris-HCl, 500 mmol / L NaCl, 500 mmol / L imidazole, pH 7.5) were prepared and purified using a 5 mL nickel column (GE Healthcare). The flow rate was set to 2 mL / min, and the nickel column was equilibrated with 5-6 column volumes of buffer A1. After loading, the unbound proteins on the nickel column were eluted with buffer A1 to the elution curve. The flow rate was set to 1.5 mL / min, and 40% (v / v) buffer A1 and 60% (v / v) buffer B1 were used for elution. The eluate was collected to measure the enzyme activity and verified by SDS-PAGE protein electrophoresis (as shown in Figure 1 ).

[0074] The purified enzyme protein was dialyzed in a dialysis bag, and the buffer was 10 mmol / L Tris-HCl, which was replaced every 6 h for 4 times.

[0075] The original enzyme, linear maltooligosaccharide-forming enzyme MFA PS purified enzyme, linear maltooligosaccharide-forming enzyme MFA PS - ΔCBM purified enzyme.

[0076] The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit.

[0077] Table 3: Truncated MFA - ΔCBM PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit.

[0078]

[0079] The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit.

[0080] Example 4: Optimum temperature and thermal stability of the purified MFA - ΔCBM enzyme PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit.

[0081] The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit.

[0082] (1) Detection of optimum temperature

[0083] The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. Figure 2

[0084] The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit.

[0085] (2) Detection of thermal stability

[0086] The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS The purified MFA - ΔCBM enzyme prepared in Example 2 was respectively detected for its enzyme activity and specific enzyme activity using Detergent Compatible Bradford Protein Assay Kit (Betotime Biotechnol, Shanghai, China) kit. PS ​The pure enzyme assay method is as follows: the enzyme solution is incubated at different temperatures (50-90°C), and every certain time, the enzyme solution is taken out, quickly cooled, and the residual enzyme activity is measured, with the activity of the unincubated enzyme solution as 100%.

[0087] The results show that the enzyme activity is stable when incubated at 50°C, and after 28 min of incubation at 50°C, the activity decreases to less than 50% (shown in the figure). Figure 3

[0088] (3) Detection of optimal pH

[0089] Linear malt oligosaccharide generating enzyme MFA PS -ΔCBM, MFA PS The optimal pH of the pure enzyme is determined as follows: first, prepare the substrate with pH 5-8 using 10 mmol / L NaH2PO4-Na2HPO4 buffer as solvent, and then prepare the substrate with pH 8-10 using 10 mmol / L Gly-NaOH buffer as solvent. Then, according to the enzyme activity determination method, the enzyme activity of the enzyme solution at pH 5-10 is determined, with the highest enzyme activity as 100%, and the results are shown in the figure. Figure 4

[0090] The results show that the mutant and wild type exhibit high enzyme activity at pH 6.0-7.5 (10 mmol / L NaH2PO4-Na2HPO4 buffer), and the optimal pH is 6.5.

[0091] When the pH is lower than 6.0 or higher than 7.5, the enzyme activity decreases significantly. In addition, at the same pH, the hydrolysis activity of the enzyme in Gly-NaOH buffer is slightly higher than that in NaH2PO4-Na2HPO4 buffer.

[0092] Example 5: Application of linear malt oligosaccharide generating enzyme MFA PS -ΔCBM

[0093] In this example, linear malt oligosaccharide generating enzyme MFA PS -ΔCBM is used to hydrolyze malt dextrin; the specific steps are as follows:

[0094] (1) Prepare 100 g of DE 7-9 malt dextrin solution with a concentration of 20% (w / w).

[0095] (2) Place the DE 7-9 malt dextrin solution with a concentration of 20% (w / w) in a 90°C water bath shaker for 0.5 h to pre-gelatinize the substrate completely, then reduce the system temperature to 50°C, and then add 20 U / g of linear malt oligosaccharide generating enzyme MFA PS ​​- ΔCBM, reaction in 50℃ water bath shaker for 24h, then transfer to boiling water to terminate the reaction for 20min.

[0096] (3) After the termination of the enzyme, the reaction solution was centrifuged at 12000rpm for 10min, and the supernatant was diluted 10000 times after removing the impurities through a 0.22μm water membrane, so that the distribution of different polymerization degrees in the reaction solution could be analyzed by ion chromatography.

[0097] Meanwhile, the original enzyme linear malt oligosaccharide generating enzyme MFA prepared in Example 2 was used as a control. PS As a control, the enzyme reaction was carried out with corn starch, waxy corn starch, malt dextrin (DE 4), malt dextrin (DE 7-9), malt dextrin (DE 16) as substrates, and the results are shown in Table 4.

[0098] Table 4: Truncated MFA PS - ΔCBM and wild type MFA PS G4 ratio and conversion rate

[0099]

[0100] The results show that when malt dextrin (DE 7-9) is used as a substrate, the substrate conversion rate and G4 ratio of the truncated MFA PS - ΔCBM are significantly higher than those of the wild type, and when corn starch and waxy corn starch are used as substrates, the substrate conversion rate of the truncated MFA PS - ΔCBM is significantly lower than that of the wild type.

[0101] Then, DE 7-9 malt dextrin was used as a substrate, and the results are shown in Table 5

[0102] Table 5: Truncated MFA PS - ΔCBM and wild type MFA PS G4 ratio and conversion rate

[0103]

[0104] The results show that the G4 ratio and conversion rate of the mutant increase rapidly at the early stage, and then the growth rate slows down, and at the end of the reaction, the G4 ratio and conversion rate of the truncated MFA PS - ΔCBM are increased by 9.4% and 11.3% respectively compared with the wild type.

[0105] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing maltodextrose, characterized in that, The method is as follows: using a linear maltodextrin-derived oligosaccharide generating enzyme with an amino acid sequence as shown in SEQ ID NO.1 as a catalyst, or using a recombinant Bacillus subtilis expressing a linear maltodextrin-derived oligosaccharide generating enzyme with an amino acid sequence as shown in SEQ ID NO.1 as a catalyst, or using a recombinant enzyme prepared by fermentation of a recombinant Bacillus subtilis expressing a linear maltodextrin-derived oligosaccharide generating enzyme with an amino acid sequence as shown in SEQ ID NO.1 as a catalyst, and using maltodextrin and / or starch as substrates, maltodextrin and / or starch are reacted to prepare maltodextrin-derived oligosaccharide.

2. The preparation method according to claim 1, characterized in that, The nucleotide sequence encoding the linear maltodextrin generating enzyme is shown in SEQ ID NO.

2.

3. The preparation method according to claim 1 or 2, characterized in that, by Bacillus subtilis WB600 is the expression host.

4. The preparation method according to claim 3, characterized in that, The recombinant Bacillus subtilis was expressed using the pP43nmk plasmid as an expression vector.

5. The preparation method according to claim 4, characterized in that, The starches include corn starch, tapioca starch, potato starch, rice starch, and wheat starch.

6. The preparation method according to claim 5, characterized in that, The expression vector pP43nmk plasmid also contains SP Bgls Signal peptides.

7. The application of linear maltodextrin-producing enzyme with the amino acid sequence shown in SEQ ID NO.1 or recombinant Bacillus subtilis expressing linear maltodextrin-producing enzyme with the amino acid sequence shown in SEQ ID NO.1 in increasing the yield of maltodextrin.

8. The application as described in claim 7, characterized in that, by Bacillus subtilis WB600 is the expression host.

9. The application as described in claim 8, characterized in that, The recombinant Bacillus subtilis was expressed using the pP43nmk plasmid as an expression vector.

10. The use of a linear maltodextrin-producing enzyme with the amino acid sequence shown in SEQ ID NO.1 or a recombinant Bacillus subtilis expressing a linear maltodextrin-producing enzyme with the amino acid sequence shown in SEQ ID NO.1 in the preparation of products with increased maltodextrin yield.

Images