Bacillus licheniformis-sourced maltogenic amylase as well as coding gene and application thereof

Abstract

The invention discloses maltogenic amylase derived from bacillus licheniformis as well as a coding gene and application of the maltogenic amylase in the technical field of genetic engineering and enzymes. The maltogenic amylase can be efficiently hydrolyzed, the optimum temperature is 35-45 DEG C, the enzyme activity can reach 471 U / mL, an enzyme resource library is enriched, and the maltogenic amylase can be obtained by taking cheap lactose as an inducer and is expected to be applied to industrialization.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and enzymes, and particularly to a maltogenic amylase derived from Bacillus licheniformis, its coding gene and applications. Background Art

[0002] Maltogenic amylase is an important class of industrial enzymes that can hydrolyze the α-1,4-glycosidic bonds in starch molecules, and can catalyze the hydrolysis of starch and its derivatives to produce maltose, which is widely used in fields such as food, medicine, feed and bioenergy. Maltogenic amylase is mainly derived from bacteria such as Bacillus subtilis, Geobacillus stearothermophilus, Bacillus licheniformis, etc. Bacillus licheniformis is a Gram-positive bacterium widely present in nature, with good environmental adaptability and high protein secretion ability. The enzymes produced by it usually have high catalytic activity, good thermal stability and wide pH adaptability. With the progress of molecular biology and enzyme engineering technologies, gene cloning and expression have become important ways to obtain functional enzymes. Exploring maltogenic amylase resources and cloning and expressing their coding genes not only helps to enrich the enzyme resource library, but also provides optional enzyme preparations for industrial applications.

[0003] The present application provides a maltogenic amylase derived from Bacillus licheniformis, efficiently produces maltogenic amylase through gene cloning and expression techniques, and explores its enzymatic properties. In addition, the inducer IPTG commonly used in laboratories has the characteristics of high efficiency and stability, but its high price and potential cytotoxicity make it only suitable for small-scale laboratory preparations. Lactose is a disaccharide and a natural inducer of the lactose operon. Its low cost and non-toxic characteristics make it have great advantages in industrial production. The present application compares the differences in the expression of maltogenic amylase when using lactose and IPTG as inducers, laying a foundation for industrial application. Summary of the Invention

[0004] The present invention aims to provide a maltogenic amylase derived from Bacillus licheniformis, its coding gene and applications, efficiently produce maltogenic amylase through gene cloning and expression techniques, and explore its enzymatic properties.

[0005] To achieve the above object, the present invention provides the following technical solutions: The present invention provides the maltogenic amylase coding gene SEQ ID NO1, with a full length of 1758 bp, and the present invention provides the maltogenic amylase amino acid sequence SEQ ID NO2, with a full length of 586 amino acids.

[0006] The present invention provides a recombinant expression vector, and the expression vector contains the aforementioned gene. Specifically, the vector is plasmid pET30a(+), and the maltogenic amylase gene is inserted into plasmid pET30a(+) through Nde I and Xho I restriction enzyme cleavage sites.

[0007] The present invention provides a recombinant engineering bacterium, and the recombinant engineering bacterium contains the aforementioned recombinant expression vector. Specifically, the recombinant engineering bacterium is Escherichia coli BL21(DE3), and the aforementioned recombinant expression vector is transferred into the competent Escherichia coli BL21(DE3) by heat shock method.

[0008] The present invention provides a method for detecting gene expression. Specifically, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is used for detection.

[0009] The present invention provides a method for producing maltogenic amylase in Escherichia coli. Specifically, the aforementioned recombinant engineering bacterium preserved in a glycerol tube is inoculated into an LB liquid medium, cultured at 37°C and 200 r / min for 12 h, transferred to a fresh LB liquid medium, and after continuing to culture at 37°C and 200 r / min for 2.5 h, IPTG with a final concentration of 0.5 mM is added, and induced expression is carried out at 30°C and 150 r / min for 8 - 10 h.

[0010] The present invention provides a protein purification method, which includes the following steps: (1) The step of induced expression of the recombinant engineering bacterium: The aforementioned recombinant engineering bacterium is inoculated into an LB liquid medium, cultured at 37°C and 200 r / min for 12 h, transferred to a fresh LB liquid medium, and after continuing to culture at 37°C and 200 r / min for 2.5 h, IPTG with a final concentration of 0.5 mM is added, and induced expression is carried out at 30°C and 150 r / min for 8 - 10 h.

[0011] (2) Centrifuge to collect the aforementioned recombinant engineering bacterium, resuspend it with Lysis buffer, break the cells using a cell disruptor, and centrifuge to collect the supernatant, thus obtaining a crude enzyme solution of maltogenic amylase; (3) Purify the aforementioned maltogenic amylase using nickel column affinity chromatography.

[0012] The present invention provides a method for detecting the activity of maltogenic amylase. The specific process is as follows: Using soluble starch as a substrate, the purified maltogenic amylase is used to catalyze the hydrolysis of soluble starch. The resulting reducing sugar reacts with DNS at 100 °C to present a reddish-brown color. The color depth is related to the content, and there is a maximum absorption value at 540 nm. By calculating the change in absorbance at 540 nm within a specific time and combining with the maltose standard curve, the production amount of maltose can be calculated.

[0013] The optimum temperature of the maltogenic amylase described in the present invention is 40 °C, and the optimum pH is 6. Mn 2+ and Fe 3 + can promote the improvement of the activity of maltogenic amylase. Under the optimum reaction conditions, the enzyme activity of maltogenic amylase can reach 471 U / mL.

[0014] The present invention provides a method for industrial production of maltose. Specifically: The above-mentioned recombinant engineering bacteria preserved in a glycerol tube are inoculated into an LB liquid medium and cultured at 37 °C and 200 r / min for 12 h. Then it is transferred to a fresh LB liquid medium and continuously cultured at 37 °C and 200 r / min for 2.5 h. After that, lactose with a final concentration of 0.25 mM - 4 mM is added, and induced expression is carried out at 30 °C and 150 r / min for 8 - 10 h. Description of the Drawings

[0015] Figure 1 Schematic diagram for constructing a recombinant expression vector; Figure 2 Electrophoresis diagram of maltogenic amylase gene expression at different induction temperatures; Figure 3 Electrophoresis diagram of the effect of induction duration on maltogenic amylase expression; Figure 4 Electrophoresis diagram of maltogenic amylase purified by nickel column; Figure 5 Curve of the effect of temperature on the activity of maltogenic amylase; Figure 6 Curve of the effect of pH on the activity of maltogenic amylase; Figure 7 Bar chart of the effect of metal ions on the activity of maltogenic amylase; Figure 8 Electrophoresis diagram of maltogenic amylase gene expression at different lactose concentrations. Detailed Description of the Invention

[0016] The following is a further detailed description through specific embodiments: The instruments and reagents used in the examples can be obtained through regular commercial channels without special instructions. The experimental methods without specific steps and conditions in the examples follow the conventional experimental procedures and conditions. These examples are intended to illustrate the present invention and not to limit the scope and application of the present invention.

[0017] Example 1 Construction of Recombinant Expression Vector 1. Sequence Analysis of Maltogenic Amylase The present invention provides a gene encoding maltogenic amylase derived from Bacillus licheniformis, and the gene is obtained by the following method: (1) A strain of Bacillus licheniformis was isolated and screened from the koji of a winery in Renhuai. (2) The whole genome of the screened Bacillus licheniformis was sequenced by Sangon Biotech (Shanghai) Co., Ltd. Through bioinformatics analysis, it was found that its genome contains a maltogenic amylase gene. (3) Through BLAST alignment in NCBI, the amino acid encoded by the maltogenic amylase gene is identical to the amino acid sequence of glycoside hydrolase of Bacillus licheniformis DSM 13 strain in the NCBI database (GenBank: AAU22247.1). The maltogenic amylase encoding gene SEQ ID NO1 and the maltogenic amylase amino acid sequence SEQ ID NO2 of the present invention are as follows: The sequence of SEQ ID NO1 is as follows: The sequence of SEQ ID NO2 is as follows: MEYAAIHHQPFSSDAYSYNGRTLHIKIRTKKDDAEHVRLVWGDPYEYTGGTWKANELAMAKIAATSTHDYWFAEVAPPFRRLQYGFILTGADDRDTFYGSNGACPFAGKAADIGKHCFKFPFVHEADTFDAPDWVKSTVWYQIFPERFASGREDLSPENALPWGSKDPEAHDFFGGDLQGIMDKLDYLEDLGVGGIYLTPIFAAPSNHKYDTLDYCSIDPHFGDEELFRTLVSRIHERGMKIMLDAVFNHIGSASQEWQDVVKNGETSRYKDWFHIHSFPVKEGSYDTFAFTPEMPKLNTANPEVQAYLLDIALYWIREFDIDGWRLDVANEVDHAFWKKFRQAVTAEKPDIFILGEIWHQADPWLRGDEFHSVMNYPFTEPMIDYFADGSISASQMASRINSHLMSGMKQVNEVMFNLLDSHDTKRILTRCGGDDKKVRSLLAFMFAQTGSPCIYYGTEVGLDGGDDPLCRKCMVWEEEKQNQEMLAFMKRLIALRKQENDVLTYGALEWKLVDDQNDFVSFSRTHEGKELIYFFHQGSEVRRVRLRDLKIASDKRIYDAWTEEALQHDDVVDIQPGGFFILGAV 2. Construction of the recombinant expression vector Based on the sequence of the maltogenic amylase coding gene SEQ ID NO1, it was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and ligated into the plasmid pET30a(+) through the Nde I and Xho I restriction enzyme cleavage sites to obtain the recombinant expression vector pET30a(+)-L6M (see attachment Figure 1 ).

[0018] The above recombinant expression vector was transformed into Escherichia coli BL21(DE3) by heat shock method, and screened using LB solid plates containing 50 μg / mL kanamycin. Single colonies were picked from the screening plates and inoculated into LB liquid medium supplemented with kanamycin at a final concentration of 50 μg / mL, and cultured at 37°C and 200 r / min for about 12 h. After the culture was completed, the bacterial cells were collected, and the plasmid pET30a(+)-L6M was extracted using a plasmid extraction kit. Clones with correct sequencing were selected and stored in glycerol tubes. The recombinant engineering bacteria were named BL21-pET30a(+)-L6M. Example 2 Expression and purification of maltogenic amylase gene 1. Expression of maltogenic amylase gene The recombinant engineering bacteria BL21-pET30a(+)-L6M stored in glycerol obtained in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C and 200 r / min for about 12 h, then inoculated into fresh LB liquid medium (containing 50 μg / mL kanamycin) and continued to be cultured at 37°C and 200 r / min for about 2.5 h. IPTG was added at a final concentration of 0.5 mM and induced at 16°C, 20°C, 30°C, and 37°C respectively. The induction rotation speed was set at 150 r / min and the induction time was 6 - 8 h. After the induction was completed, the bacterial cells were collected by centrifugation, suspended in 1×PBS buffer, and then the cells were lysed using a cell disruptor. The cell disruption conditions of the cell disruptor were: disrupt for 2 s, stop for 5 s, and the total time was 15 min. After the disruption was completed, the disrupted supernatant and precipitate were separated by centrifugation, and SDS-PAGE was used to detect the expression.

[0019] Figure 2Electrophoresis diagram of the expression of maltogenic amylase gene at different induction temperatures. Lane 1 is the whole cell before induction. Lanes 2-5 are the whole cells after induction at 16 °C, 20 °C, 30 °C, and 37 °C for 6-8 h respectively. M is the protein Marker. Lanes 6-9 are the supernatants of cell lysates after induction at 16 °C, 20 °C, 30 °C, and 37 °C respectively. Lanes 10-13 are the precipitates of cell lysates after induction at 16 °C, 20 °C, 30 °C, and 37 °C respectively. It can be seen from the electrophoresis diagram that the expression of maltogenic amylase can be induced at each temperature. The molecular weight of the induced protein is in the range of 50-70 kD, close to 70 kD. The theoretical relative molecular weight of the target maltogenic amylase plus its histidine tag at the carboxyl terminus is 68.4 kD, and the experimental results are consistent with the expectations. Analyzing the electrophoresis diagram, as the induction temperature increases, the content of maltogenic amylase in the whole cell after induction gradually increases, and the content of the target protein is basically the same when induced at 30 °C and 37 °C. This maltogenic amylase has good solubility, most of it is located in the supernatant of the cell lysate, and a small part is located in the precipitate of the cell lysate to form inclusion bodies. When the induction temperature is 20 °C, 30 °C, and 37 °C, the content of maltogenic amylase in the supernatant is basically the same. Combining the content of maltogenic amylase in the whole cell and the supernatant of the cell lysate, the subsequent induction temperature is determined to be 30 °C.

[0020] 2. Determination of induction duration According to the above steps of inducing expression of the recombinant engineering bacteria, inoculate BL21-pET30a(+)-L6M. After 2.5 h of subculture, add IPTG with a final concentration of 0.5 mM, and perform induction at 30 °C and 150 r / min. The induction durations are set to 2 h, 4 h, 6 h, 8 h, and 10 h respectively. Determine the induction duration of the subsequent experiment according to the content of maltogenic amylase in the whole cell and the supernatant of the cell lysate.

[0021] Figure 3 Electrophoresis diagram of the effect of induction duration on the expression of maltogenic amylase. Lane 1 is the whole cell before induction. Lanes 2-6 are the whole cells after induction for 2 h, 4 h, 6 h, 8 h, and 10 h respectively. M is the protein Marker. Lanes 7-11 are the supernatants of cell lysates after induction for 2 h, 4 h, 6 h, 8 h, and 10 h respectively. It can be seen from the electrophoresis diagram that when the induction duration reaches 8 h, further extending the induction duration does not result in a significant increase in the content of maltogenic amylase in the whole cell and the supernatant of the cell lysate. Given that time cost is a crucial factor in scientific research and production, in order to maximize efficiency, the induction duration is set to 8-10 h.

[0022] 3. Purification of maltogenic amylase When constructing the expression vector, a histidine tag was introduced at the carboxyl terminus of maltogenic amylase, so maltogenic amylase can be purified by nickel column affinity chromatography.

[0023] According to the method described in 2 above, inoculate BL21-pET30a(+)-L6M. After 2.5 h of subculture, add IPTG with a final concentration of 0.5 mM, and induce at 30 °C and 150 r / min for 8 - 10 h. Obtain at least 1 L of induced cells according to this process, centrifuge to collect the thalli, suspend the thalli with Lysis buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole), then break the cells with a cell disruptor, centrifuge at 12000 r / min for 90 min to collect the broken supernatant, and obtain the crude maltogenic amylase solution.

[0024] Purify maltogenic amylase using nickel column affinity chromatography. The specific steps are as follows: (1)Equilibration: Add 5 column volumes of Lysis buffer to equilibrate the column.

[0025] (2)Loading: Add the crude maltogenic amylase solution to the equilibrated column. The carboxyl terminus of maltogenic amylase contains a histidine tag and is adsorbed on the nickel column by binding to nickel ions.

[0026] (3)Washing: Add 10 column volumes of Washing buffer (50 mM Tris, 300 mM NaCl, 30 mM imidazole) to wash out the non-specifically bound miscellaneous proteins from the column, and use Coomassie Brilliant Blue to detect the washing effect.

[0027] (4)Elution: Add 2 column volumes of Elution buffer (50 mM Tris, 300 mM NaCl, 150 mM imidazole) to elute the target protein, and collect this part of the eluate.

[0028] The purification result of maltogenic amylase is shown in Figure 4 , M represents protein Marker, and lane 1 is the purified maltogenic amylase. It can be seen from the electrophoresis pattern that after purification, maltogenic amylase shows a single band in the range of 50 - 70 kD, which is consistent with the expected molecular weight.

[0029] After protein concentration determination, the concentration of the purified maltogenic amylase can reach 3.48 mg / mL.

[0030] Example 3 Enzymatic Properties of Maltogenic Amylase 1. Determination of Maltogenic Amylase Activity Maltose amylase can catalyze the hydrolysis of soluble starch into maltose and other reducing sugars. The reducing sugars react with 3,5-dinitrosalicylic acid (DNS) at 100 °C to form a brown-red complex. The color depth is related to the content of reducing sugars. The higher the content of reducing sugars, the deeper the color, indicating that the activity of maltose amylase is higher. Since the generated brown-red complex has a maximum absorption value at 540 nm, by calculating the change in absorbance at 540 nm within a specific time and combining with the maltose standard curve, the amount of maltose produced can be calculated.

[0031] The enzyme activity assay process is as follows: 990 μL of 0.5% soluble starch is preheated at the reaction temperature for 5 min, then 10 μL of purified maltose amylase is added, and the reaction is accurately timed for 10 min at the reaction temperature. After the reaction ends, 1.0 mL of DNS solution is added to the reaction solution, and it is developed at 100 °C for 5 min. Then 10 mL of deionized water is added. After waiting for 30 min, the absorbance of the reaction solution at 540 nm is measured using a spectrophotometer, and the amount of reducing sugar produced is calculated by referring to the maltose standard curve.

[0032] The enzyme activity is defined as: under the optimal reaction conditions, the amount of enzyme required to catalyze the production of 1 μmol of maltose per minute is defined as one unit U.

[0033] 2. Effect of temperature on the activity of maltose amylase According to the reaction system in 1 above, the reaction is accurately timed for 10 min at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C respectively. After the reaction ends, 1.0 mL of DNS solution is added to the reaction solution, and it is reacted at 100 °C for 5 min. Then 10 mL of deionized water is added. After waiting for 30 min, the absorbance of the reaction solution at 540 nm is measured using a spectrophotometer.

[0034] Figure 5 It is a graph showing the effect of temperature on the activity of maltose amylase. From the graph, it can be seen that as the temperature increases, the activity of maltose amylase gradually increases, and the enzyme activity reaches the maximum value at 40 °C. Continuing to increase the temperature, maltose amylase gradually inactivates, so the enzyme activity gradually decreases.

[0035] 3. Effect of pH on the activity of maltose amylase Perform according to the reaction system in 1 above, and adjust the pH values of the reaction system to 4, 5, 6, 7, 8, 9, and 10 respectively. The buffer solution used for pH 4 - 8 is disodium hydrogen phosphate - citric acid buffer solution, and the buffer solution used for pH 8 - 10 is glycine - sodium hydroxide buffer solution. React different pH reaction solutions at 40 °C for 10 min. After the reaction ends, add 1.0 mL of DNS solution to the reaction solution, react at 100 °C for 5 min, then add 10 mL of deionized water, wait for 30 min, and use a spectrophotometer to measure the absorbance value of the reaction solution at 540 nm.

[0036] Figure 6 It is a graph showing the effect of pH on the activity of maltose amylase. Maltose amylase has no activity when the pH of the reaction solution is relatively low or high, and only has activity in the neutral reaction solution with a pH of 6 - 8, and the activity reaches the maximum at pH 6.0.

[0037] 4. Effect of metal ions on the activity of maltose amylase Perform according to the reaction system in 1 above, adjust the pH value of the reaction system to 6, and add EDTA, Ca 2+ , K + , Na + , Mg 2+ , Mn 2+ , Zn 2+ , Fe 3+ with a final concentration of 5 mM to the reaction system respectively. React the reaction solutions at 40 °C for 10 min. After the reaction ends, add 1.0 mL of DNS solution to the reaction solution, react at 100 °C for 5 min, then add 10 mL of deionized water, wait for 30 min, and use a spectrophotometer to measure the absorbance value of the reaction solution at 540 nm.

[0038] Figure 7 It is a bar graph showing the effect of metal ions on the activity of maltose amylase. It can be seen from the bar graph that Mn 2+ and Fe 3 + can promote the improvement of the activity of maltose amylase. Compared with the control group, the relative enzyme activities of the two metal ions are 125% and 120% respectively. Other metal ions have no obvious inhibitory effect on the activity of maltose amylase.

[0039] Under the above - mentioned optimal reaction conditions, the enzyme activity of the purified maltose amylase can reach 471 U / mL, and it is expected to be applied in industrialization.

[0040] Example 4 Induce the expression of maltose amylase with lactose as an inducer Although IPTG has the characteristics of high efficiency and stability as an inducer, its high cost and potential cytotoxicity make it only suitable for small-scale laboratory preparation. Lactose is a disaccharide and the natural inducer of the lactose operon. Its low cost and non-toxic properties give it great advantages in industrial production. Therefore, this invention compares the differences in maltose expression induced by lactose and IPTG.

[0041] According to the recombinant engineering bacteria induction expression steps of Example 2 above, the expression of maltogenic amylase was induced at a final concentration of 0.5 mM IPTG and at final concentrations of 0.25 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM lactose respectively. The induction temperature was 30 °C and the induction time was 8 - 10 h.

[0042] Figure 8 It is the electrophoresis diagram of the effect of lactose concentration on the expression of maltogenic amylase. Lane 1 is the whole cell before induction, Lane 2 is the whole cell after induction with 0.5 mM IPTG at the final concentration, Lanes 3 - 7 are the whole cells after induction with 0.25 mM, 0.5 mM, 1 mM, 2 mM, and 4 mM lactose at the final concentration respectively, M is the protein Marker, Lane 8 is the supernatant of cell disruption after induction with 0.5 mM IPTG at the final concentration, and Lanes 9 - 13 are the supernatants of cell disruption after induction with 0.5 mM, 1 mM, 2 mM, and 4 mM lactose at the final concentration respectively. It can be seen from the electrophoresis diagram that when lactose is used as the inducer, the content of maltose in the whole cell and the disrupted supernatant is slightly lower than that when IPTG is used as the inducer. However, the low cost and non-toxic properties of lactose make it have the potential to replace IPTG in industrial applications for production.

Claims

1. A gene encoding maltogenic amylase, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

2. A maltogenic amylase, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

3. A vector comprising the gene according to claim 1.

4. A cell expressing the maltogenic amylase according to claim 2.

5. A genetically engineered bacterium comprising the vector according to claim 3.

6. The carrier according to claim 3, characterized in that: The vector is a plasmid pET30a(+), and the maltogenic amylase gene is inserted into the plasmid pET30a(+) through Nde I and Xho I restriction enzyme cutting sites.

7. The genetically engineered bacterium according to claim 5, characterized in that: The recombinant engineering bacteria was Escherichia coli BL21 (DE3), and the vector was transformed into the competent Escherichia coli BL21 (DE3) by heat shock method.

8. Use of the genetically engineered bacteria according to claim 5 or 7 in producing maltogenic amylase.

9. The use according to claim 8, characterized in that: Application for the production of maltose.

10. The use according to claim 9, characterized in that: The expression of recombinant engineered bacteria was induced by lactose.

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