L-AI (L-arabinose isomerase) and application thereof

Abstract

The invention discloses L-AI (L-arabinose isomerase). The L-AI is prepared through the step as follows: 279th phenylalanine of L-AI of Bacillus coagulans NL01 is mutated into isoleucine, and the L-AI is obtained, wherein the GenBank registration number of a nucleotide sequence of an L-AI gene araA is KX356659. The invention further discloses escherichia coli containing the L-AI as well as a construction method and an application of the escherichia coli. According to the constructed recombinant escherichia coli, F279I expressed by the escherichia coli has high catalytic efficiency on D-galactose. The optimal reaction temperature of a whole-cell catalysis reaction system is mild, a browning reaction can be avoided, and the L-AI better facilitates industrial production of D-tagatose when compared with wild type L-AI and other common L-AI from thermophilic bacteria and thermoacidophiles. Meanwhile, the whole-cell catalysis reaction system adopts simple components and has greater industrial production and application potential and economic value.

Description

technical field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an L-arabinose isomerase and application thereof. Background technique

[0002] D-tagatose is a new type of functional sweetener, which can be widely used in industries such as food, health care products, diabetes drugs and chemical synthesis. D-tagatose was first found in the gum of trees, and also exists in trace amounts in daily drinks such as milk and cocoa. Since D-tagatose shows good application prospects in human health, the industrialization of D-tagatose has been progressing rapidly in recent years, and the market development trend is good.

[0003] The commercial production of D-tagatose has long relied on chemical catalysis, but there are some inherent defects in chemical catalysis, such as severe alkaline environment will lead to the formation of by-products (such as sorbose, mannose, etc.), acid and alkali The use increases the difficulty of sewa...

Examples

Embodiment 1

[0035] Embodiment 1: Escherichia coli genetic engineering strain E.coli BL21 (pETDuet-araA F279I ) of the acquisition.

[0036] (1) Acquisition of L-arabinose isomerase gene araA and construction of recombinant plasmid pETDuet-araA.

[0037] Design primers to introduce BamH I and EcoR I restriction sites that can be inserted into the multiple cloning site of the pETDuet-1 plasmid, the sequences are as follows:

[0038] araA.f: 5'-CGC GGATCC GATGTTGAAAATAAAAGA-3'

[0039] araA.r: 5'-CCG GAATTC TCAAATTTTTACAGTTT-3'

[0040]Using the genomic DNA of Bacillus coagulans as a template, PCR amplification was carried out using the above primers. The total PCR amplification system was 100 μL, containing 64 μL of ultrapure water, 20 μL of 5×FastPfu PCR Buffer, 0.25 mmol / L of dNTPs, 0.2-0.4 μmol / L of each primer, 50-200 ng of template, and FastPfu DNA Polymerase 5U of TransGen Company. PCR amplification conditions were: pre-denaturation at 95°C for 2 minutes, 30 cycles according t...

Embodiment 2

[0046] Example 2: Expression, purification and catalytic ability analysis of original L-AI and F279I.

[0047] The recombinant bacterial strain E.coli BL21 (pETDuet-araA) that embodiment 1 obtains and E.coli BL21 (pETDuet-araA F279I ) were respectively inoculated in 50 mL of LB liquid medium containing 100 μg / mL ampicillin, cultured on a shaking table at 37°C for 12 hours, then transferred to 1L of LB liquid medium containing 100 μg / mL ampicillin, and cultured on a shaking table at 37°C. OD 600nm After reaching 0.4-0.8, add IPTG with a final concentration of 0.5-1 mmol / L to induce expression, and continue to culture at 20°C for 8 hours. After the induction culture, centrifuge at 12,000 rpm for 5 minutes to obtain cell pellets, wash twice with normal saline, and resuspend in 100 mL of phosphate buffer with pH 7.4 and 50 mmol / L. Ultrasound was used to break the cell wall in the ice-water mixture. The breaking time was 20 minutes. The solution after breaking the wall was centri...

Embodiment 3

[0050] Example 3: E.coli BL21 (pETDuet-araA F279I ) and E.coli BL21(pETDuet-araA) produced D-tagatose at different temperatures.

[0051] In the 10mL reaction system, add 20g / L substrate D-galactose and recombinant Escherichia coli wet cells with a dry weight of 4.8g / L, mix well and shake the reaction at different temperatures. The pH of the reaction system is 7.5. After 15 hours, the reaction mixture was centrifuged to remove bacteria, and the supernatant was taken to detect the amount of D-tagatose produced. The results show( figure 1 ), when the reaction system temperature is 40~70℃, E.coli BL21(pETDuet-araA F279I ) was significantly higher than E.coli BL21(pETDuet-araA); in addition, E.coli BL21(pETDuet-araA F279I ) conversion within 50-70°C. In order to prevent browning reaction, 50°C can be selected for catalytic reaction; for E.coli BL21(pETDuet-araA), the optimum catalytic temperature is 60°C.

technical field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to an L-arabinose isomerase and application thereof. Background technique

[0002] D-tagatose is a new type of functional sweetener, which can be widely used in industries such as food, health care products, diabetes drugs and chemical synthesis. D-tagatose was first found in the gum of trees, and also exists in trace amounts in daily drinks such as milk and cocoa. Since D-tagatose shows good application prospects in human health, the industrialization of D-tagatose has been progressing rapidly in recent years, and the market development trend is good.

[0003] The commercial production of D-tagatose has long relied on chemical catalysis, but there are some inherent defects in chemical catalysis, such as severe alkaline environment will lead to the formation of by-products (such as sorbose, mannose, etc.), acid and alkali The use increases the difficulty of sewa...

Examples

Embodiment 1

[0035] Embodiment 1: Escherichia coli genetic engineering strain E.coli BL21 (pETDuet-araA F279I ) of the acquisition.

[0036] (1) Acquisition of L-arabinose isomerase gene araA and construction of recombinant plasmid pETDuet-araA.

[0037] Design primers to introduce BamH I and EcoR I restriction sites that can be inserted into the multiple cloning site of the pETDuet-1 plasmid, the sequences are as follows:

[0038] araA.f: 5'-CGC GGATCC GATGTTGAAAATAAAAGA-3'

[0039] araA.r: 5'-CCG GAATTC TCAAATTTTTACAGTTT-3'

[0040]Using the genomic DNA of Bacillus coagulans as a template, PCR amplification was carried out using the above primers. The total PCR amplification system was 100 μL, containing 64 μL of ultrapure water, 20 μL of 5×FastPfu PCR Buffer, 0.25 mmol / L of dNTPs, 0.2-0.4 μmol / L of each primer, 50-200 ng of template, and FastPfu DNA Polymerase 5U of TransGen Company. PCR amplification conditions were: pre-denaturation at 95°C for 2 minutes, 30 cycles according t...

Embodiment 2

[0046] Example 2: Expression, purification and catalytic ability analysis of original L-AI and F279I.

[0047] The recombinant bacterial strain E.coli BL21 (pETDuet-araA) that embodiment 1 obtains and E.coli BL21 (pETDuet-araA F279I ) were respectively inoculated in 50 mL of LB liquid medium containing 100 μg / mL ampicillin, cultured on a shaking table at 37°C for 12 hours, then transferred to 1L of LB liquid medium containing 100 μg / mL ampicillin, and cultured on a shaking table at 37°C. OD 600nm After reaching 0.4-0.8, add IPTG with a final concentration of 0.5-1 mmol / L to induce expression, and continue to culture at 20°C for 8 hours. After the induction culture, centrifuge at 12,000 rpm for 5 minutes to obtain cell pellets, wash twice with normal saline, and resuspend in 100 mL of phosphate buffer with pH 7.4 and 50 mmol / L. Ultrasound was used to break the cell wall in the ice-water mixture. The breaking time was 20 minutes. The solution after breaking the wall was centri...

Embodiment 3

[0050] Example 3: E.coli BL21 (pETDuet-araA F279I ) and E.coli BL21(pETDuet-araA) produced D-tagatose at different temperatures.

[0051] In the 10mL reaction system, add 20g / L substrate D-galactose and recombinant Escherichia coli wet cells with a dry weight of 4.8g / L, mix well and shake the reaction at different temperatures. The pH of the reaction system is 7.5. After 15 hours, the reaction mixture was centrifuged to remove bacteria, and the supernatant was taken to detect the amount of D-tagatose produced. The results show( figure 1 ), when the reaction system temperature is 40~70℃, E.coli BL21(pETDuet-araA F279I ) was significantly higher than E.coli BL21(pETDuet-araA); in addition, E.coli BL21(pETDuet-araA F279I ) conversion within 50-70°C. In order to prevent browning reaction, 50°C can be selected for catalytic reaction; for E.coli BL21(pETDuet-araA), the optimum catalytic temperature is 60°C.

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