Novel thermophilic alpha-L-rhamnosidase stable at high temperature and application thereof

Abstract

The invention discloses thermophilic alpha-L-rhamnosidase, the amino acid sequence of which is as shown in SEQ ID No.1. Through comparison of the amino acid sequence, the thermophilic alpha-L-rhamnosidase is a novel alpha-L-rhamnosidase and comprises an amino acid sequence which has at least 92% of amino acid identity with the amino acid sequence as shown in SEQ ID No.1. The gene is derived from a metagenome of a hot spring in the Pu'er County of Yunnan The optimum pH (Potential of Hydrogen) of the thermophilic alpha-L-rhamnosidase provided by the invention is 5.5; the optimum temperature is 90 DEG C, and the enzyme has good thermal stability; the industrial application of efficiently removing the naringin in the citrus fruit juice under the high-temperature condition is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme genetic engineering and enzyme biochemical engineering, and specifically relates to a novel thermophilic alpha-L-rhamnosidase. Background Art

[0002] α-L-rhamnosidase is a class of enzymes that can catalyze the hydrolysis of terminal rhamnose in glycosides such as flavonoids, terpenes, and saponins. Based on the similarity of their amino acid sequences, they are divided into GH13, GH28, GH78, and GH106 families, with the GH78 family being the main one. Aspergillus tubingensis and Paenibacillus α-L-rhamnosidase from odorifer. α-L-rhamnosidase can debitter citrus juice because it can specifically hydrolyze the rhamnose residue at the end of naringin to form prunin and rhamnose.

[0003] Citrus fruits are popular because they are rich in nutrients such as vitamin C and minerals, as well as bioactive substances such as flavonoids and limonoids. According to statistics, the annual global production of citrus is 158.5 million tons, of which more than 30% is used to produce juice. However, there are bitter substances in juice, mainly naringin and limonin, and the bitterness caused by them is the main problem in juice production. Therefore, research on debittering by reducing / removing naringin or limonin has attracted much attention.

[0004] Currently, naringin and limonin can be reduced and / or removed by physical, chemical and biological methods. Enzymatic methods can specifically hydrolyze bitter substances such as naringin in juice, maintaining the natural flavor of the juice, as well as its antioxidant and biomolecule protection capabilities. Zhu et al. used Bacillus amyloliquefaciens Naringinase effectively removes naringin from pomelo juice; Li et al. used Aspergillus α-L-Rhamnosidase effectively removes naringin from grapefruit juice and retains the natural aroma of grapefruit juice; Peng et al. used Aspergillus niger α-L-rhamnosidase effectively removes naringin and increases the floral aroma of juice, and combined with β-D-glucosidase to significantly improve the taste, aroma, and antioxidant capacity of orange juice. Therefore, the use of α-L-rhamnosidase, naringinase, etc. to debitter citrus juice is of great value.

[0005] Most α-L-rhamnosidases are room-temperature enzymes below 60°C, which is not conducive to debittering juice. Because the juice needs to be quickly cooled to 8-10°C during juice processing, after filtering (screening), degassing, homogenization, washing and other processes, it is pasteurized at 92°C to avoid deterioration of the juice caused by microorganisms, enzymes, and oxidation. If a high-temperature enzyme is used to debitter the juice during the preheating stage before sterilization, the juice does not need to be exposed to a 30-60°C environment for a long time, reducing the risk of deterioration. Li et al. treated orange juice containing α-L-rhamnosidase by linear heating (40-85°C), and the concentration of naringin was reduced to below the bitterness threshold in 50 minutes. This shows that it is feasible to use high-temperature resistant α-L-rhamnosidase to solve the debittering problem of juice during the preheating process, and it is valuable to explore the application of α-L-rhamnosidase with good thermal stability in debittering juice.

[0006] Thermophilic α-L-rhamnosidase has good thermal stability and can effectively catalyze at higher temperatures, far exceeding the thermal stability of traditional α-rhamnosidase. Its stability at high temperatures gives it broad application prospects in a variety of industrial processes, especially in high-temperature processing processes such as citrus juice production, food debittering, and agricultural product processing. Summary of the invention

[0007] The first object of the present invention is to provide a thermophilic α-L-rhamnosidase, whose amino acid sequence is shown in SEQ ID No. 1, which is derived from the hot spring metagenome of Eryuan County, Yunnan Province. Amino acid sequence similarity analysis shows that the enzyme is a new type of thermophilic α-L-rhamnosidase.

[0008] The second object of the present invention is to provide an amino acid sequence encoding the thermophilic α-L-rhamnosidase. It includes an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID No: 1, or an amino acid sequence that is at least 93% identical to SEQ ID No: 2, or an amino acid sequence that is at least 91% identical to SEQ ID No: 3, or an amino acid sequence that is at least 90% identical to SEQ ID No: 4, or an amino acid sequence that is at least 85% identical to SEQ ID No: 5, or an amino acid sequence that is at least 82% identical to SEQ ID No: 6, or an amino acid sequence that is at least 93% identical to SEQ ID No: 7, or an amino acid sequence that is at least 88% identical to SEQ ID No: 8, or an amino acid sequence that is at least 89% identical to SEQ ID No: 9, or an amino acid sequence that is at least 87% identical to SEQ ID No: 10, or an amino acid sequence that is at least 90% identical to SEQ ID No: 11, or an amino acid sequence that is at least 93% identical to SEQ ID No: 12, or an amino acid sequence that is at least 96% identical to SEQ ID No: 13. No.: 13 has an amino acid sequence that is at least 91% identical to SEQ ID No: 13, or an amino acid sequence that is at least 91% identical to SEQ ID No: 14, or an amino acid sequence that is at least 88% identical to SEQ ID No: 15, or an amino acid sequence that is at least 83% identical to SEQ ID No: 16, or an amino acid sequence that is at least 95% identical to SEQ ID No: 17.

[0009] The third object of the present invention is to provide the use of the amino acid sequence in the preparation of α-L-rhamnosidase.

[0010] The present invention claims to protect the amino acid sequence of the α-L-rhamnosidase, and the DNA sequence encoding the amino acid sequence.

[0011] The thermophilic α-L-rhamnosidase provided by the invention has an optimum reaction temperature of 90° C., 60-110° C., good thermal stability, an optimum reaction pH of 5.5, and important economic value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the SDS-PAGE electrophoresis result of the protein obtained by expressing α-L-rhamnosidase in Escherichia coli in Example 1. From left to right are the marker protein bands; E. coliBL21 (DE3) fermentation supernatant preheated sample; containing pET-28a (+) -EyRha82 recombinant plasmid E. coli BL21 (DE3) fermentation supernatant preheated sample; 3, pure α-L-rhamnosidase.

[0013] Figure 2 The effect of temperature on the activity of α-L-rhamnosidase in Example 2. The abscissa is temperature (°C), and the ordinate is relative enzyme activity, in %.

[0014] Figure 3 The effect of pH on the activity of α-L-rhamnosidase in Example 2. The abscissa is pH, and the ordinate is relative enzyme activity, in %.

[0015] Figure 4 This is the application of thermophilic α-L-rhamnosidase to hydrolyze naringin in Example 3. The horizontal axis is time, and the vertical axis is conversion rate, in %. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with the accompanying drawings and embodiments so that those skilled in the art can understand the present invention, but the present invention is not limited in any way. Any changes or improvements made based on the teachings of the present invention fall within the scope of protection of the present invention. The methods in this embodiment are operated according to conventional methods unless otherwise specified.

[0017] The invention relates to a novel natural thermophilic alpha-L-rhamnosidase. It includes an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID No: 1, or an amino acid sequence that is at least 93% identical to SEQ ID No: 2, or an amino acid sequence that is at least 91% identical to SEQ ID No: 3, or an amino acid sequence that is at least 90% identical to SEQ ID No: 4, or an amino acid sequence that is at least 85% identical to SEQ ID No: 5, or an amino acid sequence that is at least 82% identical to SEQ ID No: 6, or an amino acid sequence that is at least 93% identical to SEQ ID No: 7, or an amino acid sequence that is at least 88% identical to SEQ ID No: 8, or an amino acid sequence that is at least 89% identical to SEQ ID No: 9, or an amino acid sequence that is at least 87% identical to SEQ ID No: 10, or an amino acid sequence that is at least 90% identical to SEQ ID No: 11, or an amino acid sequence that is at least 93% identical to SEQ ID No: 12, or an amino acid sequence that is at least 96% identical to SEQ ID No: 13. No.: 13 has an amino acid sequence that is at least 91% identical to SEQ ID No.: 13, or an amino acid sequence that is at least 91% identical to SEQ ID No.: 14, or an amino acid sequence that is at least 88% identical to SEQ ID No.: 15, or an amino acid sequence that is at least 83% identical to SEQ ID No.: 16, or an amino acid sequence that is at least 95% identical to SEQ ID No.: 17.

[0018] The theoretical molecular weight of the thermophilic α-L-rhamnosidase is 100 kDa.

[0019] The optimum reaction temperature of the thermophilic α-L-rhamnosidase is 90° C., and the optimum pH is 5.5.

[0020] Example 1 Expression of natural α-L-rhamnosidase gene Acquisition of natural α-L-rhamnosidase gene The structure and function of metagenomic data collected from hot spring samples in Eryuan County, Yunnan, China were annotated by bioinformatics, and the α-L-rhamnosidase gene was mined based on this. The gene was synthesized by Sangon Biotech Co., Ltd. using codon-optimized Escherichia coli to obtain a recombinant vector.

[0021] Plasmid extraction The recombinant plasmid pET-28a(+)-EyRha82 for transformation was extracted using the SanPrep column plasmid DNA small-scale extraction kit according to the instructions.

[0022] Conversion Using the heat shock transformation method, take the recombinant plasmid dry powder (about 4 µg) pET-28a(+)-EyRha82 after gene synthesis and place it in a centrifuge at 12000 r / min for 2 min, add 40 µL ddH2O, and the final concentration of the recombinant plasmid is 100 ng / µL. Transfer it into the prepared E. coli competent cells. When the E. coli competent cells are in an ice-water mixture, immediately take 20 µL of the recombinant plasmid and add it to the BL21 (DE3) competent cells, mix it with a pipette and then put it on ice for 30 min. After the ice bath, put the competent cells of the E. coli in a 42 ℃ water bath for 90 s (open the cell membrane and the plasmid enters the cell), immediately take it out and put it back on ice for 5 min (close the cell membrane). Add 500 µL of anti-antibody LB liquid culture medium to the centrifuge tube, place the culture medium in a constant temperature shaker at 37 ℃, 220 r / min, and culture it for 45 min. Mix well and take 200 µL of the above culture medium, use a sterile spreading rod to spread it on the solid LB medium containing kanamycin sulfate (final concentration 50 µg / mL), and place the inoculated culture medium upright in a 37 °C culture medium for 30 min, then invert and culture for 12 h-16 h to obtain the Escherichia coli BL21 (DE3) strain containing the recombinant plasmid.

[0023] Inducible expression of recombinant α-L-rhamnosidase Single clone small volume (5 mL) culture: Pick a single clone of recombinant E. coli BL21 (DE3) into a 10 mL centrifuge tube, add 5 mL LB resistance liquid medium (Kan 50 µg / mL), and culture overnight at 37 °C, 220 rpm, in a constant temperature shaker for 12-16 h to obtain the E. coli BL21 (DE3) seed solution containing the recombinant plasmid.

[0024] Recombinant plasmid identification The constructed recombinant plasmid was verified by enzyme digestion and agarose gel electrophoresis. The correct band position indicated that the target gene had been inserted into the plasmid.

[0025] Plasmid extraction The recombinant plasmid pET-28a(+)-EyRha82 for transformation was extracted using the SanPrep column plasmid DNA small-scale extraction kit according to the instructions.

[0026] Recombinant plasmid transfer E. coli BL21 Using the heat shock transformation method, 10 μL of the recombinant plasmid was aspirated and inoculated into 100 μL of Escherichia coli BL21 (DE3) competent cells. The Escherichia coli BL21 (DE3) strain containing the recombinant plasmid was finally obtained through the experiment.

[0027] Inducible expression of recombinant α-L-rhamnosidase A single clone of the recombinant Escherichia coli BL21 (DE3) was picked and placed in a 10 mL centrifuge tube. 5 mL of LB resistance liquid medium (containing 50 µg / mL of Kan) was added and cultured overnight at 37 °C, 220 rpm, in a constant temperature shaker for 12-16 h to obtain the seed solution of Escherichia coli BL21 (DE3) containing the recombinant plasmid.

[0028] Expand cultivation Take 3 mL of bacterial solution (1% inoculum) and inoculate it into 300 mL of fresh LB liquid medium (Kan 50 µg / mL) and culture it in a constant temperature shaker at 37°C and 220 rpm for 4 h to 6 h until the OD 600 =0.4-0.6, add 714 µL IPTG (final concentration 0.5 mM), culture in a constant temperature shaker at 16 °C, 220 rpm for 20 h, centrifuge at 8000 rpm / min for 5 min, discard the supernatant to collect the cells, ensure that each centrifuge tube collects 4.0 g of cells, and store at -20 °C to obtain the completed natural β -Glucosidase-expressing Escherichia coli BL21 (DE3) bacteria.

[0029] Crude enzyme acquisition 4.0 g of bacteria collected in a 50 mL centrifuge tube were added to 20 mL of non-denaturing lysis buffer, pipetted and mixed, centrifuged at 8000 r / min for 5 min, the supernatant was discarded, the bacteria were collected, 20 mL of non-denaturing lysis buffer and 20 µL of lysozyme were added, pipetted and mixed, vortexed and mixed, and incubated on ice for 30 min. Ultrasonic disruption was performed using an ultrasonic disruptor, power 40 w, amplitude 6, engineering 1, ultrasonic work 2 s, stop 8 s, the whole process was broken on ice for 30 min, centrifuged at 8000 r / min for 15 min, the supernatant was taken, placed in a 70 ℃ constant temperature water bath for preheating for 2 h, centrifuged at 8000 r / min for 15 min, and the supernatant was taken to remove most of the impurities in Escherichia coli to obtain a high-temperature impurity-free α-L-rhamnosidase crude enzyme solution.

[0030] Purification of α-L-rhamnosidase The crude enzyme solution was purified by Native-PAGE and gel recovery, with acrylamide / methylenebisacrylamide concentrations of 7% for separation gel and 4% for concentration gel. The pure enzyme solution was appropriately concentrated and the enzyme purity was tested by SDS-PAGE. The results are as follows: Figure 1 As shown, a band was obtained that matched the target molecular weight, which was 100 kDa.

[0031] Example 2 Enzymatic properties and enzyme activity experiments of α-L-rhamnosidase 1. Study on the optimum temperature of thermophilic α-L-rhamnosidase like Figure 2 As shown, the optimum temperature for thermophilic α-L-rhamnosidase to hydrolyze naringin is 90°C, it is active at 60-110°C, and maintains more than 80% activity at 80-95°C.

[0032] Study on the Optimal pH of Thermophilic α-L-rhamnosidase like Figure 3 As shown, the optimum pH for thermophilic α-L-rhamnosidase to hydrolyze naringin is 5.5, and the activity remains above 90% at pH 5-6.

[0033] Example 3 Study on the conversion of naringin by thermophilic α-L-rhamnosidase In order to determine the conversion rate of EyRha82 and naringin, the HPLC method was used to measure the conversion rate of naringin within 0-60 min. Sample pretreatment: 600 μL of the reaction solution was diluted to 10 mL with methanol and filtered with a 0.45 μm filter membrane. The standard curve was prepared by analyzing pure naringin and prunin. The HPLC system was equipped with a C18 column (4.6 × 250 mm, 5 μm), the injection volume was 30 μL / time, the mobile phase was 82% water (A) and 18% acetonitrile (B), the flow rate was 1.000 mL / min 30 μL, and the detection wavelength was 280 nm.

[0034] The results are as follows Figure 4 As shown in the figure, the conversion rate of naringin by thermophilic α-L-rhamnosidase was detected within 60 min at 90℃ and pH 5.5. The results showed that the conversion rate of naringin reached 24.6% after EyRha82 reacted with naringin for 10 min, and 6.5 mM (3.77 g / L) naringin was converted to below the detection threshold after 45 min of reaction, with a conversion rate of 100% and a conversion rate of 5 g / L / h, indicating that EyRha82 can efficiently convert naringin and can be used for debittering citrus juice.

Claims

1. A novel thermophilic α-L-rhamnosidase, the amino acid sequence of which is shown in SEQ ID No.

1. It includes an amino acid sequence that is at least 92% identical to the amino acid sequence of SEQ ID No: 1, or an amino acid sequence that is at least 93% identical to SEQ ID No: 2, or an amino acid sequence that is at least 91% identical to SEQ ID No: 3, or an amino acid sequence that is at least 90% identical to SEQ ID No: 4, or an amino acid sequence that is at least 85% identical to SEQ ID No: 5, or an amino acid sequence that is at least 82% identical to SEQ ID No: 6, or an amino acid sequence that is at least 93% identical to SEQ ID No: 7, or an amino acid sequence that is at least 88% identical to SEQ ID No: 8, or an amino acid sequence that is at least 89% identical to SEQ ID No: 9, or an amino acid sequence that is at least 87% identical to SEQ ID No: 10, or an amino acid sequence that is at least 90% identical to SEQ ID No: 11, or an amino acid sequence that is at least 93% identical to SEQ ID No: 12, or an amino acid sequence that is at least 91% identical to SEQ ID No: 13, or an amino acid sequence that is at least 92% identical to SEQ ID No:

14. No.: 14 has an amino acid sequence that is at least 91% identical to SEQ ID No: 14, or an amino acid sequence that is at least 88% identical to SEQ ID No: 15, or an amino acid sequence that is at least 83% identical to SEQ ID No: 16, or an amino acid sequence that is at least 95% identical to SEQ ID No:

17.

2. A DNA sequence encoding the amino acid sequence of α-L-rhamnosidase as claimed in claim 1.

3. A recombinant vector of the DNA sequence as claimed in claim 2.

4. A host cell transformed, transduced or transfected with the recombinant vector according to claim 3.

5. Use of the DNA sequence according to any one of claim 2 in the preparation of α-L-rhamnosidase.

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