JP2022535648A - Use of thermostable β-glucosidase in the production of gentiooligosaccharides - Google Patents

Abstract

Use of thermostable β-glucosidase TSBGl in the production of gentiooligosaccharides. The thermostable β-glucosidase TSBGl is Thermotoga sp. It is derived from KOL6 and uses Bacillus subtilis WSHI I as an expression host to realize highly efficient expression of the tsbgl gene in Bacillus subtilis. The β-glucosidase TSBGl has an optimum temperature of 90°C and an optimum pH of 6.0, and has high thermostability at 90°C. When the β-glucosidase is added to a reaction system using 1200 g/L glucose as a substrate and the enzymatic reaction is carried out at pH 6.0 and 90° C., the yield of gentiooligosaccharide reaches 178.2 g/L. This enzyme is suitable for use in industries such as foods, and can be applied to industrial production of gentio-oligosaccharides. [Selection drawing] Fig. 7

Description

The present invention relates to the use of thermostable β-glucosidase in the production of gentiooligosaccharides, and belongs to the fields of genetic engineering and enzyme engineering.

β-glucosidase (EC 3.2.1.21) is a glucoside hydrolysase that can specifically hydrolyze non-reducing terminal β-D-glucosidic bonds to release glucose and the corresponding ligand group. , GH). β-Glucosidase exists widely in vivo and exerts important actions, and is mainly distributed into six families, GH1, GH3, GH5, GH9, GH30 and GH116, depending on the difference in amino acid sequence characteristics. most of the β-glucosidases belong to the GH1, GH3 family. GH1 and GH3 family β-glucosidases differ in protein folding, physicochemical properties, catalytic properties, substrate specificity, and the like. β-Glucosidase can be applied in various industries, for example, in the bioethanol industry to degrade cellobiose to remove the inhibitory effects of the product, or to enhance the taste of glycosides, which are flavor precursors in hydrolyzed fruit juices. Some β-glucosidases have a certain level of synthetic activity due to their ability to convert glycosides. It can be applied to the food and cosmetics industry, among which the gentio-oligosaccharides produced by β-glucosidase can induce the growth of intestinal probiotics in the human body, which is beneficial to the human body.

Gentio-oligosaccharides are novel functional oligosaccharides composed of glucose linked by two or more β-1.6-glucoside bonds, and mainly composed of gentiobiose and small amounts of gentiobiose and gentiotetraose. Gentioligosaccharides are low in calories and have a low risk of tooth decay when used in food. They also have the function of suppressing tumors and promoting nutrient absorption and metabolism.

There are many methods for producing gentio-oligosaccharides. In early research, it was extracted from the roots and stems of gentian plants, but after hydrolyzing the starch using the reduced bitter almond benzene method and the acid method, it was purified from its by-products. It can be obtained, but restrictions on raw materials, market prices, etc. make its industrial production difficult. Enzymatic production is currently the main means of industrial production of gentiooligosaccharides, and the usual reaction conditions are low water activity, high substrate concentration, and utilize the glycoside conversion activity of β-glucosidase. The yield of gentio-oligosaccharides obtained is low, about 50 g/L, and the conversion rate is about 8%. Two important factors affecting the yield of gentio-oligosaccharides are the reaction temperature and the glycoside-converting activity of the enzyme. The yield of gentio-oligosaccharides increases with increasing temperature, mainly because the solubility of the substrate, glucose, increases at higher temperatures, and the acceptor, water molecules, decreases accordingly. , promotes the occurrence of glycoside conversion reaction, increases the accumulation of gentio-oligosaccharides, and at the same time prevents contamination by bacteria even at high temperatures. The deactivation phenomenon becomes more pronounced, seriously affecting production. β-glucosidases that naturally withstand high temperatures are therefore of great importance for industrial production. At present, most studies mainly use GH3 family β-glucosidases for the production of gentiooligosaccharides, but there are very few studies on GH1 family β-glucosidases. use of β-glucosidase has not yet been developed.

The present invention provides a gene encoding β-glucosidase TSBGl whose nucleotide sequence is shown in SEQ ID NO:1.

In one embodiment of the invention, the amino acid sequence of said β-glucosidase TSBGl is shown in SEQ ID NO:2.

The present invention provides vectors carrying the gene encoding β-glucosidase TSBGl.

In one embodiment of the present invention, the starting vector of said vector is the expression vector pBSMμL3 and the vector sequence is described in the patent with publication number CN107058205A.

The present invention provides a recombinant bacterium that expresses the β-glucosidase TSBGl whose amino acid sequence is shown in SEQ ID NO:2 using Bacillus subtilis as an expression host.

In one embodiment of the present invention, the Bacillus subtilis is Bacillus subtilis WSH11 as described in patent publication number CN108102997A.

The present invention provides a method for producing β-glucosidase, and the specific steps of the method are as follows.
(1) The recombinant bacterium is cultured at 35-40°C for 2-5 hours to obtain a bacterial solution.
(2) Centrifuge the bacterial solution at 2500-3500 rpm for 3-6 minutes, remove 60-90% by volume of the supernatant, apply the remaining 10-40% by volume of the supernatant to the LB plate, and Culture in an incubator at 40° C. for 10-16 hours.
(3) Collect a single colony from the LB plate into LB liquid medium and culture for 7-11 hours, then inoculate 2-6 mL of the culture solution into 100 mL TB medium and heat at 35-40° C. for 1.5-3 hours. Cultivate and further culture at 30-34° C. for 45-50 hours.
(4) After completion of culturing, the cultured cell suspension is centrifuged at 7,000 to 9,000 rpm for 15 to 25 minutes to collect cells.
(5) Add 45 to 55 mL of citric acid-disodium phosphate buffer to the cells and resuspend the cells.
(6) Using a high-pressure homogenizer to disrupt cell walls, centrifuge at 9,000 to 12,000 rpm for 15 to 25 minutes, and collect the crude enzyme solution, which is the cell wall disruption supernatant.

In one embodiment of the invention, 5-15 μg/mL of tetracycline is contained in the step (2) and (3) media.

In one embodiment of the present invention, in step (5), the citrate-disodium phosphate buffer has a concentration of 40-60 mM and a pH of 5.0-7.0.

The present invention provides a method for improving the yield of gentiooligosaccharides, in which the β-glucosidase obtained by fermenting the recombinant bacteria is reacted in a system using glucose as a substrate to obtain a reaction solution, and the reaction is performed. It is to obtain gentio-oligosaccharides by purifying the liquid.

In one embodiment of the present invention, the amount of β-glucosidase added is 300-700 U/g.

In one embodiment of the present invention, the amount of β-glucosidase added is 400-600 U/g.

In one embodiment of the invention, the glucose concentration is between 800 and 1500 g/L.

In one embodiment of the invention, the glucose concentration is between 1000 and 1300 g/L.

In one embodiment of the present invention, the method is reacting at 60-100° C. for 20-30 hours.

In one embodiment of the present invention, the method is reacting at 80-100° C. for 22-25 hours.

The present invention also protects the use of said gene in the production of gentio-oligosaccharides in the food and cosmetic fields.

The present invention also protects the use of said vector pBSMμL3-tsbgl in the production of gentio-oligosaccharide-containing products in the food and cosmetic field.

The present invention also protects the use of said β-glucosidase production process in the production of gentio-oligosaccharide-containing products in the food and cosmetic fields.

The present invention also protects the use of said gentio-oligosaccharide yield enhancing method in the manufacture of gentio-oligosaccharide-containing products in the food and cosmetic fields.

The present invention also protects the use of said recombinant bacteria in the production of gentio-oligosaccharides in the field of food and cosmetics.

Beneficial effects of the present invention are as follows. The present invention provides a highly efficient method for expressing β-glucosidase. Thermotoga sp. By synthesizing a nucleotide sequence encoding β-glucosidase TSBGl derived from KOL6, using the shuttle plasmid pBSMμL3 as an expression vector and Bacillus subtilis WSH11 as an expression host, the tsbgl gene in Bacillus subtilis can be expressed with high efficiency, and β- Glucosidase TSBGl has a temperature optimum of 90-100°C, a pH optimum of 6.0, and high thermostability at 90°C. β-Glucosidase TSBGl can be converted using glucose to produce gentiooligosaccharides, and in particular, can be produced using glucose under high concentration conditions of 1200 g/L glucose. The yield reaches 178.2 g/L, which is the highest yield when synthesizing gentiooligosaccharides by the β-glucosidase method. Therefore, this enzyme is suitable for use in industries such as food and medicine, and can be applied to industrial production of gentiooligosaccharides.

Construction process of the tsbgl gene expression vector. It is an electropherogram before and after β-glucosidase purification, M is a marker, lane 1 is the β-glucosidase TSBGl crude enzyme solution before purification, and lane 2 is the β-glucosidase TSBGl pure enzyme after purification. Relative enzymatic activity of β-glucosidase at various temperatures. Relative enzymatic activity of β-glucosidase at various pH. Enzymatic activity stability of β-glucosidase at various temperatures. Gentioligosaccharide conversion of β-glucosidase at various dosages. Conversion of gentio-oligosaccharide production by β-glucosidase at various substrate concentrations.

Analysis of the enzymatic activity of β-glucosidase:
(1) Definition of enzymatic activity unit: The enzymatic activity of hydrolyzing pNPG in 1 ml of the enzyme solution for 1 minute to produce 1 μmol of p-nitrophenol is defined as 1 enzymatic activity unit.
Calculation method for relative enzyme activity: Enzyme activity = (A ₄₀₅ +0.002)*reaction system*dilution rate/(0.0074*reaction time*enzyme added amount).
(2) Enzyme activity measurement step The reaction system was made 1 mL and diluted appropriately with 960 μL of acetate buffer of pH 5.0 (preferably, when the reaction was stopped, the absorbance at 405 nm of the reaction solution was 0.2 to 1.2. 20 μL of crude enzyme solution was added, then 20 μL of 100 mmol/L pNPG was added, reacted in a constant temperature water bath at 60° C. for 10 minutes, and immediately after 10 minutes, 200 μL of 1 mol/L Na ₂ CO ₃ solution was added. The reaction is stopped with a water bath, treated with an ice bath for 5 minutes, and the light absorption value is measured at 405 nm. An enzyme solution inactivated by heating is treated in the same manner as a blank control.
LB medium: Yeast flour 5 g/L, Tryptone 10 g/L, NaCl 10 g/L.
TB medium: yeast flour 24 g/L, glycerin ₅ g/L, tryptone ₁₂ g/L, _{K2HPO4.3H2O 16.43} g/L, _KH2PO4 2.31 g/L.
RM medium: yeast extract 5.0 g/L, tryptone 10.0 g/L, NaCl 10.0 g/L, sorbitol 90.0 g/L, mannitol 70.0 g/L.

Purification of β-Glucosidase TSBGl:
(1) Add 50 mL of 35% solid ammonium sulfate to 500 mL of the cell wall-broken supernatant of recombinant bacteria and salt out overnight (12 hours).
(2) The crude enzyme solution after salting out was centrifuged at 10,000 rpm at 4° C. for 20 minutes, and the precipitate was dissolved with pH 7.4 buffer solution A containing 20 mM sodium phosphate, 0.5 M sodium chloride, and 20 mM imidazole. , overnight (12 hours) dialysis against buffer A, followed by filtration through a 0.22 μm membrane to obtain an injection (loading) sample.
(3) After equilibrating the Ni affinity column with buffer A, the injected sample was sucked onto the Ni column and after complete adsorption, 100 mL of buffer A, 100 mL of buffer A containing 20-480 mM imidazole, 480 mM By using 100 mL each of buffer A containing imidazole and eluting at a flow rate of 1 mL/min, the target protein β-glucosidase was eluted with buffer A containing 480 mM imidazole, and this fraction was eluted. Collect fluid.
(4) The above protein eluate containing 480 mM imidazole is dialyzed overnight against pH 6.0, 50 mM sodium phosphate buffer to obtain a purified β-glucosidase enzymatic product.
(5) electrophoresis of the purified recombinant β-glucosidase; An electropherogram after purification is shown in FIG.

Example 1 Construction of tsbgl Gene-Containing Expression Vector A gene whose nucleotide sequence is shown in SEQ ID NO: 1 was chemically synthesized according to the amino acid sequence of Thermotoga sp KOL6 β-glucosidase Tsbgl in the Genbank database (WP_101510358).
After enzymatic digestion of the synthesized gene fragment and pET-24a (enzymatic digestion sites: Nde I and EcoR I), ligation was performed to obtain a ligation product. coli. Transformed into JM109. A transformant was obtained, the transformant was plated on an LB solid medium (containing 0.05 mg/mL kanamycin), and inverted culture was performed in a constant temperature incubator at 37°C for 8 to 12 hours to obtain a transformant.
Heat shock transformation method:
(1) E.I. coli. JM109 competent cells were left on ice for 5 minutes, and after the competent cells were completely thawed, 10 μL of the complete plasmid or PCR product was added, gently pipetted to homogenize, and left on ice for 45 minutes. .
(2) Competent cells were placed in a water bath pan at 42° C. and heat-shocked for 90 seconds. After the heat-shock was completed, the cells were left on ice for 5 minutes.
(3) After finishing the ice bath, 0.8 mL of LB liquid medium was added to the competent cells, mixed uniformly, placed in a shaker at 37°C, and cultured with shaking for about 60 minutes.
(4) Centrifuge the competent cells that have been cultured at 3000 rpm for 5 minutes, discard a part of the supernatant, leave about 200 μL of the fermentation liquid, and resuspend the cells by pipetting again, It was plated on an LB solid plate containing 10 μg/mL ampicillin antibiotic and cultured statically in a 37° C. incubator for about 10 hours to grow a single colony on the plate.
Monoclonal colonies were inoculated into a resistant LB liquid medium containing 10 μg/mL ampicillin, shake flask culture was performed at 37° C., 120 to 180 rpm for 8 to 12 hours, and plasmids were extracted and verified by enzymatic digestion. Verification by sequencing was performed and, if the verification was correct, the recombinant plasmid pET24a-tsbgl was obtained.
Using the plasmids pET24a-tsbgl and pBSMμL3 as templates, target gene primers and vector primers containing 15 bp homology arms upstream and downstream were designed, respectively. and 4) were amplified by PCR.
Primer 1: TAAGGAGTGTCAAGAATGAGCATGAAAAAGTTTCCGGAAG (SEQ ID NO: 3);
Primer 2: TTTATTACCAAGCTTTTAATCTTCCAGGCCGTTATTTTTAATAAC (SEQ ID NO: 4);
Primer 3: AAGCTTTGGTAATAAAAAAAACACCTC (SEQ ID NO: 5);
Primer 4: CATTCTTGACACTCCTTATTTG (SEQ ID NO: 6).
PCR system: 25 μL 2×Super PfxMasterMix, 1.25 μL each of the two primers, 22 μL ddH ₂ O, 0.5 μL template.
Reaction conditions: (1) 94°C, 4 minutes, (2) 94°C, 1 minute, (3) 55°C, 1 minute, (4) 72°C, 2 minutes, 35 cycles of amplification from (2) to (4) After that, (5) 72° C. for 5 minutes, (6) holding the temperature at 4° C.; and amplified respectively to obtain the target gene fragment tsbgl and the template fragment pBSMμL3.
The amplified fragments were recovered with a gel recovery kit (Amane Seika Technology Co., Ltd.) and verified by sequencing, and the two recovered fragments for which the results of the verification by sequencing were correct were collected using the In-Fusion HD Cloning Plus kit. Ligate with a kit, use 400 ng of gene fragment, 200 ng of vector fragment, and 2 μL of 5×In-Fusion HD Enzyme Premix as a ligation system, supplement with water to 10 μL, react the ligation system at 50° C. for 25 minutes, and ligate. The product was obtained and the ligation product was transformed into clone host JM109 (see heat shock transformation method above for specific embodiments), plated on LB solid medium (containing 10 μg/mL ampicillin) and incubated at 37°C. After culturing for 8 to 10 hours, a single colony was picked and inoculated in LB liquid medium containing 100 mg/L ampicillin, cultured at 37°C for 10 hours, the cells were collected, and the plasmid was extracted (plasmid extraction kit was purchased from Tian Ne Seika Technology Co., Ltd.), pBSM μL3-tsbgl plasmid (Fig. 1) was obtained, verified by enzymatic digestion and verified by sequencing by a professional company.

Example 2: Transformation culture of Bacillus subtilis expression host and extraction of crude enzyme solution (described in patent publication number CN108102997A). The recombinant plasmid pBSMμL3-tsbgl was transformed into Bacillus subtilis WSH11 competent cells by electric shock:
(1) Leave the Bacillus subtilis WSH11 competent cells on ice for 5 minutes. After the competent cells are completely thawed, add 10 μL of the recombinant plasmid, homogenize by gentle pipetting, and then place on ice for 15 minutes. I left it.
(2) Pre-start the electroporator and preheat for 30 minutes, set the electric shock voltage to 2400 V, add the ice-bathed competent cells to a pre-cooled 2 mm diameter electroporation cuvette, After wiping off the water on the outer wall of the electroporation cuvette, the electroporation cuvette was placed in the electroporator and subjected to an electric shock (electroporation).
(3) After the end of the electric shock, quickly add 1 mL of pre-cooled RM medium, pipet evenly, transfer the bacterial solution to 1.5 mL of sterilized EP tube, place it in a shaker at 37°C, and shake it at 200 rpm for 3 times. Cultured with shaking for hours.
(4) Centrifuge the cultured bacterial solution at 3000 rpm for 5 minutes, discard a part of the supernatant, leave about 200 μL of the supernatant, resuspend the bacterial cells by pipetting again, It was plated on a tetracycline-resistant LB solid plate and cultured in an incubator at 37° C. for about 10 hours to grow a single colony on the plate.
(5) A single colony was picked and verified by sequencing to obtain a positive transformant containing plasmid pBSMμL3-tsbgl.
A positive transformant containing the recombinant plasmid pBSMμL3-tsbgl was inoculated in LB liquid medium (containing 10 μg/mL tetracycline) and cultured for 8 to 10 hours. After culturing for 2 hours and further culturing at 33° C. for 48 hours, the cells were collected by centrifugation at 8000 rpm for 20 minutes after completion of fermentation. After adding 50 mL of 50 mM pH 6.0 citric acid-disodium phosphate buffer to the cells and sufficiently resuspending the cells, the cell walls were disrupted using a high-pressure homogenizer and centrifuged at 10,000 rpm for 20 minutes. , the crude enzyme solution, which is the cell wall disruption supernatant, was collected, and the enzyme activity of the crude enzyme solution with an OD ₆₀₀ of 5 was 10.41 U/mL.
The collected crude enzyme solution was purified and subjected to electrophoresis. The electrophoretogram after purification is shown in FIG.

（２）β－グルコシダーゼＴＳＢＧｌの最適ｐＨ
ｐＮＰＧを基質として、実施例２で得た精製後のβ－グルコシダーゼを酵素活性測定反応系に加え、様々なｐＨ下、９０℃の恒温水浴にて１０分反応させた。反応後の酵素活性を測定し、その相対酵素活性を算出し、結果を図４に示し、具体的なデータを表２に示し、以上から、β－グルコシダーゼの最適ｐＨは６．０であることが分かった。

（３）β－グルコシダーゼＴＳＢＧｌの熱安定性
ｐＮＰＧを基質として、実施例３で得た精製後のβ－グルコシダーゼを酵素活性測定反応系に加え、ｐＨ６．０では、それぞれ７０℃、８０℃、９０℃の温水浴にて６０分間反応させ、６０分間内に酵素の酵素活性を測定し、その相対酵素活性を算出し、結果を図５に示し、６０分間になったときには、β－グルコシダーゼＴＳＢＧｌは、７０℃、８０℃、９０℃での相対酵素活性がそれぞれ９９．１８％、９９．３１％、９９．８１％であった。β－グルコシダーゼは、９０℃では高い熱安定性を有した。

Example 3: Determining Conditions for Using β-Glucosidase TSBGl (1) Optimum Temperature for β-Glucosidase TSBGl Using pNPG as a substrate, the purified β-glucosidase obtained in Example 2 was added to the enzyme activity measurement reaction system, and pH is 6.0 and reacted at various temperatures, the enzyme activity was measured, the relative enzyme activity was calculated, the results are shown in FIG. 3, and the specific data are shown in Table 1. The optimum temperature for is found to be 90°C, and at 90-100°C the relative enzymatic activity reaches over 85%.

(2) Optimum pH of β-glucosidase TSBGl
Using pNPG as a substrate, the purified β-glucosidase obtained in Example 2 was added to the enzyme activity measurement reaction system, and reacted in a constant temperature water bath at 90° C. for 10 minutes under various pH values. The enzymatic activity after the reaction was measured, the relative enzymatic activity was calculated, the results are shown in FIG. I found out.

(3) Thermostability of β-Glucosidase TSBGl Using pNPG as a substrate, the purified β-glucosidase obtained in Example 3 was added to the reaction system for measuring enzyme activity. ℃ warm water bath for 60 minutes, the enzymatic activity of the enzyme was measured within 60 minutes, the relative enzyme activity was calculated, the results are shown in FIG. , 70°C, 80°C and 90°C were 99.18%, 99.31% and 99.81%, respectively. β-glucosidase had high thermostability at 90°C.

Example 4: Use of β-glucosidase TSBGl in the production of gentiooligosaccharide Using 800 g/L of glucose as a substrate, the reaction was carried out at pH 6 and 90°C for 24 hours. , 600 U/g, and 700 U/g, the amount of enzyme added was studied when synthesizing gentio-oligosaccharides by reverse hydrolysis activity using high-concentration glucose as a substrate.
From the experimental results shown in FIG. 6, it can be seen that within a given range, the substrate conversion rate continued to improve as the amount of enzyme added increased, and when the added enzyme amount reached 500 U/g glucose, the substrate conversion rate was 10.94. % and further increasing the amount of enzyme added resulted in almost no change in conversion. Taken together, an enzyme loading of 500 U/g is selected, in which case the substrate conversion rate reaches 10.94% and the gentiooligosaccharide yield reaches 73 g/L.

Example 5: Use of β-glucosidase TSBGl in the production of gentiooligosaccharides at a high substrate concentration. The nature of the high temperature reaction differs in that it allows reverse hydrolysis synthesis reactions at higher temperatures and higher substrate concentrations, thus increasing the concentration of the glucose substrate (800 g/L, 900 g/L, 1000 g/L, 1100 g/L, respectively). , 1200 g/L) on yield and conversion for reverse hydrolytic synthesis of gentio-oligosaccharides.
From the experimental results shown in FIG. 7, when the final concentration of glucose substrate is 800 g/L, 900 g/L, 1000 g/L and 1100 g/L, the conversion of substrate is 10.42%, 12.72%, 14%, respectively. .43%, 14.82%, when the final concentration of glucose substrate is 1200 g/L, the yield of gentio-oligosaccharides reaches 178.2 g/L, the conversion of substrate is 14.85%, This was the highest yield to date for synthesizing gentio-oligosaccharides by this method.

Although the present invention has been disclosed with preferred embodiments, it is not intended to limit the invention, and various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the patentable scope of the present invention should be determined by the following claims.

Claims (12)

The β-glucosidase expressed by the recombinant bacterium is used to catalyze the production of gentiooligosaccharides from glucose, and the recombinant bacterium expresses β-glucosidase whose amino acid sequence is represented by SEQ ID NO: 2 using Bacillus subtilis as an expression host. A method for improving the yield of gentiooligosaccharides, characterized by: The method according to claim 1, wherein the amount of β-glucosidase added is 300-800 U/g glucose. The method according to claim 1, characterized in that the concentration of glucose is 800-1500 g/L. The method according to claim 1, wherein the reaction is carried out at 60-100°C and pH 5.0-7.0 for 20-30 hours. A gene encoding β-glucosidase,
A gene, wherein the nucleotide sequence of said gene is shown in SEQ ID NO:1. A vector carrying the gene of claim 5. 7. A vector according to claim 6, characterized in that it is pBSM[mu]L3. A recombinant bacterium characterized by expressing a β-glucosidase whose amino acid sequence is represented by SEQ ID NO:2 using Bacillus subtilis as an expression host. A recombinant bacterium characterized by using Bacillus subtilis as an expression host and containing the vector according to claim 5 or 7. A method for producing β-glucosidase, which comprises producing β-glucosidase by fermentation of the recombinant bacterium according to claim 8. Use of the method according to any one of claims 1 to 4 or claim 10 in the preparation of gentio-oligosaccharide-containing products in the food and cosmetic field. Use of the gene according to claim 5 or the vector according to claim 6 or 7 or the recombinant bacterium according to claim 8 or 9 in the preparation of products containing gentio-oligosaccharides in the field of food and cosmetics.

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