WO2020010644A1 - 一种球毛壳菌右旋糖酐酶的发酵制备及其应用 - Google Patents

一种球毛壳菌右旋糖酐酶的发酵制备及其应用 Download PDF

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WO2020010644A1
WO2020010644A1 PCT/CN2018/096095 CN2018096095W WO2020010644A1 WO 2020010644 A1 WO2020010644 A1 WO 2020010644A1 CN 2018096095 W CN2018096095 W CN 2018096095W WO 2020010644 A1 WO2020010644 A1 WO 2020010644A1
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dextranase
dextran
fermentation
sphaeroides
concentration
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田亚平
杨柳
周楠迪
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江南大学
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2454—Dextranase (3.2.1.11)
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01011—Dextranase (3.2.1.11)

Definitions

  • the invention relates to fermentation preparation and application of dextranase from Chaetomium globosum, and belongs to the field of fermentation technology, enzyme preparation and industrial application.
  • Enzyme preparation refers to substances with enzyme properties extracted from living organisms, and has been used in various fields such as medicine, chemical industry, food, brewing, etc., and has a wide range of applications.
  • the food processing industry has a close relationship with people's lives. Enzymes are increasingly used in the food processing industry, and their role is becoming more and more important. They are greatly reflected in meat processing, deep hydrolysis of proteins, and as food additives.
  • Dextranase is a hydrolase that specifically cleaves alpha-1,6 glucosidic bonds in dextran molecules. From the point of view of the hydrolysis of dextran by enzymes, known dextranases are divided into two categories, endo-dextranase and exo-dextranase. Endo-dextranase hydrolyzes the ⁇ (1 ⁇ 6) bond in dextran to reduce its molecular weight. Exodextranase hydrolyzes the ⁇ (1 ⁇ 6) bond in dextran from the reducing end and releases glucose.
  • Dextranase is widely used in food industry, medicine and sugar industry. It plays a vital role in the processing of molasses and beverages in the food industry. In medicine, partial hydrolysates of natural dextran are used to prepare blood substitutes and prevent dental caries. Dextranase hydrolyzes high-molecular-weight dextran to ⁇ -glucan of different molecular weights and has been used as a chromatographic medium, blood volume expander, and drug delivery vehicle.
  • the purpose of the present invention is to provide a fermentation preparation and application of dextranase from C. sphaeroides.
  • the present invention screens out strains that can produce dextranase from soil.
  • Specific endonuclease the present invention improves the production of dextranase, reduces the cost of downstream separation and preparation of dextranase, and can specifically remove dextran alpha-1,6 glycosidic bond residues by using dextranase.
  • the first object of the present invention is to provide a Chaetomium globosum screened from the soil, which has been deposited at the General Microbiology Center of the China Microbial Strain Collection Management Committee on June 11, 2018, with a deposit number of CGMCCC No. 15867, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
  • a second object of the present invention is to provide the application of the strain in the field of fermentation.
  • a third object of the present invention is to provide a method for culturing the strain.
  • the method is inoculating C. sphaeroides into a fermentation medium, and the components of the fermentation medium are: carbon source and nitrogen source concentration is 0.5 to The concentration of K 2 HPO 4 and MgSO 4 is 0 to 4%, the initial pH is 4.5 to 9.0, the inoculum volume is 1 to 6%, the volume of the shake bottle is 20 to 90 mL / 250 mL, and the speed of the shaker is 140 to 220r / min; fermentation temperature is 24-34 °C.
  • the carbon source includes any one or more of ⁇ -lactose, potato starch, glucose, fish meal peptone, maltose, dextran T20, T40, T2000, sucrose, and soluble starch.
  • the nitrogen source includes any one or more of urea, sodium nitrate, fish meal peptone, beef extract, soy peptone, yeast extract, tryptone, ammonium sulfate, and potato starch.
  • the components of the fermentation medium are: Dextran T2020g / L, yeast extract 10g / L, K 2 HPO 4 and MgSO 4 addition amounts are 2g / L and 0.5g / L, the initial pH is 7.0, inoculation The amount is 3%, the amount of liquid is 50 / 250mL, the fermentation speed is 220r / min, and the culture temperature is 26 ° C.
  • a fourth object of the present invention is to provide a method for purifying dextranase, in which a fermentation broth obtained by cultivating C. sphaeroides to produce dextranase is sequentially salted out with ammonium sulfate, subjected to dialysis and desalting, purified on a gel column, and concentrated by ultrafiltration. Perform purification.
  • a fifth object of the present invention is to provide an application of dextranase obtained by the above purification method in hydrolyzing glucan.
  • a sixth object of the present invention is to provide the application of the above-mentioned C. sphaeroides in the preparation of medicines or oral products for preventing and treating caries.
  • a seventh object of the present invention is to provide the application of the above-mentioned Streptococcus in the medical field.
  • An eighth object of the present invention is to provide the application of the above-mentioned Streptococcus in the food field.
  • the present invention screens a strain producing dextranase from the soil, and after 18s rDNA identification, this strain is Chaetomium globalospore.
  • This strain was used as the starting enzyme for fermentation.
  • the initial enzyme activity of fermentation culture was 38.01 U / mL.
  • the dextranase activity was 698.22 U / mL, which was 18.37 times the highest level before optimization.
  • the purified dextranase has a high specific activity (7535.8U / mg), and the final purification factor is 10.97 with a yield of 18.7%.
  • SDS-PAGE and active electrophoresis of the purified dextranase showed that the molecular weight of the enzyme was 53 kDa.
  • Isolated and purified dextranase is an endogenous hydrolase, which has a highly specific hydrolysis of ⁇ -1,6 glycosidic bonds, and has a high hydrolytic power for high molecular weight dextran. Dextranase will polymerize high molecular weight dextran. Sugar is hydrolyzed into low-molecular-weight dextran, which has important applications in the pharmaceutical industry.
  • Dextranase has an inhibitory effect on the growth of Streptococcus mutans, and the inhibitory effect increases with the increase of the enzyme concentration.
  • Capsoccus dextranase has a significant effect on the formation and removal of Streptococcus mutans biofilms. When the enzyme concentration reaches 50 U / mL, the inhibition rate reaches 71.58% and the clearance rate reaches 49.07%.
  • a Chaetomium globeosum has been deposited at the General Microbiology Center of the China Microbial Strain Collection Management Committee on June 11, 2018, with a deposit number of CGMCCC No. 15867 and its deposit address is Beichen West Road, Chaoyang District, Beijing No. 1 courtyard No. 3.
  • FIG. 1 is a photograph of colony characteristics of Chaetomium globosus in Example 1.
  • FIG. 1 is a photograph of colony characteristics of Chaetomium globosus in Example 1.
  • FIG. 2 is a scanning electron microscope image of a colony of C. sphaeroides in Example 1.
  • FIG. 2 is a scanning electron microscope image of a colony of C. sphaeroides in Example 1.
  • FIG. 3 is a graph showing the effect of the initial pH of the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 3 is a graph showing the effect of the initial pH of the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 4 is a graph showing the effect of inoculation amount of fermentation medium on the wet weight of bacteria and dextranase activity in Example 3.
  • FIG. 4 is a graph showing the effect of inoculation amount of fermentation medium on the wet weight of bacteria and dextranase activity in Example 3.
  • FIG. 5 is a graph showing the effect of the amount of liquid in the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 5 is a graph showing the effect of the amount of liquid in the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 6 is a curve of the effect of the stirring speed of the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 6 is a curve of the effect of the stirring speed of the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 7 is a graph showing the effect of the fermentation temperature of the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 7 is a graph showing the effect of the fermentation temperature of the fermentation medium on the wet weight of the bacteria and dextranase activity in Example 3.
  • FIG. 8 is a TLC chart of a dextranase hydrolysis product in Example 4.
  • FIG. 8 is a TLC chart of a dextranase hydrolysis product in Example 4.
  • FIG. 9 is an HPLC chart of a dextranase hydrolysis product in Example 4.
  • FIG. 10 is an SDS-PAGE chart of dextranase in Example 5.
  • FIG. 11 is an electrophoresis chart of dextranase activity in Example 5.
  • FIG. 12 is a graph showing the inhibitory effect of dextranase on the growth of Streptococcus mutans in Example 6.
  • the dextranase-producing strain is Chaetomium globalosum, which has been deposited at the General Microbiology Center of the China Microbial Species Collection Management Committee on June 11, 2018.
  • the deposit number is CGMCCC No. 15867, the deposit address It is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
  • Dextranase enzyme activity determination method Dextran T2000 was used as a substrate (the substrate storage solution was prepared with acetate buffer 5.5 and the concentration was 20 mM). The reaction mixture included 0.2 mL of diluted enzyme solution and 1.8 mL of acetate buffer 5.5. % Substrate storage solution, reacted at 50 ° C for 10 minutes in a water bath, immediately added 3 mL DNS to stop the reaction, and reacted in boiling water for 10 minutes. After cooling with ice water, the absorbance was measured at 540 nm.
  • Enzyme activity unit (U) is defined as the amount of reducing sugar equivalent to 1 umol glucose released per minute using dextran T2000 as a substrate at 50 ° C.
  • Determination of protein concentration Determined using crystalline bovine serum albumin as a protein standard. The enzyme was diluted with distilled water to an appropriate concentration, and 4 mL of Coomassie Brilliant Blue was added to 1 mL of the diluted enzyme solution to a total volume of 5 ml. The reaction was accurate for 2 min. The absorbance was observed at 595 nm and the protein concentration was calculated.
  • Dextran molecular weight distribution detection Waters1525 high performance liquid chromatograph (with differential detector and Empower workstation Waters 2410), using Ultrahydrogel TM Linear (300mm ⁇ 7.8mmid ⁇ 2), operating at 30mL at a flow rate of 0.9mL / min Chromatograph. Retention time and Mw calibration curves were prepared using 200, 30.06, 13.503, 0.9750, 0.27 kDa glucan standards (sigma, USA) and glucose (180 Da).
  • the soil sample was diluted with physiological saline to 10 -5 coated PDA medium (200g of potato, 20g of glucose, 15-20g of agar), and cultured at 28 ° C for 5 days. Pure strain was obtained after preliminary screening, and the strain was treated with blue glucan T2000 medium was re-screened, and a strain with a higher enzyme yield was obtained through a transparent circle comparison. After observation by electron microscope and 18S rDNA identification, this strain was C. globosum, and the nucleotide sequence of 18S rDNA is shown in SEQ ID NO.1. A photograph of the colony characteristics of Chaetomium globosum is shown in Figure 1 and a scanning electron microscope image is shown in Figure 2.
  • the mycelia of Pleurotus globosum was picked and inserted into the seed medium, and the culture was performed using a rotary constant temperature shaker at a speed of 220r / min, a culture temperature of 28 ° C, and a 60-hour culture to obtain a seed solution; the components of the seed medium; It is: glucose 5g / L, sodium chloride 0.5g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 0.5g / L, magnesium sulfate 0.2g / L. After sterilization, filter-killed kanamycin is added to The final concentration was 50 ⁇ g / mL.
  • the seed solution was transferred into a 250 mL shake flask containing 50 mL of fermentation medium at an inoculation amount of 1%, and the fermentation temperature was 28 ° C.
  • dextran dextran T20, T40, T2000
  • different substances glucose, maltose, lactose, soluble starch, corn dextrin, sucrose, fish meal peptone
  • yeast extract, urea, pancreas Peptone, beef extract, ammonium sulfate, and potato starch were used as nitrogen sources
  • the optimal carbon source and optimal nitrogen source concentrations were set to 0.5%, 1%, 2%, 3%, 4%, and 5%, respectively.
  • the K 2 HPO 4 and MgSO 4 concentrations were set to 0, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, and 0.4%, respectively.
  • Orthogonal experiments were performed on the carbon source concentration, nitrogen source concentration, K 2 HPO 4 , and MgSO 4 to determine the amount of enzyme production and cell growth to determine the optimal medium.
  • the mycelia of Pleurotus globosum was picked and inserted into the seed medium, and the culture was performed using a rotary constant temperature shaker at a speed of 220r / min, a culture temperature of 28 ° C, and a 60-hour culture to obtain a seed solution; the components of the seed medium; It is: glucose 5g / L, sodium chloride 0.5g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 0.5g / L, magnesium sulfate 0.2g / L. After sterilization, filter-killed kanamycin is added to The final concentration was 50 ⁇ g / mL.
  • the seed solution was inserted into a 250 mL shake flask with 50 mL of fermentation medium at a 1% inoculation amount, and the fermentation temperature was 28 ° C. Based on the optimized optimal medium, the enzyme-producing fermentation conditions were sequentially optimized. Initially, The pH was set to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0, respectively. The inoculation amount was set to 1%, 2%, 3%, 4%, 5%, and 6%, respectively. The loading volume was set to 20mL, 30mL, 40mL, 50mL, 60mL, 70mL, 80mL, and 90mL, respectively.
  • the stirring speed was set to 140 rpm, 160 rpm, 180 rpm, 200 rpm, and 220 rpm, respectively.
  • the fermentation temperatures were set to 24 ° C, 26 ° C, 28 ° C, 30 ° C, 32 ° C, and 34 ° C, respectively.
  • the fermentation time was 8 days. The wet weight of bacteria and dextranase activity were measured.
  • the nucleotide sequence of dextranase is shown in SEQ ID NO.2.
  • the mycelia of Pleurotus globosum was picked and inserted into the seed medium, and the culture was performed using a rotary constant temperature shaker at a speed of 220r / min, a culture temperature of 28 ° C, and a 60-hour culture to obtain a seed solution; the components of the seed medium; It is: glucose 5g / L, sodium chloride 0.5g / L, yeast powder 5g / L, dipotassium hydrogen phosphate 0.5g / L, magnesium sulfate 0.2g / L. After sterilization, filter-killed kanamycin is added to The final concentration was 50 ⁇ g / mL.
  • the components of the fermentation medium were: dextran T2025g / L, yeast extract 10g / L, and the addition amounts of dipotassium hydrogen phosphate and magnesium sulfate were 2.5g / L and 2.5g / L, respectively.
  • the seed liquid was inserted into a 250 mL shake flask containing 50 mL of fermentation medium at an inoculation amount of 1%, the fermentation speed was 220 r / min, and the fermentation temperature was 28 ° C. The initial pH was naturally. After 8 days of fermentation, the enzyme activity was 38.01 U / mL.
  • Dextran T2000 (3%) was prepared with 0.2M acetic acid buffer (pH 5.5) and incubated at 50 ° C. C. streptococcus dextranase was added to a final concentration of 5 U / mL. The reaction solution was mechanically stirred at 50 ° C for 90 minutes, and samples were taken at 30min, 60min, and 90min, respectively. The sample was heated in a boiling water bath for 30 minutes to terminate the reaction, and then analyzed by thin layer chromatography (TLC) using 7: 5: 4: 2 (v / v / v / v) silica gel plate with n-butanol: isopropanol: acetic acid: water solvent system.
  • TLC thin layer chromatography
  • Glucose, isomaltose, and isomaltriose were used as standards, and a mixture of heat-inactivated dextranase was used as a control sample.
  • the carbohydrates were developed by spraying a 200 mL acetone solution containing dianiline (4 g), aniline (4 mL) and 85% phosphoric acid (20 mL) on a TLC plate, and then heating at 95 ° C for 10 minutes.
  • reaction mixture was treated as a sample and analyzed by HPLC-MS (Waters, USA) using a WATERS ACQUITY UPLC chromatograph and a BEAHAMIDE (2.1 ⁇ 100mm1.7um) analysis column at a column temperature of 45 ° C at a flow rate of 0.3 mL / min. ,
  • the injection volume is 2 ⁇ L, and it is connected to the WATERS MALDI SYNAPT Q-TOF MS mass spectrometer, and the mass spectrometry is performed by ESI-mode.
  • dextran T2000 (3%) was prepared from 0.2M acetate buffer solution (pH 5.5), and incubated at 50 ° C for 10 minutes. The concentration is 2U / mL, and stir uniformly. Samples of the reaction mixture were obtained at intervals of 1 to 120 minutes, boiled for 30 minutes to stop the reaction, the impurities in the reaction solution were repeatedly filtered out with filter paper, the reaction supernatant was heated and boiled for 15 minutes, and the impurities were filtered again.
  • the diluted sample was filtered through a mixed cellulose ester membrane (a filter with a pore size of 0.22 ⁇ m and a diameter of 25 mm) and analyzed by HPLC (Waters 1525, Milford, USA) using UltrahydrogelTM Linear (300 mm ⁇ 7.8 mmid ⁇ 2), The chromatograph was operated at a flow rate of 0.9 mL / min at 30 ° C, and then connected to a differential detector and an Empower workstation (Waters 2410).
  • dextranase hydrolytic ability of dextranase to dextran of different molecular weights was investigated, and 3% dextran T20, T40, T70, T2000 were prepared with 0.2M acetate buffer solution (pH 5.5), and incubated at 50 ° C for 10 minutes Add dextranase to a final concentration of 1 U / mL, and react at 50 ° C for 15 minutes, after which the operation is as described above. Retention time and Mw calibration curves were prepared using 200, 30.06, 13.503, 0.9750, 0.27 kDa glucan standards (sigma, USA) and glucose (180 Da).
  • the TLC and HPLC results of the dextranase hydrolysis product are shown in Figure 8.
  • the results show that the final products of dextran T2000 hydrolysis by Dextranase catalyzed by dextran T2000 are isomaltose, isomaltotriose and some oligoisomaltose. Glucose is produced. It is shown that globose shell dextranase is a typical endoglucanase.
  • the results of dextranase hydrolysis of Dextran T2000 are shown in Table 1. The molecular weight of Dextran decreased to 41,000 Da within 15 minutes.
  • the dextran molecular weight of the reaction for 15 minutes decreased faster than the subsequent time period, indicating that the enzyme has a higher affinity for the higher molecular weight ⁇ -glucan.
  • the Mw of the glucan obtained after the hydrolysis reaction for 120 minutes was 2506 Da.
  • the results in Table 2 show that different enzyme concentrations have different hydrolytic powers to ⁇ -glucan. As the enzyme concentration increases, the degradation rate of dextran increases. The above results comprehensively show that dextranase has a high hydrolysis rate for high molecular weight dextran, and the higher the enzyme concentration, the faster the hydrolysis rate.
  • dextran T2000 is hydrolyzed by dextranase.
  • the obtained hydrolysate does not contain glucose, and the molecular weight of dextran decreases with the increase of reaction time and enzyme concentration, and can effectively degrade high molecular weight dextran. There is great potential in industrial applications.
  • the dialysis-concentrated enzyme sample was filtered through a 0.22 ⁇ m pore size membrane filter and loaded on a Sephadex G75 column (1.5cm ⁇ 60cm) pre-equilibrated with a pre-cooled 0.2M acetate buffer solution (pH 5.5) at low temperature.
  • the flow rate was 12 mL / h.
  • Peak collection was performed with 2 mL collection fractions per tube.
  • the absorbance of each fraction was measured at 280 nm to monitor the protein during chromatographic separation to determine the protein concentration of each fraction. Fractions with high dextranase activity were collected to determine dextranase activity and protein concentration.
  • the purity and molecular weight of the enzyme were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and SDS-PAGE was performed using a 12% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie G-250. The SDS-PAGE and active electrophoresis of dextranase are shown in Figure 9.
  • dextranase from Chaetomium sphaeroides has a significant inhibitory effect on the growth of Streptococcus mutans.
  • concentration reached 50U / mL
  • inhibition rate of Streptococcus mutans biofilm formation reached 71.58%
  • clearance rate of Streptococcus mutans biofilm formation reached 49.07%.

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Abstract

一种球毛壳菌右旋糖酐酶的发酵制备及其应用。一种通过正交试验优化培养基成分和发酵条件确定球毛壳菌高产右旋糖酐酶的发酵方法。球毛壳菌在水解葡聚糖中的应用。球毛壳菌在制备防治龋齿的药物或口腔用品方面的应用。球毛壳菌在食品领域的应用。

Description

一种球毛壳菌右旋糖酐酶的发酵制备及其应用 技术领域
本发明涉及一种球毛壳菌右旋糖酐酶的发酵制备及其应用，属于发酵技术、酶制剂、工业应用领域。
背景技术
酶制剂是指从生物中提取的具有酶类性质的物质，现已应用在医药、化工、食品、酿造等各领域，应用范围非常的广泛。食品加工业与人们的生活关系紧密，酶在食品加工业中的应用越来越多，作用也越来越重要，在肉类加工、蛋白质的深度水解和作为食品添加剂中有很大体现。
右旋糖酐酶是专一性地裂解右旋糖酐分子中的α-1,6葡萄糖苷键的水解酶。从酶对葡聚糖的水解作用来看，将已知的右旋糖酐酶分为两类，内切右旋糖酐酶和外切右旋糖酐酶。内切右旋糖酐酶水解右旋糖酐中的α(1→6)键，使其分子量变小。外切右旋糖酐酶，从还原端水解右旋糖酐中的α(1→6)键，并释放出葡萄糖。
右旋糖酐酶广泛应用于食品工业，医药和糖业。在食品工业中对糖蜜和饮料加工中起着至关重要的作用，在医学上，天然葡聚糖部分水解产物被用于制备血液代用品以及预防龋齿。右旋糖酐酶将高分子量的葡聚糖水解为不同分子量的α-葡聚糖，已被用作色谱介质，血容量扩展器和药物运载工具。
但目前，右旋糖酐酶的发酵产量还比较低，不能满足工业制备的需求，导致没有大量右旋糖酐酶的商业化产品。因此，如何通过改善发酵策略来提高右旋糖酐酶的发酵产量成为亟待解决的问题。
发明内容
为解决上述技术问题，本发明的目的是提供一种球毛壳菌右旋糖酐酶的发酵制备及其应用，本发明从土壤中筛选出可以产右旋糖酐酶的菌株，生产的右旋糖酐酶是具有严格底物特异性的内切酶，本发明提高了右旋糖酐酶的产量，减轻了下游分离制备右旋糖酐酶的成本，能够利用右旋糖酐酶特异性地切除葡聚糖α-1,6糖苷键残基。
本发明的第一个目的是提供一种从土壤中筛选出的球毛壳菌(Chaetomium globosum)，已于2018年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCC No.15867，保藏地址是北京市朝阳区北辰西路1号院3号。
本发明的第二个目的是提供所述菌株在发酵领域的应用。
本发明的第三个目的是提供一种培养所述菌株的方法，所述方法是将球毛壳菌接种于发酵培养基中，发酵培养基的成分为：碳源和氮源浓度为0.5～5％，K 2HPO 4和MgSO 4浓度为0～4％，初始pH分别为4.5～9.0，接种量为1～6％，摇瓶装液量为20～90mL/250mL，摇床转速为140～220r/min；发酵温度为24～34℃。
进一步的，发酵培养基中，碳源包括α-乳糖，马铃薯淀粉，葡萄糖，鱼粉蛋白胨，麦芽糖，葡聚糖T20、T40、T2000，蔗糖、可溶性淀粉中的任意一种或多种。
进一步的，发酵培养基中，氮源包括尿素、硝酸钠、鱼粉蛋白胨、牛肉浸膏、大豆蛋白胨、酵母提取物、胰蛋白胨、硫酸铵、马铃薯淀粉中的任意一种或多种。
进一步的，发酵培养基的成分为：葡聚糖T2020g/L，酵母提取物10g/L，K 2HPO 4和MgSO 4添加量分别为2g/L和0.5g/L，初始pH为7.0，接种量为3％，装液量为50/250mL，发酵转速220r/min，培养温度为26℃。
本发明的第四个目的是提供了一种右旋糖酐酶的纯化方法，将培养球毛壳菌生产右旋糖酐酶所得的发酵液依次用硫酸铵盐析，透析除盐，凝胶柱纯化，超滤浓缩进行纯化。
本发明的第五个目的是提供上述纯化方法得到的右旋糖酐酶在水解葡聚糖中的应用。
本发明的第六个目的是提供上述球毛壳菌在制备防治龋齿的药物或口腔用品方面的应用。
本发明的第七个目的是提供上述球毛壳菌在医药领域的应用。
本发明的第八个目的是提供上述球毛壳菌在食品领域的应用。
借由上述方案，本发明至少具有以下优点：本发明从土壤中筛选出一株产右旋糖酐酶的菌株，经18s rDNA鉴定，此菌株为球毛壳菌Chaetomium globosum。以此菌株为发酵产酶出发菌株，发酵培养最初酶活为38.01U/mL，经发酵优化后得到右旋糖酐酶的酶活达698.22U/mL，是优化前最高水平的18.37倍。经过粗酶液的逐步纯化，纯化的右旋糖酐酶具有很高的比活力(7535.8U/mg)，最终纯化倍数为10.97，收率为18.7％。纯化的右旋糖酐酶的SDS-PAGE和活性电泳显示该酶的分子量为53kDa。
分离纯化的右旋糖酐酶是一种内切型水解酶，对α-1,6糖苷键有高度特异性水解作用，对高分子量的葡聚糖有高效的水解力，右旋糖酐酶将高分子量的葡聚糖水解为低分子量的葡聚糖，低分子量的葡聚糖在医药工业中有重要应用。
右旋糖酐酶对变异链球菌的生长有抑制作用，随着酶浓度的增加，抑制作用增强。球毛壳菌右旋糖酐酶对变异链球菌生物膜的形成和清除作用很明显，当酶浓度达到50U/mL时，抑制率达到71.58％，清除率达到49.07％。
生物材料保藏
一种球毛壳菌(Chaetomium globosum)，已于2018年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCC No.15867，保藏地址是北京市朝阳区北辰西路1号院3号。
附图说明
图1是实施例1中球毛壳菌的菌落特征照片。
图2是实施例1中球毛壳菌的菌落的扫描电镜图。
图3是实施例3中发酵培养基的初始pH对菌体湿重和右旋糖酐酶活力的影响曲线。
图4是实施例3中发酵培养基的接种量对菌体湿重和右旋糖酐酶活力的影响曲线。
图5是实施例3中发酵培养基的装液量对菌体湿重和右旋糖酐酶活力的影响曲线。
图6是实施例3中发酵培养基的搅拌转速对菌体湿重和右旋糖酐酶活力的影响曲线。
图7是实施例3中发酵培养基的发酵温度对菌体湿重和右旋糖酐酶活力的影响曲线。
图8是实施例4中右旋糖酐酶酶解产物的TLC图。
图9是实施例4中右旋糖酐酶酶解产物的HPLC图。
图10是实施例5中右旋糖酐酶的SDS-PAGE图。
图11是实施例5中右旋糖酐酶的活性电泳图。
图12是实施例6中右旋糖酐酶对变异链球菌生长的抑制效果图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。
生物材料样品：产右旋糖酐酶的菌株为球毛壳菌(Chaetomium globosum)，已于2018年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCC No.15867，保藏地址是北京市朝阳区北辰西路1号院3号。
右旋糖酐酶酶活力测定方法：以葡聚糖T2000为底物(底物储藏液用acetate buffer 5.5配制，浓度为20mM)，反应混合物包括0.2mL稀释后的酶液，1.8mL acetate buffer 5.5配制的1％的底物储藏液，50℃水浴反应10min，立即加入3mL DNS终止反应，并于沸水中反应10min，冰水冷却后在540nm处测定吸光值。
酶活单位(U)定义为：50℃以葡聚糖T2000为底物每分钟释放相当于1umol葡萄糖的还原糖的量。
蛋白质浓度的测定：使用结晶牛血清白蛋白作为蛋白质标准来确定。将酶用蒸馏水稀释 至适当的浓度，并将4mL考马斯亮蓝加入到1mL稀释的酶液中使总体积为5ml。准确反应2min，在595nm处观察吸光度并计算蛋白质浓度。
葡聚糖分子量分布检测：Waters1525高效液相色谱仪(配示差检测器和Empower工作站Waters 2410)，使用Ultrahydrogel TM Linear(300mm×7.8mmid×2)，在30℃下以0.9mL/分钟的流速操作色谱仪。保留时间和Mw的校准曲线用200,30.06,13.503,0.9750,0.27kDa葡聚糖标准品(sigma，USA)和葡萄糖(180Da)制备。
实施例1：产右旋糖酐酶的菌株的筛选与鉴定
将土壤样品用生理盐水稀释至10 -5涂布PDA培养基(马铃薯200g，葡萄糖20g，琼脂15-20g)，28℃培养5天，经过初筛得到纯菌株，将菌株用蓝色葡聚糖T2000培养基进行复筛，通过透明圈比较，得到一株产酶量较高的菌株。经过电镜观察和18S rDNA鉴定，此菌株为球毛壳菌，18S rDNA的核苷酸序列如SEQ ID NO.1所示。球毛壳菌的菌落特征照片见图1，扫描电镜图见图2。
实施例2：发酵培养基的优化
挑取球毛壳菌丝体接入到种子培养基中，使用回旋式恒温摇床进行培养，摇床转速为220r/min，培养温度28℃，培养60h，制得种子液；种子培养基成分为：葡萄糖5g/L、氯化钠0.5g/L、酵母粉5g/L、磷酸氢二钾0.5g/L、硫酸镁0.2g/L，灭菌后加入过滤除菌的卡那霉素至终浓度为50μg/mL。
将种子液以1％的接种量接入装有50mL发酵培养基的250mL摇瓶中，发酵温度为28℃。
用不同类型的葡聚糖(葡聚糖T20，T40，T2000)和不同物质(葡萄糖，麦芽糖，乳糖，可溶性淀粉，玉米糊精，蔗糖，鱼粉蛋白胨)作为碳源；酵母提取物，尿素，胰蛋白胨，牛肉膏，硫酸铵，马铃薯淀粉作为氮源；设置最佳碳源和最佳氮源的浓度分别为0.5％、1％、2％、3％、4％、5％。设置K 2HPO 4和MgSO 4浓度分别为0、0.05％、0.1％、0.15％、0.2％、0.25％、0.3％、0.4％。对碳源浓度，氮源浓度，K 2HPO 4，MgSO 4做正交实验，测定产酶量和细胞生长量，以确定最佳培养基。
结果表明，不同物质作为碳源，菌株有不同的右旋糖酐酶产生能力。葡聚糖用作发酵碳源时的产酶量最高。与其他碳源相比，葡聚糖T20是右旋糖酐酶形成的最佳诱导剂。发酵培养基的正交试验结果表明：葡聚糖T20，酵母提取物，K 2HPO 4和MgSO 4的最佳浓度分别为20g/L，10g/L，2g/L和0.5g/L。在最佳条件下进行三个平行实验，酶活达到329.8920U/mL(相对标准偏差为3％)。
实施例3：发酵产酶条件的优化
挑取球毛壳菌丝体接入到种子培养基中，使用回旋式恒温摇床进行培养，摇床转速为220r/min，培养温度28℃，培养60h，制得种子液；种子培养基成分为：葡萄糖5g/L、氯化钠0.5g/L、酵母粉5g/L、磷酸氢二钾0.5g/L、硫酸镁0.2g/L，灭菌后加入过滤除菌的卡那霉素至终浓度为50μg/mL。
将种子液以1％的接种量接入装有50mL发酵培养基的250mL摇瓶中，发酵温度为28℃，以优化过的最优培养基为基础，对产酶发酵条件进行依次优化，初始pH分别设置为4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、9.0。接种量分别设置为1％、2％、3％、4％、5％、6％。装液量分别设置为20mL、30mL、40mL、50mL、60mL、70mL、80mL、90mL。搅拌转速分别设置140rpm、160rpm、180rpm、200rpm、220rpm。发酵温度分别设置为24℃、26℃、28℃、30℃、32℃、34℃。发酵时间为8d。测定菌体湿重、右旋糖酐酶活力等。
结果见图3-7，经过发酵条件的依次优化，发酵的最佳条件为：发酵初始pH值为7.0，接种量为3％，体积为50mL/250mL，转速为200r/min，培养温度为26℃。在最佳条件下进行三个平行实验，酶活达到698.22U/mL(相对标准偏差为3％)。
右旋糖酐酶的核苷酸序列如SEQ ID NO.2所示。
对比例1
挑取球毛壳菌丝体接入到种子培养基中，使用回旋式恒温摇床进行培养，摇床转速为220r/min，培养温度28℃，培养60h，制得种子液；种子培养基成分为：葡萄糖5g/L、氯化钠0.5g/L、酵母粉5g/L、磷酸氢二钾0.5g/L、硫酸镁0.2g/L，灭菌后加入过滤除菌的卡那霉素至终浓度为50μg/mL。
发酵培养基的成分为：葡聚糖T2025g/L，酵母提取物10g/L，磷酸氢二钾和硫酸镁添加量分别为2.5g/L和2.5g/L。
将种子液以1％的接种量接入装有50mL发酵培养基的250mL摇瓶中，发酵转速为220r/min，发酵温度为28℃，初始pH自然。发酵培养8天，得到酶活为38.01U/mL。
实施例4：右旋糖酐酶的酶解产物分析和对葡聚糖的水解作用
用0.2M乙酸缓冲液(pH 5.5)制备葡聚糖T2000(3％)，并在50℃温育。加入球毛壳菌右旋糖酐酶至终浓度为5U/mL。将反应液在50℃下机械搅拌90分钟，分别在30min,60min,和90min时取样，将样品在沸水浴中加热30分钟以终止反应，然后用薄层色谱(TLC)分析， 使用以7:5:4:2(v/v/v/v)正丁醇：异丙醇：乙酸：水溶剂体系展开的硅胶板。葡萄糖，异麦芽糖和异麦芽三糖作标准品，热灭活的右旋糖酐酶的混合物作为对照样品。通过含二苯胺(4g)，苯胺(4mL)和85％磷酸(20mL)的200mL丙酮溶液喷雾在TLC板上使碳水化合物显色，然后在95℃下加热10分钟。此外，将反应混合物作为样品处理并通过HPLC-MS(Waters，USA)分析，使用WATERS ACQUITY UPLC色谱仪，BEHAMIDE(2.1×100mm1.7um)分析柱，柱温45℃下以0.3mL/min的流速，进样量2μL操作，连接WATERS MALDI SYNAPT Q-TOF MS质谱仪，以ESI-方式进行质谱测定。
考察右旋糖酐酶对高分子量的葡聚糖的水解能力，葡聚糖T2000(3％)由0.2M乙酸盐缓冲液(pH 5.5)配制，50℃孵育10min，加入球毛壳菌右旋糖酐酶，终浓度为2U/mL，均匀搅拌。反应混合物的样品以1至120分钟的时间间隔获得，煮沸30分钟以停止反应，反应溶液中的杂质用滤纸重复滤出，反应上清液加热煮沸15分钟，并再次过滤掉杂质。将稀释的样品通过混合纤维素酯膜(具有0.22μm孔径和25mm直径的滤膜)过滤，并通过HPLC(Waters1525，Milford，USA)进行分析，使用Ultrahydrogel TM Linear(300mm×7.8mmid×2)，在30℃下以0.9mL/分钟的流速操作色谱仪，然后连接到示差检测器和Empower工作站(Waters 2410)。
考察不同酶浓度对高分子量的葡聚糖的水解能力，用同样浓度的底物使酶的终浓度分别达到1U/ml、3U/ml、5U/ml、7U/ml，于50℃反应15min，之后操作如上所述；考察右旋糖酐酶对不同分子量葡聚糖的水解能力，用0.2M乙酸盐缓冲液(pH 5.5)分别配制3％葡聚糖T20、T40、T70、T2000，50℃保温10min，加入右旋糖酐酶使其终浓度为1U/mL，于50℃反应15min，之后操作如上所述。保留时间和Mw的校准曲线用200,30.06,13.503,0.9750,0.27kDa葡聚糖标准品(sigma，USA)和葡萄糖(180Da)制备。
右旋糖酐酶酶解产物的TLC和HPLC结果见图8，结果显示，来自球毛壳菌的右旋糖酐酶催化葡聚糖T2000水解的最终产物是异麦芽糖，异麦芽三糖和一些低聚异麦芽糖，不产生葡萄糖。表明球毛壳右旋糖酐酶是一种典型的内切葡聚糖酶。右旋糖酐酶水解葡聚糖T2000的结果如表1显示，在15分钟内葡聚糖分子量降至41000Da。反应15分钟的葡聚糖分子量比之后时间段下降更快，表明酶对分子量更高的α-葡聚糖有更高的亲和力。水解反应120分钟后得到的葡聚糖的Mw为2506Da。表2结果表明不同酶浓度对α-葡聚糖有不同的水解力，随着酶浓度的增大，葡聚糖降解速率增大。以上结果综合表明右旋糖酐酶对高分子量葡聚糖具有高的水解速率，且酶浓度越高，水解速率越快。
表1 不同反应时间右旋糖酐酶的酶解效果
Figure PCTCN2018096095-appb-000001
表2 不同右旋糖酐酶浓度对葡聚糖的酶解效果
Figure PCTCN2018096095-appb-000002
本发明通过考察右旋糖酐酶水解葡聚糖T2000，得到的水解物中不含有葡萄糖，且葡聚糖的分子量随反应时间和酶浓度的增大而变小，能有效地降解高分子量葡聚糖，在工业应用中有很大潜力。
实施例5：右旋糖苷酶的分离纯化方法
室温下添加50％至70％饱和度的固体硫酸铵于磁力搅拌器上不断搅拌来浓缩粗酶提取物，10,000×g离心10min收集沉淀蛋白。将沉淀蛋白置于透析袋内(最大透过8kD)于0.2M乙酸盐缓冲液(pH5.5)中，4℃透析过夜。
透析浓缩的酶样品用0.22μm孔径的膜滤器过滤后低温下上样到用预冷的0.2M乙酸盐缓冲液(pH5.5)预平衡的Sephadex G75柱(1.5cm i.d.×60cm)上，流速为12mL/h。以每管2mL收集级分进行峰收集。在280nm处测量每个级分的吸光度用以在色谱分离期间监测蛋白质，确定每个部分的蛋白质浓度。收集具有高右旋糖酐酶活性的级分测定右旋糖酐酶活性和蛋白质浓度。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)在还原条件下测定酶的纯度及其分子量，使用12％聚丙烯酰胺凝胶进行SDS-PAGE。电泳后，将凝胶用考马斯蓝 G-250染色。右旋糖酐酶的SDS-PAGE及活性电泳图见图9。
经测定，右旋糖酐酶的比酶活达到7535.8U/mg，最终纯化倍数为10.97，收率为18.7％，分子量为53kDa。
实施例6：右旋糖酐酶在防治龋齿方面的研究
(1)将过滤除菌的右旋糖酐酶用含1％蔗糖的脑心浸液肉汤培养基稀释，使其终浓度分别为0、10、20、30、40、50、60、70U/mL。取4.5mL酶液分别加入500uL变异链球菌的菌悬液(OD600nm＝1.0)于37℃，220r/min培养24h后测定600nm处的吸光值，未接菌液的培养基为对照。
(2)在96深孔细胞培养板中分别加入含不同浓度右旋糖酐酶的1％蔗糖脑心浸液肉汤培养基180uL，接种上述菌悬液20uL，对照组不含酶液。37℃、10％CO 2培养箱培养24h后用生理盐水清洗孔板去除未附着的菌体。晾干后用1％的结晶紫染色10min后用蒸馏水洗去染色液，加入200uL无水乙醇于150r/min震荡40min后在酶标仪上测600nm处吸光值。生物膜形成抑制率＝(1-实验组/对照组)×100％。
(3)在96深孔细胞培养板中分别加入含1％蔗糖脑心浸液肉汤培养基180uL，接种上述菌悬液20uL，37℃、10％CO 2培养箱培养24h后移去菌液，加入400μL无菌水轻轻洗涤数次，加入以含1％蔗糖培养基稀释至不同浓度的右旋糖酐酶200μL，37℃、10％CO 2培养箱培养24h后按照上述方法测600nm处吸光值。生物膜清除率＝(1-实验组/对照组)×100％。
从图10可以看出，来自球毛壳菌的右旋糖酐酶对变异链球菌的生长有明显的抑制作用，随着右旋糖酐酶浓度的增大，对变异链球菌生长的抑制作用增强，当右旋糖酐酶的浓度达到50U/mL时，对变异链球菌生物膜形成的抑制率达到71.58％，对变异链球菌形成的生物膜的清除率达到49.07％。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2018096095-appb-000003
Figure PCTCN2018096095-appb-000004
Figure PCTCN2018096095-appb-000005

Claims (11)

  1. 一种培养球毛壳菌的方法，其特征在于：将所述球毛壳菌接种于发酵培养基中，发酵培养基的成分为：碳源和氮源浓度为0.5～5％，K 2HPO 4和MgSO 4浓度为0～4％，初始pH分别为4.5～9.0，接种量为1～6％，摇瓶装液量为20～90mL/250mL，摇床转速为140～220r/min；发酵温度为24～34℃，所述球毛壳菌已于2018年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCC No.15867，保藏地址是北京市朝阳区北辰西路1号院3号。
  2. 根据权利要求1所述的培养方法，其特征在于：所述碳源包括α-乳糖，马铃薯淀粉，葡萄糖，鱼粉蛋白胨，麦芽糖，葡聚糖T20、T40、T2000，蔗糖、可溶性淀粉中的任意一种或多种。
  3. 根据权利要求1所述的培养方法，其特征在于：所述氮源包括尿素、硝酸钠、鱼粉蛋白胨、牛肉浸膏、大豆蛋白胨、酵母提取物、胰蛋白胨、硫酸铵、马铃薯淀粉中的任意一种或多种。
  4. 根据权利要求1-3任一项所述的培养方法，其特征在于：发酵培养基的成分为：葡聚糖T2020g/L，酵母提取物10g/L，K 2HPO 4和MgSO 4添加量分别为2g/L和0.5g/L，初始pH为7.0，接种量为3％，装液量为50/250mL，发酵转速220r/min，培养温度为26℃。
  5. 一种球毛壳菌(Chaetomium globosum)，其特征在于：菌株已于2018年6月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCC No.15867，保藏地址是北京市朝阳区北辰西路1号院3号。
  6. 权利要求5所述的菌株在水解葡聚糖中的应用。
  7. 一种右旋糖酐酶的纯化方法，其特征在于：将权利要求5所述的球毛壳菌生产右旋糖酐酶所得的发酵液依次用硫酸铵盐析，透析除盐，凝胶柱纯化，超滤浓缩进行纯化。
  8. 权利要求7所述纯化方法得到的右旋糖苷酶在水解葡聚糖中的应用。
  9. 权利要求5所述球毛壳菌在制备防治龋齿的药物或口腔用品方面的应用。
  10. 权利要求5所述球毛壳菌在食品领域中的应用。
  11. 一种防治龋齿的药物，其特征在于：包含权利要求5所述球毛壳菌生产的右旋糖酐酶。
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