WO2022160495A1 - 一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法及其专用复合酶 - Google Patents

一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法及其专用复合酶 Download PDF

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WO2022160495A1
WO2022160495A1 PCT/CN2021/091850 CN2021091850W WO2022160495A1 WO 2022160495 A1 WO2022160495 A1 WO 2022160495A1 CN 2021091850 W CN2021091850 W CN 2021091850W WO 2022160495 A1 WO2022160495 A1 WO 2022160495A1
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galactomannan
galactomannans
enzyme
melibiose
oligosaccharides
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French (fr)
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王欣妍
廖吉丽
黄曹兴
勇强
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南京林业大学
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    • C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase

Definitions

  • the invention belongs to the technical field of microbial culture, and in particular relates to a method for preparing small-molecule galactomannans (galactomannans with a molecular weight of less than 20,000) and galactomanno-oligosaccharides by hydrolyzing galactomannans and a special method thereof complex enzyme.
  • Dietary fiber has become one of the research hotspots in the context of "Healthy China” due to its unique physiological functions. Dietary fibers include natural polysaccharides, oligosaccharides, and small-molecule polysaccharides, among which oligosaccharides and small-molecule polysaccharides have gained more and more attention due to their outstanding effects on proliferating intestinal beneficial bacteria, increasing intestinal biodiversity and enhancing immunity. more and more people's attention.
  • small-molecule saccharides and oligosaccharides are obtained by degrading polysaccharides, so how to increase the content of low-polymerization degree components has become one of the key technologies for the preparation of small-molecule saccharides and oligosaccharides.
  • the main production processes include: dilute acid hydrolysis, enzymatic hydrolysis, Physical degradation method and combined degradation method, biological enzymatic method are favored by more and more people due to mild preparation conditions, high selectivity, strong controllability and simplified product separation and purification procedures.
  • Using microbial fermentation technology to control the synthesis of enzyme components can also effectively control the generation of by-products, thereby simplifying the extraction process and production costs. Therefore, the production of small molecular galactomannans and galactomannans based on biological enzymes has broad development. prospect.
  • the method of enzymatic preparation of small molecule galactomannan and galactomannan oligosaccharide is based on galactomannan-containing locust bean gum, fenugreek gum, succulent gum and wild acacia, etc. - It is prepared by partial enzymatic hydrolysis under the action of mannanase.
  • ⁇ -mannanase obtained by microorganism is a complex enzyme system composed of ⁇ -mannanase (also called endo- ⁇ -mannanase) and ⁇ -mannosidase, among which ⁇ -mannanase
  • the enzyme mainly degrades the ⁇ -1,4-glycosidic bonds on the main chain of galactomannan, and degrades macromolecular polysaccharides into small molecules.
  • ⁇ -mannosidase degrades small molecular polysaccharides or oligosaccharides into monosaccharides.
  • ⁇ -mannosidase will lead to an increase in the monosaccharide content of the degradation product and a decrease in the yield of small molecule galactomannans and galactomannosaccharides.
  • the content of ⁇ -mannosidase (usually expressed by enzyme activity) should be as low as possible.
  • Galactomannan is a highly branched polymer whose main chain structure is formed by mannose through ⁇ -1,4-glycosidic bonds, and galactose is connected with the main chain by ⁇ -1,6-glycosidic bonds.
  • the molar ratio of mannose and galactose molecules is 1.6, that is, there are 2 galactose molecules connected to every 3 mannose molecules on the main chain on average.
  • the galactose branched chain forms a steric hindrance effect on ⁇ -mannanase, which hinders the degradation of ⁇ -mannanase.
  • the fractions of small molecular glycans and galactomannooligosaccharides with high biological activity in the sugar degradation products were not high in the degradation products.
  • Alpha-galactosidase is an enzyme that can specifically hydrolyze the alpha-1,6-glycosidic bond formed between mannose and galactose in galactomannan molecules.
  • ⁇ -mannanase and ⁇ -galactosidase to synergistically hydrolyze galactomannan with a high degree of branching can reduce the effect of galactose branched chains on the hydrolysis of galactomannan main chain by ⁇ -mannanase.
  • the content of small molecule galactomannan and galactomannanosaccharide components in the degradation product is increased.
  • Trichoderma reesei also has the ability to synthesize ⁇ -mannosidase. Therefore, when synthesizing an enzyme solution with the optimal ratio of ⁇ -mannanase and ⁇ -galactosidase, it is required that the ⁇ -mannosidase in the enzyme solution be The activity of mannosidase should be as low as possible, so that the enzyme liquid obtained by fermentation can be directly used for the production of small molecular galactomannans and galactomannosaccharides without purification, thereby further reducing the production cost. At this stage, it has not achieved very good results.
  • a technical problem to be solved by the present invention is to provide a preparation method of a special compound enzyme for preparing small molecular galactomannans and galacto-oligosaccharides by hydrolyzing galactomannans, Using Trichoderma reesei as an enzyme-producing strain, and using microcrystalline cellulose and/or melibiose as a carbon source for fermentation and culture, an enzyme liquid with better enzyme activity can be obtained in one step, the operation is simple, and the production cost is greatly reduced.
  • Another technical problem to be solved by the present invention is to provide a special compound enzyme for preparing small molecular galactomannans and galactomannosaccharides by hydrolyzing galactomannans, and the ⁇ -mannosidase activity in the enzyme solution is relatively low Low molecular weight, galactomannan can be directly hydrolyzed to prepare small molecule galactomannan and galactomanno-oligosaccharide without purifying and removing ⁇ -mannosidase.
  • Another technical problem to be solved by the present invention is to provide the application of the above-mentioned enzyme solution in enzymatic hydrolysis to prepare small molecule galactomannans and galactomannan oligosaccharides, and the enzyme solution can effectively improve the small molecule galactomannans and galactomannooligosaccharide yield, reduce production costs, and have very good application prospects.
  • a preparation method of a special compound enzyme for hydrolyzing galactomannan to prepare small molecule galactomannan and galactomannan oligosaccharide, using Trichoderma reesei as an enzyme-producing strain, using microcrystalline cellulose and melibiose Fermentation culture is carried out for carbon source. After the fermentation culture is completed, the culture solution is processed to obtain a special compound enzyme for hydrolyzing galactomannans to prepare small molecular galactomannans and galactomannanosaccharides.
  • the total concentration of microcrystalline cellulose and melibiose is 20.0-35.0 g/L.
  • the preparation method of the special compound enzyme for preparing small molecular galactomannan and galactomannooligosaccharide by hydrolyzing galactomannan the concentration ratio of microcrystalline cellulose and melibiose is 1:0.1-1:6 .
  • the preparation method of the special compound enzyme for preparing small-molecule galactomannan and galacto-oligosaccharide by hydrolyzing galactomannan when the total concentration is 20.0g/L, the concentration of microcrystalline cellulose and melibiose is 20.0 g/L.
  • the concentrations are 15g/L and 5g/L, 10g/L and 10g/L, 5g/L and 15g/L respectively; when the total concentration is 25.0g/L, the concentrations of microcrystalline cellulose and melibiose are 20g respectively /L and 5g/L, 15g/L and 10g/L, 10g/L and 15g/L, 5g/L and 20g/L; when the total concentration was 30.0g/L, the concentrations of microcrystalline cellulose and melibiose were The concentrations are 25g/L and 5g/L, 20g/L and 10g/L, 15g/L and 15g/L, 10g/L and 20g/L, 5g/L and 25g/L respectively; when the total concentration is 35.0g/L When L, the concentrations of microcrystalline cellulose and melibiose were 30g/L and 5g/L, 25g/L and 10g/L, 20g/L and 15g/L, 15g/L and 20g/L
  • the preparation method of the special compound enzyme for preparing small-molecule galactomannan and galacto-oligosaccharide by hydrolyzing galactomannan the concentration of microcrystalline cellulose is 20g/L, and the concentration of melibiose is 5g/L L.
  • the preparation method of the special compound enzyme for preparing small-molecule galactomannans and galacto-oligosaccharides by hydrolyzing galactomannans comprises the following steps:
  • Enzyme production medium composed of the following components: glucose 1.0g/L, carbon source, ammonium sulfate 4.72g/L, urea 2.15g/L, potassium dihydrogen phosphate 2.0g/L, anhydrous calcium chloride 0.3g/L, magnesium sulfate heptahydrate 0.3g/L, ferrous sulfate heptahydrate 0.005g/L, manganese sulfate heptahydrate 0.0016g/L, zinc sulfate heptahydrate 0.0014g/L, cobalt chloride 0.002g/L; 50 mL of sodium citrate buffer solution with a concentration of 1 mol/L was added thereto to adjust the pH of the medium to 4.8.
  • Fermentation put 50 mL of the enzyme production medium in a 250 mL conical flask with a cotton stopper, insert the Trichoderma reesei spores according to 10% of the inoculum, and place it at 28-30°C and shake at a constant temperature of 170 rpm. After culturing in bed for 4 days, the culture medium was centrifuged at 3000 rpm for 10 minutes to obtain a supernatant, which was a special solution for hydrolyzing galactomannans to prepare small-molecule galactomannans and galactomanno-oligosaccharides. complex enzyme.
  • the special compound enzyme obtained by the preparation method for hydrolyzing galactomannan to prepare small molecule galactomannan and galactomannan oligosaccharide obtained by the preparation method for hydrolyzing galactomannan to prepare small molecule galactomannan and galactomannan oligosaccharide.
  • a method for preparing small-molecule galactomannans and galacto-oligosaccharides by hydrolyzing galactomannans the steps are as follows:
  • step 2) Using the compound enzyme solution obtained in step 1), without separation and compounding, directly using galactomannan as a substrate, and performing enzymatic hydrolysis to prepare small-molecule galactomannan and galactomannan oligosaccharide.
  • step 1) the weight ratio of microcrystalline cellulose and melibiose is 2:1, and the enzymatic activity ratio of ⁇ -mannanase and ⁇ -galactosidase is not less than 7.
  • step 2) during the enzymatic hydrolysis reaction, the substrate concentration is 2%, the enzyme dosage is 20 U/g galactomannan, and the pH value is 4.8.
  • the advantages of the present invention include:
  • the present invention adopts Trichoderma reesei as an enzyme-producing strain, and uses microcrystalline cellulose and melibiose as carbon sources and inducers to ferment and produce hydrolyzed galactomannans to prepare small-molecule galactomannans and galactomannans Special compound enzyme for oligosaccharides; at the same time, the content (activity) of ⁇ -mannosidase in the enzyme solution is not higher than 0.05U/mL.
  • the preparation of the present invention can directly hydrolyze galactomannans without purifying and removing ⁇ -mannosidase to prepare small-molecule galactomannans and galacto-oligosaccharides, which can effectively improve small-molecule galactomannans and galactomannooligosaccharide yield, and reduce production costs.
  • Figure 1 shows the results of complex enzymatic hydrolysis of ⁇ -mannanase and ⁇ -galactosidase
  • Figure 2 shows the enzymatic activity results of ⁇ -mannanase and ⁇ -galactosidase under different ratios of microcrystalline cellulose and melibiose when the total substrate concentration is 20, 25, 30, and 35 g/L, respectively. ;
  • Figure 3 shows the enzymatic hydrolysis results of enzyme solutions with different ⁇ -mannanase and ⁇ -galactosidase activity ratios.
  • chromatographic conditions are as follows: chromatograph: Agilent high performance liquid chromatograph 1260, chromatographic column: Waters Ultrahydrogel TM 2000 (7.8 ⁇ 300mm), Waters Ultrahydrogel TM 250 (7.8 ⁇ 300mm) and Waters Ultrahydrogel TM 120 (7.8 ⁇ 300mm) three columns in sequence In series, guard column: Waters Ultrahydrogel TM Guard Column (6 ⁇ 40mm), detector: differential detector, mobile phase: water, mobile phase flow rate: 0.60mL/min, column temperature: 65°C, injection volume: 10.0 ⁇ L, Molecular weight determination was carried out using polyethylene glycol as a standard sample.
  • the determination method is as follows: take 0.3 g of small molecule galactomannan and galactomannan oligosaccharide sample and place it in a hydrolysis bottle, add 87 mL of 4% H 2 SO 4 to react at 121 ° C for 1 h, and take 1 mL of liquid after the reaction is completed. The pH of the reaction solution was adjusted to neutrality with 50% NaOH, and the supernatant was collected by centrifugation (10,000 rpm, 5 min).
  • Ion chromatography test conditions are as follows: Chromatograph: Dionex Ion Chromatograph ICS-5000, chromatographic column: 2 ⁇ 250mm Dionex AminoPac PA10, guard column: 2 ⁇ 50mm Dionex AminoPac PA10, detector: conductivity detector, mobile phase: 3mmol hydrogen Sodium oxide; flow rate: 0.20 mL/min; column temperature: 30 °C; injection volume: 10.0 ⁇ L, measured by external standard method. Then the purity of small molecule galactomannans and galactomannosaccharides in the sample is calculated as follows:
  • ⁇ -mannanase activity unit (U) is calculated as the enzyme amount of ⁇ -mannanase required to hydrolyze the substrate to produce 1 ⁇ mol of reducing sugar (calculated as mannose) per minute.
  • pNPG p-nitrophenol- ⁇ -D-galactopyranoside
  • pNPM p-nitrophenol- ⁇ -D-mannopyranoside
  • Trichoderma reesei uses microcrystalline cellulose and melibiose as carbon sources to ferment and produce enzymes, including the following steps:
  • composition of enzyme production medium (g/L): glucose 1.0, microcrystalline cellulose 25.0 or melibiose 25.0, ammonium sulfate 4.72, urea 2.15, potassium dihydrogen phosphate 2.0, anhydrous calcium chloride 0.3, heptahydrate Magnesium sulfate 0.3, ferrous sulfate heptahydrate 0.005, manganese sulfate heptahydrate 0.0016, zinc sulfate heptahydrate 0.0014, cobalt chloride 0.002. Add 50mL of 1mol/L sodium citrate buffer to adjust the pH of the medium to 4.8.
  • the above-mentioned 50 mL culture medium was placed in a 250 mL conical flask with a cotton stopper, inoculated with Trichoderma reesei spores according to 10% of the inoculum, and placed in a constant temperature shaker at 28-30°C and 170 rpm for 4 days. After the incubation, the culture medium was centrifuged at 3000 rpm for 10 min, and the supernatant (enzyme liquid) was taken to measure the enzymatic activities of ⁇ -galactosidase, ⁇ -mannosidase and ⁇ -mannanase, respectively.
  • Trichoderma reesei used microcrystalline cellulose as the carbon source to ferment and produce enzymes
  • the ⁇ -mannanase activity of the obtained enzyme solution 1 was 3.917 U/mL
  • the ⁇ -galactosidase activity was 0.099 U/mL.
  • ⁇ -mannanase activity was 0.02U/mL.
  • Trichoderma reesei fermented melibiose as carbon source to produce enzyme, and the ⁇ -galactosidase activity in the obtained enzyme solution 2 was 0.452 U/mL.
  • the galactomannan-containing leguminous seeds were mechanically pulverized to 20-100 mesh, added with distilled water at a solid-to-liquid ratio of 1:50, extracted at 50 °C for 24 hours, and centrifuged at 10,000 rpm for 10 minutes The supernatant was obtained, and absolute ethanol was added to the supernatant, and the obtained precipitate was vacuum-dried to obtain a powdery solid of galactomannan.
  • the enzyme liquid 1 and the enzyme liquid 2 obtained in Example 1 were compounded, so that the enzyme activity ratios of ⁇ -mannanase and ⁇ -galactosidase were 2, 4, 6, 8, 10, 12, 15, 20, 30, 40. Subsequently, 20.0 g of the above galactomannan was weighed into a 2L enzyme reaction tank, distilled water, enzyme solution, and 1 mol/L citric acid buffer were added to make the volume of the reaction solution 1000 mL. Add 20U/g galactomannan, pH 4.8, and react for 24h at 50°C.
  • the enzymatic hydrolyzate was treated at 100°C for 10 minutes to inactivate the enzyme, and the inactivated enzymatic hydrolyzate was centrifuged at 10,000 rpm for 10 minutes. Enzymatic hydrolysate of sugars and galactomanno-oligosaccharides.
  • step (1) Take 1000 mL of the supernatant of the enzymatic hydrolysis solution containing small molecule galactomannans and galactomanno-oligosaccharides in step (1), add absolute ethanol under stirring conditions, and make the ethanol concentration in the system 40% (v/v), centrifuge at 10,000 rpm for 10 min to obtain supernatant and precipitate. The precipitate was washed three times with 40% (v/v) ethanol aqueous solution, centrifuged (10,000 rpm, 10 min), and freeze-dried to obtain a component, which was named GalM40, and the small molecule galactomannan was determined by gel chromatography. The molecular weight of the glycan component GalM40, and the content of its galactomannan degradation products were determined by acid hydrolysis and ion chromatography. The supernatant was continued for the next stage of fractionation.
  • step (3) Take the supernatant liquid after the solid-liquid separation in step (2), add absolute ethanol under stirring conditions to make the ethanol concentration in the system 50% (v/v), and centrifuge at 10,000 rpm for 10 min A supernatant and a pellet were obtained. The precipitate was washed 3 times with a 50% (v/v) ethanol aqueous solution, centrifuged (10,000 rpm, 10 min), and freeze-dried to obtain a component, which was named GalM50, and the small molecule galactomannan was determined by gel chromatography. The molecular weight of the glycan component GalM50, and the content of its galactomannan degradation products were determined by acid hydrolysis and ion chromatography. The supernatant was continued for the next stage of fractionation.
  • step (3) Take the supernatant after the solid-liquid separation in step (3), add absolute ethanol under stirring conditions to make the ethanol concentration in the system 65% (v/v), and centrifuge at 10,000 rpm for 10 min A supernatant and a pellet were obtained.
  • the precipitate was washed 3 times with a 65% (v/v) ethanol aqueous solution, centrifuged (10000 rpm, 10 min), and freeze-dried to obtain a component, which was named GalM65, and the small molecule galactomannan was determined by gel chromatography.
  • the molecular weight of the glycan component GalM65, and the content of its galactomannan degradation products were determined by acid hydrolysis and ion chromatography. The supernatant was continued for the next stage of fractionation.
  • step (4) take the supernatant after the solid-liquid separation in step (4), remove the ethanol by rotary evaporation under reduced pressure at 70° C. and 160 mbar, and take a part of the supernatant to measure its galactose by acid hydrolysis and ion chromatography
  • the content of mannan degradation products, the monosaccharide in the remaining liquid was removed by nanofiltration (200Da), and then the retentate was concentrated by rotary evaporation under reduced pressure at 70 ° C and 160 mbar, and the obtained concentrated liquid was dried to obtain the component GalMOS, which was obtained by condensation.
  • Gel chromatography was used to determine the molecular weight of the galactomannooligosaccharide component GalMOS.
  • Figure 1 shows the results of complex enzymatic hydrolysis of ⁇ -mannanase and ⁇ -galactosidase.
  • Figure 1 shows the results of complex enzymatic hydrolysis of ⁇ -mannanase and ⁇ -galactosidase.
  • Figure 1 shows the results of complex enzymatic hydrolysis of ⁇ -mannanase and ⁇ -galactosidase.
  • Figure 1 shows the results of complex enzymatic hydrolysis of ⁇ -mannanase and ⁇ -galactosidase.
  • the total yield and GalMOS yield of the three small molecule galactomannans GalM40, GalM50 and GalM65 showed a slow upward trend, and the total sugar The yield also showed a slow upward trend.
  • the later stage with the continuous increase of ⁇ -galactosidase, the total yield of the three small-molecule galactomannans GalM40, GalM50 and GalM65 and the GalMOS yield began to decrease, and the total
  • the compound production enzyme of microcrystalline cellulose and melibiose the steps are as follows:
  • the enzyme production medium is the same as in Example 1, wherein the substrate is replaced by a mixture of microcrystalline cellulose and melibiose in different proportions, and the sum of the concentrations of the two substrates is 20.0, 25.0, 30.0, and 35.0 g/L, respectively.
  • Small molecule galactomannan and galactomannan oligosaccharide are prepared by enzymatic hydrolysis of microcrystalline cellulose and melibiose compound enzyme liquid, and the steps are as follows:
  • the directional enzymatic hydrolysis of galactomannan is the same as in Example 2, wherein the enzyme solution used is the enzyme solution in Example 3, and the enzyme solution with different ⁇ -mannanase and ⁇ -galactosidase enzyme activity ratios is selected to carry out Enzymatic hydrolysis test.

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Abstract

本发明公开了一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法及其专用复合酶。该方法以微晶纤维素和蜜二糖为碳源，以里氏木霉为产酶菌株进行发酵培养，发酵培养获得上清液，即为具有β-甘露聚糖酶和α-半乳糖苷酶两种酶活的复合酶液。以上述复合酶液，不经分离复配，直接以半乳甘露聚糖为底物，进行酶水解制备出小分子半乳甘露聚糖和半乳甘露低聚糖。

Description

一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法及其专用复合酶 技术领域
本发明属于微生物培养技术领域，具体涉及一种水解半乳甘露聚糖制备小分子半乳甘露聚糖(分子量2万以下的半乳甘露聚糖)和半乳甘露低聚糖的方法及其专用复合酶。
背景技术
目前，我国人口的老龄化和疾病的年轻化引起了人们对健康问题的重视。在2016年，国家提出了“健康中国2030”规划纲要，旨在发展中国健康产业，改善人民健康。膳食纤维以其独特的生理功能成为“健康中国”背景下的研究热点之一。膳食纤维包括天然多糖，低聚糖和小分子聚糖等，其中低聚糖和小分子聚糖由于其突出的增殖肠道有益菌、增加肠道生物多样性以及增强免疫力的效果得到了越来越多的人的重视。通常来说，小分子聚糖和低聚糖是由多糖降解得到，因此如何提高低聚合度组分的含量成为小分子聚糖和低聚糖制备的关键技术之一。
目前，小分子半乳甘露聚糖和半乳甘露低聚糖因其出色的增强免疫力的作用得到了越来越多人的关注，其主要生产工艺包括：稀酸水解法、酶水解法、物理降解法及联合降解法，生物酶法由于制备条件温和、高选择性、可控性强并可简化产品分离纯化程序受到越来越多人的青睐。利用微生物发酵技术控制酶组分的合成还能够有效控制副产物的生成，进而简化提取工艺和生产成本，因此基于生物酶法生产小分子半乳甘露聚糖和半乳甘露低聚糖具有广阔发展前景。目前，酶法制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法是以含半乳甘露聚糖的洋槐豆胶、葫芦巴胶、田菁胶和野皂荚等为原料，在β-甘露聚糖酶的作用下部分酶水解而制得。通常，通过微生物得到β-甘露聚糖酶是由β-甘露聚糖酶(也称内切β-甘露聚糖酶)和β-甘露糖苷酶组成的复合酶系，其中，β-甘露聚糖酶主要降解半乳甘露聚糖主链上的β-1,4-糖苷键，将大分子多糖降解成小分子，β-甘露糖苷酶是将小分子的多糖或低聚糖降解成单糖。因而，β-甘露糖苷酶的存在将引起降解产物中单糖含量提高和小分子半乳甘露聚糖和半乳甘露低聚糖的得率降低。为此，在β-甘露聚糖酶降解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的反应体系中，β-甘露糖苷酶的含量(通常用酶活力表示)应尽可能低。
半乳甘露聚糖是一种高分支度的聚合物，其主链结构由甘露糖通过β-1,4-糖苷键形成，半乳糖以α-1,6-糖苷键与主链连接。例如，来源于田菁种子的半乳甘露聚糖分子中，甘露糖和半乳糖分子的摩尔比为1.6，即主链上平均每3个甘露糖分子上连有2个半乳糖分子。在β-甘露聚糖酶降解半乳甘露聚糖过程中，半乳糖支链对β-甘露聚糖酶形成空间位阻效应，阻碍β-甘露聚糖酶的降解，具体表现为半乳甘露聚糖降解产物中生物活性高的小分子聚糖和半乳 甘露低聚糖组分在降解产物中所占的比例不高。
破解β-甘露聚糖酶降解分支度高的半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖过程中由半乳糖支链对β-甘露聚糖酶形成的空间位阻效应的策略是在β-甘露聚糖酶反应体系中添加适量的α-半乳糖苷酶。α-半乳糖苷酶是一种能够特异性水解半乳甘露聚糖分子中甘露糖和半乳糖之间形成的α-1,6-糖苷键的酶。利用β-甘露聚糖酶和α-半乳糖苷酶协同水解分支度高的半乳甘露聚糖，可以降低半乳糖支链对β-甘露聚糖酶水解半乳甘露聚糖主链的影响，从而提高降解产物中小分子半乳甘露聚糖和半乳甘露低聚糖组分的含量。
但是，现阶段人们研究的重点是在甘露聚糖酶中加入α-半乳糖苷酶来提高降解产物中小分子半乳甘露聚糖和半乳甘露低聚糖的含量，这需要通过两步方法来实现，即，分别通过微生物发酵法获得α-半乳糖苷酶和β-甘露聚糖酶，再将α-半乳糖苷酶按配比加入β-甘露聚糖酶水解半乳甘露聚糖的体系中。通过一步法获得具有最优的β-甘露聚糖酶和α-半乳糖苷酶配比的酶液将极大的降低生产成本，同时应该注意的是应采用人体安全的微生物来制备，如里氏木霉(Trichoderma reesei)。但里氏木霉也具有合成β-甘露糖苷酶的能力，因此，在合成具有最优的β-甘露聚糖酶和α-半乳糖苷酶配比的酶液时，要求酶液中β-甘露糖苷酶的活力应尽可能低，使发酵获得的酶液无需纯化即可直接用于小分子半乳甘露聚糖和半乳甘露低聚糖的生产，从而进一步降低生产成本，而这一技术在现阶段并没有取得很好的效果。
发明内容
针对现有技术中存在的问题，本发明要解决的一个技术问题在于提供一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，以里氏木霉为产酶菌株，以微晶纤维素和/或蜜二糖为碳源进行发酵培养，一步处理即可得到酶活比较优的酶液，操作简单，生产成本大大降低。本发明要解决的另一个技术问题在于提供一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶，该酶液中β-甘露糖苷酶活力较低，无需纯化除去β-甘露糖苷酶即可直接水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖。本发明要解决的技术问题还有一个在于提供上述酶液在酶解制备小分子半乳甘露聚糖和半乳甘露低聚糖中的应用，该酶液可有效提高小分子半乳甘露聚糖和半乳甘露低聚糖的得率，降低生产成本，具有非常好的应用前景。
为了解决上述问题，本发明所采用的技术方案如下：
一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，以里氏木霉为产酶菌株，以微晶纤维素和蜜二糖为碳源进行发酵培养，发酵培养结束后，培养液经处理得到水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶。
所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，微晶纤维素和蜜二糖的总浓度为20.0～35.0g/L。
所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，微晶纤维素和蜜二糖的浓度比为1:0.1～1:6。
所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，当总浓度为20.0g/L时，微晶纤维素和蜜二糖的浓度分别为15g/L与5g/L、10g/L与10g/L、5g/L与15g/L；当总浓度为25.0g/L时，微晶纤维素和蜜二糖的浓度分别为20g/L与5g/L、15g/L与10g/L、10g/L与15g/L、5g/L与20g/L；当总浓度为30.0g/L时，微晶纤维素和蜜二糖的浓度分别为25g/L与5g/L、20g/L与10g/L、15g/L与15g/L、10g/L与20g/L、5g/L与25g/L；当总浓度为35.0g/L时，微晶纤维素和蜜二糖的浓度分别为30g/L与5g/L、25g/L与10g/L、20g/L与15g/L、15g/L与20g/L、10g/L与25g/L、5g/L与30g/L。
所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，微晶纤维素的浓度为20g/L，蜜二糖的浓度为5g/L。
所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，包括以下步骤：
(1)产酶培养基：由以下组分组成：葡萄糖1.0g/L，碳源，硫酸铵4.72g/L，尿素2.15g/L，磷酸二氢钾2.0g/L，无水氯化钙0.3g/L，七水合硫酸镁0.3g/L，七水合硫酸亚铁0.005g/L，七水合硫酸锰0.0016g/L，七水合硫酸锌0.0014g/L，氯化钴0.002g/L；向其中加入浓度为1mol/L的柠檬酸钠缓冲液50mL，调节培养基的pH至4.8。
(2)发酵：将产酶培养基50mL置于250mL带棉塞的三角瓶中，按10％的接种量接入里氏木霉孢子，置于28-30℃、170转/分的恒温摇床中培养4天，培养结束后培养液于3000转/分下离心10min，得到上清液，即为水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶。
所述制备方法所获得的用于水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶。
一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法，步骤如下：
1)以微晶纤维素和蜜二糖为碳源，以里氏木霉为产酶菌株进行发酵培养，发酵培养获得上清液，即为具有β-甘露聚糖酶和α-半乳糖苷酶两种酶活的复合酶液；
2)以步骤1)获得的复合酶液，不经分离复配，直接以半乳甘露聚糖为底物，进行酶水解制备出小分子半乳甘露聚糖和半乳甘露低聚糖。
步骤1)中，微晶纤维素与蜜二糖的重量比为2:1，β-甘露聚糖酶与α-半乳糖苷酶的酶活 比不低于7。
步骤2)中，酶解反应时底物浓度2％、酶加量20U/g半乳甘露聚糖、pH值4.8。
有益效果：与现有的技术相比，本发明的优点包括：
(1)本发明采用里氏木霉为产酶菌株，以微晶纤维素和蜜二糖为碳源和诱导物发酵产生水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶；同时，酶液中β-甘露糖苷酶含量(活力)不高于0.05U/mL。
(2)本发明制备的无需纯化除去β-甘露糖苷酶即可直接水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖，可有效提高小分子半乳甘露聚糖和半乳甘露低聚糖的得率，并降低生产成本。
附图说明
图1为β-甘露聚糖酶与α-半乳糖苷酶复配酶解结果；
图2为当总底物浓度分别为20、25、30、35g/L时，微晶纤维素和蜜二糖不同配比下β-甘露聚糖酶与α-半乳糖苷酶的酶活结果；
图3为具有不同β-甘露聚糖酶与α-半乳糖苷酶酶活比的酶液的酶解结果。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。
对以下实施例中使用的产品性能测试如下：
(1)小分子半乳甘露聚糖和半乳甘露低聚糖的分子量分布采用凝胶渗透色谱法(GPC)测定：
色谱条件如下：色谱仪：安捷伦高效液相色谱仪1260，色谱柱：Waters Ultrahydrogel TM 2000(7.8×300mm)、Waters Ultrahydrogel TM 250(7.8×300mm)和Waters Ultrahydrogel TM 120(7.8×300mm)三柱依次串联，保护柱：Waters Ultrahydrogel TM Guard Column(6×40mm)，检测器：示差检测器，流动相：水，流动相流速：0.60mL/min，柱温：65℃，进样体积：10.0μL，采用聚乙二醇作为标准样品进行分子量测定。
(2)小分子半乳甘露聚糖和半乳甘露低聚糖的糖含量采用酸水解法和离子色谱法测定：
测定方法如下：取小分子半乳甘露聚糖和半乳甘露低聚糖样品0.3g置于水解瓶中，加入87mL 4％H 2SO 4于121℃下反应1h，反应结束后取1mL液体用50％NaOH调节反应液pH至中性，并离心(10000转/分，5min)取上清液，最后用ICS-5000离子交换色谱仪测定反应液中甘露糖和半乳糖浓度。
离子色谱测试条件如下：色谱仪：戴安离子色谱仪ICS-5000，色谱柱：2×250mm Dionex  AminoPac PA10，保护柱：2×50mm Dionex AminoPac PA10，检测器：电导检测器，流动相：3mmol氢氧化钠；流速：0.20mL/min；柱温：30℃；进样体积：10.0μL，外标法测定。则样品中小分子半乳甘露聚糖和半乳甘露低聚糖的纯度计算如下：
Figure PCTCN2021091850-appb-000001
(3)β-甘露聚糖酶活力测定：
在25mL刻度试管中加入0.9mL 5g/L洋槐豆胶底物溶液，50℃预热5min，加入0.1mL适当稀释的酶液，于50℃下反应30min，立即加入3.0mL DNS试剂终止反应，随后沸水浴中处理7min，冷却后定容到25mL，充分摇匀，于540nm下测定反应混合物的吸光度，并根据吸光度与还原糖的相关关系，计算反应生成的还原糖的浓度。1个β-甘露聚糖酶活力单位(U)以每分钟水解底物产生1μmol还原糖(以甘露糖计)所需β-甘露聚糖酶的酶量进行计算。
(4)α-半乳糖苷酶活力测定：
于15mL试管中加入0.1mL适当稀释的酶液和0.9mL 1mmol/L pNPG(对硝基苯酚-α-D-吡喃半乳糖苷)溶液于50℃下保温10min，立即加入2.0mL 1mol/L的Na 2CO 3溶液终止反应，加入10mL蒸馏水，充分摇匀，于400nm下测定反应混合物的吸光度，并依据吸光度与对硝基苯酚的相关关系，计算反应生成的对硝基苯酚的浓度。1个α-半乳糖苷酶活力单位(U)以每分钟水解pNPG释放1μmol对硝基苯酚所需α-半乳糖苷酶的酶量进行计算。
(5)β-甘露糖苷酶活力测定：
于15mL试管中加入0.1mL适当稀释的酶液和0.9mL 1mmol/L pNPM(对硝基苯酚-β-D-吡喃甘露糖苷)溶液，于50℃下保温10min，立即加入2.0mL 1mol/L的Na 2CO 3溶液终止反应，加入10mL蒸馏水，充分摇匀，于400nm下测定反应混合物的吸光度，并依据吸光度与对硝基苯酚的相关关系，计算酶水解反应过程生成的对硝基苯酚的浓度。1个β-甘露糖苷酶酶活力单位(U)以每分钟水解pNPM释放1μmol对硝基苯酚所需β-甘露糖苷酶的酶量进行计算。
实施例1
里氏木霉分别以微晶纤维素和蜜二糖为碳源发酵产酶，包括以下步骤：
(1)产酶培养基组成(g/L)：葡萄糖1.0，微晶纤维素25.0或蜜二糖25.0，硫酸铵4.72，尿素2.15，磷酸二氢钾2.0，无水氯化钙0.3，七水合硫酸镁0.3，七水合硫酸亚铁0.005，七水合硫酸锰0.0016，七水合硫酸锌0.0014，氯化钴0.002。加入50mL 1mol/L的柠檬酸钠缓冲液调节培养基的pH至4.8。
(2)发酵产酶
将上述50mL培养基置于250mL带棉塞的三角瓶中，按10％的接种量接入里氏木霉孢子，置于28-30℃、170转/分的恒温摇床中培养4天。培养结束后，培养液于3000转/分下离心10min，取上清液(酶液)分别测定α-半乳糖苷酶、β-甘露糖苷酶和β-甘露聚糖酶的酶活。
结果表明，里氏木霉以微晶纤维素为碳源发酵产酶，所得酶液1中β-甘露聚糖酶酶活力为3.917U/mL，α-半乳糖苷酶活力为0.099U/mL，β-甘露聚糖酶活力为0.02U/mL。里氏木霉以蜜二糖为碳源发酵产酶，所得酶液2中α-半乳糖苷酶活力为0.452U/mL。
实施例2
将甘露聚糖酶和半乳糖苷酶复配酶解制备小分子半乳甘露聚糖和半乳甘露低聚糖，其步骤如下：
(1)半乳甘露聚糖定向酶水解
含半乳甘露聚糖的豆科种子(田菁)经机械粉碎至20～100目，按1:50固液比加入蒸馏水，于50℃抽提24h后，于10000转/分条件下离心10min获得上清液，并向上清液中加入无水乙醇，所得沉淀物经真空干燥得到半乳甘露聚糖粉状固体。
将实施例1中得到的酶液1和酶液2进行复配，使得β-甘露聚糖酶和α-半乳糖苷酶的酶活比分别为2、4、6、8、10、12、15、20、30、40。随后称取上述半乳甘露聚糖20.0g于2L酶反应罐中，加入蒸馏水、酶液、1mol/L柠檬酸缓冲液使反应液体积为1000mL，充分混合均匀，于底物浓度2％、酶加量20U/g半乳甘露聚糖、pH值4.8、50℃条件下反应24h。酶水解反应结束后，将酶水解物置于100℃下处理10min从而使酶失活，失活的酶解液于10000转/分条件下离心10min，上清液即为含小分子半乳甘露聚糖和半乳甘露低聚糖的酶解液。
(2)取步骤(1)中的含小分子半乳甘露聚糖和半乳甘露低聚糖的酶解液上清1000mL，在搅拌条件下加入无水乙醇，使体系中乙醇浓度为40％(v/v)，于10000转/分条件下离心10min得到上清液以及沉淀。沉淀用40％(v/v)的乙醇水溶液洗涤3次、离心(10000转/分、10min)、冷冻干燥得到组分，并将之命名为GalM40，采用凝胶色谱法测定小分子半乳甘露聚糖组分GalM40的分子量，并采用酸水解法和离子色谱法测定其半乳甘露聚糖降解产物含量。上清液继续用于下一级的分级分离。
(3)取步骤(2)中固液分离后的上清液，在搅拌条件下加入无水乙醇，使体系中乙醇浓度为50％(v/v)，于10000转/分条件下离心10min得到上清液以及沉淀。沉淀用50％(v/v)的乙醇水溶液洗涤3次、离心(10000转/分、10min)、冷冻干燥得到组分，并将之命名为GalM50，采用凝胶色谱法测定小分子半乳甘露聚糖组分GalM50的分子量，并采用酸水解法 和离子色谱法测定其半乳甘露聚糖降解产物含量。上清液继续用于下一级的分级分离。
(4)取步骤(3)中固液分离后的上清液，在搅拌条件下加入无水乙醇，使体系中乙醇浓度为65％(v/v)，于10000转/分条件下离心10min得到上清液以及沉淀。沉淀用65％(v/v)的乙醇水溶液洗涤3次、离心(10000转/分、10min)、冷冻干燥得到组分，并将之命名为GalM65，采用凝胶色谱法测定小分子半乳甘露聚糖组分GalM65的分子量，并采用酸水解法和离子色谱法测定其半乳甘露聚糖降解产物含量。上清液继续用于下一级的分级分离。
(5)取步骤(4)中固液分离后的上清液，于70℃、160mbar下减压旋转蒸发除去其中的乙醇，取一部分上清液采用酸水解法和离子色谱法测定其半乳甘露聚糖降解产物含量，其余液体通过纳滤(200Da)除去其中的单糖，然后将截留液于70℃、160mbar下减压旋转蒸发浓缩，获得的浓缩液经干燥得到组分GalMOS，采用凝胶色谱法测定半乳甘露低聚糖组分GalMOS的分子量。
图1为β-甘露聚糖酶与α-半乳糖苷酶复配酶解结果。由如图1可知，前期随着α-半乳糖苷酶活力的不断增加，3种小分子半乳甘露聚糖GalM40、GalM50和GalM65的总得率和GalMOS得率呈现出缓慢上升的趋势，总糖得率也呈现出缓慢上升的趋势，后期随着α-半乳糖苷酶的继续增加，3种小分子半乳甘露聚糖GalM40、GalM50和GalM65的总得率和GalMOS得率开始下降，总糖得率也开始下降。从图1中可知，当β-甘露聚糖酶和α-半乳糖苷酶的酶活比为8时，总糖得率最高，3种小分子半乳甘露聚糖GalM40、GalM50和GalM65的总得率和GalMOS组分得率也最高。同时，对得到的小分子半乳甘露聚糖的分子量进行测量，发现各组分的平均分子量分别为:GalM40:13100Da，GalM50:8930Da，GalM65:4310Da，GalMOS：1630Da。
实施例3
微晶纤维素和蜜二糖的复配产酶，其步骤如下：
(1)产酶培养基同实施例1，其中底物替换为微晶纤维素和蜜二糖不同比例的混合物，两种底物浓度之和分别为20.0、25.0、30.0、35.0g/L。
(2)发酵产酶方法同实施例1。
结果如图2所示，当总底物浓度为20g/L时，随着蜜二糖的不断增加，β-甘露聚糖酶的活力不断降低。但是当总底物浓度为25、30、35g/L时，随着蜜二糖的不断增加，β-甘露聚糖酶的酶活呈现一种先升后降的趋势。当总底物浓度为25g/L且微晶纤维素为20g/L和蜜二糖为5g/L时，β-甘露聚糖酶与α-半乳糖苷酶的酶活比达到最高。总体来说随着蜜二糖的增加，α-半乳糖苷酶酶活不断升高，当蜜二糖浓度过高时，α-半乳糖苷酶酶活逐步下降。
实施例4
利用微晶纤维素和蜜二糖复配产酶的酶液酶解制备小分子半乳甘露聚糖和半乳甘露低聚糖，其步骤如下：
半乳甘露聚糖的定向酶水解同实施例2，其中所用酶液为实施例3中的酶液，选择具有不同β-甘露聚糖酶与α-半乳糖苷酶酶活比的酶液进行酶水解测试。
结果如图3所示，微晶纤维素15g/L和蜜二糖10g/L所得酶液的酶解效果最好，总糖得率为86.47％，GalM OS的得率为16.51％，3种小分子半乳甘露聚糖GalM40、GalM50和GalM65的总得率为55.63％，此时β-甘露聚糖酶与α-半乳糖苷酶的酶活比为7.235。这个结果和前期β-甘露聚糖酶与α-半乳糖苷酶复配酶解的结果相呼应。

Claims (10)

  1. 一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，其特征在于，以里氏木霉为产酶菌株，以微晶纤维素和蜜二糖为碳源进行发酵培养，发酵培养结束后，培养液经处理得到水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶。
  2. 根据权利要求1所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，其特征在于，微晶纤维素和蜜二糖的总浓度为20.0～35.0g/L。
  3. 根据权利要求1所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，其特征在于，微晶纤维素和蜜二糖的浓度比为1:0.1～1:6。
  4. 根据权利要求1所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，其特征在于，当总浓度为20.0g/L时，微晶纤维素和蜜二糖的浓度分别为15g/L与5g/L、10g/L与10g/L、5g/L与15g/L；当总浓度为25.0g/L时，微晶纤维素和蜜二糖的浓度分别为20g/L与5g/L、15g/L与10g/L、10g/L与15g/L、5g/L与20g/L；当总浓度为30.0g/L时，微晶纤维素和蜜二糖的浓度分别为25g/L与5g/L、20g/L与10g/L、15g/L与15g/L、10g/L与20g/L、5g/L与25g/L；当总浓度为35.0g/L时，微晶纤维素和蜜二糖的浓度分别为30g/L与5g/L、25g/L与10g/L、20g/L与15g/L、15g/L与20g/L、10g/L与25g/L、5g/L与30g/L。
  5. 根据权利要求1所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，其特征在于，微晶纤维素的浓度为20g/L，蜜二糖的浓度为5g/L。
  6. 根据权利要求1所述水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶的制备方法，其特征在于，包括以下步骤：
    (1)产酶培养基：由以下组分组成：葡萄糖1.0g/L，碳源，硫酸铵4.72g/L，尿素2.15g/L，磷酸二氢钾2.0g/L，无水氯化钙0.3g/L，七水合硫酸镁0.3g/L，七水合硫酸亚铁0.005g/L，七水合硫酸锰0.0016g/L，七水合硫酸锌0.0014g/L，氯化钴0.002g/L；向其中加入浓度为1mol/L的柠檬酸钠缓冲液50mL，调节培养基的pH至4.8。
    (2)发酵：将产酶培养基50mL置于250mL带棉塞的三角瓶中，按10％的接种量接入里氏木霉孢子，置于28-30℃、170转/分的恒温摇床中培养4天，培养结束后培养液于3000转/分下离心10min，得到上清液，即为水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶。
  7. 权利要求1-6任一项所述制备方法所获得的用于水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的专用复合酶。
  8. 一种水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法，其特征在 于，步骤如下：
    1)以微晶纤维素和蜜二糖为碳源，以里氏木霉为产酶菌株进行发酵培养，发酵培养获得上清液，即为具有β-甘露聚糖酶和α-半乳糖苷酶两种酶活的复合酶液；
    2)以步骤1)获得的复合酶液，不经分离复配，直接以半乳甘露聚糖为底物，进行酶水解制备出小分子半乳甘露聚糖和半乳甘露低聚糖。
  9. 根据权利要求8所述的水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法，其特征在于，步骤1)中，微晶纤维素与蜜二糖的重量比为2:1，β-甘露聚糖酶与α-半乳糖苷酶的酶活比不低于7。
  10. 根据权利要求8所述的水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法，其特征在于，步骤2)中，酶解反应时底物浓度2％、酶加量20U/g半乳甘露聚糖、pH值4.8。
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