Method and special complex enzyme for hydrolyzing galactomannan (GM) to prepare small-molecule GM and galactomannan oligosaccharide (GMOS)

Abstract

A method and special complex enzyme for hydrolyzing a galactomannan (GM) to prepare a small-molecule GM and a galactomannan oligosaccharide (GMOS) is provided. The method includes: conducting fermentation with microcrystalline cellulose (MCC) and melibiose as carbon sources and Trichoderma reesei (T. reesei) as an enzyme-producing strain to obtain a supernatant, which is a complex enzyme solution with enzymatic activities of ?-mannanase and ?-galactosidase; and directly using the complex enzyme solution for enzymatic hydrolysis of a GM as a substrate to prepare the small-molecule GM and the GMOS.

Claims

1. A preparation method of an enzyme solution for hydrolyzing a galactomannan (GM) to prepare a small-molecule GM and a galactomannan oligosaccharide (GMOS), wherein the enzyme solution comprises enzymatic activities of ?-mannanase and ?-galactosidase, wherein an enzymatic activity ratio of the of the ?-mannanase to the ?-galactosidase is no less than 7:1, and wherein the small-molecule GM has a molecular weight of 20,000 Da or less, comprising: conducting a fermentation with Trichoderma reesei (T. reesei) as an enzyme-producing strain and microcrystalline cellulose (MCC) and melibiose as carbon sources, wherein the total concentration of the MCC and the melibiose is 20.0 g/L to 35.0 g/L; and after the fermentation is completed, centrifuging a resulting culture solution, wherein the supernatant comprises the enzyme solution for hydrolyzing the GM to prepare the small-molecule GM and the GMOS.

2. The preparation method of the enzyme solution for hydrolyzing the GM to prepare the small-molecule GM and the GMOS according to claim 1, wherein a concentration ratio of the MCC to the melibiose is 1:0.1 to 1:6.

3. The preparation method of the enzyme solution for hydrolyzing the GM to prepare the small-molecule GM and the GMOS according to claim 1, wherein when a total concentration is 20.0 g/L, concentrations of the MCC and the melibiose are 15 g/L and 5 g/L, 10 g/L and 10 g/L, or 5 g/L and 15 g/L, respectively; wherein when the total concentration is 25.0 g/L, concentrations of the MCC and the melibiose are 20 g/L and 5 g/L, 15 g/L and 10 g/L, 10 g/L and 15 g/L, or 5 g/L and 20 g/L, respectively; wherein when the total concentration is 30.0 g/L, concentrations of the MCC and the melibiose are 25 g/L and 5 g/L, 20 g/L and 10 g/L, 15 g/L and 15 g/L, 10 g/L and 20 g/L, or 5 g/L and 25 g/L, respectively; and wherein when the total concentration is 35.0 g/L, concentrations of the MCC and the melibiose are 30 g/L and 5 g/L, 25 g/L and 10 g/L, 20 g/L and 15 g/L, 15 g/L and 20 g/L, 10 g/L and 25 g/L, or 5 g/L and 30 g/L, respectively.

4. The preparation method of the enzyme solution for hydrolyzing the GM to prepare the small-molecule GM and the GMOS according to claim 1, wherein a concentration of the MCC is 20 g/L, and a concentration of the melibiose is 5 g/L.

5. The preparation method of the enzyme solution for hydrolyzing the GM to prepare the small-molecule GM and the GMOS according to claim 1, comprising the following steps: (1) an enzyme-producing medium: comprising the following components: glucose: 1.0 g/L, MCC and melibiose, ammonium sulfate: 4.72 g/L, urea: 2.15 g/L, monopotassium phosphate (MKP): 2.0 g/L, anhydrous calcium chloride: 0.3 g/L, magnesium sulfate heptahydrate: 0.3 g/L, ferrous sulfate heptahydrate: 0.005 g/L, manganese sulfate heptahydrate: 0.0016 g/L, zinc sulfate heptahydrate: 0.0014 g/L, and cobalt chloride: 0.002 g/L; and adding 50 mL of a sodium citrate buffer with a concentration of 1 mol/L to adjust a pH of the enzyme-producing medium to 4.8; and (2) the fermentation: adding 50 mL of the enzyme-producing medium to a 250 ml Erlenmeyer flask with a cotton stopper, inoculating T. reesei spores into the enzyme-producing medium at an inoculum size of 10%, and cultivating the T. reesei spores in a thermostatic shaker at 28? C. to 30? C. and 170 rpm for 4 d; and after a cultivation is completed, centrifuging the resulting culture solution at 3,000 rpm for 10 min to obtain a supernatant, which is the enzyme solution for hydrolyzing the GM to prepare the small-molecule GM and the GMOS.

6. A method for hydrolyzing a GM to prepare a small-molecule GM and a GMOS, wherein the small-molecule GM has a molecular weight of 20,000 Da or less, comprising the following steps: 1) conducting a fermentation with MCC and melibiose as carbon sources and T. reesei as an enzyme-producing strain to obtain a supernatant, which is an enzyme solution with enzymatic activities of ?-mannanase and ?-galactosidase, wherein an enzymatic activity ratio of the of the ?-mannanase to the ?-galactosidase is no less than 7:1, and wherein the total concentration of the MCC and the melibiose is 20.0 g/L to 35.0 g/L; and after the fermentation is completed, centrifuging a resulting culture solution, wherein the supernatant comprises the enzyme solution; and 2) directly using the enzyme solution obtained in step 1) for an enzymatic hydrolysis of the GM as a substrate to prepare the small-molecule GM and the GMOS.

7. The method for hydrolyzing the GM to prepare the small-molecule GM and the GMOS according to claim 6, wherein in step 1), a weight ratio of the MCC to the melibiose is 2:1.

8. The method for hydrolyzing the GM to prepare the small-molecule GM and the GMOS according to claim 6, wherein in step 2), during the enzymatic hydrolysis, a substrate concentration is 2%, the enzyme solution is added at an amount of 20 U/g relative to the GM, and a pH is 4.8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows results of enzymatic hydrolysis by a combination of ?-mannanase and ?-galactosidase;

(2) FIGS. 2A-2D show enzymatic activity results of ?-mannanase and ?-galactosidase at different MCC-to-melibiose ratios when a total substrate concentration is 20 g/L, 25 g/L, 30 g/L, and 35 g/L; and

(3) FIG. 3 shows enzymatic hydrolysis results of enzyme solutions with different enzymatic activity ratios of ?-mannanase to ?-galactosidase.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) To make the objectives, features, and advantages of the present disclosure clear and comprehensible, specific implementations of the present disclosure will be described in detail below in conjunction with specific examples.

(5) The performance of a product used in the following examples is tested as follows:

(6) (1) A molecular weight distribution (MWD) is determined by gel permeation chromatography (GPC) for a small-molecule GM and a GMOS.

(7) Chromatography conditions are as follows: chromatographic instrument: Agilent High-Performance liquid Chromatography (HPLC) 1260; chromatographic column: Waters Ultrahydrogel? 2000 (7.8?300 mm), Waters Ultrahydrogel? 250 (7.8?300 mm), and Waters Ultrahydrogel? 120 (7.8?300 mm) sequentially connected in series; guard column: Waters Ultrahydrogel? Guard Column (6?40 mm); detector: differential detector: mobile phase: water; mobile phase flow rate: 0.60 mL/min; column temperature: 65? C.; injection volume: 10.0 ?L; and standard sample for molecular weight determination: polyethylene glycol (PEG).

(8) (2) A saccharide content is determined by acid hydrolysis and ion chromatography for a small-molecule GM and a GMOS.

(9) A determination method is as follows: 0.3 g of a small-molecule GM and GMOS sample is taken and added to a hydrolysis flask, 87 mL of 4% H.sub.2SO.sub.4 is added, and a reaction is conducted at 121? C. for 1 h; after the reaction is completed, 1 mL of a reaction solution is collected, and a pH of the reaction solution is adjusted with 50% NaOH to 7.0: the reaction solution is centrifuged at 10,000 rpm for 5 min, and a resulting supernatant is collected; and finally an ICS-5000 ion exchange chromatograph is used to determine concentrations of mannose and galactose in the supernatant.

(10) Ion chromatography conditions are as follows: chromatographic instrument: Dionex ion chromatography ICS-5000; chromatographic column: 2?250 mm Dionex AminoPac PA10; guard column: 2?50 mm Dionex AminoPac PA10 detector: conductivity detector; mobile phase: 3 mmol sodium hydroxide; flow rate: 0.20 mL min; column temperature: 30? C.; injection volume: 10.0 ?L; and determination is conducted by an external standard method. Purities of a small-molecule GM and a GMOS in a sample are calculated as follows:

(11) $\mathrm{GM}\mathrm{degradation}\mathrm{product}\mathrm{content}=\frac{\left(\mathrm{Galactose}\mathrm{concentration}+\mathrm{mannose}\mathrm{concentration}\right)?0.9?0.087}{0.3}?100\%$

(12) (3) Determination of an activity of ?-mannanase:

(13) 0.9 mL of a 5 g/L locust bean gum substrate solution is added to a 25 mL graduated test tube and pre-heated at 50? C. for 5 min, 0.1 mL of an appropriately-diluted enzyme solution is added, and a reaction is conducted at 50? C. for 30 min; 3.0 mL of a DNS reagent is immediately added to terminate the reaction, and a resulting reaction solution is treated in a boiling water bath for 7 min, cooled, diluted to 25 mL, and thoroughly shaken; the absorbance of the reaction solution is determined at 540 nm; and according to a correlationship between the absorbance and a reducing sugar, a concentration of the reducing sugar generated is calculated. One activity unit (U) of ?-mannanase is calculated according to an amount of ?-mannanase required for hydrolysis of a substrate to produce 1 ?mol of a reducing sugar (based on mannose) per minute.

(14) (4) Determination of an activity of ?-galactosidase:

(15) 0.1 mL of an appropriately-diluted enzyme solution and 0.9 mL of a 1 mmol/L p-nitrophenol-?-D-galactopyranoside (pNPG) solution are added to a 15 mL test tube and incubated at 50? C. for 10 min, and then 2.0 mL of a 1 mol/L Na.sub.2CO.sub.3 solution is immediately added to terminate a reaction; 10 mL of distilled water is added, and a resulting reaction solution is thoroughly shaken; the absorbance of the reaction solution is determined at 400 nm; and according to a correlationship between the absorbance and p-nitrophenol, a concentration of the p-nitrophenol generated is calculated. One activity unit (U) of ?-galactosidase is calculated according to an amount of ?-galactosidase required for hydrolysis of pNPG to release 1 ?mol of p-nitrophenol per minute.

(16) (5) Determination of an activity of ?-mannosidase:

(17) 0.1 mL of an appropriately-diluted enzyme solution and 0.9 mL of a 1 mmol/L p-nitrophenol-?-D-mannopyranoside (pNPM) solution are added to a 15 mL test tube and incubated at 50? C. for 10 min, and then 2.0 mL of a 1 mol/L Na.sub.2CO.sub.3 solution is immediately added to terminate a reaction; 10 mL of distilled water is added, and a resulting reaction solution is thoroughly shaken; the absorbance of the reaction solution is determined at 400 nm; and according to a correlationship between the absorbance and p-nitrophenol, a concentration of the p-nitrophenol generated by enzymatic hydrolysis is calculated. One activity unit (U) of ?-mannosidase is calculated according to an amount of ?-mannosidase required for hydrolysis of pNPM to release 1 ?mol of p-nitrophenol per minute.

Example 1

(18) Fermentation was conducted with T. reesei as an enzyme-producing strain and MCC or melibiose as carbon sources to produce an enzyme, including the following steps:

(19) (1) Composition of an enzyme-producing medium (g/L): glucose: 1.0, MCC or melibiose: 25.0, ammonium sulfate: 4.72, urea: 2.15, MKP: 2.0, anhydrous calcium chloride: 0.3, magnesium sulfate heptahydrate: 0.3, ferrous sulfate heptahydrate: 0.005, manganese sulfate heptahydrate: 0.0016, zinc sulfate heptahydrate: 0.0014, and cobalt chloride: 0.002. 50 mL of a 1 mol/L sodium citrate buffer was added to adjust a pH of the medium to 4.8.

(20) (2) Fermentation to Produce the Enzyme

(21) 50 mL of the medium was added to a 250 ml Erlenmeyer flask with a cotton stopper, and T. reesei spores were inoculated into the medium at an inoculum size of 10% and cultivated in a thermostatic shaker at 28? C. to 30? C. and 170 rpm for 4 d; and after the cultivation was completed, a resulting culture solution was centrifuged at 3,000 rpm for 10 min to obtain a supernatant (enzyme solution), and enzymatic activities of ?-galactosidase, ?-mannosidase, and ?-mannanase were determined.

(22) Results showed that, in enzyme solution 1 produced through fermentation with T. reesei as an enzyme-producing strain and MCC as a carbon source, an enzymatic activity of ?-mannanase was 3.917 U/mL, an enzymatic activity of ?-galactosidase was 0.099 U/mL, and an enzymatic activity of ?-mannosidase was 0.02 U/mL; and in enzyme solution 2 produced through fermentation with T. reesei as an enzyme-producing strain and melibiose as a carbon source, an enzymatic activity of ?-galactosidase was 0.452 U/mL.

Example 2

(23) A small-molecule GM and a GMOS were prepared through enzymatic hydrolysis by a combination of mannanase and galactosidase, specifically including the following steps:

(24) (1) Directed Enzymatic Hydrolysis of a GM

(25) GM-containing leguminous seeds (sesbania) were mechanically crushed to 20-100 mesh, distilled water was added according to a solid-to-liquid ratio of 1:50, and extraction was conducted at 50? C. for 24 h; a resulting extraction solution was centrifuged at 10,000 rpm for 10 min; a resulting supernatant was collected, and absolute ethanol was added to the supernatant; and a resulting precipitate was vacuum-dried to obtain a GM powdered solid.

(26) The enzyme solution 1 and the enzyme solution 2 obtained in Example 1 were mixed to allow the following enzymatic activity ratios of ?-mannanase to ?-galactosidase: 2, 4, 6, 8, 10, 12, 15, 20, 30, and 40. Then, 20.0 g of the GM was weighed and added to a 2 L enzyme reaction tank, distilled water, an enzyme solution, and a 1 mol/L citric acid buffer were added in the tank to obtain 1,000 mL of a reaction solution: the reaction solution was thoroughly mixed, and a reaction was conducted for 24 h under the following conditions: substrate concentration: 2%, enzyme amount relative to the GM: 20 U/g, pH: 4.8, and temperature: 50? C.; and after the enzymatic hydrolysis reaction was completed, an enzymatic hydrolysate was treated at 100? C. for 10 min to inactivate enzymes, and an inactivated enzymatic hydrolysate was centrifuged at 10,000 rpm for 10 min to obtain a supernatant, which was an enzymatic hydrolysate including the small-molecule GM and the GMOS.

(27) (2) 1,000 mL of the enzymatic hydrolysate including the small-molecule GM and the GMOS obtained in step (1) was taken, absolute ethanol was added under stirring until an ethanol concentration in a resulting system was 40% (v/v), and the system was centrifuged at 10,000 rpm for 10 min to obtain a supernatant and a precipitate; the precipitate was washed 3 times with a 40% (v/v) ethanol aqueous solution, collected through centrifugation at 10,000 rpm for 10 min, and lyophilized to obtain a small-molecule GM component named GalM40: a molecular weight of the small-molecule GM component GalM40 was determined by gel chromatography, and a content of a GM degradation product was determined by acid hydrolysis and ion chromatography: and the supernatant was further used for the next fractionation.

(28) (3) The supernatant obtained after solid-liquid separation (SLS) in step (2) was taken, absolute ethanol was added under stirring until an ethanol concentration in a resulting system was 50% (v/v), and the system was centrifuged at 10,000 rpm for 10 min to obtain a supernatant and a precipitate; the precipitate was washed 3 times with a 50% (v/v) ethanol aqueous solution, collected through centrifugation at 10,000 rpm for 10 min, and lyophilized to obtain a small-molecule GM component named GalM50; a molecular weight of the small-molecule GM component GalM50 was determined by gel chromatography, and a content of a GM degradation product was determined by acid hydrolysis and ion chromatography; and the supernatant was further used for the next fractionation.

(29) (4) The supernatant obtained after SLS in step (3) was taken, absolute ethanol was added under stirring until an ethanol concentration in a resulting system was 65% (v/v) and the system was centrifuged at 10,000 rpm for 10 min to obtain a supernatant and a precipitate: the precipitate was washed 3 times with a 65% (v/v) ethanol aqueous solution, collected through centrifugation at 10,000 rpm for 10 min, and lyophilized to obtain a small-molecule GM component named GalM65; a molecular weight of the small-molecule GM component GalM65 was determined by gel chromatography, and a content of a GM degradation product was determined by acid hydrolysis and ion chromatography: and the supernatant was further used for the next fractionation.

(30) (5) The supernatant obtained after SLS in step (4) was taken and subjected to vacuum rotary evaporation at 70? C. and 160 mbar to remove ethanol; a part of a resulting supernatant was taken, and a content of a GM degradation product in the supernatant was determined by acid hydrolysis and ion chromatography; monosaccharides in the remaining part of the supernatant were removed through nanofiltration (200 Da), then a resulting filtrate was concentrated through vacuum rotary evaporation at 70? C. and 160 mbar to obtain a concentrate; and the concentrate was dried to obtain a GMOS component GalMOS, and a molecular weight of the GMOS component GalMOS was determined by gel chromatography.

(31) FIG. 1 shows results of enzymatic hydrolysis by a combination of ?-mannanase and ?-galactosidase. It can be seen from FIG. 1 that, at an early stage, with the continuous increase of an enzymatic activity of ?-galactosidase, a total yield of the three small-molecule GMs GalM40, GalM50, and GalM65 and a yield of GalMOS tend to increase slowly, and a total yield of saccharides also tends to increase slowly; and at a later stage, with the further increase of an enzymatic activity of ?-galactosidase, a total yield of the three small-molecule GMs GalM40, GalM50, and GalM65 and a yield of GalMOS start to decrease, and a total yield of saccharides also starts to decrease. It can be seen from FIG. 1 that, when an enzymatic activity ratio of ?-mannanase to ?-galactosidase is 8, a total yield of saccharides is the highest, and a total yield of the three small-molecule GMs GalM40, GalM50, and GalM65 and a yield of GalMOS are also the highest. In addition, a molecular weight of a small-molecule GM obtained was measured, and an average molecular weight of each component was as follows: GalM40: 13,100 Da, GalM50: 8,930 Da, GalM65: 4,310 Da, and GalMOS: 1,630 Da.

Example 3

(32) An enzyme was prepared with a combination of MCC and melibiose, including the following steps:

(33) (1) An enzyme-producing medium was the same as in Example 1. The substrate was replaced by a mixture of MCC and melibiose in different ratios, where a total concentration of the two substrates was 20.0 g/L, 25.0 g/L, 30.0 g/L, and 35.0 g/L.

(34) (2) An enzyme-producing fermentation method was the same as in Example 1.

(35) Results were shown in FIGS. 2A-2D. When a total substrate concentration is 20 g/L, an activity of ?-mannanase continuously decreases with the continuous increase of melibiose. However, when a total substrate concentration is 25 g/L, 30 g/L, and 35 g/L, an enzymatic activity of ?-mannanase tends to increase first and then decrease with the continuous increase of melibiose. When a total substrate concentration is 25 g/L with an MCC concentration of 20 g/L and a melibiose concentration of 5 g/L, an enzymatic activity ratio of ?-mannanase to ?-galactosidase is the highest. In general, an enzymatic activity of ?-galactosidase continuously increases with the increase of melibiose; and when a melibiose concentration is too high, an enzymatic activity of ?-galactosidase gradually decreases.

Example 4

(36) A method for preparing a small-molecule GM and a GMOS through enzymatic hydrolysis with an enzyme solution produced by a combination of MCC and melibiose was provided, including the following steps:

(37) directed enzymatic hydrolysis of a GM was the same as in Example 2, where an enzyme solution used was the enzyme solution in Example 3; and enzyme solutions with different enzymatic activity ratios of ?-mannanase to ?-galactosidase were used to conduct an enzymatic hydrolysis test.

(38) Results were shown in FIG. 3. An enzyme solution produced with 15 g/L MCC and 10 g/L melibiose has the optimal enzymatic hydrolysis effect, where a total yield of saccharides is 86.47%, a yield of GalM OS is 16.51%, a total yield of the three small-molecule GMs GalM40, GalM50, and GalM65 is 55.63%, and an enzymatic activity ratio of ?-mannanase to ?-galactosidase is 7.235. These results correspond to the results of the previous enzymatic hydrolysis by the combination of ?-mannanase and ?-galactosidase.