# US 11,999,984 B2 · Method and special complex enzyme for hydrolyzing galactomannan (GM) to prepare small-molecule GM and galactomannan oligosaccharide (GMOS)

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- Number: US 11,999,984 B2
- Document ID: US11999984B2
- Status: Granted
- Kind: B2
- Application number: 18/274,770
- Filed: 2021-05-06
- Published: 2024-02-01
- Granted: 2024-06-04
- Earliest claimed: 2021-01-29
- Assignees: Nanjing Forestry University
- Applicants: Nanjing Forestry University
- Inventors: Caoxing Huang; Qiang Yong; Yuheng Tao; Chenhuan Lai; Yong Xu; Xin Li
- Web page: https://patentgrep.com/patent/US11999984B2

## CPC

- C12P 19/04: Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
- C12N 9/2465: {acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)}
- C12N 9/2491: {Beta-mannosidase (3.2.1.25), i.e. mannanase}
- C12N 9/2494: {Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase}
- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12Y 302/01022: Alpha-galactosidase (3.2.1.22)
- C12Y 302/01025: Beta-mannosidase (3.2.1.25), i.e. mannanase
- C12Y 302/01078: Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## 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 β-mannanase and α-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

### Claim 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 β-mannanase and α-galactosidase, wherein an enzymatic activity ratio of the of the β-mannanase to the α-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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 1)

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.

### Claim 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 β-mannanase and α-galactosidase, wherein an enzymatic activity ratio of the of the β-mannanase to the α-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.

### Claim 7 (depends on 6)

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.

### Claim 8 (depends on 6)

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.