CN108060149B

CN108060149B - Preparation method of konjac mannan-oligosaccharide and special beta-mannase mutant thereof

Info

Publication number: CN108060149B
Application number: CN201810006702.9A
Authority: CN
Inventors: 闫巧娟; 李延啸; 江正强; 李斌; 王楠楠; 易萍; 游鑫
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2018-01-04
Filing date: 2018-01-04
Publication date: 2020-08-18
Anticipated expiration: 2038-01-04
Also published as: CN108060149A

Abstract

The invention discloses a preparation method of konjac mannan oligosaccharide and a special beta-mannase mutant thereof. The beta-mannase mutant provided by the invention is derived from Mucor miehei, has the advantages of good stability, high specific enzyme activity and the like, and has great application value in the industries of food, feed and the like. The beta-mannase mutant is introduced into engineering bacteria formed by pichia pastoris to be fermented in a 5L fermentation tank at high density, and the enzyme activity of the fermentation liquid can reach 72600U/mL (the protein content is 9.1 mg/mL). The beta-mannase mutant is used for hydrolyzing konjac flour to prepare konjac mannan oligosaccharide, mannan oligosaccharide with the polymerization degree of 2-6 is mainly contained in the hydrolysate, the konjac hydrolysis rate is 90.2%, and the reducing sugar yield is 69.9%. The invention provides an important basis for preparing konjac mannan oligosaccharide by utilizing konjac flour.

Description

Preparation method of konjac mannan-oligosaccharide and special beta-mannase mutant thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a preparation method of konjac mannan oligosaccharide and a special beta-mannase mutant thereof.

Background

Depending on the constituent monomers and the manner of linkage, mannans can be classified into four classes, namely linear mannans, galactomannans, glucomannans and galactoglucomannans (Malgas et al, world Journal of microbiology and Biotechnology 2015,31: 1167-. The main chain is formed by connecting mannose (and glucose) through beta-1, 4-glycosidic bond, and the main chain also contains galactose residue side chain connected by alpha-1, 6-glycosidic bond. These mannans are widely present as structural and energy-storing polysaccharides in plant tissue cell walls and in the endosperm of dicotyledonous plants, such as: agricultural waste (linear mannan) such as palm meal and coffee grounds, seed endosperm (galactomannan) such as guar bean, and rhizoma Amorphophalli tuber (glucomannan). Mannan is complex in structure, and the complete degradation requires the synergistic hydrolysis of various glycoside hydrolases, such as: beta-mannanase (EC 3.2.1.78), beta-mannosidase (EC 3.2.1.25), beta-glucosidase (EC3.2.1.21), alpha-galactosidase (EC 3.2.1.23), and mannan acetylesterase (EC 3.1.1.6) (Moreirae al. applied Microbiology and Biotechnology,2008,79: 165-178).

As the most important hydrolase in the mannanase system, β -mannanase is capable of randomly degrading β -1, 4-glycosidic bonds in the mannan backbone to produce low molecular weight mannooligosaccharides with varying degrees of polymerization (Chauhan et al applied microbiology and Biotechnology,2012,93: 1817-. Based on their amino acid sequence similarity, the β -mannanases belong to the glycoside hydrolases families 5, 26, 113 and 134 (Dhawan et al. critical Reviews in Biotechnology,2007,27: 197-216). At present, a plurality of beta-mannase enzymes are cloned and expressed, but the existing beta-mannase has certain defects (poor temperature stability, low expression level and the like) and limits the application of the beta-mannase in actual production (vanZyl et al process Biochemistry,2010,45: 1203-. Therefore, the molecular modification of the beta-mannase is carried out, and the development of the beta-mannase with better enzymological properties is of great significance for the application of the beta-mannase in the industries of food, feed and the like.

Konjac is one of the important sources of glucomannan, which accounts for more than 52% of the dry weight of the konjac tuber, has a Man/Glc ratio of about 1.6:1 and a degree of polymerization of about 6000 (Behera and ray. food reviews international,2017,33: 22-43). As an important prebiotic and dietary fiber, konjac mannan has been identified by the U.S. food and drug administration and the Canadian Ministry of health as a "Generally recognized as Safe" (GRAS) food additive (Tester and Al-Ghazzewi. journal of the Science of food and Agriculture,2016,96, 3283-. The konjac mannan is easily dissolved in water, the viscosity of the water solution is high, the konjac mannan is in a gel state, and excessive konjac flour added into food can influence the absorption of nutrient substances and destroy the taste of the food. The problem can be effectively avoided by degrading konjac mannan into konjac mannan oligosaccharide by using beta-mannase. Konjak mannan-oligosaccharide is oligosaccharide formed by connecting 2-10 mannose (and glucose) through beta-1, 4-glycosidic bond. The product has the characteristics of good water solubility, low viscosity, acid and alkali resistance, good stability and the like, does not cause decayed teeth, can effectively control blood sugar, is a new generation of functional food, and is widely concerned by researchers at home and abroad. Currently, there are many reports on the use of konjac flour as a substrate for the preparation of konjac mannan oligosaccharides, with low concentrations of substrate used in hydrolysis (< 5%) (Zang et al, enzyme and Microbial Technology,2015,78: 1-9). Chinese patent application No. 200910014349.X discloses a method for preparing mannan oligosaccharide by hydrolyzing konjac flour with beta-mannase from bacillus, but the concentration of the konjac flour is very low, only 1% -1.5%, and the hydrolysis time is as long as 30 h; chinese patent application No. 201310428885.0 discloses a preparation method of high-purity mannanoligosaccharide, but the concentration of konjac powder used is lower, 15% -25%; chinese patent application No. 201510465107.8 discloses a method for preparing konjac oligosaccharide by hydrolyzing konjac refined powder with acidic mannase, wherein the concentration of the acting substrate (konjac refined powder) is only 15% -25% as well. It can be seen that the above patents are slightly insufficient in terms of practicality, and there is a need to develop a method for efficiently producing konjac mannan oligosaccharide with high substrate concentration and short reaction time.

Mucor miehei CAU432 is a thermophilic filamentous fungus which can secrete a plurality of glycoside hydrolases, and the secreted beta-mannanase has a plurality of excellent enzymological properties (Katrolia et al, journal of Agricultural and food Chemistry,2013,61: 394-401). The invention carries out molecular modification on beta-mannase derived from rhizomucor miehei CAU432 to obtain a beta-mannase mutant gene, and the beta-mannase mutant has the following advantages: neutral pH, good thermal stability, excellent hydrolysis characteristic and the like. In addition, the obtained beta-mannase gene is transformed into pichia pastoris GS115 for high-density secretory expression. Compared with the beta-mannase reported before, the newly invented beta-mannase mutant has larger application value in the industries of food, feed and the like.

Disclosure of Invention

The invention aims to provide konjac mannan-oligosaccharide, wherein the content of mannose-disaccharide to mannose-hexaose is more than 80 percent in percentage by mass.

The invention also aims to provide a beta-mannase mutant and a coding gene thereof, wherein the beta-mannase mutant is a protein consisting of an amino acid sequence shown as a sequence 1 in a sequence table; the coding gene of the beta-mannase mutant is shown as a sequence 2 in a sequence table.

The invention also aims to provide a preparation method of konjac mannan oligosaccharide, which comprises the following steps:

s1: performing molecular modification on beta-mannase by using an directed evolution technology;

s2: preparing a recombinant bacterium containing the beta-mannase mutant;

s3: fermenting by using recombinant bacteria to prepare a beta-mannase mutant;

s4: hydrolyzing the konjac flour solution by using the beta-mannase mutant;

in step S1, the coding gene of the beta-mannanase is derived from Mucor miehei CAU 432.

In step S2, the recombinant bacteria are pichia pastoris GS115 and/or escherichia coli BL 21.

In the fermentation liquor of the step S3, the enzyme activity of the beta-mannase mutant reaches 72600U/mL.

In step S4, the mixing ratio of the β -mannanase mutant to the konjac flour is: adding 250 units of beta-mannase mutant and 1000 units of beta-mannase mutant into each gram of konjac flour.

In step S4, the concentration of the konjac flour aqueous solution is 10% to 50%.

In step S4, the hydrolysis time is 1-24h, the hydrolysis temperature is 30-90 ℃, and the hydrolysis pH value is 3.5-11.0.

The invention provides an important basis for the application of the beta-mannase in the preparation of konjac mannan oligosaccharide by hydrolyzing konjac flour.

Drawings

The invention has the following drawings:

FIG. 1 is a purified electrophoretogram of a mutant β -mannanase.

FIG. 2 is a graph showing the determination of the optimum pH of the mutant β -mannanase. Wherein (●) a citrate phosphate buffer (pH3.0-7.0), (■) an acetate buffer (pH4.0-6.0), (. diamond. -O) a phosphate buffer (pH6.0-8.0), (. diamond. -Tris-HCl buffer (pH7.0-9.0), (□) a CHES buffer (pH8.0-10.0), (. smallcircle.) a glycine-sodium hydroxide buffer (pH 9.5-10.5).

FIG. 3 is a graph showing the pH stability assay of the beta-mannanase mutants. Wherein (●) a citrate phosphate buffer (pH3.0-7.0), (■) an acetate buffer (pH4.0-6.0), (. diamond. -O) a phosphate buffer (pH6.0-8.0), (. diamond. -Tris-HCl buffer (pH7.0-9.0), (□) a CHES buffer (pH8.0-10.0), (. diamond. -glycine-sodium hydroxide buffer (pH 9.5-10.5).

FIG. 4 is a graph showing the optimum temperature determination of the mutant β -mannanase.

FIG. 5 is a graph showing the temperature stability assay of the β -mannanase mutants.

FIG. 6 is a graph of the hydrolysis profile of the β -mannanase mutants.

FIG. 7 is a process chart of Pichia pastoris mutant for high-density fermentation and secretion of beta-mannase.

FIG. 8 is a high performance liquid chromatography analysis chart of konjac mannan oligosaccharide prepared by hydrolyzing konjac flour with a beta-mannase mutant.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The enzyme activity of the beta-mannanase is determined as follows in the following examples:

0.1mL of an appropriately diluted enzyme solution was added to 0.9mL of 0.5% (by mass/volume) locust bean gum substrate solution (prepared with 50mM, pH7.0 phosphate buffer citrate), reacted in a water bath at 65 ℃ for 10min, and the amount of the released reducing sugar was measured by the 3, 5-dinitrosalicylic acid (DNS) method using mannose as a standard.

The specific enzyme activity is defined as the unit of enzyme activity possessed by 1mg of protein and is expressed as U/mg.

Definition of 1 β -mannanase enzyme activity unit: the enzyme amount required for decomposing 0.5 percent of locust bean gum substrate to release 1 mu mol of mannose per minute at the conditions of pH7.0 and 65 ℃, and the calculation formula of the enzyme activity is as follows: h ═ Cx × n/(T × V), where H represents the enzyme activity (U/mL), Cx represents the amount of mannose-producing substance (μmol), n represents the dilution factor of the enzyme solution, T represents the reaction time (min), and V represents the volume of the enzyme solution (mL) after the dilution was added.

Example 1 construction of beta-mannanase mutant Gene

1. Error prone PCR

A signal peptide error-prone PCR primer pair was designed based on the wild-type gene of Mucor miehei beta-mannanase (Katrolia et al. journal of agricultural and Food Chemistry,2013,61: 394-401). The primer sequences are as follows:

an upstream primer:

5′-CGCGGATCCGCTTCTTGGTTTGTCCAGACAAG-3′；

a downstream primer:

5′-CCGCTCGAGCTACTTCTTCTTGGCCATGGCATCAGC-3′。

error-prone PCR system (50. mu.L): 7mM Mg²⁺，0.2mM Mn²⁺0.2mM dATP, 0.2mM dAGP, 1mM dCTP, 1mM dTTP, 0.2 μ M RmMan5AF, 0.2 μ M RmMan5AR, 2.5U Taq DNA polymerase, 20ng cDNA.

Error-prone PCR reaction conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, renaturation at 55 ℃ for 30s, extension at 72 ℃ for 1.5min, 30 cycles, and total extension at 72 ℃ for 5 min.

2. DNA fragmentation

And (3) carrying out enzyme digestion treatment on the error-prone PCR product by using DNase I.

Enzyme digestion system (100. mu.L): 8mM Mg²⁺，0.67mM Mn ²⁺8 μ g of PCR amplification product, 0.0252U DNase I.

Enzyme cutting conditions are as follows: the reaction was stopped by adding 2.5mM EDTA at 20 ℃ for 15min and inactivated DNaseI by incubation at 90 ℃ for 10 min.

Recovering and purifying DNA fragment of about 40-60 bp.

3. Pool of random mutants

And (3) performing overlap extension PCR by using the DNA fragment as a template to obtain an overlap extension PCR product. And using the PCR product as a template, and carrying out full-length PCR by using the primer pair in the step 1 to obtain a PCR amplification product (random mutant library).

Full-length PCR system (50. mu.L): 0.2mM dNTP, 1.5mM MgSO₄0.2. mu.M RmMan5AF, 0.2. mu.M MRmMan5AF, 1. mu.L (50-100ng) template, 1U Pfu DNA polymerase.

Full-length PCR conditions: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, renaturation at 55 ℃ for 30s, extension at 72 ℃ for 1.5min, 30 cycles, and total extension at 72 ℃ for 5 min.

4. Mutant expression libraries

The random mutant pool and pET28a vector were digested with BamHI and XhoI restriction enzymes, and the digested product was recovered. Connecting the enzyme digestion frameworks, transforming the connection product into escherichia coli BL21(DE3) by using an electrical transformation method, and collecting all transformants to obtain a mutant expression library. Randomly 10 transformants were selected for sequencing analysis with a mutation rate of about 0.72%.

5. Directed screening of mutant expression libraries

The above-mentioned mutant expression library was diluted appropriately, spread evenly on LB plates (containing 50. mu.g/mL kanamycin), and cultured at 37 ℃ for 24 hours. Single colonies were collected, numbered, transferred to a screening plate (LB plate containing 0.5% locust bean gum, 1mM IPTG and 50. mu.g/mL kanamycin), and cultured at 37 ℃ for 1 day. After the culture is finished, placing the screening plate at 70 ℃ for 1h to crack thalli, then dyeing the thalli for 15min by using a 0.1% Congo red solution, and then decoloring the thalli by using a 1M NaCl solution; corresponding to the corresponding colony number through the transparent ring, picking the corresponding colony from the original plate, and re-screening.

6. Rescreening of mutant expression libraries

Centrifuging the fermentation liquid of each strain capable of generating transparent circles at 10000 Xg for 5min, resuspending the thallus in distilled water, centrifuging at 10000 Xg for 10min after ultrasonic wall breaking, and taking supernatant (crude enzyme liquid). The enzyme activity of the crude enzyme solution is measured at different temperatures respectively, and the pH is measured to be 7.0. Under the condition that the temperature is higher than 60 ℃, if the crude enzyme liquid enzyme activity of the mutant strain is higher than that of the wild beta-mannase, the strain is a positive mutant. After re-screening, 1 positive mutant was obtained.

7. Sequence analysis of Positive mutants

Sequencing the positive mutant extracted plasmid, and comparing with a wild beta-mannanase gene, wherein 7 nucleotides are mutated, namely T → A (365 th nucleotide from 5 ' tail end of sequence 2 in a sequence table), T → C (384 th nucleotide from 5 ' tail end of sequence 2 in the sequence table), A → G (485 th nucleotide from 5 ' tail end of sequence 2 in the sequence table), A → C (582 th nucleotide from 5 ' tail end of sequence 2 in the sequence table), T → C (840 th nucleotide from 5 ' tail end of sequence 2 in the sequence table), A → G (914 th nucleotide from 5 ' tail end of sequence 2 in the sequence table), and G → A (918 th nucleotide from 5 ' tail end of sequence 2 in the sequence table); the above nucleotide mutations cause mutation of three amino acid residues of the wild-type β -mannanase protein, which are L → H (122 th amino acid residue from N-terminus of sequence 1 in the sequence listing), K → R (162 th amino acid residue from N-terminus of sequence 1 in the sequence listing), E → D (194 th amino acid residue from N-terminus of sequence 1 in the sequence listing), and D → G (305 th amino acid residue from N-terminus of sequence 1 in the sequence listing). The recombinant plasmid carrying the positive mutant is transformed into Escherichia coli BL21(DE3) to obtain recombinant bacterium A.

Example 2 preparation of beta-mannanase mutants and determination of enzymatic Properties

1. Inducible expression of beta-mannanase mutants

Inoculating the recombinant Escherichia coli into liquid LB culture medium (containing 50. mu.g/mL kanamycin), performing shake culture at 37 ℃ until OD600 reaches 0.6-0.8, adding IPTG (final concentration of 1mM), performing induction culture at 30 ℃ overnight, collecting thalli at 10000 Xg, resuspending with buffer solution A (20mM phosphate buffer solution pH8.0, 300mM NaCl, 20mM imidazole), performing ultrasonication, centrifuging at 10000 Xg for 10min, and collecting supernatant to obtain crude enzyme solution.

2. Purification of beta-mannanase mutants

The beta-mannanase mutants were purified using agarose Ni-IDA affinity columns. The method comprises the following specific steps:

the Ni-IDA column was equilibrated with buffer A for 5 to 10 column volumes, the above crude enzyme solution was loaded at a flow rate of 0.5mL/min, eluted with buffer A and buffer B (20mM phosphate buffer pH8.0, 300mM NaCl, 50mM imidazole) at a flow rate of 1mL/min to OD280<0.05, and finally eluted with buffer C (20mM phosphate buffer pH8.0, 300mM NaCl, 200mM imidazole) and the target protein was collected to obtain a purified product.

The SDS-PAGE purification of the beta-mannanase mutants is shown in FIG. 1. In FIG. 1, lane M is the molecular weight standard, 1 is the crude enzyme solution of recombinant bactericidium, and 2 is the purified product of the crude enzyme solution of recombinant bactericidium, i.e., the beta-mannanase mutant. The results in FIG. 1 show that the mutant β -mannanase has a size of 44kDa, which is consistent with the expected size.

3. Determination of optimum pH of beta-mannase mutant

And taking the prepared purified product as enzyme solution to be detected, and performing enzyme activity determination on the enzyme solution under different buffer solution conditions. The various buffers were as follows:

1) a citrate phosphate buffer (pH 3.0-7.0);

2) acetic acid buffer solution (pH4.0-6.0);

3) phosphate buffer (pH6.0-8.0);

4) Tris-HCl (Tris-hydroxymethyl-aminomethane-hydrochloric acid) buffer (pH7.0-9.0);

5) CHES (1-cyclohexylaminoethanesulfonic acid) buffer solution (pH8.0-10.0);

6) glycine-sodium hydroxide buffer (pH 9.5-10.5).

When the determination is carried out, the reaction temperature is 65 ℃, the enzyme activity of the beta-mannase mutant under the optimum pH value is taken as 100%, and the relative enzyme activities under other pH values are calculated.

The results are shown in FIG. 2: the optimum pH of the beta-mannase mutant is 7.0.

4. Determination of pH stability of beta-mannanase mutants

Diluting the beta-mannase mutant with the buffer solution, treating in a water bath kettle at 50 ℃ for 30min, rapidly cooling in ice water for 30min, and measuring the enzyme activity. And respectively taking the untreated beta-mannase mutants as 100 percent to calculate the relative enzyme activity of the beta-mannase mutants after treatment at different pH values. The enzyme activity determination conditions are as follows: 50mmol/L citrate phosphate buffer (pH7.0) reaction temperature 65 ℃.

The results are shown in FIG. 3: the beta-mannase mutant has good pH stability and is kept stable at a pH value of 4.0-10.0.

5. Determination of optimum temperature of beta-mannanase mutant

After the beta-mannase mutant is diluted properly by using an optimal pH buffer solution, the enzyme activity of the beta-mannase mutant is measured at different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90 ℃). The enzyme activity of the enzyme at the optimum temperature is taken as 100 percent, and the relative enzyme activities at other pH values are calculated.

The results are shown in FIG. 4: the optimum temperature of the beta-mannase mutant is 65 ℃.

6. Temperature stability assay for beta-mannanase mutants

Properly diluting the beta-mannase mutant by using an optimal pH buffer solution, respectively preserving the temperature for 30min at different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 and 90 ℃), quickly placing the beta-mannase mutant in ice water for cooling for 30min, and then measuring the enzyme activity. And taking the untreated beta-mannase mutant as 100 percent, and calculating the relative enzyme activity of the beta-mannase mutant after treatment at different temperatures. The enzyme activity determination conditions are as follows: 50mmol/L citrate phosphate buffer (pH7.0) reaction temperature 65 ℃.

The results are shown in FIG. 5: the beta-mannase mutant has good temperature stability and is stable at 60 ℃.

7. Hydrolytic Properties of beta-mannanase mutants

Locust bean gum or konjac flour (1%, w/v) was dissolved in 50mM citrate phosphate buffer (pH7.0), the beta-mannanase mutant (5U/mL) was added, and the mixture was hydrolyzed at 50 ℃ for 12 hours, samples were taken at intervals, and all samples were inactivated in a boiling water bath for 10 min. All samples were subjected to thin layer chromatography. The sample loading amount is 2 mu L, and the spreading agent is n-butyl alcohol: acetic acid: water (2: 1: 1), and the developer is methanol: sulfuric acid (95: 5). The results of thin layer chromatography are shown in FIG. 6. As can be seen, the beta-mannase mutant hydrolyzes locust bean gum to mainly produce mannobiose, mannotriose and other mannan oligosaccharide with polymerization degree; it hydrolyzes konjac flour to mainly produce mannan oligosaccharide with polymerization degree of 2-4.

Example 3 high Density fermentation of Pichia pastoris expressing beta-mannanase mutants

1. Construction of recombinant bacterium

The beta-mannase mutant obtained in example 1 was used as a template, and the following primer pairs were used for PCR amplification to obtain PCR amplification products. The primer sequences are as follows:

an upstream primer:

5′-CCATGTACGTAGCTTCTTCGTTTGTCCAGACAAG-3′；

a downstream primer: 5'-CCGCCTAGGCTACTTCTTGGCCATGGCATC-3' are provided.

And carrying out double enzyme digestion on the PCR amplification product and the pGAPZ alpha A vector by using restriction enzymes SnaBI and AvrII, recovering the enzyme digestion product, and connecting the enzyme digestion product to obtain the recombinant plasmid. And (3) transforming the recombinant plasmid into pichia pastoris GS115 to obtain a recombinant strain B containing the recombinant plasmid.

2. High density fermentation

The Fermentation method is described in "Pichia Fermentation Process Guidelines (Version B,053002, Invitrogen)". The fermentation was carried out in a 5L fermenter. The seed medium, the fermentation minimal medium and the glycerol fed-batch medium were prepared according to the methods described in the above documents. The whole fermentation process adopts two stages of batch culture and glycerol fed-batch induction culture.

3. Results of fermentation

And detecting the enzyme activity of the beta-mannase mutant in the supernatant in the fermentation process. Enzyme activity determination conditions: 50mM citrate phosphate buffer (pH7.0), reaction temperature 65 ℃.

The process of the recombinant strain B growing and secreting the beta-mannase mutant in the fermentation process is shown in figure 7. The result shows that after 168 hours of fermentation, the enzyme activity of the beta-mannase mutant in the fermentation supernatant of the recombinant bacterium B reaches 72600U/mL, the protein content of the fermentation liquid reaches 9.1mg/mL, and the wet weight of the bacterium reaches 460 g/L.

Example 4 application of beta-mannase mutant in preparation of konjac mannan oligosaccharide

1. Beta-mannase mutant hydrolyzes konjaku flour with different addition amounts

Respectively weighing 20g, 30 g, 40 g and 50g of konjac flour, completely dissolving in 100mL of distilled water (the distilled water can be replaced by phosphate buffer with pH of 7.0 or citrate phosphate buffer, etc.), adding beta-mannase mutant according to the proportion of 1000U/g of konjac flour, hydrolyzing at 50 ℃ for 8h, and inactivating in boiling water bath for 10min after enzymolysis to obtain enzymatic hydrolysate. Centrifuging the obtained enzymolysis liquid at 10000rpm for 10min, and collecting supernatant, i.e. crude sugar liquid. And (3) measuring the content of reducing sugar in the crude sugar solution by using a 3, 5-dinitrosalicylic acid method and calculating the yield of the reducing sugar. Washing the precipitate twice with pure water, drying, weighing, and calculating the hydrolysis rate of the konjac flour. The hydrolysis rate and the reducing sugar yield are calculated according to the following formulas:

hydrolysis rate (dry raw material weight-dry hydrolysis residue weight)/dry raw material weight × 100%;

the yield of reducing sugar is (concentration of reducing sugar in crude sugar solution x volume)/dry weight of raw material x 100%.

The hydrolysis rate and the reducing sugar yield of the beta-mannase mutant after hydrolyzing konjac flour with different addition amounts are shown in table 1.

TABLE 1 hydrolysis rate and reducing sugar yield of rhizoma Amorphophalli powder hydrolyzed by beta-mannase mutant

Addition amount (%, w/v)	Hydrolysis ratio (%)	Reducing sugar yield (%)
			20	90.9	70.1
30	90.2	69.9
			40	86.9	68.2
50	88.6	64.6

As can be seen from table 1, as the addition amount of konjac flour was gradually increased, the hydrolysis rate and reducing sugar yield of konjac flour were gradually decreased. When the addition amount of the konjac flour is 20% (w/v), the hydrolysis rate of the konjac flour is 90.9%, and the yield of reducing sugar is 70.1%; when the addition amount of the konjac flour is 30% (w/v), the hydrolysis rate of the konjac flour is 90.2%, and the yield of reducing sugar is 69.9%.

2. Hydrolyzed konjaku flour with different addition amounts of beta-mannase mutants

Weighing 100mL of distilled water (the distilled water can be replaced by phosphate buffer with pH7.0 or citrate phosphate buffer), adding 20g of konjac flour, stirring, adding the beta-mannase mutant according to the proportion of 250U/g, 500U/g, 750U/g and 1000U/g of konjac flour, hydrolyzing at 50 ℃ for 8h, and inactivating in boiling water bath for 20min after enzymolysis to obtain an enzymolysis solution. Centrifuging the obtained enzymolysis liquid at 10000rpm for 10min, and collecting supernatant, i.e. crude sugar liquid. And (3) measuring the content of reducing sugar in the crude sugar solution by using a 3, 5-dinitrosalicylic acid method and calculating the yield of the reducing sugar. Washing the precipitate twice with pure water, drying, weighing, and calculating the hydrolysis rate of the konjac flour. The hydrolysis rate and the reducing sugar yield are calculated according to the following formulas:

The hydrolysis rate and the reducing sugar yield of the konjac flour hydrolyzed by the beta-mannase mutants with different addition amounts are shown in table 2.

Table 2. hydrolysis rate and reducing sugar yield of konjac flour hydrolyzed by beta-mannase mutants with different addition amounts

Enzyme dosage (U/g konjak flour)	Hydrolysis ratio (%)	Reducing sugar yield (%)
			250	84.7	62.6
500	88.6	64.3
			750	90.2	69.2
1000	90.9	70.1

As can be seen from Table 2, as the amount of enzyme added was increased, the hydrolysis rate of konjac flour and the yield of reducing sugar were increased. When the enzyme dosage is 750U/g of konjak powder, the hydrolysis rate and the reducing sugar yield of the konjak powder reach 90.2 percent and 69.2 percent respectively after the konjak powder is hydrolyzed by the beta-mannase mutant for 8 hours. When the enzyme amount is increased to 1000U/g konjaku flour, the hydrolysis rate and the reducing sugar yield of the konjaku flour are 90.9% and 70.1%, respectively.

Example 5 high Performance liquid chromatography analysis of konjak mannan-oligosaccharide

Weighing 20g of konjac flour, dissolving in 100mL of distilled water, adding 750U/g substrate beta-mannase mutant, hydrolyzing at 50 ℃ for 8h, inactivating in boiling water bath for 20min after enzymolysis, obtaining enzymolysis liquid, centrifuging at 10000rpm for 10min, and collecting supernatant, namely crude sugar liquid. And (4) carrying out vacuum freeze drying on the crude sugar solution to obtain a powdery product, namely the konjac mannan oligosaccharide.

6mg of konjac mannan oligosaccharide sample is weighed, dissolved in 3mL of distilled water respectively, filtered by a 0.22 mu m filter membrane and then subjected to high performance liquid chromatography analysis. The chromatographic column was Shodex Sugar KS802, the column temperature was 80 ℃, the mobile phase was pure water at a flow rate of 0.6 min/mL. Mannose, mannose disaccharide, mannose trisaccharide, mannose tetrasaccharide, mannose pentasaccharide and mannose hexasaccharide are used as standard substances.

The high performance liquid chromatogram of konjac mannan oligosaccharide is shown in FIG. 8. As can be seen from the figure, the peak area of the mannooligosaccharides with the polymerization degree of 2-6 in the sample accounts for more than 80% of the total peak area, which indicates that the mass of the mannooligosaccharides accounts for more than 80% of the total mass of the product.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Those not described in detail in this specification are within the skill of the art.

Claims

1. A mutant β -mannanase enzyme characterized by: the beta-mannase mutant is a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table.

2. The gene encoding the protein according to claim 1, wherein: the coding gene of the beta-mannase mutant is shown as a sequence 2 in a sequence table.

3. A preparation method of konjak mannan oligosaccharide is characterized by comprising the following steps: the method specifically comprises the following steps:

s1: obtaining the beta-mannanase mutant of claim 1 by using a DNA amplification technique;

s2: preparing a recombinant bacterium containing the beta-mannase mutant;

s3: fermenting by using recombinant bacteria to prepare a beta-mannase mutant;

s4: hydrolyzing the konjac flour solution by using the beta-mannase mutant;

in step S4, the mixing ratio of the β -mannanase mutant to the konjac flour is: adding 250 and 1000U of beta-mannase mutant into per gram of konjac flour;

4. The method for producing konjac mannan oligosaccharide as claimed in claim 3, wherein: in step S1, the coding gene of the beta-mannanase is derived from Mucor miehei CAU 432.

5. The method for producing konjac mannan oligosaccharide as claimed in claim 3, wherein: in step S2, the recombinant bacteria are pichia pastoris GS115 and/or escherichia coli BL 21.

6. The method for producing konjac mannan oligosaccharide as claimed in claim 3, wherein: in the fermentation liquor of the step S3, the enzyme activity of the beta-mannase mutant reaches 72600U/mL.

7. The method for producing konjac mannan oligosaccharide as claimed in claim 3, wherein: in step S4, the hydrolysis time is 1-24h, the hydrolysis temperature is 30-90 ℃, and the hydrolysis pH value is 3.5-11.0.

8. The konjac mannan oligosaccharide produced by the process for producing konjac mannan oligosaccharide according to any one of claims 3 to 7, wherein: the content of the mannose-disaccharide to the mannose-hexaose is more than 80 percent by mass percentage.