CN112029751B - Production method and application of thermophilic fungus mannase - Google Patents

Production method and application of thermophilic fungus mannase Download PDF

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CN112029751B
CN112029751B CN201910476109.5A CN201910476109A CN112029751B CN 112029751 B CN112029751 B CN 112029751B CN 201910476109 A CN201910476109 A CN 201910476109A CN 112029751 B CN112029751 B CN 112029751B
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mannase
partially hydrolyzed
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闫巧娟
李延啸
江正强
刘瑜
杨绍青
刘军
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China Agricultural University
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Abstract

The invention discloses a production method and application of mannase. The method for producing the mannase provided by the invention comprises the following steps: introducing a mannanase gene derived from the cladosporium camphorata S168 into a receptor yeast to obtain a recombinant yeast; and sequentially carrying out basic fermentation culture, glycerol feeding fermentation stage and methanol feeding induction expression stage on the recombinant yeast to obtain the mannanase from the fermentation product. The expression level of mannase prepared by high-density fermentation of engineering bacteria reaches 42200U/mL, the most suitable pH of the prepared enzyme is 7.5, the most suitable temperature is 75 ℃, the enzyme has the characteristics of acid and alkali resistance, good thermal stability and excellent hydrolysis characteristic, in addition, the invention widens the application range of mannase, the partially hydrolyzed konjac glucomannan prepared by the enzyme has great application value in the industries of food, feed and the like, enriches the types of prebiotic products, promotes the development of prebiotic industry in China, and has great social and economic benefits.

Description

Production method and application of thermophilic fungus mannase
Technical Field
The invention relates to the technical field of biology, in particular to a production method and application of thermophilic fungus mannase.
Background
Mannanase (EC 3.2.1.78) is an endohydrolase that specifically hydrolyzes the β -1, 4-mannosidic bonds in mannans. Mannanases can be classified into glycoside hydrolases families 5, 26, 113 and 134, based on their amino acid sequence similarity. As an important glycoside hydrolase, mannanases are capable of hydrolyzing different kinds of mannans (linear mannans, glucomannans, galactomannans) to produce mannooligosaccharides with a low degree of polymerization (Srivastava and Kapoor. Biotechnology Advances,2017,35: 1-19). Thus, mannanases have been widely used in various industries such as food, feed, etc. (Chauhan et al applied Microbiology and Biotechnology,2012,93: 1817-. Mannanases are widely available and are found in many microorganisms, plants, and lower animals. Improving the expression level of the mannase, expanding the source of the mannase and exploring more applications of the mannase are still hot spots of current research.
Dietary fiber generally refers to a class of polysaccharides that are not digested and absorbed by the human body, and mainly includes inulin, pectin, partially hydrolyzed guar gum, and the like. It can effectively promote the proliferation of beneficial flora in organisms, inhibit the generation of pathogenic microorganisms, improve the flora structure in intestinal tracts and reduce the generation of toxic metabolites (Sevinc et al. journal of Drug Targeting,2014,22: 262-. In addition, dietary fiber has various Functional activities such as promoting gastrointestinal motility, improving constipation, regulating human metabolism, etc. (Kapoor et al. journal of Functional Foods,2016,24: 207-. Konjac gum is an important dietary fiber, the main component of which is glucomannan, and the konjac gum has the characteristics of high viscosity, water solubility, good stability and the like (Behera et al, food Reviews International,2017,33: 22-43). As a thickener and emulsifier, konjac gum has been widely used in the food field. However, adding too much konjac gum in food can destroy the taste of food, even block the absorption of nutrient substances, and limit the application of konjac gum in food to a certain extent.
Currently, vegetable gums such as guar gum are the main raw material used to produce partially hydrolyzed mannans. Sunfiber, a partially hydrolyzed guar product, was successfully prepared by hydrolyzing guar by Sun Japan K.K., the product having a weight average molecular weight of 2.0X 104Da and mass production has been achieved (Theertham. physiology and Behavior,2016,164: 277-283). Chinese patent application No. 201610808817.0A method for preparing dietary fiber (partially hydrolyzed guar gum) by hydrolyzing guar gum with mannanase is provided, and the obtained product has a weight average molecular weight of 2.5 × 104Da; chinese patent application No. (201610808818.5) discloses a method for preparing dietary fiber by hydrolyzing guar gum with mannase and galactosidase in a composite manner, and the weight average molecular weight of the obtained product is 1.5 multiplied by 104Da. At present, konjac gum is mainly used for producing mannan-oligosaccharide, and Chinese patent application Nos. 200910014349.X, 201310428885.0 and 201510465107.8 disclose various methods for producing mannan-oligosaccharide by using konjac gum as raw material, and the concentration of the substrate used is usually less than 20%. There are no reports and patent publications on the production of partially hydrolyzed konjac gum having a high weight average molecular weight from konjac gum as a raw material.
Disclosure of Invention
The invention aims to provide a production method and application of mannase.
In a first aspect, the invention claims a method for producing mannanase.
The method for producing the mannanase can comprise the following steps:
(A) introducing a coding gene of mannase from the cladosporium camphorate into a receptor yeast to obtain a recombinant yeast;
(B) fermenting and culturing the recombinant yeast according to the following steps to obtain the mannanase from a fermentation product:
B1) basic fermentation culture: inoculating the recombinant yeast into a BSM culture medium for culture, and performing a glycerol feeding fermentation stage when glycerol is completely consumed;
B2) and (3) glycerol feeding and fermenting stage: on the basis of the step B1), glycerol is fed into the fermentation system at the speed of 18.4mL/h/L initial fermentation liquor until the light absorption value of the fermentation liquor at 600nm reaches 180-220, starvation is carried out for 0.5h, and then a methanol feeding induced expression stage is carried out;
B3) methanol feeding induction expression stage: based on step B2), methanol was fed to the fermentation system, the flow rate was increased from 3.6mL/h/L of the starting fermentation broth to 10.9mL/h/L of the starting fermentation broth over 4h (the flow rate was increased linearly), and then methanol was fed to the end of the fermentation while maintaining the flow rate of the starting fermentation broth of 10.9 mL/h/L.
In step (a), the mannanase derived from cladosporium camphorata may be a mannanase derived from cladosporium camphorata S168.
Further, the mannanase derived from the cladosporium camphorata specifically can be any one of the following proteins:
a1) a protein consisting of amino acid residues 18 to 355 from the N-terminus of SEQ ID No.1 (without a signal peptide);
a2) a protein (containing a signal peptide) consisting of the amino acid sequence shown in SEQ ID No. 1;
a3) the protein with mannanase activity is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein a1) or a 2).
a4) A protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in any one of a1) -a3) and having a mannanase activity.
a5) A fusion protein obtained by attaching a label to the N-terminal and/or C-terminal of the protein defined in any one of a1) -a 4).
SEQ ID No.1 consists of 355 amino acid residues in total, wherein the 1 st to 17 th positions are signal peptide sequences. The protein shown in SEQ ID No.1 is named McMan 5A.
In the step (A), the gene encoding the mannanase derived from M.camphorata is introduced into the recipient yeast in the form of a recombinant vector.
Furthermore, the recombinant vector is a recombinant plasmid obtained by inserting the encoding gene of the mannase from the Cladosporium camphorata into the multiple cloning site of the pPIC9K vector.
In the present invention, the yeast is pichia pastoris.
Further, the pichia is specifically pichia GS 115.
In the step (A), the coding gene of the mannanase derived from the Cladosporium camphoratum is a DNA molecule as follows:
b1) a DNA molecule of SEQ ID No.2 from position 52 to 1068 of the 5' terminus (without signal peptide);
b2) a DNA molecule (containing a signal peptide) shown in SEQ ID No. 2;
b3) a DNA molecule which hybridizes under stringent conditions with the DNA molecule defined in b1) or b2) and which encodes the mannanase from Leptospira camphorata;
b4) a DNA molecule which has more than 99 percent, more than 95 percent, more than 90 percent, more than 85 percent or more than 80 percent of homology with the DNA sequence defined in any one of b1) -b3) and codes the mannanase derived from the cladosporium laurentii.
The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.
The SEQ ID No.2 consists of 1068 nucleotides in total, and the 1 st to 51 st bases from the 5 'end are signal peptide coding sequences, and the 52 nd to 1068 th bases from the 5' end are mature protein coding sequences.
In step B1), the recombinant yeast inoculated in the BSM medium may be a seed solution of the recombinant yeast.
Further, the seed liquid is prepared by inoculating the recombinant yeast obtained in the step (A) into a BMGY medium, and culturing at 30 ℃ and 200rpm with shaking until the absorbance value of the fermentation liquid at 600nm reaches 10.
Wherein the composition of the BMGY medium is as follows: 1% yeast extract, 2% peptone, 1.34% nitrogen source YNB without amino yeast, 4X 10-5% biotin, 1% glycerol, balance 100mmol/L pH 6.0 phosphate buffer. Wherein,% represents the mass-to-volume ratio (e.g., 1% represents 1g/100 mL).
In the step B1), the seed solution is inoculated into the BSM medium and is inoculated into a fermentation tank filled with the BSM medium (the inoculation amount is 1/10 volume of the seed solution of the BSM medium), and the liquid filling amount of the BSM medium in the fermentation tank is 1.5/5 of the volume of the fermentation tank (for example, 1.5L of the BSM medium is filled in a 5-L fermentation tank).
In the step B1), the solvent of the BSM culture medium is water, and the solutes and the concentrations are as follows: 2.67% phosphoric acid, 0.093% CaSO4，1.82％K2SO4，1.49％MgSO4·H2O, 0.413% KOH, 4% glycerol. Wherein,% represents the mass-to-volume ratio (e.g., 1% represents 1g/100 mL).
Step B1), the method also comprises the step of adding a PTM1 solution (pichia pastoris trace metal salt solution).
Further, the PTM1 solution was added in an amount of 6.535mL of the PTM1 solution per 1.5L of the BSM medium. The solvent of the PTM1 solution is water, and the solutes and the concentrations are as follows: 0.6% CuSO4·5H2O，0.008％NaI，0.3％MnSO4·H2O，0.02％Na2MoO4·2H2O，0.002％H3BO3，0.05％CoCl2，2％ZnCl2，6.5％FeSO4·7H2O, 0.02% biotin, 0.5% concentrated sulfuric acid. Wherein,% represents the mass-to-volume ratio (e.g., 1% represents 1g/100 mL).
In the step B1), the temperature of the culture is 30 ℃, the pH is 4.0, the rotating speed is 600rpm, the ventilation amount is 1.0vvm, and the culture time is 18-24 h.
In the step B2), the glycerol is dissolved in water to form 500g/L of glycerol aqueous solution; the culture temperature of the glycerol feeding fermentation stage is 30 ℃, the pH value is 4.0, and the dissolved oxygen is controlled to be more than 20%.
In step B3), the methanol is anhydrous methanol; the culture temperature of the methanol feeding induction expression stage is 30 ℃, the pH value is 6.0, and the dissolved oxygen is controlled to be more than 15%.
In a particular embodiment of the invention, the time of the fermentation culture in step (B) is in particular 168 hours.
In the step (B), the fermentation culture of the recombinant yeast further comprises the following steps: dialyzing the supernatant of the fermentation liquor to remove salt, then carrying out anion exchange chromatography, carrying out linear elution by using 20mM Tris-HCl pH 8.0 buffer solution containing 0-500mM NaCl, and collecting the purified mannanase.
Further, the anion exchange chromatography is Q-sepharose FF ion exchange chromatography.
The elution procedure for the linear elution with 20mM Tris-HCl pH 8.0 buffer containing 0-500mM NaCl was: the concentration of NaCl in the eluate increased linearly from 0 to 500mM within 100 min.
The supernatant of the fermentation broth was dialyzed and equilibrated to 20mM Tris-HCl buffer pH 8.0. The anion exchange chromatography (Q-Sepharose FF, Q-Sepharose FF ion exchange chromatography) was carried out by column equilibration using 20mM Tris-HCl buffer pH 8.0. The anion exchange chromatography (Q-Sepharose FF, Q-Sepharose FF ion exchange chromatography) is carried out by eluting with 20mM Tris-HCl buffer solution at pH 8.0 for 20min, washing unbound protein, and performing linear elution with 0-500mM NaCl-containing Tris-HCl buffer solution at pH 8.0.
In a second aspect, the invention claims mannanases prepared by the method of the first aspect hereinbefore.
In a third aspect, the invention claims the use of any of the following:
(C1) use of a mannanase enzyme as hereinbefore described in the second aspect for hydrolysing mannans and/or plant gums;
(C2) use of a mannanase enzyme according to the second aspect hereinbefore in the preparation of a product for hydrolysing mannans and/or plant gums;
(C3) Use of a mannanase enzyme as hereinbefore described in the second aspect for the production of mannooligosaccharides;
(C4) use of a mannanase enzyme as hereinbefore described in the second aspect in the preparation of a product for the production of mannooligosaccharides;
(C5) use of a mannanase enzyme as hereinbefore described in the second aspect for the production of partially hydrolysed konjac gum;
(C6) use of a mannanase enzyme as hereinbefore described in the second aspect for the manufacture of a product for the production of partially hydrolysed konjac gum.
Further, in the (C1) and the (C2), the hydrolysis pH is 4.0-10.5 (specifically, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5; or a range between any two of the above values, such as 4.5-8; or 5.5-7.5; or a pH value within 7-7.5); and/or the hydrolysis temperature is 30-85 deg.C (30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, 80 deg.C, 85 deg.C), or a range between any two of the above values such as 30-65 deg.C, or 65-80 deg.C, or a temperature within 70-75 deg.C).
Further, in (C3) and (C4), the mannooligosaccharides are linked by 2 to 10 mannose and galactose or glucose.
Further, in the (C3) and the (C4), the production substrate is mannan or vegetable gum.
Further, in the (C5) and the (C6), a production substrate is konjac gum; and/or the initial concentration of the konjac glucomannan in the reaction system is 1-20g/100 mL; and/or the hydrolysis time is 0.5-12 h; and/or the addition amount of the mannase is 20-800U/g konjac glucomannan.
Wherein the mannan may be a linear mannan or a glucomannan or a galactomannan or a galactoglucomannan. The vegetable gum can be locust bean gum, konjac gum, cassia gum and/or guar gum.
In a fourth aspect, the invention claims any of the following methods or products prepared by said methods:
(D1) a method for preparing medium molecular weight partially hydrolyzed konjac gum comprises the following steps: hydrolyzing konjac gum with the mannase of claim 8, wherein the ratio of konjac gum to the mannase is 1g konjac gum: 20U of the mannase is hydrolyzed at 70 ℃ for 8 hours to obtain the medium molecular weight partially hydrolyzed konjac glucomannan; the weight average molecular weight of the medium molecular weight partially hydrolyzed konjac gum is 1.6 × 105D(2×103To 6X 105Da range).
(D2) A method for preparing low molecular weight partially hydrolyzed konjac gum comprises the following steps: hydrolyzing konjac gum with the mannase of claim 8, wherein the ratio of konjac gum to the mannase is 1g konjac gum: hydrolyzing 200U of the mannase at 70 deg.C for 8h to obtain the low molecular weight partially hydrolyzed konjac glucomannan; the low molecular weight partially hydrolyzed konjac gum has a weight average molecular weight of 2.0 × 10 4Da(1×103To 7X 104Da range).
The present invention utilizes genetic engineeringThe technology clones beta-mannase of a glucoside hydrolase 5 family from the Cladosporium camphorata S168, and the beta-mannase is efficiently expressed in Pichia pastoris GS115, and the expression level reaches 42200U/mL. The enzyme prepared by the invention has the characteristics of 7.5 optimal pH, 75 optimal temperature, acid and alkali resistance, good thermal stability and excellent hydrolysis characteristic, can stably and simultaneously play a catalytic role in a wide pH range and high temperature condition, is obviously different from the prior mannase, is mainly used for hydrolyzing mannan to generate mannan oligosaccharides with higher polymerization degree and has larger difference with beta-mannase from other sources, and is used for hydrolyzing konjac glucomannan to prepare two different partially hydrolyzed konjac glucomannan with the weight average molecular weights of 1.6 multiplied by 10 respectively5And 2.0X 104Da, the content ratio of the total dietary fiber is 93.2 percent and 90.6 percent respectively. The invention widens the application range of the mannase, and the obtained partially hydrolyzed konjac glucomannan has great application value in the industries of food, feed and the like, enriches the variety of prebiotics products, promotes the development of the prebiotics industry in China, and has great social and economic benefits.
Drawings
FIG. 1 shows the enzyme production history of mannanase in a 5L fermentor. Wherein (■) is mannanase activity in the fermentation broth, (●) is protein content in the fermentation broth, and (a-solidup) is wet weight of thallus in the fermentation broth.
FIG. 2 is a purified electrophoretogram of mannanase. Wherein Lane M is a low molecular weight standard protein, Lane 1 is the supernatant of fermentation broth of a fermentation tank, Lane 2 is a pure enzyme purified by a Q-sepharose column, and Lane 3 is an enzyme deglycosylated by N-deglycosylation enzyme.
FIG. 3 is the pH optimum of mannanase. Wherein (●) citrate phosphate buffer (pH3.0-7.0), (■) citrate buffer (pH3.0-6.0), (. diamond-solid.) phosphate buffer (pH6.0-8.0), (. tangle-solidup.) MOPS buffer (6.5-7.5), (□) CHES buffer (pH8.0-10.0), (. smallcine-sodium hydroxide buffer (pH9.0-10.5), (. smallcine-sodium hydroxide buffer (pH10.0-11.0) and (CAPS) buffer (pH 10.0-11.0).
FIG. 4 shows the pH stability of mannanase. Wherein (●) citrate phosphate buffer (pH3.0-7.0), (■) citrate buffer (pH3.0-6.0), (. diamond-solid.) phosphate buffer (pH6.0-8.0), (. tangle-solidup.) MOPS buffer (6.5-7.5), (□) CHES buffer (pH8.0-10.0), (. smallcine-sodium hydroxide buffer (pH9.0-10.5), (. smallcine-sodium hydroxide buffer (pH10.0-11.0) and (CAPS) buffer (pH 10.0-11.0).
FIG. 5 is a graph showing the optimum temperature measurement of mannanase.
FIG. 6 is a graph showing the temperature stability assay of mannanase.
FIG. 7 is a thin layer chromatography of mannanase hydrolyzed locust bean gum and konjac gum product. Wherein Mn is a mannan oligosaccharide standard (M is mannose, M2 is mannose, M3 is mannotriose, M4 is mannotetraose, M5 is mannopentaose, and M6 is mannohexaose).
FIG. 8 is a graph of gel exclusion chromatography analysis of medium molecular weight partially hydrolyzed konjac gum.
FIG. 9 is a graph of gel exclusion chromatography analysis of low molecular weight partially hydrolyzed konjac gum.
Detailed Description
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 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% (mass/volume ratio, i.e., 0.5g/100mL) locust bean gum substrate solution (prepared with 50mM MOPS buffer solution, pH 7.5), reacted in a water bath at 75 ℃ 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 units of mannanase activity are defined as: under the above reaction conditions, the amount of enzyme required for the reaction to produce 1. mu. mol of reducing sugar (in terms of mannose) per minute was one enzyme activity unit (1U).
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 mannanase enzyme activity unit: the enzyme amount required for decomposing 0.5% locust bean gum (mass to volume ratio, i.e., 0.5g/100mL) substrate to release 1. mu. mol of mannose per minute at pH7.5 and 75 ℃. The enzyme activity calculation formula 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.
Delirium camphoratum (Malbranchea cinnamonmea) S168: obtained by screening in the laboratory, and stored in China general microbiological culture Collection center (CGMCC), the preservation number is as follows: 6022. the method is also described in the article of purification and property research of low molecular weight xylanase in the branch mould of Naematoloma camphorata, such as plum Yan Shao, the science and technology of food industry, No. 17 2014.
Example 1 cloning and high expression of the mannanase Gene
1. Cloning of the mannanase Gene
Amplifying the mannosidase gene from the cladosporium camphorata by using a specific primer, wherein the specific primer has the following sequence:
an upstream primer: 5'-ATGAAGTTATCATCCTTCGCT-3';
a downstream primer: 5'-CTAGCTTGCGTTTCGAGTAGC-3' is added.
The PCR reaction was carried out using Ex taq DNA polymerase (Takara) using a cDNA of A. camphorata (Malbranchea cinnamomea) S168 as a template and the above primer set as primers. The procedure is as follows: pre-denaturation at 95 ℃ for 5 min; 30 cycles of amplification at 94 ℃ for 30s, at 55 ℃ for 30s and at 72 ℃ for 1 min; total extension was 10 min. After the PCR product was detected by 1% agarose gel electrophoresis, the plasmid ligated to pMD-18T vector (Takara Co., Ltd.) was recovered, transformed into E.coli by heat shock, and single colonies were selected for sequencing.
The analysis shows that the total length of the mannanase gene is 1068bp (SEQ ID No.2) and codes 355 amino acids (SEQ ID No. 1). The 1 st to 17 th amino acid residues from the amino terminal (N terminal) are predicted to be signal peptide by SignalP 3.0(http:// www.cbs.dtu.dk/services/SignalP /), and the 18 th to 355 th amino acid residues from the amino terminal are predicted to be mature protein. The protein shown in SEQ ID No.1 is named McMan 5A.
2. Construction of engineering bacteria for expressing mannanase
Designing an expression primer according to sequences of a pichia pastoris expression vector and mannase, wherein the upstream primer and the downstream primer are respectively added with an EcoRI restriction enzyme site and a NotI restriction enzyme site as follows:
An upstream primer: 5' -CTCAGGAATTCGCTCCCTTGGAATCGCGG-3′；
A downstream primer: 5' -TGACTGCGGCCGCCCTAGCTTGCGTTTCGAGTAGC-3′。
PCR amplification is carried out by using the primer pair and taking cDNA of the Cladosporium camphorata S168 as a template, the amplified product is cut by restriction enzymes EcoRI and NotI, and the amplified product is connected to a pPIC9K vector which is cut by corresponding enzyme in advance after being recovered by agarose gel electrophoresis. The sequence was verified using PCR and double digestion, and the already correct ligation into the Pichia expression vector was designated pPIC9K-McMan 5A. And transforming the recombinant expression vector into pichia pastoris GS115 to obtain the recombinant pichia pastoris.
3. High-density fermentation of recombinant bacterium pichia pastoris GS115
The fermentation was carried out in a 5L fermenter. The whole fermentation process adopts three stages of batch culture, glycerol fed-batch culture and 100% methanol induction culture. The culture medium and the operation process are as follows:
the media and their components used in the experimental procedure were as follows:
BMGY medium: 1% yeast extract powder, 2% peptone, 1.34% YNB, 4X 10-5% Biotin, 1% glycerol, 100mmol/L pH 6.0 potassium phosphate buffer. Wherein,% represents the mass-to-volume ratio (e.g., 1% represents 1g/100 mL).
BSM medium: the solvent is water; the solutes and the concentrations are as follows: 2.67% phosphoric acid, 0.093% CaSO 4，1.82％K2SO4，1.49％MgSO4·H2O, 0.413% KOH, 4% glycerol. Wherein,% represents a mass-to-volume ratio (e.g., 1% represents 1g/100 mL).
Seed culture: and (3) sucking 150 mu L of the recombinant pichia pastoris bacterial solution obtained in the step (2) from a preserved glycerol tube, inoculating the bacterial solution into 150mL of BMGY culture medium, and performing shake culture at the temperature of 30 ℃ and the rpm of 200 until OD600 is about 10.0.
Batch culture: the solution was fermented and canned with 1.5L BSM medium, sterilized, adjusted to pH 4.0 with concentrated ammonia, and added with 6.535mL PTM1 solution (solvent is water, solutes and concentrations are as follows:0.6％CuSO4·5H2O，0.008％NaI，0.3％MnSO4·H2O，0.02％Na2MoO4·2H2O，0.002％H3BO3，0.05％CoCl2，2％ZnCl2，6.5％FeSO4·7H2o, 0.02% biotin, 0.5% concentrated sulfuric acid. Wherein the percentages are mass-volume ratios, such as 1% represents 1g/100mL), 150mL of seed solution is inoculated, the inoculation amount is 10% (V/V), the temperature is 30 ℃, the rotation speed is 600rpm, the ventilation volume is 1.0vvm, and the fermentation is carried out for 18-24 h.
Glycerol fed-batch culture: after the batch culture was completed until the glycerol was exhausted, the feed of glycerol (500 g/L aqueous glycerol solution obtained by dissolving glycerol in water) was started, and DO (dissolved oxygen) was constantly monitored at a flow rate of 18.4mL/h/L for the starting fermentation solution, and the dissolved oxygen was maintained at more than 20% by adjusting the rotation speed, the aeration rate, and the like. The feeding time is 4 hours, and the light absorption value of the fermentation liquor at 600nm reaches about 180-220. The culture temperature in this step was 30 ℃ and pH was 4.0.
100% methanol induction culture: after stopping the glycerol feed, starving for about 30min, adding anhydrous methanol, increasing the flow rate from 3.6mL/h/L of the starting fermentation broth to 10.9mL/h/L of the starting fermentation broth over 4h (the flow rate is linearly increased and finally maintained at 10.9mL/h/L), and monitoring the DO for > 15%. And sampling and analyzing the wet weight of the thallus, the mannase activity and the protein content of the fermentation liquor in the fermentation process. The culture temperature in this step was 30 ℃ and pH 6.0.
The high density fermentation process is shown in FIG. 1. Wherein (■) is mannanase activity in the fermentation broth, (●) is protein content in the fermentation broth, and (a-solidup) is wet weight of thallus in the fermentation broth. Along with the gradual extension of the fermentation time, the activity of the mannase, the protein content and the wet weight of the thalli in the fermentation liquid are gradually increased. When the fermentation is carried out till the 7 th day, the activity of the mannanase reaches 42200U/mL, the protein content reaches 10.2mg/mL, and the wet weight of the thalli reaches 479.5 g/L.
Example 2 purification and enzymatic Properties of mannanase
1. Purification of mannanase
Q-Sepharose FF (Q-Sepharose FF) ion exchange chromatography: 10mL of the fermentation broth subjected to the high-density fermentation in example 1 was centrifuged at 10000rpm for 5min to obtain a supernatant, which was dialyzed overnight against 20mmol/L Tris-HCl buffer (pH8.0), and then applied to a Q-sepharose column equilibrated with 20mmol/L Tris-HCl buffer (pH 8.0). Eluting with 20mmol/L Tris-HCl buffer (pH8.0) for 20min, eluting unbound protein, linearly eluting with 20mmol/L Tris-HCl buffer (pH8.0) containing 0-500mmol/L NaCl for 100min, combining eluates containing the target protein, and dialyzing with 20mmol/L MOPS buffer (pH7.5) overnight.
The SDS-PAGE purification pattern is shown in FIG. 2. Wherein, Lane 1 is the fermentation supernatant, Lane 2 is the purified enzyme purified by Q-Sepharose column, Lane 3 is the enzyme deglycosylated with N-deglycosylation enzyme (the protein band at 30kDa in Lane is N-deglycosylation enzyme). After deglycosylation, the tailing of the mannase in an electrophoretogram is obviously reduced, and the molecular weight is reduced, which indicates that the mannase is expressed in pichia pastoris and has glycosylation.
2. Determination of optimum pH
And taking the prepared pure enzyme solution as an enzyme solution to be detected, respectively carrying out enzyme activity determination on the enzyme solution in different buffer solution systems at 75 ℃, and calculating the relative enzyme activity by taking the highest enzyme activity as 100%. The various buffers are as follows:
1) a citrate phosphate buffer (pH 3.0-7.0);
2) a citric acid buffer (pH 3.0-6.0);
3) phosphate buffer (pH6.0-8.0);
4) MOPS buffer (pH6.5-7.5);
5) CHES buffer solution (pH8.0-10.0)
6) Glycine-sodium hydroxide buffer (pH 9.0-10.5).
7) CAPS buffer (pH10.0-11.0).
The results are shown in FIG. 3: the optimum pH of the mannanase McMan5A was 7.5.
3. Determination of pH stability
Diluting the mannase pure enzyme solution with the above buffer solution, treating in 50 deg.C water bath for 30min, rapidly cooling in ice water for 30min, and measuring enzyme activity. The relative enzyme activities of McMan5A after different pH treatments were calculated using the untreated mannanase as 100%.
The results are shown in FIG. 4: McMan5A has good pH stability, and 80% of enzyme activity is remained after 30min of heat preservation at pH 4.0-10.5.
4. Determination of optimum temperature
The mannase pure enzyme solution is diluted properly with 50mmol/L MOPS buffer solution (pH7.5), and the enzyme activity is measured at different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 ℃). The relative enzyme activity is calculated by taking the highest enzyme activity as 100 percent.
The results are shown in FIG. 5: the optimum temperature for McMan5A was 75 ℃.
5. Determination of temperature stability
Properly diluting the mannase pure enzyme solution with 50mmol/L MOPS buffer solution (pH7.5), respectively preserving the temperature for 30min at different temperatures (30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 ℃), quickly placing the mannase pure enzyme solution in ice water for cooling for 30min, and then measuring the enzyme activity. The relative enzyme activities of McMan5A after different pH treatments were calculated using the untreated mannanase as 100%.
The results are shown in FIG. 6: McMan5A has excellent thermal stability, and has enzyme activity of 80% or more after 30min at 85 deg.C or below, and 50% after 30min at 100 deg.C.
6. Determination of substrate specificity
Different mannan substrates are selected, and the enzyme activity of the mannase on the substrates is measured, wherein the substrate concentration is 0.5g/100 mL. The results are shown in Table 1, and the enzyme has the highest enzyme activity on locust bean gum, and the second is konjac gum and cassia gum.
TABLE 1 substrate specificity of mannanase McMan5A
Substrate Relative enzyme activity (%)
Sophora bean gum 100
Konjak glue 88.4
Guar gum 63.5
Cassia seed gum 76.8
Fragrant bean gum 20.3
7. Analysis of hydrolysis characteristics
Locust bean gum and konjac gum (1g/100mL) are dissolved in 50mmol/L MOPS buffer solution (pH7.5), McMan5A (5U/mL) is added, the mixture is placed at 70 ℃ for hydrolysis for 12h, samples are taken at intervals, and all samples are placed in a boiling water bath for 10min to terminate the reaction. 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, volume ratio), and the developer is methanol: sulfuric acid (95: 5, volume ratio).
The results of thin layer chromatography are shown in FIG. 7. It can be seen that McMan5A hydrolyzes locust bean gum to produce mainly mannobiose, mannotriose and some mannooligosaccharides with higher degree of polymerisation. McMan5A hydrolyzed konjac gum, hardly produced mannooligosaccharides, mainly some partially degraded components with higher degree of polymerization.
Example 3 hydrolysis of konjac gum at various substrate concentrations
Weighing 100mL MOPS buffer solution 50mmol/L pH7.5, adding 1g, 5g, 10g, 20g, and 30g konjac glucomannan, stirring, adding mannase McMan5A at a ratio of 200U/g konjac glucomannan, hydrolyzing at 70 deg.C for 8 hr, and boiling water bathTerminating the reaction for 20min to obtain the enzymolysis liquid. The viscosity of the enzymatic hydrolysate was measured at 25 ℃ using a DV-1 rotational viscometer. The weight average molecular weight of the product was determined by gel exclusion chromatography. Diluting the product with distilled water to 10mg/mL, centrifuging at 10000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, and collecting the supernatantXLThe column temperature was 35 ℃ and the mobile phase was water at a flow rate of 0.6 mL/min. And (3) measuring the molecular weight and the molecular weight distribution curve of the sample by using a dextran standard substance with the molecular weight of 1200-375500Da as a standard.
The viscosity and the product weight average molecular weight of the obtained enzymatic hydrolysate after hydrolyzing konjac glucomannan with different concentrations are shown in table 2. It can be seen that as the concentration of the substrate is increased, the viscosity of the enzymolysis solution is gradually increased, and the weight average molecular weight of the product is also gradually increased. When the substrate concentration is increased to 30g/100mL, the viscosity of the zymolytic fluid and the weight average molecular weight of the product are both increased remarkably. When the concentration of konjac glucomannan is 20g/100mL, after enzymolysis for 8h, the viscosity of the enzymolysis liquid reaches 42720mPa s, and the weight average molecular weight of the product reaches 2.0 multiplied by 10 4Da. This is in contrast to the weight average molecular weight (2.5X 10) of partially hydrolyzed guar products which are currently commercially available4Da) are similar, and has better application prospect.
TABLE 2 viscosity of the enzymatic hydrolysate and weight-average molecular weight of the product obtained by hydrolyzing konjac glucomannan with mannanase
Konjak glue (g/100mL) Viscosity (mPa. s) Weight average molecular weight (Da)
1 480 3.5×103
5 5090 9.1×103
10 26840 1.8×104
20 42720 2.0×104
30 110030 2.4×105
Example 4 hydrolysis of konjac gum with different amounts of mannanase added
Weighing 100mL of 50mmol/L MOPS buffer solution with pH7.5, adding 20g of konjac glucomannan, fully stirring, adding mannase McMan5A according to the proportion of 20U/g, 50U/g, 100U/g, 200U/g, 400U/g and 800U/g of konjac glucomannan, hydrolyzing at 70 ℃ for 8h, and boiling water bath for 20min after enzymolysis to stop reaction to obtain an enzymolysis solution. The viscosity of the enzymatic hydrolysate was measured at 25 ℃ using a DV-1 rotational viscometer. The weight average molecular weight of the product was determined by gel exclusion chromatography. Diluting the product with distilled water to 10mg/mL, centrifuging at 10000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, and collecting the supernatantXLThe column temperature was 35 ℃ and the mobile phase was water at a flow rate of 0.6 mL/min. And (3) measuring the molecular weight and the molecular weight distribution curve of the sample by using a dextran standard substance with the molecular weight of 1200-375500Da as a standard.
The viscosity and the product weight average molecular weight of the obtained enzymatic hydrolysate after hydrolyzing konjac gum with different amounts of added enzyme are shown in table 3. It can be seen that the viscosity of the enzymolysis solution gradually decreases and the weight average molecular weight of the product gradually decreases as the amount of the enzyme added gradually increases. MannanThe addition amount of enzyme is 20-800U/g, after enzymolysis for 8h, the viscosity of the enzymolysis liquid is reduced from 110210 mPas to 560 mPas, and the weight average molecular weight of the product is 1.6 × 105Da is reduced to 6.5X 103Da。
TABLE 3 viscosity of the enzymatic hydrolysate and weight-average molecular weight of the product obtained by hydrolyzing konjac gum with different amounts of enzymes
Figure BDA0002082301320000111
Figure BDA0002082301320000121
Example 5 hydrolysis of konjac gum at different hydrolysis times
Weighing 100mL of MOPS buffer solution with 50mmol/L and pH7.5, adding 20g of konjac glucomannan, fully stirring, adding mannase McMan5A according to the proportion of 200U/g of konjac glucomannan, hydrolyzing at 70 ℃ for 0.5, 1, 2, 4, 8 and 12 hours, and terminating the reaction in a boiling water bath for 20min after enzymolysis to obtain an enzymolysis solution. The viscosity of the enzymatic hydrolysate was measured at 25 ℃ using a DV-1 rotational viscometer. The weight average molecular weight of the product was determined by gel exclusion chromatography. Diluting the product with distilled water to 10mg/mL, centrifuging at 10000rpm for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, and collecting the supernatantXLThe column temperature was 35 ℃ and the mobile phase was water at a flow rate of 0.6 mL/min. And (3) measuring the molecular weight and the molecular weight distribution curve of the sample by using a dextran standard substance with the molecular weight of 1200-375500Da as a standard.
The viscosity and the product weight average molecular weight of the resulting enzymatic hydrolysate at different hydrolysis times are shown in table 4. It can be seen that the viscosity of the enzymatic hydrolysate and the weight average molecular weight of the product are gradually decreased as the hydrolysis time is gradually prolonged. Hydrolyzing konjac gum with mannanase for 0.5-8h, reducing viscosity of enzymolysis solution from 395000 mPas to 38600 mPas, and adjusting weight average molecular weight of the product from 1.3 × 106Da is reduced to 1.8X 104Da。
TABLE 4 viscosity of the enzymatic solution and weight-average molecular weight of the product obtained by hydrolyzing konjac glucomannan at different hydrolysis times
Time of hydrolysis (h) Viscosity (mPa. s) Weight average molecular weight (Da)
0.5 395000 1.3×106
1 201600 2.6×105
2 105800 7.7×104
4 53500 3.1×104
8 42720 2.0×104
12 38600 1.8×104
Example 6 preparation of medium molecular weight partially hydrolyzed konjac gum
Weighing 20g of konjac glucomannan, dissolving in 100mL of 50mmol/L MOPS buffer solution with pH of 7.5, adding 20U/g of konjac glucomannan, hydrolyzing at 70 deg.C for 8h, and stopping reaction in boiling water bath for 20min to obtain enzymatic hydrolysate. Subsequently, the viscosity of the enzymatic hydrolysate was determined to be 110210mPa · s, which was significantly lower than the viscosity of the konjac gum solution before hydrolysis. And (3) performing vacuum freeze drying on the enzymolysis liquid to obtain a powdery product, namely the partially hydrolyzed konjac glucomannan with medium molecular weight, wherein the yield of the product is 93.2%.
Weighing lyophilized partial hydrolyzed konjac glucomannan 6mg, dissolving in 3mL distilled water, centrifuging at 10000rpm for 5min, filtering the supernatant with 0.22 μm filter membrane, and analyzing with gel exclusion chromatography. The chromatographic column is TSKgel GMPW XLThe mobile phase was pure water, and the flow rate was 0.6 mL/min. And (3) measuring the molecular weight and the molecular weight distribution curve of the sample by using a dextran standard substance with the molecular weight of 1200-375500Da as a standard. Gel exclusion chromatography of medium molecular weight partially hydrolyzed konjac gum is shown in fig. 8. The product is mainly distributed at 2 × 103To 6X 105In Da range, the molecular weight distribution is wider, and the product is richer. The weight average molecular weight of the medium molecular weight partially hydrolyzed konjac gum is 1.6 × 105Da。
Example 7 preparation of Low molecular weight partially hydrolyzed Konjac Gum
Weighing 20g of konjac glucomannan, dissolving in 100mL of 50mmol/L MOPS buffer solution with pH of 7.5, adding 200U/g of konjac glucomannan, hydrolyzing at 70 deg.C for 8h, and stopping reaction in boiling water bath for 20min to obtain enzymatic hydrolysate. The viscosity of the enzymolysis liquid is reduced to 42720 mPa.s, which is obviously lower than the viscosity of the original konjak gum solution and the partial hydrolyzed konjak gum solution with medium molecular weight. And (3) performing vacuum freeze drying on the enzymolysis liquid to obtain a powdery product, namely the low-molecular-weight partially hydrolyzed konjac glucomannan, wherein the yield of the product is 96.8%.
Weighing 6mg of lyophilized low molecular weight partially hydrolyzed konjac glucomannan, dissolving in 3mL distilled water, centrifuging at 10000rpm for 5min, filtering the supernatant with 0.22 μm filter membrane, and analyzing with gel exclusion chromatography. The chromatographic column is TSKgel GMPW XLThe mobile phase was pure water, and the flow rate was 0.6 mL/min. By usingThe dextran standard substance with the molecular weight of 1200-375500Da is taken as a standard, and the molecular weight distribution curve of the sample are measured. The results of gel exclusion chromatography of low molecular weight partially hydrolyzed konjac gum are shown in fig. 9. It can be seen that the molecular weight distribution of the product is relatively narrow, mainly centered at 1X 103To 7X 104Within the Da range. The weight average molecular weight of the low molecular weight partially hydrolyzed konjac gum is calculated to be 2.0 × 104Da。
Example 8 measurement of Total dietary fiber of partially hydrolyzed konjac glucomannan
The total dietary fiber content of the two partially hydrolyzed konjac gums of the two examples was determined by the method of AOAC2009.01, and the results are shown in table 5. It can be seen that the non-soluble dietary fiber and the alcohol-precipitable soluble dietary fiber, which account for 84.5% and 78.2%, respectively, were predominant in both products, with less non-precipitable soluble dietary fiber content. The total dietary fiber content was 93.2% and 90.6% in both products, respectively.
TABLE 5 Total dietary fiber content in two partially hydrolyzed konjac gums
Figure BDA0002082301320000131
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.
Comparative examples 1,
The "100% methanol induction culture" condition in the fermentation condition in step 3 of example 1 was changed as follows:
100% methanol induction culture: after stopping the glycerol feed, 100% methanol induction medium was immediately fed in, and the flow rate was increased from 3.6mL/h/L of the starting fermentation broth to 20mL/h/L of the starting fermentation broth over 4h (the flow rate was increased linearly and finally maintained at 20mL/h/L), and DO was monitored to be > 0%. And sampling and analyzing the wet weight of the thallus, the mannase activity and the protein content of the fermentation liquor in the fermentation process. The culture temperature in this step was 30 ℃ and pH 6.0.
Other conditions were exactly the same as in example 1.
The experimental results are as follows:
along with the gradual extension of the fermentation time, the enzyme activity, the protein content and the wet weight of the bacteria of the mannanase in the fermentation liquid are gradually increased, but the expression level is obviously lower than that of the mannanase in the example 1. When the fermentation is carried out till the 7 th day, the activity of the mannanase is only 10200U/mL, the protein content reaches 2.6mg/mL, and the wet weight of the thallus reaches 351.2 g/L.
Comparative examples 2,
The elution conditions for mannanase in step 1 of example 2 were changed to:
eluting with 20mmol/L Tris-HCl buffer (pH8.0) for 20min to elute unbound protein, eluting with 20mmol/L Tris-HCl buffer (pH8.0) containing 500mmol/L NaCl for 20min, combining eluates containing the protein of interest, and dialyzing with 20mmol/L MOPS buffer (pH7.5) overnight.
Other conditions were exactly the same as in example 2.
The experimental results are as follows:
after SDS-PAGE analysis, the mannanase eluate collected under the condition still contains more hybrid proteins, and the mannanase cannot be purified and separated.
<110> university of agriculture in China
<120> production method and application of thermophilic fungus mannase
<130> GNCLN191133
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 355
<212> PRT
<213> Malbranchea cinnamonmea
<400> 1
Met Lys Leu Ser Ser Phe Ala Leu Pro Leu Leu Ala Gly Leu Ser Ser
1 5 10 15
Ser Ala Pro Leu Glu Ser Arg Gln Ser Gly Leu Ser Pro Phe Ala Gly
20 25 30
Thr Asn Ala Tyr Trp Leu Pro Phe Leu Thr Asn Asp Ala Asp Val Glu
35 40 45
Ala Ser Phe Gln Ala Met Lys Asn Ala Gly Met Lys Val Val Arg Thr
50 55 60
Trp Ala Phe Asn Asp Asn Thr Glu Cys Gln Glu Ile His Phe Gln Cys
65 70 75 80
Trp Lys Asn Gly Gln Pro Thr Ile Asn Thr Gly Glu Asn Gly Leu Gln
85 90 95
Arg Leu Asp Val Ile Val Arg Thr Ala Glu Ala His Gly Ile Gln Leu
100 105 110
Ile Leu Pro Phe Val Asn Asn Trp Gly Asp Tyr Gly Gly Met Asp Val
115 120 125
Tyr Val Gln Gln Leu Gly Gly Asn Gly His Ser Ser Phe Tyr Thr Asp
130 135 140
Ala Ala Ile Gln Asp Ala Tyr Lys Asn Tyr Val Arg Thr Ile Val Asn
145 150 155 160
Arg Tyr Lys Asn Ser Ser Ala Ile Phe Ala Trp Glu Leu Ala Asn Glu
165 170 175
Pro Arg Cys Gln Gly Cys Asp Thr Ser Ile Ile Thr Glu Trp Ala Ser
180 185 190
Asn Met Ser Ala Phe Val Lys Ser Leu Asp Pro Ser His Tyr Val Val
195 200 205
Leu Gly Asp Glu Gly Phe Phe Asn Arg Pro Gly Asp Pro Ser Tyr Pro
210 215 220
Tyr Gln Gly Gly Glu Gly Val Asp Phe Glu Ala Asn Leu Lys Ile Ser
225 230 235 240
Thr Leu Asp Phe Gly Ile Phe His Met Tyr Ile Thr Pro Trp Gly Gln
245 250 255
Thr Tyr Asp Trp Gly Asn Gln Trp Ile Ala Asp His Ser Ala Ala Cys
260 265 270
Glu Ala Ala Gly Lys Pro Cys Ile Leu Glu Glu Tyr Gly Val Asp Gln
275 280 285
Asp Gly Asp Phe Arg Thr Thr Trp Met Thr Asn Trp His Asn Thr Leu
290 295 300
Leu Glu Ser Pro Gly Val Pro Ala Asp Met Phe Trp Gln Phe Gly Leu
305 310 315 320
Gln Leu Ser Tyr Gly Pro Asn His Asp Asp Gly Tyr Thr Ile Tyr Asn
325 330 335
Phe Glu Asp Asn Phe Gln Pro Val Val Val Asp Trp Ala Ala Thr Arg
340 345 350
Asn Ala Ser
355
<210> 2
<211> 1068
<212> DNA
<213> Malbranchea cinnamonmea
<400> 2
atgaagttat catccttcgc tttacccctg ttagcagggc taagttcttc agctcccttg 60
gaatcgcggc aaagtggctt gtctcccttc gcaggtacca atgcctactg gcttccgttc 120
ctcacaaatg acgcagacgt ggaggccagc ttccaggcta tgaaaaacgc cggtatgaaa 180
gtggttcgaa cctgggcgtt caacgacaat actgagtgcc aggaaatcca ttttcagtgc 240
tggaaaaatg gacaacctac aatcaacacc ggagaaaacg ggctgcaacg tcttgatgta 300
attgttcgaa ccgcagaagc ccatggaata cagttgattc tgccatttgt aaacaactgg 360
ggtgattatg gcgggatgga tgtctatgtc caacagcttg gtggcaacgg tcattcttcc 420
ttttataccg atgctgcaat tcaggatgcg tataagaact atgtcagaac tattgtgaac 480
cgctacaaaa attcgtccgc cattttcgct tgggaacttg ccaatgagcc acgctgccaa 540
ggctgcgaca cgtccatcat cacggaatgg gcgtcgaaca tgagcgcatt cgttaaatct 600
ctcgacccat cgcactatgt tgtccttggt gacgagggtt ttttcaatcg acctggtgac 660
ccgtcgtacc cttaccaagg cggcgaaggt gttgacttcg aggccaatct gaagatcagc 720
acactagact tcgggatctt ccacatgtac attaccccct ggggacagac gtacgattgg 780
ggaaatcagt ggatcgccga ccactctgcg gcttgcgagg ctgctggaaa gccatgcatc 840
ttagaagagt atggagtgga tcaggatggt gactttagaa ccacttggat gacaaattgg 900
cacaacacct tgctggagag ccccggtgtc ccagccgata tgttttggca gtttggttta 960
cagttgagtt atgggcccaa ccatgatgat gggtacacca tttacaattt cgaggacaac 1020
tttcagccgg tcgttgtcga ttgggccgct actcgaaacg caagctag 1068

Claims (5)

1. The application of mannase in the production of partially hydrolyzed konjac glucomannan;
the mannase is protein shown in any one of the following formulas:
a1) a protein consisting of amino acid residues 18 to 355 from the N-terminus of SEQ ID No. 1;
a2) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
a3) a protein having more than 99% homology with the amino acid sequence defined in any one of a1) -a2) and having mannanase activity derived from cladosporium camphorata;
a4) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein defined in any one of a1) -a 3);
the partially hydrolyzed konjac gum is medium molecular weight partially hydrolyzed konjac gum or low molecular weight partially hydrolyzed konjac gum;
the weight average molecular weight of the medium molecular weight partially hydrolyzed konjac gum is 1.6 × 105Da; the low molecular weight partially hydrolyzed konjac gum has a weight average molecular weight of 2.0 × 10 4Da。
2. Use of mannanase in the preparation of a product for the production of partially hydrolysed konjac gum;
the mannase is protein shown in any one of the following formulas:
a1) a protein consisting of amino acid residues 18 to 355 from the N-terminus of SEQ ID No. 1;
a2) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
a3) a protein having more than 99% homology with the amino acid sequence defined in any one of a1) -a2) and having mannanase activity derived from cladosporium camphorata;
a4) a fusion protein obtained by attaching a label to the N-terminal and/or C-terminal of the protein defined in any one of a1) -a 3);
the partially hydrolyzed konjac gum is medium molecular weight partially hydrolyzed konjac gum or low molecular weight partially hydrolyzed konjac gum;
the weight average molecular weight of the medium molecular weight partially hydrolyzed konjac gum is 1.6 × 105Da; the low molecular weight partially hydrolyzed konjac gumThe weight average molecular weight is 2.0X 104Da。
3. Use according to claim 1 or 2, characterized in that: in the process of producing the partially hydrolyzed konjac glucomannan, a production substrate is konjac glucomannan; the initial concentration of the konjac glucomannan in the reaction system is 1-20g/100 mL; the hydrolysis time is 0.5-12 h; the addition amount of the mannase is 20-800U/g konjac glucomannan.
4. A method for preparing medium molecular weight partially hydrolyzed konjac gum comprises the following steps: hydrolyzing konjac glucomannan by using mannase, wherein the proportion of the konjac glucomannan to the mannase is 1g of konjac glucomannan: 20U of the mannase is hydrolyzed for 8 hours at 70 ℃ to obtain the medium molecular weight partially hydrolyzed konjac glucomannan; the weight average molecular weight of the medium molecular weight partially hydrolyzed konjac gum is 1.6 × 105Da；
The mannase is protein shown in any one of the following formulas:
a1) a protein consisting of amino acid residues 18 to 355 from the N-terminus of SEQ ID No. 1;
a2) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
a3) a protein having more than 99% homology with the amino acid sequence defined in any one of a1) -a2) and having mannanase activity derived from cladosporium camphorata;
a4) a fusion protein obtained by attaching a label to the N-terminal and/or C-terminal of the protein defined in any one of a1) -a 3).
5. A method for preparing low molecular weight partially hydrolyzed konjac gum comprises the following steps: hydrolyzing konjac glucomannan with mannase, wherein the proportion of konjac glucomannan to mannase is 1g of konjac glucomannan: hydrolyzing 200U of the mannase at 70 deg.C for 8h to obtain the low molecular weight partially hydrolyzed konjac glucomannan; the low molecular weight partially hydrolyzed konjac gum has a weight average molecular weight of 2.0 × 10 4Da；
The mannase is protein shown in any one of the following formulas:
a1) a protein consisting of amino acid residues 18 to 355 from the N-terminus of SEQ ID No. 1;
a2) a protein consisting of an amino acid sequence shown in SEQ ID No. 1;
a3) a protein having more than 99% homology with the amino acid sequence defined in any one of a1) -a2) and having mannanase activity derived from cladosporium camphorata;
a4) a fusion protein obtained by attaching a label to the N-terminal and/or C-terminal of the protein defined in any one of a1) -a 3).
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