CN104630189B - A kind of carbohydrase Pullulanase joint product and its production method - Google Patents

A kind of carbohydrase Pullulanase joint product and its production method Download PDF

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CN104630189B
CN104630189B CN201510062545.XA CN201510062545A CN104630189B CN 104630189 B CN104630189 B CN 104630189B CN 201510062545 A CN201510062545 A CN 201510062545A CN 104630189 B CN104630189 B CN 104630189B
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liquid
pullulanase
filter aid
carbohydrase
fermentation medium
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CN104630189A (en
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孙利鹏
高璇璇
管宁
刘仲敏
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HENAN YANGSHAO BIOCHEMICAL ENGINEERING Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2428—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01135—Neopullulanase (3.2.1.135)

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Abstract

The invention belongs to microbial technology field, and in particular to a kind of novel saccharification enzyme Pullulanase joint product and its production method.The novel saccharification enzyme Pullulanase joint product, during it is per 1kg joint products, liquid saccharifying enzyme containing 400-800g and 200-600g liquid Pullulanases, wherein, the U/mL of liquid saccharifying enzyme vigor 6-11 ten thousand, liquid Pullulanase vigor 600-1800U/mL.The compound enzyme product of high-yield and high-efficiency of the present invention is combined by liquid saccharifying enzyme and liquid Pullulanase, starch saccharification α 1 can be respectively used to, 4 glycosidic bonds and α 1,6 two kinds of glycosidic bond liquid monomer system biological products, such as it is used for glucose syrup conversion production glucose, maltose, saccharified liquid DX values are up to more than 99%.

Description

A kind of carbohydrase Pullulanase joint product and its production method
Technical field
The invention belongs to microbial technology field, and in particular to a kind of novel saccharification enzyme Pullulanase joint product and its life Production method.
Background technology
Carbohydrase is also known as glucoamylase, is that yield is maximum in the world and the most wide enzyme of purposes.Carbohydrase it is main Effect is from the non reducing end in the carbochains such as starch, dextrin, the glycogen successively glycosidic bond of hydrolyzing alpha -1,4.For amylopectin, When running into branch point, it can also hydrolyzing alpha -1,6 glycosidic bond, amylopectin is all thus hydrolyzed into glucose.Carbohydrase Fastest, energy weakly hydrolyse α -1 of hydrolyzing alpha-Isosorbide-5-Nitrae glycosidic bond, the carbochains of 3 connections.
Pullulanase is a kind of starch debranching enzymes, because it can selectivity hydrolysis pulullan（Maltotriose is with α -1,6 glucosides The polymer that key connection is got up）And gain the name.Can selectivity cut amylopectin branch point in α -1,6 glycosidic bonds, cut whole Individual branched structure forms amylose.Pullulanase can decompose the side chain of least unit, former using starch to greatest extent Material, so having the function that in using starch as the industrial production of raw material important.
At present, carbohydrase is widely used in the fermentation industries such as brewing industry, sugar industry, food processing and organic acid, amino acid With industry light industry textile industry industry, but two drawbacks are still suffered from its production application：
1）Glucose present in saccharification enzyme product turns glycosides enzyme（Referred to as turn glycosides enzyme）Have a strong impact on product in saccharification production Purity and yield.The carbohydrase of production application mainly passes through aspergillus niger in industry（Asp.niger）Fermenting and producing obtains.Black song Mould fermenting and producing carbohydrase has enzyme activity stability height, the advantages that being applied under the conditions of higher temperature and relatively low pH, still Purity is not high, during fermentation of Aspergillus niger in addition to having glucoamylase, is also mixed with a small amount of other enzymes, as glucose turns Glycosides enzyme, protease, cellulase etc., and the presence of these enzymes produces negatively influencing to the action effect of carbohydrase.
2）Hydrolysis of the glucoamylase to α-Isosorbide-5-Nitrae glycosidic bond is highly effective, but α -1, the hydrolysis rate of 6 keys are dropped significantly It is low.Therefore effectively reduce carbohydrase production transfer glycosides enzyme content, improve carbohydrase product hydrolysis action effect have it is important Practical significance.
The content of the invention
Present invention aims to overcome that prior art defect, there is provided a kind of novel saccharification enzyme Pullulanase joint product and its Production method.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of novel saccharification enzyme Pullulanase joint product, often in 1kg joint products, liquid saccharifying enzyme containing 400-800g With 200-600g liquid Pullulanases, wherein, the U/mL of liquid saccharifying enzyme vigor 6-11 ten thousand, liquid Pullulanase vigor 600- 1800U/mL。
The production method of the novel saccharification enzyme Pullulanase joint product, it comprises the following steps：
1）Produce liquid saccharifying enzyme：Aspergillus niger strain is first subjected to Liquid Culture, then in 32-35 DEG C, pH 4.0- 3-12% is pressed under conditions of 5.0（It is preferred that 5-10%）Inoculum concentration be inoculated in fermented and cultured 120-180h in fermentation medium（Turn Fast 100-200 revs/min）；The fermentation medium forms（W/v, g/L）：Cornstarch 1.5-3.0%, dregs of beans 0.5- 2.0%, ammonium sulfate 0.1-0.30%, potassium dihydrogen phosphate 0.1-0.2%, fermented and cultured add filter aid, filtrate warp after terminating After ultrafiltration concentration, take concentrate standby；
2）Produce liquid Pullulanase：Bacillus subtilis strain is first subjected to Liquid Culture, then in 34-38 DEG C, pH Under 6.0-7.5 temperature conditionss fermented and cultured 35-45h in fermentation medium is inoculated in by 2-6% inoculum concentration（Rotating speed 150-250 revs/min）；The fermentation medium forms（W/v, g/L）：Cornstarch 1.0-3.0%, dregs of beans 0.1- 2.0%, ammonium sulfate 0.1-0.3%, dipotassium hydrogen phosphate 0.05-0.2%；Fermented and cultured adds filter aid, filtrate warp after terminating After ultrafiltration concentration, take concentrate standby；
3）By step 1）With 2）Gained filtrate, which mixes in proportion, to be produced.
Specifically, step 1）Described in liquid culture condi be：In 32-35 DEG C of temperature, pH 4.0-5.0 condition Lower culture 2-3 days（100-200 revs/min of rotating speed）；Fluid nutrient medium forms（W/v, g/L）：Cornstarch 1.5- 3.0%, peptone 0.5-2.0%, ammonium sulfate 0.1-0.30%, potassium dihydrogen phosphate 0.1-0.2%.
Step 2）Described in liquid culture condi be：Cultivated under conditions of 34-38 DEG C of temperature, pH 6.0-7.5 1-2 days（150-250 revs/min of rotating speed）；Fluid nutrient medium forms（W/v, g/L）：Cornstarch 1.0-3.0%, albumen Peptone 0.1-2.0%, ammonium sulfate 0.1-0.3%, dipotassium hydrogen phosphate 0.05-0.2%.
Step 1）The addition of middle filter aid is 0.6-2.0%（W/v, g/L）, each raw material weight percentage of the filter aid It is than composition：Perlite 20-35%, diatomite 35-45%, bentonite 20-45%.
Step 2）Middle filter aid addition is 0.6-2.0%（W/v, g/L）, each raw material weight percentage of the filter aid Form and be：Perlite 35-70%, diatomite 30-65%.
In the present invention, Aspergillus niger strain（Asp.niger）Purchased from China General Microbiological culture presevation administrative center, strain Numbering is CGMCC 3.1858.Bacillus subtilis strain（Bacillus subtilis）Purchased from Chinese industrial microorganism fungus kind Preservation administrative center, bacterium numbering are CICC 20872.Step 1）Middle addition filter aid is to remove thalline, glucose turns Glycosides enzyme etc., gained filtrate are carbohydrase, and its concentrated rear vigor reaches the U/mL of 10-14 ten thousand, and glucose turns glycosides enzymatic activity in 50U/mL Below.Step 2）To remove other impurity such as thalline, gained filtrate is Pullulanase for middle filtering, and its concentrated rear vigor reaches 1000-5000U/mL.
Compared to the prior art, beneficial effects of the present invention：
The compound enzyme product of high-yield and high-efficiency of the present invention is combined by liquid saccharifying enzyme and liquid Pullulanase, can be acted on respectively Two kinds of liquid monomer system biological products of starch α -1,4 glycosidic bonds and α -1,6 glycosidic bonds.It is contained in carbohydrase used in the present invention to turn Glycosides enzymatic activity turns glycosides enzymatic activity then in 7000U/mL or so in below 50U/mL contained by other commercially available carbohydrase.It is of the invention high The glucose syrup conversion production crystal glucose after compound enzyme product acts on starch liquefacation, fructose syrup etc. are imitated, by GB/ 6.2 clauses determine saccharified liquid DX values up to more than 99% in T20880-2007, higher than other commercially available saccharification enzyme products（Saccharified liquid DX values Highest only up to 96%）Action effect.
Embodiment
The present invention is described further by the following examples, but protection scope of the present invention not limited to this.
In following embodiments, Aspergillus niger strain（Asp.niger）Purchased from China General Microbiological culture presevation administrative center, Bacterium numbering is CGMCC 3.1858.Bacillus subtilis strain（Bacillus subtilis）Purchased from Chinese industrial microorganism Culture presevation administrative center, bacterium numbering are CICC 20872.
Embodiment 1
A kind of novel saccharification enzyme Pullulanase joint product, often in 1kg joint products, liquid saccharifying enzyme containing 600g and 400g Liquid Pullulanase, wherein, the U/mL of liquid saccharifying enzyme vigor 60,000, the U/mL of liquid Pullulanase vigor 720.
The production method of above-mentioned novel saccharification enzyme Pullulanase joint product, it comprises the following steps：
1）Produce liquid saccharifying enzyme：Aspergillus niger strain is first subjected to Liquid Culture, then under conditions of 34 DEG C, pH 4.8 Fermented and cultured 160h in fermentation medium is inoculated in by 8% inoculum concentration（120 revs/min of rotating speed）；The fermentation medium composition For（W/v, g/L）：Cornstarch 2.5%, dregs of beans 1.0%, ammonium sulfate 0.2%, potassium dihydrogen phosphate 0.15%, fermented and cultured add after terminating Add filter aid, filtrate takes concentrate standby after ultrafiltration concentration；
Above-mentioned liquid culture condi is：Cultivated 2 days under conditions of 34 DEG C of temperature, pH 4.8（120 revs/min of rotating speed Clock）；Fluid nutrient medium forms（W/v, g/L）：Cornstarch 2.5%, peptone 1.0%, ammonium sulfate 0.20%, potassium dihydrogen phosphate 0.15%；The addition of the filter aid is 1.0%（W/v, g/L）, each raw material weight percentage of filter aid, which forms, is：Perlite 25%, diatomite 40%, bentonite 35%.
2）Produce liquid Pullulanase：Bacillus subtilis strain is first subjected to Liquid Culture, then in 37 DEG C, pH 6.7 Under conditions of by 5% inoculum concentration be inoculated in fermented and cultured 40h in fermentation medium（200 revs/min of rotating speed）；The fermentation training Supporting base composition is（W/v, g/L）：Cornstarch 1.5%, dregs of beans 1.8%, ammonium sulfate 0.15%, dipotassium hydrogen phosphate 0.05%；Fermentation training Support and add filter aid after terminating, filtrate takes concentrate standby after ultrafiltration concentration；
Above-mentioned liquid culture condi is：Cultivated 1 day under conditions of 37 DEG C of temperature, pH 6.7（200 revs/min of rotating speed Clock）；Fluid nutrient medium forms（W/v, g/L）：Cornstarch 1.5%, peptone 1.8%, ammonium sulfate 0.15%, dipotassium hydrogen phosphate 0.05%；The addition of the filter aid is 1.2%（W/v, g/L）, each raw material weight percentage of filter aid, which forms, is：Perlite 65%, diatomite 35%.
3）By step 1）With 2）Gained filtrate, which mixes in proportion, to be produced.
The glucose syrup production crystallization grape that above-mentioned gained carbohydrase Pullulanase joint product is acted on after starch liquefacation Sugar, addition 0.06%（v/v）, the pH 4.2 of glucose syrup, after 58 DEG C are reacted 48h, saccharified liquid DX values reach in products obtained therefrom 99.2%。
Embodiment 2
A kind of novel saccharification enzyme Pullulanase joint product, often in 1kg joint products, liquid saccharifying enzyme containing 500g and 500g Liquid Pullulanase, wherein, the U/mL of liquid saccharifying enzyme vigor 70,000, the U/mL of liquid Pullulanase vigor 800.
The production method of above-mentioned novel saccharification enzyme Pullulanase joint product, it comprises the following steps：
1）Produce liquid saccharifying enzyme：Aspergillus niger strain is first subjected to Liquid Culture, then under conditions of 35 DEG C, pH 4.3 Fermented and cultured 120h in fermentation medium is inoculated in by 10% inoculum concentration（150 revs/min of rotating speed）；The fermentation medium group Turn into（W/v, g/L）：Cornstarch 3.0%, dregs of beans 0.5%, ammonium sulfate 0.1%, potassium dihydrogen phosphate 0.2%, after fermented and cultured terminates Filter aid is added, filtrate takes concentrate standby after ultrafiltration concentration；
Above-mentioned liquid culture condi is：Cultivated 3 days under conditions of 35 DEG C of temperature, pH 4.3（150 revs/min of rotating speed Clock）；Fluid nutrient medium forms（W/v, g/L）：Cornstarch 3.0%, peptone 0.5%, ammonium sulfate 0.10%, potassium dihydrogen phosphate 0.2%；The addition of the filter aid is 0.6%（W/v, g/L）, each raw material weight percentage of filter aid, which forms, is：Perlite 20%, diatomite 35%, bentonite 45%.
2）Produce liquid Pullulanase：Bacillus subtilis strain is first subjected to Liquid Culture, then in 34 DEG C, pH 6.0 Under conditions of by 2% inoculum concentration be inoculated in fermented and cultured 40h in fermentation medium（200 revs/min of rotating speed）；The fermentation training Supporting base composition is（W/v, g/L）：Cornstarch 1.0%, dregs of beans 2.0%, ammonium sulfate 0.1%, dipotassium hydrogen phosphate 0.05%；Fermented and cultured Filter aid is added after end, filtrate takes concentrate standby after ultrafiltration concentration；
Above-mentioned liquid culture condi is：Cultivated 1 day under conditions of 34 DEG C of temperature, pH 6.0（200 revs/min of rotating speed Clock）；Fluid nutrient medium forms（W/v, g/L）：Cornstarch 1.0%, peptone 2.0%, ammonium sulfate 0.1%, dipotassium hydrogen phosphate 0.05%；The addition of the filter aid is 1.6%（W/v, g/L）, each raw material weight percentage of filter aid, which forms, is：Perlite 70%, diatomite 30%.
3）By step 1）With 2）Gained filtrate, which mixes in proportion, to be produced.
Above-mentioned gained carbohydrase Pullulanase joint product is used for the production of fructose syrup, addition 0.2%（v/v）, grape 4.5,60 DEG C of syrup pH reaction 52h, gained saccharified liquid DX values are up to more than 99%.Saccharified liquid is final through glucose isomerase enzyme reaction again Sugar is grouped into fructose more than 55%, glucose 45% or so in gained fructose syrup.

Claims (4)

  1. A kind of 1. carbohydrase Pullulanase joint product, it is characterised in that in every 1kg joint products, liquid sugar containing 400-800g Change enzyme and 200-600g liquid Pullulanases, wherein, the U/mL of liquid saccharifying enzyme vigor 6-11 ten thousand, liquid Pullulanase vigor 600-1800U/mL；The joint product acts on the conversion production of the glucose syrup after starch liquefacation crystal glucose, high fructose corn Slurry, saccharified liquid DX values are determined up to more than 99% by 6.2 clauses in GB/T20880-2007；It is contained in carbohydrase used to turn glycosides enzyme activity Property is in below 50U/mL；
    The production method of the carbohydrase Pullulanase joint product, comprises the following steps：
    1）Produce liquid saccharifying enzyme：Aspergillus niger strain is first subjected to Liquid Culture, then connect under 32-35 DEG C of temperature conditionss Kind fermented and cultured 120-180h in fermentation medium；The fermentation medium forms：Cornstarch 1.5-3.0 w/v %, dregs of beans 0.5-2.0 w/v %, ammonium sulfate 0.1-0.30 w/v %, potassium dihydrogen phosphate 0.1-0.2 w/v %, fermented and cultured Filter aid is added after end, filtrate takes concentrate standby after ultrafiltration concentration；
    2）Produce liquid Pullulanase：Bacillus subtilis strain is first subjected to Liquid Culture, then in 34-38 DEG C of temperature Under the conditions of be inoculated in fermented and cultured 35-45h in fermentation medium；The fermentation medium forms：Cornstarch 1.0-3.0 W/v %, dregs of beans 0.1-2.0 w/v %, ammonium sulfate 0.1-0.3 w/v %, dipotassium hydrogen phosphate 0.05-0.2 w/v %；Culture Filter aid is added after end, filtrate takes concentrate standby after ultrafiltration concentration；
    3）By step 1）With 2）Gained filtrate, which mixes in proportion, to be produced；
    Step 1）The addition of middle filter aid is 0.6-2.0 w/v %, and each raw material weight percentage of the filter aid forms For：Perlite 20-35%, diatomite 35-45%, bentonite 20-45%；
    Step 2）Middle filter aid addition is 0.6-2.0 w/v %, and each raw material weight percentage composition of the filter aid is： Perlite 35-70%, diatomite 30-65%.
  2. 2. the production method of carbohydrase Pullulanase joint product described in claim 1, it is characterised in that comprise the following steps：
    1）Produce liquid saccharifying enzyme：Aspergillus niger strain is first subjected to Liquid Culture, then connect under 32-35 DEG C of temperature conditionss Kind fermented and cultured 120-180h in fermentation medium；The fermentation medium forms：Cornstarch 1.5-3.0 w/v %, dregs of beans 0.5-2.0 w/v %, ammonium sulfate 0.1-0.30 w/v %, potassium dihydrogen phosphate 0.1-0.2 w/v %, fermented and cultured Filter aid is added after end, filtrate takes concentrate standby after ultrafiltration concentration；
    2）Produce liquid Pullulanase：Bacillus subtilis strain is first subjected to Liquid Culture, then in 34-38 DEG C of temperature Under the conditions of be inoculated in fermented and cultured 35-45h in fermentation medium；The fermentation medium forms：Cornstarch 1.0-3.0 W/v %, dregs of beans 0.1-2.0 w/v %, ammonium sulfate 0.1-0.3 w/v %, dipotassium hydrogen phosphate 0.05-0.2 w/v %；Culture Filter aid is added after end, filtrate takes concentrate standby after ultrafiltration concentration；
    3）By step 1）With 2）Gained filtrate, which mixes in proportion, to be produced；
    Step 1）The addition of middle filter aid is 0.6-2.0 w/v %, and each raw material weight percentage of the filter aid forms For：Perlite 20-35%, diatomite 35-45%, bentonite 20-45%；
    Step 2）Middle filter aid addition is 0.6-2.0 w/v %, and each raw material weight percentage composition of the filter aid is： Perlite 35-70%, diatomite 30-65%.
  3. 3. the production method of carbohydrase Pullulanase joint product as claimed in claim 2, it is characterised in that step 1）Described in Liquid culture condi be：32-35 DEG C of temperature, pH 4.0-5.0, incubation time 2-3 days；Fluid nutrient medium forms：It is beautiful Rice starch 1.5-3.0%, peptone 0.5-2.0%, ammonium sulfate 0.1-0.30%, potassium dihydrogen phosphate 0.1-0.2%.
  4. 4. the production method of carbohydrase Pullulanase joint product as claimed in claim 2, it is characterised in that step 2）Described in Liquid culture condi be：34-38 DEG C of temperature, pH 6.0-7.5, incubation time 1-2 days；Fluid nutrient medium forms：It is beautiful Rice starch 1.0-3.0%, peptone 0.1-2.0%, ammonium sulfate 0.1-0.3%, dipotassium hydrogen phosphate 0.05-0.2%.
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CN105002113B (en) * 2015-07-24 2018-07-17 河南仰韶生化工程有限公司 A kind of mixed fodder additive and its production method containing microorganism and biological enzyme
CN107557410B (en) * 2017-08-01 2021-03-30 华南理工大学 Method for preparing low amylose starch nanocrystal by enzymolysis pretreatment and acid method
CN107365730B (en) * 2017-09-08 2021-12-10 河南新仰韶生物酶制剂有限公司 Bacillus subtilis strain and method for producing pullulanase by using same
CN109266632A (en) * 2018-10-16 2019-01-25 郑州力酷生物科技有限公司 Removal sugar turns the method for glycosides enzyme in a kind of glucoamylase fermentation broth
CN110628747B (en) * 2019-08-30 2021-12-21 广东溢多利生物科技股份有限公司 Combined amylase and application thereof

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