CN101831416A - Pullulanase and production method thereof - Google Patents
Pullulanase and production method thereof Download PDFInfo
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- CN101831416A CN101831416A CN201010101281A CN201010101281A CN101831416A CN 101831416 A CN101831416 A CN 101831416A CN 201010101281 A CN201010101281 A CN 201010101281A CN 201010101281 A CN201010101281 A CN 201010101281A CN 101831416 A CN101831416 A CN 101831416A
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Abstract
The invention relates to pullulanase and a production method thereof, and belongs to an amylolytic enzyme acted on an alpha-1,6-glucosidic bond. The pullulanase and the production method are characterized in that: a, zymologic characteristic is that the optimal pH is 3.8 to 4.4, the stable pH is 3.5 to 4.5, and the optimal action temperature is 50 to 65 DEG C; and b, Bacillus licheniformis is selected as an enzyme producing strain and derived from China Center of Industrial Culture Collection (CICC No.10185), and the pullulanase is prepared by a liquid state fermentation process in a mode of compositely feeding a backing material and a feed supplement and the method meets the following technological indexes that: (1) the enzymatic activity of the product is more than or equal to 1,100u/ml; (2) the fermentation period is 72 to 80 hours; and (3) the liquid enzyme recovery rate is more than or equal to 86.5 percent. The pullulanase product has the advantages of high heat resistance and acid resistance, high enzymatic activity, stable performance and low cost; the liquid biological fermentation production method has the advantages of high fermentation enzymatic activity, high extracting yield and low manufacturing cost; and the pullulanase and the production method are suitable for the fermentation industry using starch as a raw material and manufacturing of modified starch products, amylose starch and high-amylose starch.
Description
Technical field
The present invention is a kind of Pullulanase and production method thereof.Belong to and act on α-1, the amylolytic enzyme on the 6-glycosidic link.
Background technology
Starch is the photosynthetic product of green plants, and in plant amylum, the content of amylopectin accounts for 70%～95%, as in the corn 74%, in the potato 76%, in the wheat 75%, account for 81% in the paddy, accounts for 100% in the glutinous rice.The relative molecular weight of amylopectin is very big, and about 1,000,000～6,000,000.In the amylopectin, as in amylose starch, also by α-1, the 4-glycosidic bond connects into a straight chain to the D-glucose unit, only passes through α-1 on this straight chain again, and the 6-glycosidic bond forms side chain, another branched building block can occur again on side chain.Therefore structure is very complicated, becomes tree-like branched structure.Owing to contain 4%～5% α-1,6-glycosidic link approximately in the amylopectin, and traditional amylase to the hydrolysis ability of the α in the starch-1,6-glycosidic link a little less than, affect the raising of the final degree of hydrolysis of starch always.
(Pullulanase EC.3.2.1.41) can specificity cuts α-1,6-glycosidic link in the amylopectin tapping point to Pullulanase, cuts whole side shoot, and makes it become amylose starch.Effectively remedied the deficiency of saccharifying enzyme in the starch hydrolysis, the compound use of Pullulanase and saccharifying enzyme has good synergy, is lower than at the saccharifying enzyme consumption under 50% the condition of conventional amount, can make the DE value up to more than 98%.Pullulanase mixes with beta-amylase when using, and almost can all starch be converted into maltose.Thereby can produce the superhigh maltose syrup more than 80%.In process for beer production, add Pullulanase and when improving saccharification result, shortened saccharification time, in dry beer is produced, use it and reduce dextrin content, improved the quality of beer.In addition, with Pullulanase be used for amylose starch production, brew alcohol, monosodium glutamate, resistant starch, disproportionation cyclodextrin, slowly digestible starch, high amylose starch manufacturing etc. be the Industrial products industry of raw material with starch, can obtain very high productive rate, improve the utilization ratio of starch resource greatly.And then for food, beverage, healthcare products, medicine and the weaving of human living needs, food product pack, and environment protection high quality raw material are provided.
But also there is following deficiency in Pullulanase of the prior art:
1. poor heat resistance, optimum temperuture surpass 60 ℃ of vigor and descend rapidly about 60 ℃;
2. acid resistance is poor, and pH is lower than 5.0 instabilities;
3. enzyme activity is low, and manufacturing cost is too high;
4. cost an arm and a leg.
Summary of the invention
The objective of the invention is to avoid above-mentioned weak point of the prior art, a strain is heat-resisting, acid resistance good and provide, the Pullulanase product that enzyme activity height, stable performance and price are lower.
The present invention also aims to provide the liquid biological fermentation production method of Pullulanase of a kind of fermenting enzyme vigor height, extract yield height, low cost of manufacture.
Purpose of the present invention can reach by following measure:
Pullulanase of the present invention is characterized in that:
A. enzymatic property
Optimal pH 3.8～4.4 is stablized pH 3.5～4.5; Optimum temperature is 50 ℃-65 ℃;
B. select for use Bacillus licheniformis (Bacillus licheniformis) to be the zymogenic bacteria kind, derive from Chinese industrial microbial strains preservation administrative center (CICC numbering 10185), adopt the compound reinforced liquid-state fermentation technology preparation of bed material and feed supplement, reach following technical indicator:
1. product enzyme activity: 〉=1100u/ml
2. fermentation period is 72～80 hours
3. the liquid enzymes rate of recovery 〉=86.5%.
The Bacillus licheniformis that the contriver selects for use, its optimal pH and thermotolerance and saccharifying enzyme are more approaching, and compatibleness is preferably arranged, and can be applied to better with starch is the food-processing industry of raw material.
The zymogenic bacteria kind that is fit to combines with liquid-state fermentation technology is ingenious, has produced beyond thought technique effect, thereby has finished task of the present invention.
Purpose of the present invention can also reach by following measure:
The present inventor improves enzyme activity by changing the strain fermentation condition, makes the Pullulanase of production active high, stable performance.
Disclose a kind of preparation method of Pullulanase of the present invention below, it is characterized in that comprising the steps:
A. spawn culture
Substratum comprises following according to component materials in parts by weight:
1. liquid substratum
Glutinous rice starch 30
Peptone 5
KH
2PO
4 12
K
2H?PO
4 5
MgSO
4·7H
2O 1
pH 7.0
2. slant medium
Glutinous rice starch 1.0
Peptone 5
Yeast extract paste 5
KH
2PO
4 4.5
MgSO
4·7H
2O 1
Agar 2.0
pH 7.0
3. isolation medium
Red pulullan 3
Yeast extract paste 5
KH
2PO
4 4.5
MgSO
4·7H
2O 1
Agar 2.0
pH 7.0
4. fermention medium
Liquid A component
Soybean cake powder 50
Corn steep liquor 20
Sticky rice flour 30
CaCl
2 0.45
pH 7.0
Liquid B component
Trisodium citrate 20
K
2H?PO 16
KH
2PO
4 4
pH 7.5
Liquid A wherein: liquid B=9: 1 (volume ratio), mix before the inoculation;
The spawn culture processing condition
Separating plate: 34 ℃ 36 hours;
Liquid seeds: 34 ℃ of 24 hours shaking speed 200r/m;
34 ℃ of slant culture 2～3 days;
Fermentation shake flask: 34 ℃ of 3 days shaking speed 200r/m;
B. seeding tank enlarged culturing
1. in the culture medium raw material component of parts by weight
Starch 6
Soybean cake powder 5
Corn steep liquor 2
CaCl
2 0.15
Trisodium citrate 0.2
K
2H?PO 1.2
KH
2PO
4 0.4
Ammonium sulfate 0.4
Defoamer 0.04
pH 7.0
2. sterilization process condition: 121 ℃-124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
3. raise craft condition
Tank pressure Mpa 0.05～0.08
Temperature ℃ 34
Air quantity m
3/ h 30
Mixing speed r/min 180
4. culture transferring condition: thalline dyeing is dark, sturdy, does not have assorted bacterium, and enzyme activity is about 10u/ml.
C. Pullulanase is produced in liquid state fermentation
1. comprise following according to component materials in parts by weight:
Starch 2
Sticky rice flour 3
Soybean cake powder 5
Corn steep liquor 2
CaCl
2 0.15
Trisodium citrate 0.2
K
2H?PO 1.6
KH
2PO
4 0.5
Ammonium sulfate 0.4
Defoamer 0.04
pH 7.0
2. the hydrolysis of liquefying
Add alpha-amylase 100 weight parts and be warming up to 90 ℃, be incubated 30 minutes;
3. sterilization process condition
121 ℃～124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
4. technological condition for fermentation
Tank pressure Mpa 0.05～0.08
Temperature ℃ 34
Air quantity m
3/ h 0～24 hour 1: 0.3
24～48 hours 1: 0.5
48 hours～discharging 1: 0.6
Mixing speed r/min 180:
pH 5～6.7；
D. feed supplement
1. comprise following according to component materials in parts by weight:
Starch 20
Sticky rice flour 6
CaCl
2 0.15
Defoamer 0.04
pH 7.0
2. the hydrolysis of liquefying
Add alpha-amylase 500 weight parts and be warming up to 90 ℃, be incubated 30 minutes;
3. sterilization process condition
121 ℃～124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
4. feed supplement method
When DO reduce to minimum when rising to 35% again, the beginning feed supplement, DO is at 30%-40% in control;
E. discharging
Cultivated about 72 hours, enzyme activity increasess slowly, and thalline begins the part self-dissolving, can put;
F. Pullulanase extracts
Fermented liquid → pre-treatment → press filtration → ultrafiltration → stdn → essence filter → allotment → can → detection → finished product.
The contriver finds that the bacillus licheniformis that amylopectin adopts for the present invention produces enzyme and has more powerful inducing action.This is because amylopectin is compared other carbon source and had more side chain, and the generation of Pullulanase is had stronger inducing action.Amylopection content is 100% in the sticky rice flour structure, all is amylopectin, and an amount of sticky rice flour component helps bacillus licheniformis and produces enzyme.
Nitrogenous source has material impact to enzymatic production, and experiment shows that soybean cake powder and yeast extract paste effect are preferable, is extractum carnis and peptone secondly.Ammonium sulfate has certain promoter action to producing enzyme in inorganic nitrogen-sourced.Ammonium nitrate then has restraining effect to enzyme synthetic.
PH is the metabolic important parameter of microorganism growth, and pH has tangible influence to producing enzyme activity.Experiment shows, it is the highest to live at pH6.5 ± 0.2 enzymatic production enzyme, and along with rising gradually or the reduction of pH, enzyme work then all can reduce low, and in pH5.5 and pH8.0, enzyme is lived and just dropped to about 20%, and in pH 4.5 and pH 9.0, just do not had enzyme activity basically.
In addition, the component of ventilation, inorganic salt and content, culture temperature, fermentation time etc. all produce enzyme activity to bacterial classification material impact.Culture medium raw material component in the present inventor's liquid towards zymotechnique has been carried out repeatedly experiment, and carbon source, nitrogenous source, pH value, ventilation, leavening temperature, fermentation period and feed way have all been carried out careful, careful, rigorous optimization screening.The cooperation of above-mentioned all liquid-state fermentation technology conditions has improved the enzymatic productivity that adopts bacterial strain greatly.
Pullulanase of the present invention, be applicable to beer plus enzyme Mashing process, alcohol, monosodium glutamate, amino acid, lactic acid etc. are the fermentation industry of raw material with starch, the superhigh maltose syrup process industry, modified starch products such as resistant starch, disproportionation cyclodextrin, slowly digestible starch, and amylose starch, high amylose starch manufacturing.
The disclosed technical scheme of Pullulanase of the present invention and production method thereof has outstanding substantive distinguishing features and obvious improvement compared to existing technology, and can produce following positively effect:
1 provide a kind of heat-resisting, acid resistance good, the Pullulanase product that enzyme activity height, stable performance and price are lower.
2. the liquid biological fermentation production method of Pullulanase of a kind of fermenting enzyme vigor height, extract yield height, low cost of manufacture is provided.
3. end-use is extensive.
Embodiment
The present invention will now be further detailed embodiment:
Embodiment 1
The preparation method of a kind of Pullulanase of the present invention comprises the steps:
A. spawn culture
Substratum comprises following according to component materials in parts by weight:
1. liquid substratum
Glutinous rice starch 30
Peptone 5
KH
2PO
4 12
K
2H?PO
4 5
MgSO
4·7H
2O 1
pH 7.0
2. slant medium
Glutinous rice starch 1.0
Peptone 5
Yeast extract paste 5
KH
2PO
4 4.5
MgSO
4·7H
2O 1
Agar 2.0
pH 7.0
3. isolation medium
Red pulullan 3
Yeast extract paste 5
KH
2PO
4 4.5
MgSO
4·7H
2O 1
Agar 2.0
pH 7.0
4. fermention medium
Liquid A component
Soybean cake powder 50
Corn steep liquor 20
Sticky rice flour 30
CaCl
2 0.45
pH 7.0
Liquid B component
Trisodium citrate 20
K
2H?PO 16
KH
2PO
4 4
pH 7.5
Liquid A wherein: liquid B=9: 1 (volume ratio), mix before the inoculation;
The spawn culture processing condition
Separating plate: 34 ℃ 36 hours;
Liquid seeds: 34 ℃ of 24 hours shaking speed 200r/m;
34 ℃ of slant culture 2～3 days;
Fermentation shake flask: 34 ℃ of 3 days shaking speed 200r/m;
B. seeding tank enlarged culturing
1. in the culture medium raw material component of parts by weight
Starch 6
Soybean cake powder 5
Corn steep liquor 2
CaCl
2 0.15
Trisodium citrate 0.2
K
2H?PO 1.2
KH
2PO
4 0.4
Ammonium sulfate 0.4
Defoamer 0.04
pH 7.0
2. sterilization process condition: 121 ℃-124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
3. raise craft condition
Tank pressure Mpa 0.05～0.08
Temperature ℃ 34
Air quantity m
3/ h 30
Mixing speed r/min 180
4. culture transferring condition: thalline dyeing is dark, sturdy, does not have assorted bacterium, and enzyme activity is about 10u/ml.
C. Pullulanase is produced in liquid state fermentation
1. comprise following according to component materials in parts by weight:
Starch 2
Sticky rice flour 3
Soybean cake powder 5
Corn steep liquor 2
CaCl
2 0.15
Trisodium citrate 0.2
K
2H?PO 1.6
KH
2PO
4 0.5
Ammonium sulfate 0.4
Defoamer 0.04
pH 7.0
2. the hydrolysis of liquefying
Add alpha-amylase 100 weight parts and be warming up to 90 ℃, be incubated 30 minutes;
3. sterilization process condition
121 ℃～124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
4. technological condition for fermentation
Tank pressure Mpa 0.05～0.08
Temperature ℃ 34
Air quantity m
3/ h 0～24 hour 1: 0.3
24～48 hours 1: 0.5
48 hours～discharging 1: 0.6
Mixing speed r/min 180:
pH 5～6.7；
D. feed supplement
1. comprise following according to component materials in parts by weight:
Starch 20
Sticky rice flour 6
CaCl
2 0.15
Defoamer 0.04
pH 7.0
2. the hydrolysis of liquefying
Add alpha-amylase 500 weight parts and be warming up to 90 ℃, be incubated 30 minutes;
3. sterilization process condition: 121 ℃～124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
4. feed supplement method
When DO reduce to minimum when rising to 35% again, the beginning feed supplement, DO is at 30%-40% in control;
E. discharging
Cultivated about 72 hours, enzyme activity increasess slowly, and thalline begins the part self-dissolving, can put;
F. Pullulanase extracts
Fermented liquid → pre-treatment → press filtration → ultrafiltration → stdn → essence filter → allotment → can → detection → finished product.
Technical indicator:
1. product enzyme activity: 〉=1100u/ml
2. fermentation period 72-80 hour
3. the liquid enzymes rate of recovery 〉=86.5%.
Claims (3)
1. Pullulanase is characterized in that:
A. enzymatic property
Optimal pH 3.8～4.4 is stablized pH 3.5～4.5; Optimum temperature is 50 ℃-65 ℃
B. select for use Bacillus licheniformis (Bacillus licheniformis) to be the zymogenic bacteria kind, derive from Chinese industrial microbial strains preservation administrative center (CICC numbering 10185), adopt bed material and the compound feed way of feed supplement, the liquid-state fermentation technology preparation reaches following technical indicator:
1. product enzyme activity: 〉=1100u/ml;
2. fermentation period is 72～80 hours
3. the liquid enzymes rate of recovery 〉=86.5%.
2. the preparation method of the Pullulanase of a claim 1 is characterized in that comprising the steps:
A. spawn culture
Substratum comprises following according to component materials in parts by weight:
1. liquid substratum
Glutinous rice starch 30
Peptone 5
KH
2PO
4 12
K
2H?PO
4 5
MgSO
4·7H
2O 1
pH 7.0
2. slant medium
Glutinous rice starch 1.0
Peptone 5
Yeast extract paste 5
KH
2PO
4 4.5
MgSO
4·7H
2O 1
Agar 2.0
pH 7.0
3. isolation medium
Red pulullan 3
Yeast extract paste 5
KH
2PO
4 4.5
MgSO
4·7H
2O 1
Agar 2.0
pH 7.0
4. fermention medium
Liquid A component
Soybean cake powder 50
Corn steep liquor 20
Sticky rice flour 30
CaCl
2 0.45
pH 7.0
Liquid B component
Trisodium citrate 20
K
2HPO 16
KH
2PO
4 4
pH 7.5
Liquid A wherein: liquid B=9: 1 (volume ratio), mix before the inoculation;
The spawn culture processing condition
Separating plate: 34 ℃ 36 hours;
Liquid seeds: 34 ℃ of 24 hours shaking speed 200r/m;
34 ℃ of slant culture 2～3 days;
Fermentation shake flask: 34 ℃ of 3 days shaking speed 200r/m;
B. seeding tank enlarged culturing
1. in the culture medium raw material component of parts by weight
Starch 6
Soybean cake powder 5
Corn steep liquor 2
CaCl
2 0.15
Trisodium citrate 0.2
K
2H?PO 1.2
KH
2PO
4 40.4
Ammonium sulfate 0.4
Defoamer 0.04
pH 7.0
2. sterilization process condition: 121 ℃-124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
3. raise craft condition
Tank pressure Mpa 0.05～0.08
Temperature ℃ 34
Air quantity m
3/ h 30
Mixing speed r/min 180
4. culture transferring condition: thalline dyeing is dark, sturdy, does not have assorted bacterium, and enzyme activity is about 10u/ml.
C. Pullulanase is produced in liquid state fermentation
1. comprise following according to component materials in parts by weight:
Starch 2
Sticky rice flour 3
Soybean cake powder 5
Corn steep liquor 2
CaCl
2 0.15
Trisodium citrate 0.2
K
2HPO 1.6
KH
2PO
4 0.5
Ammonium sulfate 0.4
Defoamer 0.04
pH 7.0
2. the hydrolysis of liquefying
Add alpha-amylase 100 weight parts and be warming up to 90 ℃, be incubated 30 minutes;
3. sterilization process condition
121 ℃～124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
4. technological condition for fermentation
Tank pressure Mpa 0.05～0.08
Temperature ℃ 34
Air quantity m
3/ h 0～24 hour 1: 0.3
24～48 hours 1: 0.5
48 hours～discharging 1: 0.6
Mixing speed r/min 180:
pH 5～6.7；
D. feed supplement
1. comprise following according to component materials in parts by weight:
Starch 20
Sticky rice flour 6
CaCl
2 0.15
Defoamer 0.04
pH 7.0
2. the hydrolysis of liquefying
Add alpha-amylase 500 weight parts and be warming up to 90 ℃, be incubated 30 minutes;
3. sterilization process condition
121 ℃～124 ℃, under 0.11～0.12Mpa, sterilized 30 minutes;
4. feed supplement method
When DO reduce to minimum when rising to 35% again, the beginning feed supplement, DO is at 30%-40% in control;
E. discharging
Cultivated about 72 hours, enzyme activity increasess slowly, and thalline begins the part self-dissolving, can put;
F. Pullulanase extracts
Fermented liquid → pre-treatment → press filtration → ultrafiltration → stdn → essence filter → allotment → can → detection → finished product.
3. the Pullulanase of claim 1, it is characterized in that being applicable to beer plus enzyme Mashing process, alcohol, monosodium glutamate, amino acid, lactic acid etc. are the fermentation industry of raw material with starch, the superhigh maltose syrup process industry, modified starch products such as resistant starch, disproportionation cyclodextrin, slowly digestible starch, and amylose starch, high amylose starch manufacturing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101012811A CN101831416B (en) | 2010-01-22 | 2010-01-22 | Pullulanase and production method thereof |
Applications Claiming Priority (1)
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|---|---|---|---|
| CN2010101012811A CN101831416B (en) | 2010-01-22 | 2010-01-22 | Pullulanase and production method thereof |
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| CN101831416B CN101831416B (en) | 2012-11-21 |
Family
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102120971A (en) * | 2010-12-02 | 2011-07-13 | 天津工业生物技术研究所 | Pullulanase-producing bacterium, heat-resisting pullulanase produced from same, and coding gene of pullulanase-producing bacterium |
| CN102604861A (en) * | 2012-02-16 | 2012-07-25 | 复旦大学 | Pullulanase, pullulanase producing strain and application of the pullulanase |
| CN103834629A (en) * | 2014-01-07 | 2014-06-04 | 长江大学 | Recombinant high-temperature pullulanase and preparation method thereof |
| CN105063133A (en) * | 2015-08-10 | 2015-11-18 | 江苏宝宝宿迁国民生物科技有限公司 | Preparation method of resistant starch with glutinous rice starch as raw material |
| CN106520734A (en) * | 2016-11-28 | 2017-03-22 | 山东正德食品有限公司 | Method for improving heat resistance of pullulanase for catalyzing maltosyl-beta-cyclodextrin synthesis |
| CN107058168A (en) * | 2017-01-21 | 2017-08-18 | 淮海工学院 | A kind of bacillus megaterium and its method and product for producing Pullulanase |
| CN108504645A (en) * | 2018-05-24 | 2018-09-07 | 长春鸿成生物化工材料技术开发有限公司 | A kind of preparation method of Pullulanase |
Family Cites Families (3)
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| CN1321179C (en) * | 2004-09-07 | 2007-06-13 | 云南师范大学 | Process for preparing Promozyme through gene recombination of Pichia pastoris |
| CN100351372C (en) * | 2005-11-09 | 2007-11-28 | 云南师范大学 | Prolan enzyme bacterial and preparation process |
| CN101195819B (en) * | 2007-12-15 | 2010-12-15 | 淮海工学院 | Method for Pyrococcus producing high temperature pullulanuse and product thereof |
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2010
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102120971A (en) * | 2010-12-02 | 2011-07-13 | 天津工业生物技术研究所 | Pullulanase-producing bacterium, heat-resisting pullulanase produced from same, and coding gene of pullulanase-producing bacterium |
| CN102120971B (en) * | 2010-12-02 | 2014-04-09 | 天津工业生物技术研究所 | Pullulanase-producing bacterium, heat-resisting pullulanase produced from same, and coding gene of pullulanase-producing bacterium |
| CN102604861A (en) * | 2012-02-16 | 2012-07-25 | 复旦大学 | Pullulanase, pullulanase producing strain and application of the pullulanase |
| CN103834629A (en) * | 2014-01-07 | 2014-06-04 | 长江大学 | Recombinant high-temperature pullulanase and preparation method thereof |
| CN105063133A (en) * | 2015-08-10 | 2015-11-18 | 江苏宝宝宿迁国民生物科技有限公司 | Preparation method of resistant starch with glutinous rice starch as raw material |
| CN106520734A (en) * | 2016-11-28 | 2017-03-22 | 山东正德食品有限公司 | Method for improving heat resistance of pullulanase for catalyzing maltosyl-beta-cyclodextrin synthesis |
| CN107058168A (en) * | 2017-01-21 | 2017-08-18 | 淮海工学院 | A kind of bacillus megaterium and its method and product for producing Pullulanase |
| CN108504645A (en) * | 2018-05-24 | 2018-09-07 | 长春鸿成生物化工材料技术开发有限公司 | A kind of preparation method of Pullulanase |
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| Publication number | Publication date |
|---|---|
| CN101831416B (en) | 2012-11-21 |
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