CN101831416B - Pullulanase and production method thereof - Google Patents

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

A kind of Pullulanase and working method thereof

Technical field

The present invention is a kind of Pullulanase and working 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 pulullan accounts for 70%～95%, as in the corn 74%, in the yam 76%, in the wheat 75%, account for 81% in the paddy, accounts for 100% in the glutinous rice.The relative molecular weight of pulullan is very big, and about 1,000,000～6,000,000.In the pulullan, as the same in amylose starch, also through α-1, the 4-glycosidic bond connects into a straight chain to the D-glucose unit, only on this straight chain, passes through α-1 again, and the 6-glycosidic bond forms side chain, on side chain, another branched building block can occur again.Therefore structure is very complicated, becomes tree-like branched structure.Owing to contain 4%～5% α-1,6-glycosidic link approximately in the pulullan, and traditional glycase 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 cut α-1,6-glycosidic link in the pulullan 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 SANMALT-S.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 Schardinger dextrins, 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 the food of human living needs, beverage, healthcare products, medicine and weaving, 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, enzyme activity height, stable performance and the lower Pullulanase product of price.

The present invention also aims to provide a kind of fermenting enzyme vigor height, the liquid biological fermentation working method of extract yield Pullulanase high, low cost of manufacture.

The object of the invention can reach through following measure:

Pullulanase of the present invention is characterized in that:

A. enzymatic property

Ph optimum 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 recovery >=86.5%.

The Bacillus licheniformis that the contriver selects for use, its ph optimum 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 accomplished task of the present invention.

The object of the invention can also reach through following measure:

Contriver of the present invention improves enzyme activity through 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 as follows 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

Steeping water 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

Steeping water 2

CaCl ₂ 0.15

Trisodium citrate 0.2

K ₂H?PO 1.2

KH ₂PO ₄ 0.4

Ammonium sulfate 0.4

Skimmer 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 as follows according to component materials in parts by weight:

Starch 2

Sticky rice flour 3

Soybean cake powder 5

Steeping water 2

CaCl ₂ 0.15

Trisodium citrate 0.2

K ₂H?PO 1.6

KH ₂PO ₄ 0.5

Ammonium sulfate 0.4

Skimmer 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 as follows according to component materials in parts by weight:

Starch 20

Sticky rice flour 6

CaCl ₂ 0.15

Skimmer 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 pulullan adopts for the present invention produces enzyme and has more powerful inducing action.This is because pulullan 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 pulullan, 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 Carnis Bovis seu Bubali cream and peptone secondly.Ammonium sulfate has certain promoter action to producing enzyme in inorganic nitrogen-sourced.An 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 that it is the highest to live at pH6.5 ± 0.2 enzymatic production enzyme, and along with the rising gradually of pH perhaps reduces; 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 has material impact.Culture medium raw material component in contriver's liquid towards zymotechnique of the present invention 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 Schardinger dextrins, slowly digestible starch, and amylose starch, high amylose starch manufacturing.

The disclosed technical scheme of Pullulanase of the present invention and working method thereof is compared prior art and is had outstanding substantive distinguishing features and obvious improvement, and can produce following positively effect:

1 provide a kind of heat-resisting, acid resistance good, enzyme activity is high, stable performance and the lower Pullulanase product of price.

2. a kind of fermenting enzyme vigor height, the liquid biological fermentation working method of extract yield Pullulanase high, low cost of manufacture are provided.

3. end-use is extensive.

Embodiment

The present invention below will combine embodiment to make further detailed description:

Embodiment 1

The preparation method of a kind of Pullulanase of the present invention comprises the steps:

A. spawn culture

Substratum comprises as follows 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

Steeping water 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

Steeping water 2

CaCl ₂ 0.15

Trisodium citrate 0.2

K ₂H?PO 1.2

KH ₂PO ₄ 0.4

Ammonium sulfate 0.4

Skimmer 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 as follows according to component materials in parts by weight:

Starch 2

Sticky rice flour 3

Soybean cake powder 5

Steeping water 2

CaCl ₂ 0.15

Trisodium citrate 0.2

K ₂H?PO 1.6

KH ₂PO ₄ 0.5

Ammonium sulfate 0.4

Skimmer 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 as follows according to component materials in parts by weight:

Starch 20

Sticky rice flour 6

CaCl ₂ 0.15

Skimmer 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 recovery >=86.5%.

Claims (1)

1. the preparation method of a Pullulanase; It is characterized in that: select for use the Bacillus licheniformis (Bacillus licheniformis) of Chinese industrial microbial strains preservation administrative center CICC numbering 10185 to be the zymogenic bacteria kind; 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 recovery >=86.5%

Its preparation method comprises the steps:

A. spawn culture

Substratum comprises as follows according to component materials in parts by weight:

1. liquid substratum

2. slant medium

3. isolation medium

4. fermention medium

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

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 as follows according to component materials in parts by weight:

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

D. feed supplement

1. comprise as follows according to component materials in parts by weight:

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; Make the enzymatic property of Pullulanase: ph optimum 3.8～4.4, stablize pH 3.5～4.5; Optimum temperature is 50 ℃-65 ℃.