CN106906153A - One plant of Pichia pastoris recombinant bacterium for producing endo-inulinase and its application in high density fermentation produces inulinase - Google Patents
One plant of Pichia pastoris recombinant bacterium for producing endo-inulinase and its application in high density fermentation produces inulinase Download PDFInfo
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- CN106906153A CN106906153A CN201710286554.6A CN201710286554A CN106906153A CN 106906153 A CN106906153 A CN 106906153A CN 201710286554 A CN201710286554 A CN 201710286554A CN 106906153 A CN106906153 A CN 106906153A
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- inulinase
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- 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)
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- 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)
- C12Y302/01007—Inulinase (3.2.1.7)
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Abstract
The invention discloses one plant of Pichia pastoris recombinant bacterium of product endo-inulinase, gene order shown in SEQ ID NO.1 is cloned into pPIC9K, recombinant plasmid is obtained, then by recombinant plasmid transformed Pichia pastoris GS115, both obtains producing the Pichia pastoris recombinant bacterium of endo-inulinase.Method the invention also discloses inulinase is produced using above-mentioned Pichia pastoris recombinant bacterium high density fermentation, is carried out high density fermentation and produces inulinase, the characteristics of the endoinulase for obtaining has good stability, enzyme activity is high using the bacterial strain.
Description
Technical field
The invention belongs to technical field of biological fermentation, and in particular to Pichia pastoris recombinant bacterium and its high density fermentation produce chrysanthemum
Application in powder restriction endonuclease.
Background technology
At present, the FOS of inulin is come from, as a kind of functional sugar, is widely used in medicines and health protection industry, food industry
Deng field.FOS can promote propagation, low cariogenicity, indigestible and similar dietary fiber of Bifidobacterium etc. to act on,
A kind of food ingredients with unique physiologically active are turned into.FOS is general by inulin, through endoinulase biology
It is prepared from after degraded.Therefore, endoinulase is the core and key for preparing FOS, directly affects the product of FOS
Product quality.
Endoinulase, is that can hydrolyze β -2 as the biological assistant that a kind of crucial FOS is produced, and 1-D- is really
The class of enzymes of glycosidic bond, scientific name is β -2,1-D- levanases.The method that inulin can be degraded by inulinase prepares fructose
And FOS.Inulinase is the compound enzyme of class catalysis synanthrin hydrolysis, is made up of with exoinulinase endoinulase.Chrysanthemum
Powder is to be completely degraded into monose under the synergy of endoinulase and exoinulinase in inulin enzyme system.And inulinase
The mechanism that hydrolytic inulin prepares FOS is that Inulin polysaccharide primarily forms FOS in the presence of restriction endonuclease.Inulinase
Source is very wide, wherein microbe-derived inulinase species is more, heat endurance is good, is suitable to fermenting and producing.In recent years, scientific research personnel
It is devoted to screening and building new producing enzyme strain, existing strain is transformed, it is using advanced enzymatic production technology and right
The modification of enzyme molecule, the industrial production FOS biological assistant good to obtain enzyme activity high, heat endurance.High-cell density is sent out
Ferment (high cell density fermentation, HCDF), is the improved fermentation technique in traditional zymotic technology, and it is adopted
With increasing the growth time of engineering bacteria logarithmic phase, shortening the decline time relatively to improve the fermentation density of thalline, greatly improve
Zymotechnique, the final specific production rate (yield of product in the unit volume unit interval) for improving product, can not only reduce training
Volume is supported, reinforcing downstream separation is extracted, and can also shorten the production cycle, reduces equipment investment so as to reduce production cost, greatly
Improve competitiveness commercially.At present, high cell density fermentation has turned into the master that scale up test is carried out in biotechnology
Want zymotechnique.Certainly, for endoinulase, the Microbe synthesis of high-cell density are the substantial amounts of industry of production
With unique, the efficient method of enzyme.
The content of the invention
The technical problem to be solved in the present invention is to provide one plant of Pichia pastoris gene recombination bacterium.
The technical problem also to be solved of the invention is to provide above-mentioned Pichia pastoris gene recombination bacterium and produces inulin inscribe in fermentation
Application in enzyme.
To solve above technical problem, the present invention is adopted the following technical scheme that：
One plant of Pichia pastoris recombinant bacterium of product endo-inulinase, the gene order shown in SEQ ID NO.1 is cloned into
In pPIC9K, recombinant plasmid is obtained, then by recombinant plasmid transformed Pichia pastoris GS115, both obtain producing the complete red of endo-inulinase
Yeast recombinant strain.
A kind of method that utilization Pichia pastoris recombinant bacterium high density fermentation produces inulinase, comprises the following steps：
(1) prepared by seed liquor：The Pichia pastoris recombinant bacterium for producing endo-inulinase is inoculated on activation medium and is activated, then
It is transferred in BMGY fluid nutrient mediums, 12~18h is cultivated under the conditions of 28~30 DEG C, 200~250r/min, obtains seed liquor；
(2) high density fermentation：The seed liquor that step (1) is obtained is transferred in basal medium, is cultivated in fermentation tank,
Fermentation condition is：28~30 DEG C of temperature, 800~1000r/min of mixing speed, dissolved oxygen amount 20%~30%, throughput be 5~
6L/min, it is 5.5~6 to control fermentation pH, and stream plus supplementing culture medium are started after culture 6h, and methyl alcohol to whole body is added every 12h
Product concentration is 0.5%, cultivates 64~72h.
In step (1), the formula of the activation medium is as follows：Yeast extract 10g/L, peptone 20g/L, glucose 20g/
L。
In step (1), the formula of the BMGY Liquid Cultures is as follows：Tryptone 20g/L, yeast extract 10g/L121
DEG C, after sterilizing 20min, the potassium phosphate of 100mmol/L pH=6 is added, without amino nitrogen source 13.4g/L, biotin 4 × 10-4G/L,
Glycerine 10g/L.
In step (2), the formula of the basal medium is as follows：Ammonium sulfate 10g/L, potassium dihydrogen phosphate 6g/L, magnesium sulfate
1g/L, EDTA 3mg/L, zinc sulfate 1g/L, ferrous sulfate 5mg/L, copper sulphate 1mg/L, calcium chloride 1g/L, manganese chloride 0.5mg/
L, cobalt chloride 0.8mg/L, KI 0.2mg/L, sodium molybdate 0.05mg/L, nicotinic acid 5mg/L, biotin 0.8mg/L, glucose
10g/L。
In step (2), the formula of the supplementing culture medium is as follows：Ammonium sulfate 10g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate
0.05g/L, EDTA 2mg/L, zinc sulfate 0.08g/L, ferrous sulfate 2mg/L, copper sulphate 0.5mg/L, calcium chloride 0.5g/L, chlorine
Change manganese 0.3mg/L, cobalt chloride 0.8mg/L, KI 0.1mg/L, sodium molybdate 0.03mg/L, nicotinic acid 5mg/L, biotin 1mg/L,
Glucose 50g/L, defoamer 0.05ml/L.
Beneficial effect：The invention discloses one plant of Pichia pastoris recombinant bacterium, and high density fermentation product is carried out using the bacterial strain
Inulinase, the characteristics of the endoinulase for obtaining has good stability, enzyme activity is high.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should also without limitation on sheet described in detail in claims
Invention.
Enzyme activity is defined as in the present invention：Enzyme amount needed for 1 micromole's hexose of generation per minute is 1 enzymatic activity in reaction system
Unit.
The formula of activation medium is as follows：Yeast extract 10g/L, peptone 20g/L, glucose 20g/L.
The formula of BMGY Liquid Cultures is as follows：121 DEG C of tryptone 20g/L, yeast extract 10g/L, after sterilizing 20min,
The potassium phosphate of 100mmol/L pH=6 is added, without amino nitrogen source 13.4g/L, biotin 4 × 10-4G/L, glycerine 10g/L.
The formula of basal medium is as follows：Ammonium sulfate 10g/L, potassium dihydrogen phosphate 6g/L, magnesium sulfate 1g/L, EDTA 3mg/
L, zinc sulfate 1g/L, ferrous sulfate 5mg/L, copper sulphate 1mg/L, calcium chloride 1g/L, manganese chloride 0.5mg/L, cobalt chloride 0.8mg/
L, KI 0.2mg/L, sodium molybdate 0.05mg/L, nicotinic acid 5mg/L, biotin 0.8mg/L, glucose 10g/L.
The formula of culture medium is as follows：Ammonium sulfate 10g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate 0.05g/L, EDTA 2mg/
L, zinc sulfate 0.08g/L, ferrous sulfate 2mg/L, copper sulphate 0.5mg/L, calcium chloride 0.5g/L, manganese chloride 0.3mg/L, cobalt chloride
0.8mg/L, KI 0.1mg/L, sodium molybdate 0.03mg/L, nicotinic acid 5mg/L, biotin 1mg/L, glucose 50g/L, defoamer
0.05ml/L。
Embodiment 1：The pure enzyme Enzyme activity assay of endoinulase.
His6 sequences are added in the afterbody of the endoinulase sequence shown in SEQ ID NO.1, pET28a is then cloned into
On plasmid, bacillus coli DH 5 alpha is converted, ni-sepharose purification endoinulase is used in culture, and the enzyme activity for detecting pure enzyme is 25.6U/mL.
Embodiment 2：The structure of the Pichia pastoris recombinant bacterium of restriction endonuclease.
Gene order shown in SEQ ID NO.1 is cloned into pPIC9K, recombinant plasmid is obtained, then recombinant plasmid is turned
Change Pichia pastoris GS115, both obtain producing the Pichia pastoris recombinant bacterium of endo-inulinase.
Embodiment 3：Pichia pastoris recombinant bacterium high density fermentation produces endo-inulinase.
(1) prepared by seed liquor：The Pichia pastoris recombinant bacterium for producing endo-inulinase is inoculated on activation medium and is activated, then
It is transferred in BMGY fluid nutrient mediums, 12h is cultivated under the conditions of 28~30 DEG C, 200~250r/min, obtains seed liquor；
(2) high density fermentation：The seed liquor that step (1) is obtained is transferred in basal medium, is cultivated in fermentation tank,
Fermentation condition is：28~30 DEG C of temperature, mixing speed 800r/min, dissolved oxygen amount 20%, throughput are 6L/min, control fermentation pH
It is 5.5, stream plus supplementing culture medium is started after culture 6h, it is 0.5% to add methyl alcohol to final volume concentration every 12h, culture
72h。
Under the fermentation condition, the expression quantity of endo-inulinase is 250mg/L.
Embodiment 4：Pichia pastoris recombinant bacterium high density fermentation produces endo-inulinase.
(1) prepared by seed liquor：The Pichia pastoris recombinant bacterium for producing endo-inulinase is inoculated on activation medium and is activated, then
It is transferred in BMGY fluid nutrient mediums, 12h is cultivated under the conditions of 28~30 DEG C, 200r/min, obtains seed liquor；
(2) high density fermentation：The seed liquor that step (1) is obtained is transferred in basal medium, is cultivated in fermentation tank,
Fermentation condition is：30 DEG C of temperature, mixing speed 1000r/min, dissolved oxygen amount 30%, throughput be 5L/min, control ferment pH be
6, start stream plus supplementing culture medium after culture 6h, it is 0.5% to add methyl alcohol to final volume concentration every 12h, cultivates 72h.
Under the fermentation condition, the expression quantity of endo-inulinase is 275mg/L.
Embodiment 5：Pichia pastoris recombinant bacterium high density fermentation produces endo-inulinase.
(1) prepared by seed liquor：The Pichia pastoris recombinant bacterium for producing endo-inulinase is inoculated on activation medium and is activated, then
It is transferred in BMGY fluid nutrient mediums, 12h is cultivated under the conditions of 28~30 DEG C, 250r/min, obtains seed liquor；
(2) high density fermentation：The seed liquor that step (1) is obtained is transferred in basal medium, is cultivated in fermentation tank,
Fermentation condition is：28 DEG C of temperature, mixing speed 900r/min, dissolved oxygen amount 25%, throughput be 6L/min, control ferment pH be
5.8, start stream plus supplementing culture medium after culture 6h, it is 0.5% to add methyl alcohol to final volume concentration every 12h, cultivates 72h.
Under the fermentation condition, the expression quantity of endo-inulinase is 285mg/L.
Claims (6)
1. one plant product endo-inulinase Pichia pastoris recombinant bacterium, it is characterised in that by the gene order shown in SEQ ID NO.1
It is cloned into pPIC9K, obtains recombinant plasmid, then by recombinant plasmid transformed Pichia pastoris GS115, both obtains producing endo-inulinase
Pichia pastoris recombinant bacterium.
2. a kind of method that utilization Pichia pastoris recombinant bacterium high density fermentation produces inulinase, it is characterised in that comprise the following steps：
(1) prepared by seed liquor：The Pichia pastoris recombinant bacterium for producing endo-inulinase is inoculated on activation medium and is activated, then transferred
To in BMGY fluid nutrient mediums, 12~18h is cultivated under the conditions of 28~30 DEG C, 200~250r/min, obtain seed liquor；
(2) high density fermentation：The seed liquor that step (1) is obtained is transferred in basal medium, is cultivated in fermentation tank, fermentation
Condition is：28~30 DEG C of temperature, 800~1000r/min of mixing speed, dissolved oxygen amount 20%~30%, throughput are 5~6L/
Min, it is 5.5~6 to control fermentation pH, and stream plus supplementing culture medium are started after culture 6h, adds methyl alcohol every 12h dense to final volume
It is 0.5% to spend, and cultivates 64~72h.
3. the method that utilization Pichia pastoris recombinant bacterium high density fermentation according to claim 2 produces inulinase, its feature exists
In in step (1), the formula of the activation medium is as follows：Yeast extract 10g/L, peptone 20g/L, glucose 20g/L.
4. the method that utilization Pichia pastoris recombinant bacterium high density fermentation according to claim 2 produces inulinase, its feature exists
In in step (1), the formula of the BMGY Liquid Cultures is as follows：10g/L121 DEG C of tryptone 20g/L, yeast extract, sterilizing
After 20min, the potassium phosphate of 100mmol/L pH=6 is added, without amino nitrogen source 13.4g/L, biotin 4 × 10-4G/L, glycerine
10g/L。
5. the method that utilization Pichia pastoris recombinant bacterium high density fermentation according to claim 2 produces inulinase, its feature exists
In in step (2), the formula of the basal medium is as follows：Ammonium sulfate 10g/L, potassium dihydrogen phosphate 6g/L, magnesium sulfate 1g/L,
EDTA 3mg/L, zinc sulfate 1g/L, ferrous sulfate 5mg/L, copper sulphate 1mg/L, calcium chloride 1g/L, manganese chloride 0.5mg/L, chlorination
Cobalt 0.8mg/L, KI 0.2mg/L, sodium molybdate 0.05mg/L, nicotinic acid 5mg/L, biotin 0.8mg/L, glucose 10g/L.
6. the method that utilization Pichia pastoris recombinant bacterium high density fermentation according to claim 2 produces inulinase, its feature exists
In in step (2), the formula of the supplementing culture medium is as follows：Ammonium sulfate 10g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate
0.05g/L, EDTA 2mg/L, zinc sulfate 0.08g/L, ferrous sulfate 2mg/L, copper sulphate 0.5mg/L, calcium chloride 0.5g/L, chlorine
Change manganese 0.3mg/L, cobalt chloride 0.8mg/L, KI 0.1mg/L, sodium molybdate 0.03mg/L, nicotinic acid 5mg/L, biotin 1mg/L,
Glucose 50g/L, defoamer 0.05ml/L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341709A (en) * | 2001-09-11 | 2002-03-27 | 湖北大学 | Yeast gene engineering bacterium and endoinulase preparation and its application method |
| CN1594542A (en) * | 2003-08-19 | 2005-03-16 | 中国农业科学院饲料研究所 | Aspergillus niger inulin endopeptidase gene and recombinant Pichia strain for expressing same |
| CN103952326A (en) * | 2014-05-21 | 2014-07-30 | 山东大学 | Recombinant pichia pastoris bacterial strain for co-expressing inulin excision enzyme and incision enzyme as well as construction method and application of bacterial strain |
| CN106497898A (en) * | 2016-12-16 | 2017-03-15 | 丰宁平安高科实业有限公司 | The preparation method of the engineering strain and restructuring endo-inulinase of expression restructuring endo-inulinase |
-
2017
- 2017-04-25 CN CN201710286554.6A patent/CN106906153A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341709A (en) * | 2001-09-11 | 2002-03-27 | 湖北大学 | Yeast gene engineering bacterium and endoinulase preparation and its application method |
| CN1594542A (en) * | 2003-08-19 | 2005-03-16 | 中国农业科学院饲料研究所 | Aspergillus niger inulin endopeptidase gene and recombinant Pichia strain for expressing same |
| CN103952326A (en) * | 2014-05-21 | 2014-07-30 | 山东大学 | Recombinant pichia pastoris bacterial strain for co-expressing inulin excision enzyme and incision enzyme as well as construction method and application of bacterial strain |
| CN106497898A (en) * | 2016-12-16 | 2017-03-15 | 丰宁平安高科实业有限公司 | The preparation method of the engineering strain and restructuring endo-inulinase of expression restructuring endo-inulinase |
Non-Patent Citations (2)
| Title |
|---|
| 张帆 等: "Aspergillus niger菊粉内切酶的生产及应用", 《第八届中国酶工程学术研讨会论文集》 * |
| 陈敏: "菊粉酶的筛选、异源表达及固定化", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
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