CN101698840A - Method for expressing xylose isomerase by applying pGAP regulation of Pichia pastoris - Google Patents

Method for expressing xylose isomerase by applying pGAP regulation of Pichia pastoris Download PDF

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Publication number
CN101698840A
CN101698840A CN200910209772A CN200910209772A CN101698840A CN 101698840 A CN101698840 A CN 101698840A CN 200910209772 A CN200910209772 A CN 200910209772A CN 200910209772 A CN200910209772 A CN 200910209772A CN 101698840 A CN101698840 A CN 101698840A
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Prior art keywords
xylose isomerase
pgap
pichia pastoris
expressing
regulation
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CN200910209772A
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Chinese (zh)
Inventor
张爱联
易国辉
罗进贤
张添元
尹慧祥
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Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
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Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
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Priority to CN200910209772A priority Critical patent/CN101698840A/en
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Abstract

The invention relates to a method for expressing xylose isomerase by applying pGAP regulation of Pichia pastoris, belonging to the biotechnology field. Firstly, pGAP promoters are amplificated in Pichia pastoris, a Pichia pastoris expression vector of which xylose isomerase is regulated by the pGAP is built, and then the Pichia pastoris expression vector is transformed into Pichia pastoris genome. High copy recons are selected as engineering bacterial strain according to resistance genes on the Pichia pastoris expression vector, and the engineering bacterial strain is inoculated into liquid culture medium containing carbon source to ferment engineering bacteria for secreting and expressing the xylose isomerase. The invention has the advantages of low production cost and short fermenting period. The using of the pGAP regulation of Pichia pastoris to express the xylose isomerase is favorable to purifying the product, and the problems of high production cost and hard product purification caused by using natural bacterial strains produced xylose isomerase to produce xylose isomerase can be solved. The invention is favourable for mass production of xylose isomerase.

Description

Use the method for the pGAP regulating and expressing xylose isomerase of pichia spp
Technical field
The invention belongs to biological technical field, relate to the method that a kind of pGAP promoter regulation of using pichia spp is produced bioprotein, specifically is a kind of method of using the pGAP regulating and expressing xylose isomerase of pichia spp.
Background technology
Xylose isomerase (Xylose Isomerase EC5.3.1.5) by a series of microorganisms, has the enzyme of destructurization.The main effect of xylose isomerase enzyme is catalysis D-wood sugar (aldehyde pentose) and the mutual enzyme of changing of D-xylulose (ketone pentose), also acts on the D-glucose and obtains D-fructose.Xylose isomerase is mainly used in high fructose syrup industry and hemicellulose ethanol industry.Multiple microorganism has the function that produces xylose isomerase.Mainly be to produce the xylose isomerase gene bacterial strain with natural containing to carry out the fermentative production xylose isomerase at present on the industrial production.But; because the poor growth of natural product xylose isomerase bacterial strain; production cycle is long; nutritional requirement is higher; product or in born of the same parents, form inclusion body (bacterial isolates) or a large amount of self the albumen (fungal bacterial strain) of secretion are unfavorable for the purifying of target protein; production cost is higher, is unfavorable for the large-scale production and the utilization of xylose isomerase.
Pichia spp (Pichia pastoris) is a kind of eukaryotic expression host who develops rapidly recently, nutritional requirement is not high, the mass production method that is suitable for high density fermentation because the albumen of Pichiapastoris secretion self seldom, helps the purifying of expression product.PGAP (glyceraldehyde 3-phosphate dehydrogenase promotor) is the composing type strong promoter of Pichia pastoris, and comparing its superiority with the pAOX1 (alcohol oxidase 1 promotor) of Pichia pastoris mainly is not need toxic methyl alcohol as carbon source.
Summary of the invention
The objective of the invention is to produce the problem that cost is higher, product is difficult to purifying of xylose isomerase, and a kind of method of using pGAP (glyceraldehyde 3-phosphate dehydrogenase promotor) the regulating and expressing xylose isomerase of pichia spp is provided for solving the bacterial strain of using natural product xylose isomerase.
The technical solution adopted in the present invention is:
A kind of method of using the pGAP regulating and expressing xylose isomerase of pichia spp the steps include:
1, amplification pGAP promotor from pichia spp at first;
2, at 3 ' the terminal His6 label that merges of xylose isomerase enzyme sequence, make up yeast expression vector, and this carrier is transformed the pichia spp genome by pGAP regulation and control xylose isomerase gene;
3, according on the carrier with the high copy of resistant gene screening recon as engineering strain;
4, engineering strain is inoculated in the liquid nutrient medium of carbonaceous sources, in 28～30 ℃ of fermentation engineering bacterium 1～2d secreting, expressing xylose isomerases in bio-reactor.Described liquid nutrient medium is composed of the following components: 85% phosphoric acid, 25～28ml/L, CaSO 42H 2O 0.8～1.2g/L, K 2SO 417～19g/L, MgSO 47H 2O 13～16g/L, KOH 3.5～4.5g/L, carbon source 15～45g/L, peptone 18～22g/L, Yeast extract 8～12g/L, PTM4 3～5ml/L; Described PTM4 is composed of the following components: CuSO 45H 2O 2.0g/L, NaI2H 2O 0.1g/L, MnSO 4H 2O 3.0g/L, Na 2MoO 42H 2O 0.2g/L, H 3BO 30.02g/L, CoCl 26H 2O 1.05g/L, ZnCl 27.0g/L, Fe 2(SO 4) 37H 2O 22g/L, vitamin H 0.2g/L, sulfuric acid 1ml/L.
Described carbon source is glycerine, oleic acid, sorbyl alcohol, glucose, lactic acid, glycine, L-Ala, trehalose or N.F,USP MANNITOL.
The present invention has adopted the xylose isomerase of the pGAP regulating and expressing of pichia spp, has following advantage:
1, production cost is low, only needs to use common inorganic salt, carbon source and nitrogenous source as fermention medium.
2, the pichia spp that can ferment in bio-reactor, its cell can grow to cell density and reach more than the 200A, can express xylose isomerase with abundant cell quantity, help the scale operation of xylose isomerase.
3, fermentation period is short, and whole fermentation period only needs 1～2d.
4, use avirulent carbon source, do not need to use virose methyl alcohol as carbon source.
5, because pGAP is the strong promoter of pichia spp, it is higher to account for the proteic ratio of total secretion by the xylose isomerase of pGAP regulating and expressing, and owing to merged the His label at the C of xylose isomerase end, helps the purifying of product.
Embodiment
The invention will be further described to adopt indefiniteness embodiment below.
Embodiment one
1, amplification pGAP promotor from Pichia pastoris at first;
2, at 3 ' the terminal His6 label that merges of xylose isomerase enzyme sequence, make up Pichia pastoris expression vector, and this carrier is transformed Pichia pastoris genome by pGAP regulation and control xylose isomerase gene;
3, according on the carrier with the high copy of resistant gene screening recon as engineering strain;
4, engineering strain bacterium liquid is inoculated in the [prescription (every liter) of liquid nutrient medium: 85% phosphoric acid 25.7ml, CaSO in the liquid nutrient medium 42H 2O 0.87g, K 2SO 417.5g, MgSO 47H 2O 13.9g, KOH 3.73g, glycerine 40g, peptone 19g, Yeast extract 9g, PTM4 trace element 3ml] [PTM4 (every liter): the CuSO that fills a prescription 45H 2O 2.0g, NaI2H 2O 0.1g, MnSO 4H 2O 3.0g, Na 2MoO 42H 2O 0.2g, H 3BO 30.02g, CoCl 26H 2O 1.05g, ZnCl 27.0g, Fe 2(SO 4) 37H 2O 22g, vitamin H 0.2g, sulfuric acid 1ml], in bio-reactor, carry out the fermentation expression xylose isomerase.Its fermentation parameter is: 28 ℃ of temperature, pH5.0, mixing speed 700r/min.Centrifugal results supernatant carries out the purifying xylose isomerase behind the fermentation 2d.By protein electrophoresis and enzyme assay the xylose isomerase of expressing is identified (target protein of protein electrophoresis proof fermentation expression meets the xylose isomerase molecular weight of albumen, and xylose isomerase enzymic activity＞1 μ/μ g target protein) and proved that the product that this enforcement obtains is an xylose isomerase.
Embodiment two
1, amplification pGAP promotor from Pichia pastoris at first;
2, at 3 ' the terminal His6 label that merges of xylose isomerase gene sequence, make up Pichia pastoris expression vector, and this carrier is transformed Pichia pastoris genome by pGAP regulation and control xylose isomerase gene;
3, according on the carrier with the high copy of resistant gene screening recon as engineering strain;
4, engineering strain bacterium liquid is inoculated in the [prescription (every liter) of liquid nutrient medium: 85% phosphoric acid 26.7ml, CaSO in the liquid nutrient medium 42H 2O 0.93g, K 2SO 418.2g, MgSO 47H 2O 14.9g, KOH 4.13g, oleic acid 20g, peptone 20g, Yeast extract 10g, PTM4 trace element 4ml] [PTM4 (every liter): the CuSO that fills a prescription 45H 2O 2.0g, NaI2H 2O 0.1g, MnSO 4H 2O 3.0g, Na 2MoO 42H 2O 0.2g, H 3BO 30.02g, CoCl 26H 2O 1.05g, ZnCl 27.0g, Fe 2(SO 4) 37H 2O 22g, vitamin H 0.2g, sulfuric acid 1ml], in bio-reactor, carry out the fermentation expression xylose isomerase.Its fermentation parameter is: 30 ℃ of temperature, pH5.0, mixing speed 300r/min.Centrifugal results supernatant carries out the purifying xylose isomerase behind the fermentation 2d.By protein electrophoresis and enzyme assay the xylose isomerase of expressing is identified (target protein of protein electrophoresis proof fermentation expression meets the xylose isomerase molecular weight of albumen, and xylose isomerase enzymic activity＞1 μ/μ g target protein) and proved that the product that this enforcement obtains is an xylose isomerase.
Embodiment three
1, amplification pGAP promotor from Pichia pastoris at first;
2, at 3 ' the terminal His6 label that merges of xylose isomerase gene sequence, make up Pichia pastoris expression vector, and this carrier is transformed Pichia pastoris genome by pGAP regulation and control xylose isomerase gene;
3, according on the carrier with the high copy of resistant gene screening recon as engineering strain;
4, engineering strain bacterium liquid is inoculated in the [prescription (every liter) of liquid nutrient medium: 85% phosphoric acid 27.2ml, CaSO in the liquid nutrient medium 42H 2O 1.03g, K 2SO 418.2g, MgSO 47H 2O 15.2g, KOH 4.23g, sorbyl alcohol 35g, peptone 20g, Yeast extract 10g, PTM4 trace element 4ml] [PTM4 (every liter): the CuSO that fills a prescription 45H 2O 2.0g, NaI2H 2O 0.1g, MnSO 4H 2O 3.0g, Na 2MoO 42H 2O 0.2g, H 3BO 30.02g, CoCl 26H 2O 1.05g, ZnCl 27.0g, Fe 2(SO 4) 37H 2O 22g, vitamin H 0.2g, sulfuric acid 1ml], in bio-reactor, carry out the fermentation expression xylose isomerase.Its fermentation parameter is: 28 ℃ of temperature, pH5.0, mixing speed 500r/min.Centrifugal results supernatant carries out the purifying xylose isomerase behind the fermentation 2d.By protein electrophoresis and enzyme assay the xylose isomerase of expressing is identified (target protein of protein electrophoresis proof fermentation expression meets the xylose isomerase molecular weight of albumen, and xylose isomerase enzymic activity＞1 μ/μ g target protein) and proved that the product that this enforcement obtains is an xylose isomerase.
Embodiment four
1, amplification pGAP promotor from Pichia pastoris at first;
2, at 3 ' the terminal His6 label that merges of xylose isomerase gene sequence, make up Pichia pastoris expression vector, and this carrier is transformed Pichia pastoris genome by pGAP regulation and control αDian Fenmei gene;
3, according on the carrier with the high copy of resistant gene screening recon as engineering strain;
4, engineering strain bacterium liquid is inoculated in the [prescription (every liter) of liquid nutrient medium: 85% phosphoric acid 27.7ml, CaSO in the liquid nutrient medium 42H 2O 1.13g, K 2SO 418.7g, MgSO 47H 2O 15.5g, KOH 4.33g, glucose 40g, peptone 21g, Yeast extract 11g, PTM4 trace element 5ml] [PTM4 (every liter): the CuSO that fills a prescription 45H 2O 2.0g, NaI2H 2O 0.1g, MnSO 4H 2O 3.0g, Na 2MoO 42H 2O 0.2g, H 3BO 30.02g, CoCl 26H 2O 1.05g, ZnCl 27.0g, Fe 2(SO 4) 37H 2O 22g, vitamin H 0.2g, sulfuric acid 1ml], in bio-reactor, carry out the fermentation expression xylose isomerase.Its fermentation parameter is: 30 ℃ of temperature, pH5.0, mixing speed 700r/min.Centrifugal results supernatant carries out the purifying xylose isomerase behind the fermentation 1d.By protein electrophoresis and enzyme assay the xylose isomerase of expressing is identified (target protein of protein electrophoresis proof fermentation expression meets the xylose isomerase molecular weight of albumen, and xylose isomerase enzymic activity＞1 μ/μ g target protein) and proved that the product that this enforcement obtains is an xylose isomerase.
Embodiment five
1, amplification pGAP promotor from Pichia pastoris at first;
2, at 3 ' the terminal His6 label that merges of xylose isomerase gene sequence, make up Pichia pastoris expression vector, and this carrier is transformed Pichia pastoris genome by pGAP regulation and control xylose isomerase gene;
3, according on the carrier with the high copy of resistant gene screening recon as engineering strain;
4, engineering strain bacterium liquid is inoculated in the [prescription (every liter) of liquid nutrient medium: 85% phosphoric acid 27.7ml, CaSO in the liquid nutrient medium 42H 2O 0.93g, K 2SO 418.7g, MgSO 47H 2O 15.7g, KOH 4.33g, lactic acid 30g, peptone 22g, Yeast extract 12g, PTM4 trace element 5ml] [PTM4 (every liter): the CuSO that fills a prescription 45H 2O 2.0g, NaI2H 2O 0.1g, MnSO 4H 2O 3.0g, Na 2MoO 42H 2O 0.2g, H 3BO 30.02g, CoCl 26H 2O 1.05g, ZnCl 27.0g, Fe 2(SO 4) 37H 2O 22g, vitamin H 0.2g, sulfuric acid 1ml], in bio-reactor, carry out the fermentation expression xylose isomerase.Its fermentation parameter is: 28 ℃ of temperature, pH5.0, mixing speed 800r/min.Centrifugal results supernatant carries out the purifying xylose isomerase behind the fermentation 1d.By protein electrophoresis and enzyme assay the xylose isomerase of expressing is identified (target protein of protein electrophoresis proof fermentation expression meets the xylose isomerase molecular weight of albumen, and xylose isomerase enzymic activity＞1 μ/μ g target protein) and proved that the product that this enforcement obtains is an xylose isomerase.

Claims (2)

1. method of using the pGAP regulating and expressing xylose isomerase of pichia spp is characterized in that its step is as follows:
1), amplification pGAP promotor from pichia spp at first;
2), at 3 ' the terminal His6 label that merges of xylose isomerase enzyme sequence, make up yeast expression vector, and this carrier transformed the pichia spp genome by pGAP regulation and control xylose isomerase gene;
3), according on the carrier with the high copy of resistant gene screening recon as engineering strain;
4), engineering strain is inoculated in the liquid nutrient medium of carbonaceous sources, in 28～30 ℃ of fermentation engineering bacterium 1～2d secreting, expressing xylose isomerases in bio-reactor; Described liquid nutrient medium is composed of the following components: 85% phosphoric acid, 25～28ml/L, CaSO 42H 2O 0.8～1.2g/L, K 2SO 417～19g/L, MgSO 47H 2O 13～16g/L, KOH 3.5～4.5g/L, carbon source 15～45g/L, peptone 18～22g/L, Yeast extract 8～12g/L, PTM43～5ml/L; Described PTM4 is composed of the following components: CuSO 45H 2O 2.0g/L, NaI2H 2O 0.1g/L, MnSO 4H 2O 3.0g/L, Na 2MoO 42H 2O 0.2g/L, H 3BO 30.02g/L, CoCl 26H 2O 1.05g/L, ZnCl 27.0g/L, Fe 2(SO 4) 37H 2O 22g/L, vitamin H 0.2g/L, sulfuric acid 1ml/L.
2. the method for the pGAP regulating and expressing xylose isomerase of application pichia spp according to claim 1 is characterized in that: described carbon source is glycerine, oleic acid, sorbyl alcohol, glucose, lactic acid, glycine, L-Ala, trehalose or N.F,USP MANNITOL.
CN200910209772A 2009-10-25 2009-10-25 Method for expressing xylose isomerase by applying pGAP regulation of Pichia pastoris Pending CN101698840A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174549A (en) * 2011-02-22 2011-09-07 山东大学 Nucleic acid molecules for coding xylose isomerase and xylose isomerase coded by same
CN102747062A (en) * 2012-07-09 2012-10-24 中国热带农业科学院热带生物技术研究所 Method for producing enzyme system of starch hydrolyzing-generated high fructose corn syrup
CN102747063A (en) * 2012-07-09 2012-10-24 中国热带农业科学院热带生物技术研究所 Xylose isomerase producing method
US9951326B2 (en) 2015-07-13 2018-04-24 MARA Renewables Corporation Enhancing microbial metabolism of C5 organic carbon

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174549A (en) * 2011-02-22 2011-09-07 山东大学 Nucleic acid molecules for coding xylose isomerase and xylose isomerase coded by same
CN102174549B (en) * 2011-02-22 2012-10-10 山东大学 Nucleic acid molecules for coding xylose isomerase and xylose isomerase coded by same
CN102747062A (en) * 2012-07-09 2012-10-24 中国热带农业科学院热带生物技术研究所 Method for producing enzyme system of starch hydrolyzing-generated high fructose corn syrup
CN102747063A (en) * 2012-07-09 2012-10-24 中国热带农业科学院热带生物技术研究所 Xylose isomerase producing method
US9951326B2 (en) 2015-07-13 2018-04-24 MARA Renewables Corporation Enhancing microbial metabolism of C5 organic carbon
US10662418B2 (en) 2015-07-13 2020-05-26 MARA Renewables Corporation Enhancing microbial metabolism of C5 organic carbon

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Application publication date: 20100428