CN102965292A

CN102965292A - Glucose oxidase secretion enhanced bacterial strain and application thereof

Info

Publication number: CN102965292A
Application number: CN2012105279089A
Authority: CN
Inventors: 陈坚; 顾磊; 张娟; 堵国成; 沈伊娜; 闻一凡; 李梦洁; 李婷; 丁雪
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2012-12-10
Filing date: 2012-12-10
Publication date: 2013-03-13

Abstract

The invention discloses a method for enhancing secretion expression of glucose oxidase through excessively expressing a Haclp gene, and belongs to the technical field of genetic engineering. The Haclp gene of pichia pastoris is cloned and connected to a pichia pastoris expression vector pPICZalpha by adopting a gene recombination technology, and is transformed into a Pichia pastoris GS115-pPIC9K-GOX strain with a preservation number of CCTCC NO:M2012266, and a strain which is capable of enhancing the secretion expression of the glucose oxidase compared with the original strain is obtained through screening and identification. The glucose oxidase expressed by the strain has the enzyme activity of 66.24U/mL in a shake flask, which is remarkably increased compared with the enzyme activity of 55.31 U/mL before the method is used. In addition, by utilizing the method, the production efficiency can be remarkably increased, and a better foundation is laid for the large-scale production of the glucose oxidase.

Description

A kind of glucose oxidase enzyme secretion enhancement type bacterial strain and application thereof

Technical field

The present invention relates to a kind of glucose oxidase and produce bacterial strain, particularly a kind of glucose oxidase enzyme secretion enhancement type bacterial strain and application thereof belong to gene engineering technology field.

Background technology

Glucose oxidase is one of topmost toolenzyme in the biological field.From Updike in 1967 and Hicks GOD is fixed on Clark oxygen electrode surface, has been applied to since the blood sugar detection, GOD is widely used in many association areas such as food, feed, medicine.

In foodstuffs industry, because the existence of oxygen causes many chemical reactions that are unfavorable for quality product, and created condition for many microorganism growth.At present, many countries are widely used in GOD in various food and the food processing technology as workman's safe oxidation inhibitor.Although purposes is various, the effect of GOD mainly is except glucose, deoxygenation, formation hydrogen peroxide, forms four aspects of gluconic acid.Utilize its single-minded oxidasic principle, make the glucose oxidase enzyme analyser, can quick and precisely measure simply the glucose content in the various food, instruct and produce.

In medicine industry, GOD is used for the external quantitative analysis of serum (slurry), urine and cerebrospinal fluid glucose as test kit, enzyme electrodes etc.; The zymin that GOD makes also can be used for removing or the formation of alleviating dental plaque, tartar and carious tooth prevents the generation of oral disease and odontopathy.In addition, owing to can catalysis generate H202, also can be used for the treatment to the lymphadenomatous target goal of H2O2 sensitivity.

GOD or a kind of novel enzyme feed additive can improve the animal intestinal environment, regulate diet digestion, promote growth of animal.Contain the mixed fodder additive of glucose oxidase, lactic acid superoxide and lactoferrin, can be used for preventing livestock gastrointestinal tract infection, diarrhoea, and the effect of the growth of animal of promotion is arranged.

Extracting GOD from animal vegetable tissue has certain limitation, and the enzyme amount is also not abundant; Bacterium GOD yield of enzyme is few; General aspergillus niger (having the GRAS qualification) and the Penicillium bacterial strain of adopting produced bacterium as GOD.China and the U.S. all adopt a mould and Penicllium chrysogenum to produce GOD, Japan's rugged mould of Buddhist nun commonly used, Russia uses the life mould, reports that in recent years the mould genus of glue (Clioctadium), paecilomyces (Paecilomyces) and the mould genus of broom (Scopulariopsis) also can produce GOD.

Yield poorly, enzyme is lived restrictive factor low, the complicated GOD of the being industrialization of detection method, do a lot of work both at home and abroad for this reason and obtained obvious progress.The external GOD producer that produces mainly is German Boehringer and the TOYOBO of Japan at present.The highly active GOD of large-scale production also has any problem.Produce a large amount of foreign proteins in the time of fermentative production GOD, separation and Extraction is complicated, and cost is high.

This research has made up genetic engineering bacterium Pichia pastoris GS115-pPIC9K-GOX(deposit number in early-stage Study be CCTCC NO:M 2012266), its glucose oxidase production of enzyme has obtained certain raising, is the technical problem to be solved in the present invention but how further to improve its glucose oxidase production of enzyme to adapt to industrialized needs.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of glucose oxidase enzyme secretion enhancement type bacterial strain, is the pichia yeast genetic engineering bacteria of coexpression glucose oxidase gene and Haclp gene.

By pichia spp Haclp gene is connected to yeast expression vector pPICZ α, and to be transformed into Pichia pastorisGS115-pPIC9K-GOX(deposit number be CCTCC NO:M 2012266) bacterial strain in.

Described Haclp gene nucleotide series such as gene nucleotide series shown in Genbank XM_002489994.1.

The present invention also provides a kind of method that makes up above-mentioned bacterial strains, and step is as follows:

1) by pcr amplification method or the complete synthesis acquisition Haclp gene of chemistry;

2) the Haclp gene is connected to yeast expression vector pPICZ α, obtains recombinant plasmid pPICZ-Hac1;

3) recombinant vectors transforms Pichia pastoris GS115-pPIC9K-GOD and obtains secreting the enhancement type genetic engineering bacterium.

Glucose oxidase enzyme activity determination method: general ortho-, meta-or p-(two) methyl oxyaniline spectrophotometry that adopts of GOD determination of activity.Under aerobic conditions, the dehydrogenation of GOD catalysis glucose produces H ₂O ₂, under peroxidase (POD) effect, oxygen donor ortho-, meta-or p-(two) methyl oxyanilines (DH2) are oxidized to brown product.Survey the variation of 540nm place absorbancy, according to the result of typical curve, calculate the glucose oxidase enzyme activity unit.

Use that enzyme work is 66.24U/mL on the bacterial strain shaking flask provided by the invention, than using enzyme before the method 55.31U/mL that lives to be significantly improved.This haves laid a good foundation for the scale operation of glucose oxidase.

Description of drawings

Fig. 1: clonal expression plasmid map.

Fig. 2: the ability of strain fermentation malaga carbohydrate oxidase.

Embodiment

Structure and the evaluation of embodiment 1 recombinant bacterium

Obtain Haclp by chemical total synthesis method, it is cloned into intermediate carrier pPICZ α obtains recombinant plasmid pPICZ-Hac1, recombinant vectors is transformed Pichia pastoris GS115-pPIC9K-GOX, obtain recombinant bacterium Pichia pastoris GS115-pPIC9K-GOX/pPICZ-Hac1 through Screening and Identification.

Pichia pastoris GS115-pPIC9K-GOX(Pichiapastoris GS115-pPIC9K-GOX) be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M 2012266.

Electrotransformation is adopted in the conversion of pichia spp.Bacterial strain is cultured to OD in 500mL YPD ₆₀₀=1.2-1.5, the 1500g centrifugal collecting cell; Use successively ice-cold twice cell of aseptic washing of 400mL, use the ice-cold cell of 1mol/L sorb alcohol wash of 40mL again, re-suspended cell is in 1mL 1mol/L sorbyl alcohol.100 μ L protoplastiss mix with 5-10 μ g linearization plasmid DNA (SacI cuts), change ice-cold electric revolving cup over to, place 5 minutes; The mixture (1.5kv, 4.2-4.9ms) of electric shock cell and DNA; Add the ice-cold 1mol/L sorbyl alcohol of 1mL, continue to place 30min; Add 0.5mL SOS, 30 ° of C placed 2 hours again, shook once in a while the will thalline and were difficult for precipitation; With coating the solid MD substratum that contains bleomycin (200 μ g/mL) behind the 1mol/L sorbyl alcohol dilution thalline.30 ° of C cultivate picking mono-clonal after 4-6 days.Plasmid map is seen Fig. 1.

Enzyme activity determination and the protein electrophoresis of embodiment 2 secretion enhancement type bacterial strains

Substratum: seed and slant medium are YPD substratum (1L): Tryptones 20g, yeast extract 10g, glucose 20g; Slant medium adds agar 20g; Basic fermention medium is BMGY substratum (1L): Tryptones 20g, yeast extract 10g, glycerine 10mL, YNB 13.4g, 100mM phosphoric acid buffer (pH 6.0); Inducing culture is BMMY substratum (1L): Tryptones 20g, yeast extract 10g, methyl alcohol 8mL, YNB 13.4g, 100mM phosphoric acid buffer (pH 6.0);

Cultural method: will cultivate OD under 30 ℃, 200rpm ₆₀₀Seed between 1.6～1.7 changes basic fermention medium over to 2% inoculum size, cultivates under 30 ℃, 200rpm condition;

Inductive condition: when in BMGY, being cultured to the OD value for 1.2-1.5, yeast cell is changed over to the generation of inducible protein in the BMMY substratum.

Secretion enhancement type recombinant bacterium enzyme in shaking flask is lived and is 66.24U/mL(74h), than the 55.31U/mL(74h alive of the enzyme before use the method) (Fig. 2) is significantly improved.

In addition, the present invention is intended to improve the output of enzyme, yet we but find by scheme, use present method not only can improve the output of enzyme, also cocoa is obviously enhanced productivity, and at fermentation 61h, its enzyme activity can reach 55.31U/mL, production cycle has improved 13 hours than original strain, has beyond thought technique effect.

Be understandable that, for those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.

Claims

1. a glucose oxidase enzyme secretion enhancement type bacterial strain is the pichia yeast genetic engineering bacteria of coexpression glucose oxidase gene and Haclp gene.

2. bacterial strain claimed in claim 1 is characterized in that, described Haclp gene is connected to yeast expression vector pPICZ α, and described glucose oxidase gene is expressed in Pichia pastoris GS115-pPIC9K-GOX.

3. the construction process of the described bacterial strain of claim 1 comprises the steps:

1) obtains the Haclp gene by PCR method;

2) the Haclp gene that step 1) is obtained is connected to yeast expression vector pPICZ α, obtains recombinant plasmid pPICZ-Hac1;

3) with step 2) the recombinant plasmid transformed Pichia pastoris GS115-pPIC9K-GOX that obtains obtains secreting the enhancement type genetic engineering bacterium.

4. construction process claimed in claim 3 is characterized in that the Haclp gene nucleotide series is shown in GenbankXM_002489994.1.

5. the application of bacterial strain claimed in claim 1 in the fermentative production glucose oxidase is characterized in that: bacterial strain is inoculated into basic fermention medium behind seed activation, cultivates under 30 ℃, 200rpm condition; When the OD value is 1.2-1.5, bacterial strain is changed over to the generation of inducible protein in the inducing culture.

6. application claimed in claim 5 is characterized in that described inducing culture consists of (1L): Tryptones 20g, yeast extract 10g, methyl alcohol 8mL, YNB 13.4g, the 100mM phosphoric acid buffer of pH 6.0.

7. claim 5 or 6 described application is characterized in that seed activation is to OD ₆₀₀Seed between 1.6～1.7 changes basic fermention medium over to 2% inoculum size.

8. claim 5 or 6 described application, it is characterized in that inductive condition is: the methanol concentration of Induction Process is kept 1.8% (W/V), induces the glucose oxidase expression of enzymes.