CN106967737A - For producing the gene with high activity lipase - Google Patents
For producing the gene with high activity lipase Download PDFInfo
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- CN106967737A CN106967737A CN201710177364.0A CN201710177364A CN106967737A CN 106967737 A CN106967737 A CN 106967737A CN 201710177364 A CN201710177364 A CN 201710177364A CN 106967737 A CN106967737 A CN 106967737A
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- lipase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
- A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
- A21D2/24—Organic nitrogen compounds
-
- A—HUMAN NECESSITIES
- A21—BAKING; EDIBLE DOUGHS
- A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
- A21D8/00—Methods for preparing or baking dough
- A21D8/02—Methods for preparing dough; Treating dough prior to baking
- A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
- A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
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- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to biological technical field, a kind of gene for being used to produce with high activity lipase is specifically disclosed and as the high activity lipase expressed by the gene.Lipase active expressed by the gene of the present invention is high, with potentiality to be exploited, with extensive and actual application value, such as brightening available for Flour product and baking goodses.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of to be used to produce the gene with high activity lipase.
Background technology
APROL (Rhizopus oryzae lipase), comes from Rhizopus oryzae, Amazon has the fat of commercialization
Fat enzyme gene.Minning et al. (2001) are using pPIC α A/P.pastoris X-33 expression systems to Rhizopus
The gene in oryzae sources is simultaneously expressed it, and enzyme activity is 12888U/Lh (Olive oil, in 30 DEG C of pH after fermentation 103h
8.1 times reactions), it is expressed with pPICZaC/P.pastoris expression systems, enzyme activity is 110U/ml after fermentation 48h,
8571U/mg (triolein, 30 DEG C of pH 8.1), native enzyme (8.5,37 DEG C of 10000U/mg, olive oil, pH).
But the enzymatic activity is not high, it have impact on its commercialization and promote or apply.
The content of the invention
In order to solve the above problems, an object of the present invention is the provision of a kind of for producing with high activity fat
The gene of enzyme；The second object of the present invention is to provide a kind of high activity lipase as expressed by the gene.
The present invention is achieved through the following technical solutions：
A kind of to be used to produce the gene with high activity lipase, the sequence of the gene is as follows：
A kind of high activity lipase as expressed by said gene, the specific enzyme activity of the high activity lipase for 380~
430U/mg。
It is preferred that the specific enzyme activity of the high activity lipase is 409U/mg.
Using upper, the high activity lipase can be used for brightening Flour product and baking goodses, such as noodles, steamed bun.
Compared with prior art, the invention has the advantages that：Lipase active expressed by the gene of the present invention
It is high, with potentiality to be exploited, with extensive and actual application value, such as brightening available for Flour product and baking goodses.
Brief description of the drawings
Fig. 1 is linearization plasmid APROL-pPICZ α A of the present invention agarose electrophoresis detection, wherein, line M, DL15000bp
marker；Line 1, linearisation APROL-pPICZ α A.
Fig. 2 is the schematic diagram that BMMY- rhodamine Bs flat band method screens recon.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, is managed to help those skilled in the art
The solution present invention.
The sequence that the present invention is used to produce the gene (being represented with APROL-pPICZ α A) with high activity lipase is as follows：
First, the expression of gene
Host：Pichi strain X-33 and GS115.Certainly, can express other hosts of lipase gene can all apply
To the present invention.
Gene chemical synthesis：The present invention is used for gene of the production with high activity lipase and synthesized by Shanghai JaRa company.
1.1 expression strain constructions
1.1.1 plasmid extraction
With reference to the small extraction reagent kit operation instructions of Guangzhou Dongsheng plasmid.
1.1.2 recombinant plasmid APROL-pPICZ α A linearisation and recovery
The recombinant plasmid APROL-pPICZ α A of gained are subjected to endonuclease reaction 1h at linearization process, 37 DEG C with Sac I,
Reaction system is as follows：
It is positioned over after 37 DEG C of water-baths, 1h and takes out after endonuclease reaction solution is mixed, is carried out with gel-purified QIAquick Gel Extraction Kit
Reclaim (referring to Tiangeng glue reclaim kit operation instructions).
1.1.3 the preparation of Pichi strain competence and electricity conversion
Referring to Invitrogen pPICZ α A, B, and C user's operation manuals.
1.1.4 result
Correct recombinant plasmid APROL-pPICZ α A it will be linearized after digestion verification and sequence verification with Sac I
Processing, digestion products condense electrophoresis after reclaiming with 1% agarose to be identified, as a result as shown in Figure 1.
The screening of the transformant of 1.2 tool lipase actives
1.2.1 primary dcreening operation
Most fast monoclonal is grown on picking YPDS flat boards, and point is connected to YPD (being used for conservation) and BMMY- rhodamine Bs respectively
Flat board (is used to induce) flat board, and 30 DEG C are inverted incubated 3~5d；
100 μ L methanol are added on the lid of BMMY- rhodamine B flat boards every 24h induced expression is carried out to recon,
In incubation, lipase enzymatic activity most strong 10 transformants are selected according to the relative size of hydrolysis circle, number consecutively is 1#,
2#, 3#~10#, plate screening result are as shown in Figure 2.
1.2.2 secondary screening
10 selected transformants are inoculated with the fluid nutrient mediums of BMGY containing 10mL (100mL specifications shaking flask), 30 DEG C respectively
250rpm is cultivated to OD600=6.0；
3,000g centrifugation 5min collect thalline, and 50mLBMMY liquid is transferred to after cell is resuspended with 2mLBMMY fluid nutrient mediums
In body culture medium, make its OD600=1.0；
30 DEG C of 250rpm continue Fiber differentiation, and adding a methanol every 24h makes to start after its final concentration of 0.5%, 48h
Enzymatic activity is surveyed in sampling, and sampling time point is 48h, 72h, 96h, 120h, and samples taken collects supernatant after 3,000g centrifugations 3min,
Lipase enzymatic activity is surveyed using titration, it is determined that active highest transformant and its inductive condition.
1.2.3 result
10 transformants that the lipase active of selection primary dcreening operation acquisition is higher are further screened, and observe its triangular flask water
Flat expression, using the lipase active in alkaline titration measuring fermented liquid supernatant, APROL-pPICZ α A enzyme activity is surveyed
Surely it the results are shown in Table 1.
The lipase enzymatic activity of each transformant in the horizontal triangular flask of the shaking table of table 1
| Induction time (h) | APROL-pPICZ α A enzymatic activitys (U/mL) |
| 48 | 70 |
| 72 | 75 |
| 96 | 85 |
| 120 | 85 |
1.3 lipase activities are determined
This experiment uses alkali formula titration measuring lipase activity.
1.3.1 principle
Lipase under certain condition, can make triglyceride be hydrolyzed into aliphatic acid, diglyceride, monoglycerides and glycerine,
The aliphatic acid available standards aqueous slkali discharged carries out acid-base titration, is counted with PH or phenol peptide indicator solution Indicator Reaction terminal, according to
The alkali number of consumption, calculates its enzyme activity.Reaction equation is：
RCOOH+NaOH→RCOONa+H2O
1.3.2 operating procedure
1) two 50ml triangular flasks are taken, substrate solution 4.00ml olives are added respectively at each in blank tube (A) and sample cell (B)
Olive oil emulsion (olive oil：PVA=1:3, v/v) and 5.00ml phosphate buffers (100mM, pH 7.0), added in A pipes
95% ethanol 15.00ml, preheats 5min in 40 DEG C of water-baths, and enzyme liquid 1.00ml to be measured is then respectively added in A, B pipe, is mixed immediately
Timing, the accurate response 10min in 40 DEG C of water-baths adds 95% ethanol 15.0ml terminating reactions immediately in B pipes, takes out；
2) respectively add phenol peptide indicator solution 2 to drip in blank and sample solution, titrated with 50mM NaOH standard liquids, until micro-
Red simultaneously keeps 30s colour-fast for titration end-point, the volume of record consumption 50mM NaOH standard liquids.
1.3.3 enzyme activity is calculated
Enzyme activity is defined:1g solid enzyme powders (or 1mL liquid enzymes), under the conditions of 40 DEG C of temperature and pH 7.0,1min hydrolysis olives
Olive oil (olive oil) produces 1 μm of ol titratable aliphatic acid, and as 1 enzyme activity unit is represented with U/g (U/ml).
Computational methods：The enzyme activity of lipase preparation is calculated as follows
X1=(V1-V2) * c*50*n1/0.05*1/10
In formula：
The enzyme activity of X1----- samples, U/g (U/ml)
V1---- consumes the volume of standard solution of sodium hydroxide when titrating sample, unit is milliliter (ml)；
V2---- consumes the volume of standard solution of sodium hydroxide when titrating blank, unit is milliliter (ml)
C---- Concentration of Sodium Hydroxide Solution Standard, unit is mole every liter (mol/L)；
50---0.05mol/L sodium hydroxide solution 1.00ml is equivalent to 50 μm of ol of aliphatic acid；
The extension rate of n1---- samples；
0.05---- Concentration of Sodium Hydroxide Solution Standard conversion coefficients；
1/10---- reaction time 10min, in terms of 1min.
Acquired results are represented to integer.
1.3.4 result
Comparative example：LBK-B4000 (the special lipase of steamed bun, green micro- health)：46.5mg albumen/g powder (protein contents
4.65%)；CaL-B (is used for organic synthesis, Novozymes)：9.24mg albumen/mL original enzyme liquids (protein content 0.924%).
The comparison of each lipase enzymatic property is as shown in table 2
| Name feature | Specific enzyme activity (U/mg) (Oliveoil) |
| LBK-B4000 | 282.61 |
| CaL-B | 1.84 |
| APROL-pPICZαA | 409 |
As shown in Table 2：APROL-pPICZ α A specific enzyme activity is higher than 1.7 times of LBK-B4000；CaL-B hydrolysis vigor
Minimum, only 1.84U/mg is mainly used in organic synthesis.In terms of specific enzyme activity, we are the lipase A PROL- in research
PPICZ α A have potentiality to be exploited.
It is made it is noted that the enzyme activity determination of lipase uses NaOH titrations, different people are commented chromogenic reaction
The yardstick sentenced is different, and the result obtained during enzyme activity determination has certain difference.Such as：The special lipase LBK- of steamed bun
Enzyme activity 16346U/g powder (than living for 355.35U/mg albumen) is surveyed in B4000, laboratory, and this experiment surveys enzyme activity and is
13000U/g powder (than living for 282.61U/mg albumen).
2nd, apply
By the way that the Gene A PROL-pPICZ α A of the present invention to be obtained to the lipase of high activity on yeast after high efficient expression,
Two sample Fs NL-25000 and FSL-20000 are obtained by existing formulation optimization again, can be applied to respectively Flour product with
Baking goodses are brightened.Wherein, by lipase optimization to belong to prior art suitable for Flour product or baking goodses, herein not
Repeat again.
2.1 lipase FNL-25000 noodles whitening effects are tested
2.1.1, experiment purpose
Test whitening effects of the lipase FNL-25000 in white salt noodles
2.1.2 primary raw material is tested
Flour：Muscle basis powder in Shandong
Lipase：FNL-25000
2.1.3 experimental formula and technique
Table 3, noodle formula
| Raw material | Consumption/g |
| Flour | 100 |
| Salt | 1 |
| Water | 33 |
Noodles technique：Add water and face；Relaxation；Oodle maker pressure surface；Integer, is made noodles and dough sheet.
2.1.4 experimental result
Table 4, FNL-25000 test results
As can be known from the above table, with the increase of FNL-25000 concentration, noodles and dough sheet whiteness are incremented by successively.
2.1.5 experiment conclusion
Muscle basis powder adds lipase FNL-25000, noodles whiteness substantially increases, added as flour material using in Shandong
Amount is bigger, and noodles whiteness is bigger.
2.2 lipase FSL-20000 steamed buns whitening effects are tested
2.2.1, experiment purpose
Test lipase FSL-20000 whitening effects in north steamed bread
2.2.2 primary raw material is tested
Flour：Muscle basis powder in Shandong；Muscle basis powder in Hebei
Lipase：FSL-20000
2.2.3 experimental formula and technique
Table 5, steamed bun formula
| Raw material | Consumption/g |
| Flour | 100 |
| Yeast | 0.8 |
| Water | 46 |
2.2.4 steamed bun technique
Add water and proof → boiling with face → oodle maker pressure surface → manual rounding →
2.2.5 experimental result
Table 6, FSL-20000 test results
The basic powder of muscle adds the increase of concentration with FSL-20000 in muscle basis powder and Hebei in Shandong, and steamed bun epidermis is white
Degree increases successively.
2.2.6 conclusion
The basic powder of muscle, as flour material, with the addition of lipase FSL-20000, steamed bun in muscle basis powder and Hebei using in Shandong
Epidermis whiteness substantially increases, and with the increase of addition, steamed bun epidermis whiteness is in rising trend.
Applicant：Dongguan Fanyatai Biological Sci-Tech Co., Ltd.
Denomination of invention：For producing the gene with high activity lipase
Sequence signature：Gene complete sequence for producing the gene with high activity lipase
Sequence description：
GAA TTC GAC GAC AAT TTG GTC GGT GGA ATG ACC TTG GAC TTG CCT TCT GAT GCC
CCT CCT ATT TCT TTG TCT GGT TCT ACA AAT TCT GCT TCT GAC GGA GGA AAG GTT GTT
GCT GCT ACT ACT GCC CAG ATC CAG GAG TTC ACT AAA TAC GCT GGT ATT GCA GCC ACA
GCC TAT TGT AGA TCT GTT GTC CCA GGA AAT AAG TGG GAC TGC GTC CAG TGC CAG AAG
TGG GTT CCA GAC GGT AAG ATC ATT ACC ACA TTC ACC TCT TTG TTG TCT GAC ACC AAT
GGA TAC GTC TTG AGA TCT GAC AAG CAA AAA ACT ATT TAC TTG GTC TTC AGA GGA ACT
AAC TCT TTT AGA TCT GCA ATC ACC GAC ATC GTT TTC AAC TTT TCT GAC TAT AAG CCA
GTC AAA GGA GCC AAG GTT CAT GCT GGA TTC TTG TCT TCT TAC GAA CAA GTC GTC AAC
GAC TAC TTT CCA GTC GTT CAG GAG CAG TTG ACC GCA AAC CCA ACA TAC AAG GTT ATT
GTC ACC GGA CAC TCT TTG GGA GGT GCT CAG GCA TTG TTG GCA GGT ATG GAT TTG TAT
CAA AGA GAG CCT AGA TTG TCT CCT AAG AAC TTG TCT ATC TTC ACC GTT GGA GGT CCT
AGA GTC GGA AAC CCT ACC TTC GCA TAC TAT GTT GAG TCT ACA GGA ATC CCA TTC CAG
AGA ACC GTC CAC AAA AGA GAC ATT GTT CCA CAC GTT CCT CCA CAG TCT TTC GGA TTT
TTG CAC CCA GGT GTC GAA TCT TGG ATC AAG TCT GGT ACT TCT AAC GTC CAA ATC TGC
ACC TCT GAG ATC GAA ACC AAG GAT TGC TCT AAT TCT ATC GTT CCT TTC ACA TCT TTG
TTG GAC CAT TTG TCT TAC TTT GAC ATC AAC GAA GGT TCT TGC TTG TAA GCG GCC GC。
Claims (4)
1. a kind of be used to produce the gene with high activity lipase, it is characterised in that the sequence of the gene is as follows：
2. a kind of high activity lipase expressed by gene as described in claim 1, it is characterised in that the high activity fat
The specific enzyme activity of enzyme is 380~430U/mg.
3. high activity lipase according to claim 2, it is characterised in that the specific enzyme activity of the high activity lipase is
409U/mg。
4. high activity lipase according to claim 2, it is characterised in that the high activity lipase is used for whitening face system
Product and baking goodses.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710177364.0A CN106967737A (en) | 2017-03-22 | 2017-03-22 | For producing the gene with high activity lipase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710177364.0A CN106967737A (en) | 2017-03-22 | 2017-03-22 | For producing the gene with high activity lipase |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762277A (en) * | 2015-04-22 | 2015-07-08 | 江南大学 | Method for increasing lipase expression through glycosylation modification as well as mutant enzyme and application thereof |
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2017
- 2017-03-22 CN CN201710177364.0A patent/CN106967737A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104762277A (en) * | 2015-04-22 | 2015-07-08 | 江南大学 | Method for increasing lipase expression through glycosylation modification as well as mutant enzyme and application thereof |
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Application publication date: 20170721 |