CN105420205B - A kind of glutamine transaminage that secretory volume improves - Google Patents
A kind of glutamine transaminage that secretory volume improves Download PDFInfo
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- CN105420205B CN105420205B CN201511026454.7A CN201511026454A CN105420205B CN 105420205 B CN105420205 B CN 105420205B CN 201511026454 A CN201511026454 A CN 201511026454A CN 105420205 B CN105420205 B CN 105420205B
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- C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
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Abstract
The invention discloses a kind of glutamine transaminage, especially a kind of glutamine transaminage of secretory volume raising；The raising of enzyme secretory volume is realized by the short skin of the amalgamation and expression parents of the C-terminal in leader peptide.
Description
Technical field
The present invention relates to a kind of glutamine transaminage, especially a kind of glutamine transaminage of secretory volume raising.
Background technique
Glutamine transaminage (Transglutaminase, EC 2.3.2.13, TGase), is that amide can occur for one kind
Group-transfer reaction, eventually forms the albumen of ε-(γ-glutamyl) lysine covalent bond.Based on above-mentioned catalysis reaction presence,
TGase can promote between various protein molecules, the hydrolysis of intramolecular crosslinking and glutamine residue, to improve protein
Various functional properties, such as emulsibility, dissolubility etc.；Meanwhile TGase can be by such as relying ammonia for some small-molecule substances
Acid etc. introduces protein, to increase the nutritive value of protein.Therefore, TGase is widely applied in the market as having characteristic
Food additives are in the working process of the food such as Flour product, dairy products, a word used in place name baked goods, meat products and aquatic products, market demand
It is extremely huge.
Leader peptide is also known as leader sequence (pro-sequence), is intramolecular chaperone (Intramolecular
Chaperones one kind), it is main by helping albumen to form cramped construction between signal peptide and maturase, to drop
The configuration entropy of low albumen, or false folding and the aggregation of albumen are prevented to instruct protein folding to form correct structure.Leader peptide pair
The folding and secretion of TGase has important influence, is based on this, we finally obtain the C-terminal of parents' small peptide insertion leader peptide
The glutamine transaminage that secretory volume improves.
Summary of the invention
The technical problem to be solved in the present invention is to provide the glutamine transaminages that a kind of secretory volume improves, by leading
The short skin of the amalgamation and expression parents of the C-terminal of peptide realizes the raising of enzyme secretory volume, and sequence is as shown in SEQ ID NO.1 or in SEQ ID
The amino acid sequence that activity remains unchanged after being lacked, being mutated on the basis of NO.1.
The amino acid sequence of the glutamine transaminage can also be as shown in SEQ ID NO.2, shown in SEQ ID NO.3
Or shown in SEQ ID NO.4.
SEQ ID NO.2
ASGGDGEREGSYAETHGLTAEDVKNINALNKRALTAGQPGNSLAELPPSVSALFRAPDAADERVTPPAE
PLNRMPDAYRAYGGRATTVVNNYIRKWQQVYSHRDGIQQQMTEEQREKLSYGCVGVTWVNSGPYPTNKLAFAFFDED
KYKSDLENSRPRPNETQAEFEGRIVKDSFDEGKGFKRARDVASIMNKALDSAHDEGTYIDNLKKGLANKNDALRYED
SRSNFYSALRNTPSFKERDGGNYDPSKMKAVVYSKHFWSGQDQRGSSDKRKYGDPDAFRPDQGTGLVDMSKDRNIPR
SPARPGESWVNFDYGWFGAQTEADADKTIWTHANHYHAPNGGVGPMNVYESKFRNWSAGYADFDRGTYVITFIPKSW
NTAPAEVKQGWS
SEQ ID NO.3
ASGGDGEREGSYAETHGLTAEDVKNINALNKRALTAGQPGNSLAELPPSVSALFRAPDAADERVTPPAE
PLNRMPDAYRAYGGRATTVVNNYIRKWQQVYSHRDGIQQQMTEEQREKLSYGCVGVTWVNSGPYPTNKLAXAFFDED
KYKSDLENSRPRPNETQAEFEGRIVKDSFDEGKGFKRARDVASIMNKALDSAHDEGTYIDNLKKELANKNDALRYED
SRSNFYSALRNTPSFKERDGGNYDPSKMKAVVYSKHFWSGQDQRGSSDKRKYGDPDAFRPDQGTGLVDMSKDRNIPR
SPARPGESWVNFDYGWFGAQTEADADKTIWTHANHYHAPNGGVGPMNVYESKFRNWSAGYADFDRGTYVITFIPKSW
NTAPAEVKQGWS
SEQ ID NO.4
ASGGDGEREGSYAETHGLTAEDVKNINALNKRALTAGQPGNSLAELPPSVSALFRAPDAADERVTPPAE
PLNRMPDAYRAYGGRATTVVNNYIRKWQQVYSHRDGIQQQMTEEQREKLSYGCVGVTWVNSGPYPTNKLAFAFFDED
KYKSDLENSRPRPNETQAEFEGRIVKDSFDEGKGFKRARDVASIMNKALDSAHDEGTYIDNLKKELANKNDALRYED
SRSNFYSALRNTPSFKERDGGNYDPSKMKAVVYSKHFWSGQDQRGSSDKRKYGDPDAFRPDQGTGLVDMSKDRNIPR
SPARPGESWVNFDYGWFGAQTEADADKTIWTHANHYHAPNGGVGPMNVYESKFRNWSAGYADFDRGTXVITFIPKSW
NTAPAEVKQGWS
Technical problem in order to solve the above problem, the present invention the following technical solution is employed example:
1, the acquisition of the short skin gene of first step parents designs corresponding according to the amino acid sequence of the short skin of parents on primer
DNA sequence dna.
2, with S.hygroscopicus pro-TGase expression plasmid pET-22b (+)/pro-TG (Liu S, Zhang D,
Wang M,Cui W,Chen K,Du G,Chen J,Zhou Z.The order of expression is a key
factor in the production of active transglutaminase in Escherichia coli by
Co-expression with its pro-peptide.MicrobCellFact, 2011,10 (112): 1-7.) it is template, into
The full plasmid PCR of row.
3, pET-22b (+)/pro-SAP-TG connected is rotated into JM109, rotates into E.coliBL21 after sequencing is correct
(DE3)。
Culture medium:
Seed culture medium (LB): yeast powder 5g/L, tryptone 10g/L, NaCl 10g/L, (pH 7.0).
Fermentation medium (TB): yeast powder 24g/L, tryptone 12g/L, glycerol 5g/L, K2HPO472mmol/L,
KH2PO417mmol/L, (pH 7.0).
Cultural method:
Seed culture condition: LB culture medium, with 250mL shaking flask culture, liquid amount 10%, cultivation temperature is 37 DEG C, is turned
Speed is 220rpm, incubation time 10h.
Conditions of flask fermentation: TB culture medium is cultivated using 250mL shaking flask, liquid amount 10%, inoculum concentration 3%,
Cultivation temperature is 37 DEG C, and revolving speed 220rpm works as OD600When reaching 2.0, the IPTG of final concentration of 0.4mmol/mL is induced, 20 °
C Fiber differentiation 48h.
Enzyme activity determination method:
Colorimetric method for determining enzyme activity.For Pidolidone-γ-mono- Hydroxylamine HCL as standard curve, α-N-CBZ-GLN-GLY is bottom
Object.The TGase enzyme activity of 1 unit is defined as: under conditions of 37 DEG C, be catalyzed the L- paddy that above-mentioned substrate synthesizes 1 μm of ol per minute
Enzyme amount (U/mL) used in propylhomoserin-γ-mono- Hydroxylamine HCL.Enzyme activity determination condition: 10min is reacted under the conditions of 37 DEG C.
Specific embodiment
Next by the following examples the present invention is furture elucidated, and the experiment side of actual conditions is not specified in the following example
Method is substantially all and is operated according to condition described in common molecular cloning handbook.
The acquisition of 1 parents' small peptide of embodiment
The amino acid sequence of the short skin of parents obtains AEAEAKAKAEAEAKAK, DNA sequence corresponding to the sequence by amino acid
Column design is on primer:
Upstream primer M-F:
GCAGAAGCAGAAGCGAAAGCCAAAGCGGAGGCGGAAGCTAAGGCTAAACTCTTCCGGGCCCCCGACGCT
G
Downstream primer M-R:CGCACTGACGCTCGGCGGCAATTC
Embodiment 2: in the building of the recombinant bacterial strain of leader peptide C-terminal insertion parents' small peptide
Using S.hygroscopicus pro-TGase expression plasmid pET-22b (+)/pro-TG as template, with above-mentioned implementation
Primer carries out full plasmid PCR in example 1.
Embodiment 3: recombinant bacterial strain fermenting and producing TGase
Correct plasmid will be sequenced, be named as Pro-SAP-L-TG, Transformed E .coli BL 21 selects transformant and is inoculated into
In LB liquid medium, 37 DEG C, 12h is cultivated, is transferred in TB culture medium, inoculum concentration 3% works as OD600It is dense eventually when reaching 2.0
The IPTG that degree is 0.4mmol/mL is induced, 20 DEG C of Fiber differentiation 48h.By the fermentation liquid of recombinant bacterial strain after 12000rpm is centrifuged
Fermentation supernatant is collected, is detected, the full outer enzyme activity of discovery transformation bacterial strain increases 54% (table 1) compared to wild TGase, passes through
The exocytosis amount of SDS-PAGE electrophoresis, discovery transformation bacterial strain increases really compared to wild TGase.
The present invention is leading in STG based on leader peptide engineering using high efficient expression of the STG in Escherichia coli as platform
The C-terminal of peptide is inserted into parents' small peptide, has obtained the mutant strain of secretory volume raising, and secretory volume improves 54%, and improved enzyme can
Production cost is reduced, production efficiency is improved.
Embodiment 4: rite-directed mutagenesis improves TGase secretory volume
With site-directed mutagenesis kit, it is G glycine (SEQ ID that the E211 in Pro-TGase, which is done rite-directed mutagenesis,
NO.2), G F140 and Y368 are done into saturation mutation (SEQ ID NO.3-4).Shake flask fermentation according to the method described above surveys its enzyme activity,
To compare its secretory volume.It was found that mutant strain E211G and original WT do not have significant difference, and other mutant strains are in addition to Y368F
There are also outside 31.8% enzyme activity, remaining is without enzyme activity (table 1).
1 glutamine transaminage mutant secernment efficiency of table
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (1)
1. the glutamine transaminage that a kind of secretory volume improves, it is characterised in that amino acid sequence is as shown in SEQ ID NO.1.
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| CN106755000A (en) * | 2017-03-10 | 2017-05-31 | 安徽医学高等专科学校 | The aminotransierase gene of glutamine and targeting sequencing and its secreting, expressing of a kind of optimization |
| CN114634920B (en) * | 2022-03-24 | 2024-02-27 | 江南大学 | Recombinant pichia pastoris for producing human hyaluronidase PH20 and construction method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660570A (en) * | 2012-05-10 | 2012-09-12 | 江南大学 | Method for improving thermal stability of enzyme |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660570A (en) * | 2012-05-10 | 2012-09-12 | 江南大学 | Method for improving thermal stability of enzyme |
Non-Patent Citations (3)
| Title |
|---|
| High level expression of Streptomyces mobaraensis transglutaminase in Corynebacterium glutamicum using a chimeric pro-region from Streptomyces cinnamoneus transglutaminase.;Date M等;《J Biotechnol》;20140610;全文 * |
| 分子改造强化Streptomyces hygroscopicus谷氨酰胺转胺酶催化性能研究;陈康康;《中国博士学位论文全文数据库》;20140115;参见摘要和3.3.3 * |
| 基于分子改造和发酵优化提高透明质酸酶的表达;张娜;《中国硕士学位论文全文数据库》;20151215;摘要，第2.2.3和3.2节 * |
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