CN106432451B - One kind can be used as the protectant albumen of enzyme activity - Google Patents

One kind can be used as the protectant albumen of enzyme activity Download PDF

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CN106432451B
CN106432451B CN201611056582.0A CN201611056582A CN106432451B CN 106432451 B CN106432451 B CN 106432451B CN 201611056582 A CN201611056582 A CN 201611056582A CN 106432451 B CN106432451 B CN 106432451B
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albumen
dehydrin
protection
present
dehydrogenase
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CN106432451A (en
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周鹏
安渊
吕爱敏
刘星辰
樊娜娜
文武武
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Shanghai Jiaotong University
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • 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)
    • C12N9/2462—Lysozyme (3.2.1.17)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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    • C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
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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)
    • C12Y302/01017—Lysozyme (3.2.1.17)

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Abstract

The present invention relates to field of biotechnology, can be used as the protectant albumen of enzyme activity (Dehydrin-S) more particularly to one kind.The present invention provides a kind of isolated albumen, and the albumen includes: amino acid sequence polypeptide as shown in SEQ ID No.1；Amino acid sequence and SEQ ID No.1 have the function of 80% or more homology and have the polypeptide of polypeptide a) limited.Albumen provided by the present invention is a kind of dehydrin protein cloned from Bermuda grass TifwayDehydrin‑SThe albumen that gene is expressed in protokaryon bacterium, this albumen come out by gene engineering expression, which can be used as, protects the protective agent of other albumen and enzyme to use.

Description

One kind can be used as the protectant albumen of enzyme activity
Technical field
The present invention relates to field of biotechnology, can be used as the protectant albumen (Dehydrin- of enzyme activity more particularly to one kind S)。
Background technique
Bermuda grass (Cynodon dactylon) is very important warm season turf, plants for grass family perennial herb Object has flourishing rhizome and stolon, the speed of growth is fast, power of regeneration is strong, fast at level ground, heat-resisting, resistance to trample, quality are very thin, The advantages that color is good is at home and abroad widely used in sports ground, esplanade, park and soil and slope protection lawn etc..It therefore is season type One of Turf characteristic highest, most widely used grass seeds in turfgrass, enjoy the title of warm season turf " main cultivated orages ", great society Meeting, economy and the ecological value.The drought-resistant ability of Bermuda grass is very strong, and it is only the agricultures such as corn, wheat that year evapotranspiration, which is lower than 400mm, The a quarter or so of crop yearly water consumption, the Bermuda grass kinds (as ' Tifway ') of some drought resistings annual rainfall 250mm with Under remain to well grow, therefore be the excellent material on the water-saving lawn of planting.
Dehydrins (dehydrin) belong to II family of LEA-, and being that plant is intracorporal a kind of has high heat stability, hydrophilic The LEA protein (late embryogensis abundant proteins) of property, can be in Embryos Development of Plant later period and inverse Great expression under border, is widely present in plant kingdom.It can be expressed in the case where plant is in the adverse environmental factors such as arid, in adverse circumstance following table Power and the plant stress-resistance ability reached has close ties, and the expression quantity in resistant plant is higher than the plant sensitive to adverse circumstance. It has thus been shown that the drought resisting of it and plant has close relationship.
Bermuda grass ' Tifway ' is one of kind the most drought-enduring in Bermuda grass.Pass through protein immunoblot and gene expression Analysis, determines that Dehydrins are drought-enduring closely related with ' Tifway '.But currently, the drought resisting mechanism of dehydrin protein is unclear.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide one kind, to can be used as enzyme activity protectant Albumen, for solving the problems of the prior art.
In order to achieve the above objects and other related objects, the present invention provides a kind of isolated albumen, and the albumen includes:
A) amino acid sequence polypeptide as shown in SEQ ID No.1；
B) amino acid sequence and SEQ ID No.1 have the function of 80% or more homology and have polypeptide a) limited Polypeptide；
Specifically, it is described b) in polypeptide refer specifically to: amino acid sequence polypeptide as shown in SEQ ID No.1 is by taking Generation, missing or addition it is one or more (specifically can be 1-50, be also possible to 1-30, be also possible to 1-20, can also To be 1-10, it is also possible to 1-5, is also possible to 1-3) obtained from amino acid, or in the end N- and/or the end C- Addition one or more (it specifically can be 1-50, be also possible to 1-30, be also possible to 1-20, be also possible to 1-10, It is also possible to 1-5, is also possible to 1-3) obtained from amino acid, and have amino acid sequence as shown in SEQ ID No.1 Polypeptide function polypeptide.It is described b) in polypeptide amino acid sequence can with SEQ ID NO.1 have 80% or more homology, Can more specifically have 85% or more homology, can more specifically have 90% or more homology, can more specifically have 93% or more same Source property can more specifically have 95% or more homology, can more specifically have 97% or more homology, further can specifically have 99% or more homology.
Specifically, in the polypeptide, it is one or more (specifically to can be 1-10, be also possible to 1-5, be also possible to 1-3) amino acid replaced by conservative.It is specified that the conservative replacement typically refers to another similar amino acid substitution of property Amino acid, for example, be considered conservative replacement (property is similar) example include but is not limited to: a) alanine, silk Propylhomoserin and threonine；B) glutamic acid and aspartic acid；C) asparagine and glutamine；D) arginine and lysine；E) different bright Propylhomoserin, leucine, methionine and；F) phenylalanine, tyrosine and tryptophan.
Specifically, the albumen is isolated albumen, the biological activity protein more specifically separated.
Include the present invention provides the albumen with drought stress defencive function, in the albumen Bermuda grass ' Tifway ' de- Water element Dehydrin-S gene, and added before terminator the peptide fragment of 13 amino acid residues.The albumen is by such as SEQ The composition of amino acid sequence shown in ID No.1, the albumen have the function of protecting other albumen and enzyme activity.
MEHQGQYGHGTTGRVDEYGNPVAGHGTGTGEMGMGTHGTTGTGGMGMGTHGTGTGAGMGGQFQPTREE HKTGGILHRSGSSSSSSSEDDGMGGRRKKGIKDKIKEKLPGGHKDDQQQMGTGTYGQQGHTGMTGSTGTGTGTYGH ETGEKKGIMDKIKERLPGQHKLAAALEHHHHHH(SEQ ID No.1)
Second aspect of the present invention provides a kind of isolated DNA molecular, encoding said proteins.
Specifically, the isolated DNA molecular sequence is as shown in SEQ ID No.2.
ATGGAGCACCAGGGACAGTACGGCCACGGCACCACGGGCCGCGTCGACGAGTACGGTAACCCGGTTGC CGGACACGGCACCGGAACAGGAGAAATGGGCATGGGAACCCACGGCACCACCGGTACCGGCGGCATGGGCATGGGA ACGCACGGCACCGGTACCGGCGCCGGCATGGGCGGGCAGTTCCAGCCCACCAGGGAGGAGCACAAGACCGGAGGCA TCCTGCACCGCTCCGGCAGCTCCAGCTCCAGCTCGTCTGAGGATGATGGCATGGGTGGCCGGAGGAAGAAGGGAAT CAAGGACAAGATCAAGGAAAAGCTACCCGGCGGCCACAAGGACGACCAGCAGCAGATGGGTACCGGCACCTACGGT CAGCAAGGACACACCGGCATGACCGGCTCCACCGGAACTGGCACCGGCACCTACGGCCACGAGACCGGCGAGAAGA AGGGCATCATGGACAAGATCAAGGAGAGGCTCCCCGGCCAGCACAAGCTTGCGGCCGCACTCGAGCACCACCACCA CCACCACTGA(SEQ ID No.2)
Third aspect present invention provides a kind of construct, contains the isolated DNA molecular.
Specifically, the construct by the isolated DNA molecular be inserted into expression vector multiple cloning sites building and At.
Preferably, the expression vector be selected from pET systemic vectors, pETBlue TM serial carrier, pGEX serial carrier, One of pBAD serial carrier, pMAL-2c carrier, pQE-9 etc. or a variety of combinations.
Fourth aspect present invention provides a kind of host cell, and the cell includes being integrated in the construct or genome The DNA molecular of external source.
Specifically, the host cell is prokaryotic cell.
More specifically, the host cell is Escherichia coli.
More specifically, the Escherichia coli are the bacterial strains such as Rosetta series, BL21 series, Origami series.
Fifth aspect present invention provides the preparation method of the albumen, includes the following steps: be suitble to the expression albumen Under conditions of, it cultivates the host cell and is isolated and purified with the albumen to give expression to the albumen.
Sixth aspect present invention provides purposes of the albumen in enzymatic activity protection, or the use in enzymatic protective reagent preparation On the way.
Specifically, the enzyme is selected from lactic dehydrogenase, malic dehydrogenase, alcohol dehydrogenase, citrate synthetase, cell One of wall lyase, luciferase etc. or a variety of combinations.
Specifically, the protection is heat-resisting and/or cold-resistant protection.
The heat-resisting protective refer specifically to can effective protection albumen or enzyme in the item for being higher than its optimum temperature or higher temperature Activity under part.
The cold-resistant protection refer specifically to can effective protection albumen or enzyme in the item lower than its optimum temperature or lower temperature Activity under part.
A kind of enzymatic protective reagent, including the isolated albumen.
As described above, albumen provided by the present invention is a kind of dehydrin protein cloned from Bermuda grass Tifway The albumen that Dehydrin-S gene is expressed in protokaryon bacterium.This albumen come out by gene engineering expression can be used as protection The protective agent of other albumen and enzyme uses, the high temperature and/or the activity under low temperature that the albumen is capable of effective protection albumen and enzyme, With good industrialization prospect.
Detailed description of the invention
Fig. 1 is the protein amino acid sequence overall length of the Dehydrin-S gene order and coding cloned in Bermuda grass Tifway (the wherein conserved sequence area that Y, S, K1, K2 are Dehydrin-S albumen)；
Fig. 2 is the structure chart of prokaryotic expression carrier pET-21a；
Fig. 3 is that improved prokaryotic expression carrier pET-21aDSPCR identifies electrophoretogram；
Fig. 4 is the SDS-PEG electrophoresis of IPTG induction expression protein after recombinant plasmid turns Escherichia coli Rosetta (DE3) Figure；
Fig. 5 is the SDS- for the Dehydrin-S gene protein that Rosetta (DE3) thick leach protein obtains after ni-sepharose purification PEG electrophoretogram is control with the Dehydrin-L gene protein obtained after ni-sepharose purification；
Fig. 6 still has greater activity through high-temperature process 25min for Dehydrin-S protein protection alcohol dehydrogenase (ADH)；
Fig. 7 is the Dehydrin-S protein protection lactic dehydrogenase (LDH) of different component ratio through high-temperature process, and LDH is living The variation of property；
Fig. 8 is Dehydrin-S protein protection lactic dehydrogenase (LDH) through low-temperature treatment, the variation of LDH activity.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；The series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998； METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
The clone of Bermuda grass ' Tifway ' Dehydrin-S gene:
1. the acquisition of vegetable material
Bermuda grass ' Tifway ' leaf tissue is taken, for extracting RNA；
The extracting of 2.RNA
With " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (Trizol:Invitrogen), first is used Aldehyde is denaturalized the integrality of gel electrophoresis identification RNA, then in spectrophotometer (Thermo Scientific NANODROP The purity and concentration of RNA are measured on 1000Spectrophotometer)；
3. the full-length clone of gene
Kit (Primer ScriptTM 1st cDNA Synthesis is efficiently synthesized with the first chain of cDNA of Takara Kit).By specification method obtains cDNA, and -20 DEG C save backup.With primer EF-DHN and HR-DHN, using cDNA as template, PCR Amplification clone's Dehydrin-S gene, PCR reaction cycle condition are as follows: enter circular response, 94 DEG C of denaturation after 94 DEG C of denaturation 2min 1min, 54 DEG C of annealing 1min, 72 DEG C of extension 1min (circulation 30 times), 72 DEG C of extension 10min.
EF-DHN:5’-CCGGAATTCATGGAGCACCAGGGACAGTAC-3’(SEQ ID No.3)
HR-DHN:5’-CCCAAGCTTGTGCTGGCCGGGGAGCTT-3’(SEQ ID No.4)
Embodiment 2
Expression vector establishment:
It is template with plasmid pET-21a (purchased from excellent precious biology, Hunan China), by the Dehydrin-S base of embodiment 1 (3) After being handled because of sequence with HindIII and EcoRI digestion, it is connected between HindIII the and EcoRI restriction enzyme site of pET-21a, it will Dehydrin-S gene connect to form complete ORF expression cassette with His Tag.The recombinant plasmid of acquisition is named as pET-21aDS. The correctness of recombinant plasmid is verified by digestion and PCR, verification result shows construction of recombinant plasmid success (such as Fig. 3 institute Show).
Embodiment 3
Prokaryotic expression, purifying and the Western blot identification of Dehydrin-S albumen:
1. prokaryotic expression and identification
Prokaryotic expression carrier pET-21aDS containing Dehydrin-S gene is transferred in Escherichia coli Rosetta (DE3), Bacterium solution PCR screening positive clone.Rosetta (DE3) positive colony of the picking containing carrier (contains Ka Na and chloramphenicol antibiosis to 5mL Element) LB culture solution in, 37 DEG C, 220rpm concussion overnight.Culture solution is connected in the LB of 10mL in the ratio of 1:100.37 DEG C, 220rpm cultivates OD600=0.4~0.6 (about 2.5h).It is added IPTG inducer (final concentration 1mM), 26 DEG C, 220rpm oscillation training Support 4-6h.1mL bacterium solution is taken to be put into the EP pipe of 1.5mL, 12000rpm is centrifuged 2min, collects thallus.Supernatant is removed, 20ul is added PBS and 20ul 2* albumen sample-loading buffer mixes well, by 100 DEG C of heating 5min of sample solution of mixing, cracking release albumen； It is centrifuged 10min, the expression of Dehydrin-S albumen is identified in SDS-PEG gel electrophoresis, and qualification result is as shown in figure 4, in figure It is Dehydrin-S protein expression band at arrow meaning.
2. the purifying of albumen
The albumen of His label is carried out with ProteinIso Ni-NTA Resin (purchased from full formula gold biology, BeiJing, China) It isolates and purifies, step includes dress column, balance, loading, washing, elution.Balance: it is balanced with the equilibrating buffer of 5-10 times of volume Low concentration imidazoles (10-20mM) is added in equilibrium liquid in chromatographic column, improves specific binding；Loading: Sample Buffer solution is as far as possible It is consistent with equilibrium liquid, and handled through 0.45um filter membrane；
Washing: chromatographic column is washed with the equilibrium liquid of 5-10 times of volume；Elution: purpose egg is eluted with the imidazoles of various concentration It is white, collect eluent.By above-mentioned gained albumen ultra-filtration centrifuge tube deionization, BCA method measures protein concentration, SDS-PAGE detection Albumen after purification, as a result as shown in figure 5, being respectively Dehydrin-L, Dehydrin-S protein expression item at arrow meaning in figure Band, wherein the expression of Dehydrin-L is referring to Chinese patent application 201610289163.5.- 80 DEG C save backup.
Embodiment 4
Protection of the Dehydrin-S albumen to alcohol dehydrogenase (ADH) high-temperature process:
ADH((EC1.1.1.1 comes from saccharomyces cerevisiae, Sigma-Aldrich, USA), is dissolved in 10mM phosphate buffer solution (pH7.4) in, concentration 10mg/mL, spare as mother liquor, working solution concentration is 0.5mg/mL.Purifying gained egg in embodiment 3 White (DS), BSA (Sigma-Aldrich, USA) are configured to the test buffer that concentration is 0.5mg/ml.Enzyme reaction solution includes 1.25mM ethyl alcohol and 2mM NAD+(100mM NaH2PO4, pH 7.5).Isometric ADH and DS or BSA is mixed into trip temperature Processing.
High-temperature process experiment, by ADH and mixed liquid of protein in 50 DEG C of water-baths 25min, take 10ul to mix after ice bath 20min Liquid is added in 200ul reaction buffer solution.The light absorption value of 340nm in 6min is measured at 25 DEG C with microplate reader, testing result is such as Shown in Fig. 6, the results show that Dehydrin-S protein protection alcohol dehydrogenase still has greater activity through high-temperature process 25min.
Embodiment 5
Protection of the Dehydrin-S albumen to lactic dehydrogenase (LDH) high-temperature process:
According to the step in embodiment 4 by LDH and albumen respectively with 1:1,1:2；The concentration ratio of 1:5 mixes, wherein The concentration of LDH is 10 μ g/mL, and mixed liquor is placed in 50 DEG C, samples after 25 minutes, the light absorption value of 340nm is measured, as a result such as Fig. 7 institute Show, the results show that Dehydrin-S protein protection lactic dehydrogenase (LDH) is after high-temperature process, LDH is mixed with albumen with 1:2, It is best to the protection of LDH activity.
Embodiment 6
Protection of the Dehydrin-S albumen to lactic dehydrogenase (LDH) low-temperature treatment:
LDH and albumen mixes respectively with the ratio of 1:2 to (wherein, test protein is according to the step in embodiment 4 The concentration ratio of BSA and DS, test protein and LDH are 2:1；LDH concentration is 10ug/ml), mixed liquor is placed in -20 DEG C, It is sampled after 0,10,20,30 minute, measures the light absorption value of 340nm, as a result as shown in figure 8, the results show that Dehydrin- S protein protects lactic dehydrogenase (LDH) after low-temperature treatment, still has greater activity.
Embodiment 7
Dehydrin-S albumen is to malic dehydrogenase, alcohol dehydrogenase, citrate synthetase, Lysozyme, glimmering The protection of light element enzyme high-temperature process:
Malic dehydrogenase, alcohol dehydrogenase, citrate synthetase, Lysozyme, luciferase are dissolved respectively In 10mM phosphate buffer solution (pH7.4), mother liquid concentration is 10mg/mL.Purifying gained albumen (DS), BSA in embodiment 3 (Sigma-Aldrich, USA) is configured to the test buffer that working solution concentration is 0.5mg/mL, wherein the concentration of BSA and DS It is identical as the concentration of enzyme.It includes 2mM NADH, 10mM Sodium Pyruvate, 10mM phosphate buffer solution that enzyme, which tests buffer solution, (pH7.4)。
High-temperature process experiment, by the solution equal proportion mixed liquor of above-mentioned enzyme and albumen in 50 DEG C of water-baths 25min, ice bath 10ul mixed liquor is taken to be added in 200ul reaction buffer solution after 20min.340nm in 6min is measured at 25 DEG C with microplate reader Light absorption value, the results show that the malic dehydrogenase of Dehydrin-S protein protection, alcohol dehydrogenase, citrate synthetase, cell Wall lyase, luciferase still have greater activity after high-temperature process 25min.
Embodiment 8
Dehydrin-S albumen is to malic dehydrogenase, alcohol dehydrogenase, citrate synthetase, Lysozyme, glimmering The protection of light element enzyme freezing processing:
It will be respectively by malic dehydrogenase, alcohol dehydrogenase, citrate synthetase, cell according to the step in embodiment 6 Wall lyase, luciferase and mixed liquid of protein are placed in -20 DEG C, respectively at 0,30,60,90min sampling, measure the suction of 340nm Light value, the results show that the malic dehydrogenase of Dehydrin-S protein protection, alcohol dehydrogenase, citrate synthetase, cell wall Lyase, luciferase still have greater activity after cryogenic freezing is handled.
In conclusion the present invention effectively overcomes various shortcoming in the prior art and has high industrial utilization value.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as At all equivalent modifications or change, should be covered by the claims of the present invention.
SEQUENCE LISTING
<110>Shanghai Communications University
<120>one kind can be used as the protectant albumen of enzyme activity
<130> PCN
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 177
<212> PRT
<213> Cynodon dactylon
<400> 1
Met Glu His Gln Gly Gln Tyr Gly His Gly Thr Thr Gly Arg Val Asp
1 5 10 15
Glu Tyr Gly Asn Pro Val Ala Gly His Gly Thr Gly Thr Gly Glu Met
20 25 30
Gly Met Gly Thr His Gly Thr Thr Gly Thr Gly Gly Met Gly Met Gly
35 40 45
Thr His Gly Thr Gly Thr Gly Ala Gly Met Gly Gly Gln Phe Gln Pro
50 55 60
Thr Arg Glu Glu His Lys Thr Gly Gly Ile Leu His Arg Ser Gly Ser
65 70 75 80
Ser Ser Ser Ser Ser Ser Glu Asp Asp Gly Met Gly Gly Arg Arg Lys
85 90 95
Lys Gly Ile Lys Asp Lys Ile Lys Glu Lys Leu Pro Gly Gly His Lys
100 105 110
Asp Asp Gln Gln Gln Met Gly Thr Gly Thr Tyr Gly Gln Gln Gly His
115 120 125
Thr Gly Met Thr Gly Ser Thr Gly Thr Gly Thr Gly Thr Tyr Gly His
130 135 140
Glu Thr Gly Glu Lys Lys Gly Ile Met Asp Lys Ile Lys Glu Arg Leu
145 150 155 160
Pro Gly Gln His Lys Leu Ala Ala Ala Leu Glu His His His His His
165 170 175
His
<210> 2
<211> 534
<212> DNA
<213> Cynodon dactylon
<400> 2
atggagcacc agggacagta cggccacggc accacgggcc gcgtcgacga gtacggtaac 60
ccggttgccg gacacggcac cggaacagga gaaatgggca tgggaaccca cggcaccacc 120
ggtaccggcg gcatgggcat gggaacgcac ggcaccggta ccggcgccgg catgggcggg 180
cagttccagc ccaccaggga ggagcacaag accggaggca tcctgcaccg ctccggcagc 240
tccagctcca gctcgtctga ggatgatggc atgggtggcc ggaggaagaa gggaatcaag 300
gacaagatca aggaaaagct acccggcggc cacaaggacg accagcagca gatgggtacc 360
ggcacctacg gtcagcaagg acacaccggc atgaccggct ccaccggaac tggcaccggc 420
acctacggcc acgagaccgg cgagaagaag ggcatcatgg acaagatcaa ggagaggctc 480
cccggccagc acaagcttgc ggccgcactc gagcaccacc accaccacca ctga 534
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 3
ccggaattca tggagcacca gggacagtac 30
<210> 4
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223>primer
<400> 4
cccaagcttg tgctggccgg ggagctt 27

Claims (8)

1. a kind of isolated albumen, the albumen are as follows:
Amino acid sequence polypeptide as shown in SEQ ID No.1.
2. a kind of isolated DNA molecular, encodes albumen as described in claim 1.
3. a kind of construct contains the DNA molecular separated as claimed in claim 2.
4. a kind of host cell, the cell includes being integrated with external source in construct as claimed in claim 3 or genome DNA molecular as claimed in claim 2.
5. the preparation method of albumen as described in claim 1 includes the following steps: in the condition for being suitble to the expression albumen Under, host cell as claimed in claim 4 is cultivated, to give expression to the albumen, is isolated and purified with the albumen.
6. purposes of the albumen as described in claim 1 in enzymatic activity protection, the protection is heat-resisting and/or cold-resistant protection.
7. purposes as claimed in claim 6, which is characterized in that the enzyme is selected from lactic dehydrogenase, malic dehydrogenase, ethyl alcohol One of dehydrogenase, citrate synthetase, Lysozyme and luciferase or a variety of combinations.
8. a kind of enzymatic protective reagent, including the albumen separated as described in claim 1.
CN201611056582.0A 2016-11-25 2016-11-25 One kind can be used as the protectant albumen of enzyme activity Active CN106432451B (en)

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US20230287059A1 (en) * 2020-07-20 2023-09-14 Vanderbilt University A molecular strategy to protect against desiccation

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105001313A (en) * 2015-04-15 2015-10-28 上海交通大学 'C299' dehydrin protein Dehydrin-S of cynodon dactylon and encoding gene and probe thereof
CN105017394A (en) * 2015-04-15 2015-11-04 上海交通大学 Cynodon dactylon 'Tifway' dehydrin protein Dehydrin-S as well as coding gene and probe thereof
CN105037514A (en) * 2015-04-15 2015-11-11 上海交通大学 Bermuda grass 'Tifway' dehydrin-L, encoding gene and probe thereof
CN105037515A (en) * 2015-04-15 2015-11-11 上海交通大学 Bermuda grass 'C299' dehydrin-L, encoding gene and probe thereof
CN106084020A (en) * 2016-05-04 2016-11-09 上海交通大学 One can be used as enzyme protectant albumen alive

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105001313A (en) * 2015-04-15 2015-10-28 上海交通大学 'C299' dehydrin protein Dehydrin-S of cynodon dactylon and encoding gene and probe thereof
CN105017394A (en) * 2015-04-15 2015-11-04 上海交通大学 Cynodon dactylon 'Tifway' dehydrin protein Dehydrin-S as well as coding gene and probe thereof
CN105037514A (en) * 2015-04-15 2015-11-11 上海交通大学 Bermuda grass 'Tifway' dehydrin-L, encoding gene and probe thereof
CN105037515A (en) * 2015-04-15 2015-11-11 上海交通大学 Bermuda grass 'C299' dehydrin-L, encoding gene and probe thereof
CN106084020A (en) * 2016-05-04 2016-11-09 上海交通大学 One can be used as enzyme protectant albumen alive

Non-Patent Citations (1)

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Title
脱水素研究进展;孙歆等;《武汉植物学研究》;CNKI;20050625;第23卷(第3期);第299-303页

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