CN106884030A - The method for improving the fermentation yield of the fusion protein of containing cellulose binding domain - Google Patents
The method for improving the fermentation yield of the fusion protein of containing cellulose binding domain Download PDFInfo
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- CN106884030A CN106884030A CN201510946981.3A CN201510946981A CN106884030A CN 106884030 A CN106884030 A CN 106884030A CN 201510946981 A CN201510946981 A CN 201510946981A CN 106884030 A CN106884030 A CN 106884030A
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- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
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Abstract
Fermentation yield the present invention relates to improve the fusion protein of containing cellulose binding domain, ferment or prepare the fusion protein of containing cellulose binding domain, or the method for the fusion protein of separation and/or purifying containing cellulose binding domain, methods described includes the microorganism of expression vector of the fermentation containing the fusion protein that can express the containing cellulose binding domain, wherein, in fermentation process, methods described adsorbs the fusion protein in the zymotic fluid for thalline separate using cellulose, the step of being fermented with the zymotic fluid and/or separated thalline again after separation and fermentation liquid and cellulose.Present invention additionally comprises application of the cellulose in the fusion protein that fermentation prepares containing cellulose binding domain.Using the inventive method, the amount that can make the fusion protein obtained by fermentation is 2 times of normal fermentation gained Tot Prot.
Description
Technical field
The invention belongs to cellulose binding domain fusion enzyme field, it is related to improve the fusion protein of containing cellulose binding domain
Fermentation yield method.
Background technology
Cellulose binding domain (Cellulose binding domain, CBD) is to be present in fiber-like raw material
Relative molecular mass in enzyme (such as cellulase) is 0.4 × 104~2.0 × 104The fragment not waited, it can
Insertion and the separately crystal region of cellulose, by the hydrophobic plane of aromatic amino acid (hydrophobic effect) and virtue
The accumulation force of fragrant ring and glucose ring be adsorbed onto cellulose chain surface (Sugimoto N, Igarashi K,
Samejima M, Cellulose affinity purification of fusion proteins tagged
with fungal family 1 cellulose-binding domain.Protein Expression and
Purification, 2012；82:290-6〕.So the region is often used to cellulase (Naohisa
Sugimoto, Kiyohiko Igarashi, Masahiro Samejima, Cellulose affinity
purification of fusion proteins tagged with fungal family 1
Cellulose-binding domain, Protein Expression and Purification, 2012,
82,290-296；M.A.Lemos、J.A.Teixeira、M.R.M.Domingues、M.Mota、
F.M.Gama, The enhancement of the cellulolytic activity of
cellobiohydrolase I and endoglucanase by the addition of cellulose
Binding domains derived from Trichoderma reesei, Enzyme and Microbial
Technology, 2003,32:35-40；Markus Linder、Irma Salovuori、Laura Ruohonen
With Tuula T.Teeri, Characterization of a Double Cellulose-binding Domain.
The Journal of Biological Chemistry, 1996,271, No.35,21268-21272；
Jantaporn Thongekkaew, Hiroko Ikeda, Kazuo Masaki, Haruyuki Iefuji,
Fusion of cellulose binding domain from Trichoderma reesei CBHI to
Cryptococcus sp.S-2cellulase enhances its binding affinity and its
Cellulolytic activity to insoluble cellulosic substrates, Enzyme and
Microbial Technology, 2013,52:241-246), lipase (Ahn J, Choi E, Lee
H, Hwang S, Kim C, Jang H etc., Enhanced secretion of Bacillus
stearothermophilus L1 lipase in Saccharomyces cerevisiae by
Translational fusion to cellulose-binding domain, Appliedmicrobiology
And biotechnology, 2004,64:833-839；Thongekkaew J, Ikeda H, Iefuji H,
Increases thermal stability and cellulose-binding capacity of
Cryptococcus sp.S-2lipase by fusion of cellulose binding domain
Derived from Trichoderma reesei, Biochemical and Biophysical Research
Communications, 2012,420:183-187), chaperone (Ram ó n-Luing LA, Cruz-Migoni
A, Ru í z-Medrano R, Xoconostle-C á zares B, Ortega-Lopez J, One-step
purification and immobilization in cellulose of the GroEL apical domain
Fused to a carbohydrate-binding module and its use in protein refolding,
Biotechnology letters, 2006,28:301-7), fructosidase (Santiago-Hern á ndez
J、Vásquez-Bahena J、Cal ixto-Romo M、Xoconostle-Cázares G、Ortega-López
J, Ruiz-Medrano R etc., Direct immobilization of a recombinant invertase
to Avicel by E.coli overexpression of a fusion protein containing the
extracellular invertase from Zymomonas mobilis and the
Carbohydrate-binding domain CBDCex from Cellulomonas fimi, Enzyme and
Microbial technology, 2006,40:172-6), organic phosphoric acid enzyme (Richins RD,
Mulchandani A, Chen W, Expression, immobilization, and enzymatic
characterization of cellulose-binding domain-organophosphorus
Hydrolase fusion enzymes, Biotechnology and bioengineering, 2000,
69:591-6) etc. fusion so that these enzymes or albumen obtain some new characteristics or can be immobilized.
So, the yield for improving CBD fusion enzyme particularly CBD lipase is also one problem of the thing followed.
Improving the usual channel of CBD fusion enzymes has many kinds, such as promoter optimization, screen mutation equimolecular is biological
Section is learned to do, changes fermentation temperature, medium component adjusts pH or adds the modes such as some metal ions.
There is the shortcomings of cycle is long, and universality is not high in these modes, and the present invention passes through scheme optimization using absorbent cotton,
In certain hour point, the mesh for improving CBD lipase fermentation total amounts has been reached by CBD lipase adsorptions out
's.
The content of the invention
First aspect present invention provides a kind of side of the fermentation yield of the fusion protein for improving containing cellulose binding domain
Method, methods described adsorbs fusion protein, separation and fermentation in the zymotic fluid for thalline separate using cellulose
The step of being fermented with the zymotic fluid and/or separated thalline again after liquid and cellulose.
Second aspect present invention provides a kind of fermentation process of the fusion protein of containing cellulose binding domain, methods described
After the fusion protein in the zymotic fluid that cellulose absorption with thalline separate, separation and fermentation liquid and cellulose
The step of being fermented with the zymotic fluid and/or separated thalline again.
Third aspect present invention provides a kind of preparation method of the fusion protein of containing cellulose binding domain, methods described
Microorganism including expression vector of the fermentation containing the fusion protein that can express the containing cellulose binding domain, wherein,
Methods described using the fusion protein in the cellulose zymotic fluid that with thalline separate of absorption, separation and fermentation liquid with
The step of being fermented with the zymotic fluid and/or separated thalline again after cellulose.
Fourth aspect present invention provides a kind of side of the fusion protein for separating and/or purifying containing cellulose binding domain
Method, methods described includes expression vector of the fermentation containing the fusion protein that can express the containing cellulose binding domain
The step of the step of microorganism and the purifying fusion protein, it is characterised in that methods described is also using fiber
The fermentation is used again after fusion protein, separation and fermentation liquid and cellulose in the zymotic fluid that element absorption with thalline separate
The step of liquid and/or separated thalline are fermented.
In a specific embodiment of above-mentioned aspect of the invention, methods described includes：
(1) adsorb
Induced expression for a period of time after, by thalline and separation of fermentative broth, obtain separate thalline and separate
Zymotic fluid, makes zymotic fluid be contacted with cellulose, to allow fusion protein therein to be adsorbed on cellulose,
And zymotic fluid is separated with the cellulose for having adsorbed fusion protein；
(2) continue to ferment
The zymotic fluid for by the thalline of foregoing separation and/or with cellulose separate is used to ferment；With it is optional
(3) the step of repeating foregoing (1) and (2).
In a specific embodiment of above-mentioned aspect of the invention, in step (2), using selected from fresh bacterium
Body, the thalline of the thalline of the separation or its mixture and selected from fresh fermentation medium, it is described with fibre
The fermentation medium of the mixture of the zymotic fluid or both that dimension element is separate carries out continuation fermentation, and condition is, thalline
It is at least a kind of comprising thalline or zymotic fluid from step (1) with fermentation medium.
In a specific embodiment of above-mentioned aspect of the invention, the fermentation is carried out with fed-batch mode, will be flowed out
Zymotic fluid contacted with cellulose after again by it is anti-plus in the way of flow plus culture, wherein, will be with cellulose point
Zymotic fluid after individually instead plus, or after mix with fresh fermentation medium instead plus, or trained with fresh fermentation
Foster base flows add respectively.
In a specific embodiment of above-mentioned aspect of the invention, methods described includes：Induced expression is for a period of time
Afterwards, zymotic fluid is separated with thalline, separate zymotic fluid is contacted into a period of time with cellulose, then make cellulose
With separation of fermentative broth, the zymotic fluid for making separation again afterwards mixes with the thalline for being previously separated out, continues to ferment.
In a specific embodiment of above-mentioned aspect of the invention, fermented with fed-batch mode, the hair that will be flowed out
Zymotic fluid with anti-add mode flow plus culture again after being contacted with cellulose.
In a specific embodiment of above-mentioned aspect of the invention, the zymotic fluid that with cellulose will separate individually instead adds,
Or after mixing with fresh medium instead plus, or by its with fresh medium respectively flow plus.
In a specific embodiment of above-mentioned aspect of the invention, methods described includes the hair that with cellulose will separate
The step of zymotic fluid is mixed into row fermentation with new fresh thalli.
In a specific embodiment of above-mentioned aspect of the invention, methods described includes being added to separated thalline
The step of being fermented in fresh culture.
In a specific embodiment of above-mentioned aspect of the invention, methods described includes the hair that with cellulose will separate
Zymotic fluid mix with fresh fermentation broth after the step of with new fresh thalli or separated thalline mixed fermentation.
In a specific embodiment of above-mentioned aspect of the invention, methods described includes：
(a) induced expression for a period of time after, by zymotic fluid with containing merging for the containing cellulose binding domain can be expressed
The thalline of the expression vector of albumen is separated；
B () makes the zymotic fluid that step (a) is obtained that a period of time is contacted with cellulose, then make cellulose with hair
Zymotic fluid is separated；With
C () mixes the thalline that step (b) gained zymotic fluid is obtained with step (a), continue to ferment.
Fifth aspect present invention provides cellulose in the fusion protein that fermentation prepares containing cellulose binding domain
Using, wherein, the application includes making the cellulose be connect with the zymotic fluid separated during the fermentation
Tactile step.
In a specific embodiment of above-mentioned aspect of the invention, the fusion protein is cellulase, fat
The fusion protein that enzyme, chaperone, fructosidase or organic phosphoric acid enzyme and cellulose binding domain are formed.
In a specific embodiment of above-mentioned aspect of the invention, it is fine that the cellulose is selected from absorbent cotton, crystallite
Dimension element and cotton.
In a specific embodiment of above-mentioned aspect of the invention, the cellulose binding domain is in cellulase
Relative molecular mass 0.4 × 104~2.0 × 104Between fragment.
In a specific embodiment of above-mentioned aspect of the invention, the coded sequence of the cellulose binding domain contains
SEQ ID NO:1 the 16th~327, or such as SEQ ID NO:Shown in 10.
In a specific embodiment of above-mentioned aspect of the invention, the lipase is to dredge the thermophilic hyphomycete of cotton like
The lipase mutant (TTL) of (Thermomyces lanuginosus), rhizomucor miehei (Rhizomucor
Miehei lipase (RML)), the lipase B of antarctic candida (Candida antarctica)
(CALB)。
In a specific embodiment of above-mentioned aspect of the invention, the amino acid sequence such as SEQ ID of the TTL
NO:Shown in 4.
In a specific embodiment of above-mentioned aspect of the invention, the amino acid sequence of the CALB is by SEQ ID
NO:64-1014 of 9 is nucleotide sequence coded.
In a specific embodiment of above-mentioned aspect of the invention, the amino acid sequence of the RML is by SEQ ID
NO:11 codings.
In a specific embodiment of above-mentioned aspect of the invention, methods described includes, during one section of induced expression
Between after, make thalline and separation of fermentative broth, described a period of time is 15~30 hours, and preferably 18~28 is small
When, more preferably 20~24 hours.
In a specific embodiment of above-mentioned aspect of the invention, cellulose is with the time of contact of zymotic fluid
10~60 minutes.
In a specific embodiment of above-mentioned aspect of the invention, cellulose is with the Contact Temperature of zymotic fluid
4~15 DEG C.
In a specific embodiment of above-mentioned aspect of the invention, before fermentation ends, it is repeated several times described by bacterium
Body is contacted with zymotic fluid with separation of fermentative broth, by cellulose, isolates cellulose and makes zymotic fluid and thalline weight
The step of new mixing and continuation are fermented, time interval is 15~30 hours, preferably 20~26 hours.
In a specific embodiment of above-mentioned aspect of the invention, every time the consumption of contact cellulose for 0.1~
2.0g celluloses/liter zymotic fluid.
In a specific embodiment of above-mentioned aspect of the invention, methods described also includes from having adsorbed fusion egg
The step of fusion protein being isolated on white cellulose.
It is described that fusion protein is isolated from cellulose in a specific embodiment of above-mentioned aspect of the invention
The step of including using ethylene glycol or cellobiose eluting fibers element on adsorb fusion protein the step of.
In a specific embodiment of above-mentioned aspect of the invention, the cellulose of fusion protein described can be adsorbed with
Directly used as immobilization product.
In a specific embodiment of above-mentioned aspect of the invention, the fusion protein is lipase and cellulose
The fusion protein of binding domain, methods described uses fusion protein described in yeast fermenting and producing.
In one embodiment of the invention, the fusion protein is CALB-CBD, the cellulose
It is microcrystalline cellulose.
In one embodiment of the invention, the fusion protein is RML-CBD, the cellulose
It is cotton.
Brief description of the drawings
Fig. 1 display cellulose binding domains merge enzyme (TTL-CBD) expression.In figure, swimming lane 1~3 is respectively
Fusion enzyme tri- transformant Fermented Condensed liquid of TTL-CBD.
Fig. 2 display absorbent cotton is directly added into culture medium to merging the influence of expression of enzymes.In (a), swimming lane 1：
Absorbent cotton denaturing liquid (the Tris-Cl buffer solutions containing 10M urea are denatured)；Swimming lane 2：After taking out absorbent cotton
Zymotic fluid；Swimming lane 3：Control fermentation liquid (undressed normal fermentation liquid).B () shows absorbent cotton
Add the cultivation conditions after culture medium.
Fig. 3 displays absorbent cotton influence of the absorption to fusion expression of enzymes offline.In figure (a), swimming lane 1：Control
Zymotic fluid；Swimming lane 2：From the isolated denaturing liquid of absorbent cotton after the offline absorption of absorbent cotton；Swimming lane 3：From
Remaining zymotic fluid after line absorption.In figure (b), swimming lane 1 is methanol induction 48h zymotic fluids (before absorption),
2 is that, through the zymotic fluid (after absorption) of cellulose absorbed portion fusion protein, 3 is that wash-out is denatured on cellulose
Fusion protein sample.
Fig. 4 display absorbent cotton absorption fusion enzyme total amount detection electrophoretograms.Swimming lane 1 to 4 distinguishes display density
50th, the electrophoretogram of bovine serum albumin(BSA) (BSA) solution of 100,200 and 500 μ g/mL, swimming lane 5 shows
Show the electrophoretogram of absorbent cotton denaturing liquid.
Lifting result of Fig. 5 displays absorbent cotton absorption fusion enzyme to total output.
Fig. 6 shows CALB-CBD and TTL-CBD expression.A and b are respectively CALB-CBD and RML-CBD
Expression, according to sequence and predicted molecular weight size, at arrow for purpose albumen (CALB-CBD for original
Zymotic fluid loading, RML-CBD is 10 times of loadings of concentration).
Specific embodiment
The present invention relates to the preparation of the fusion protein of containing cellulose binding domain, the preparation is preferably by fermentation
Method is implemented.
Cellulose binding domain (CBD) is relative point be present in fiber-like dhdps enzyme (such as cellulase)
Protonatomic mass is 0.4 × 104~2.0 × 104Between fragment, it can be inserted into and separates the crystal region of cellulose,
Adsorbed with the accumulation force of glucose ring by the hydrophobic plane of aromatic amino acid (hydrophobic effect) and aromatic rings
To cellulose chain surface.Can well known in the art melt containing CBD for being formed suitable for CBD of the invention
The various CBD of hop protein, including but not limited to aforementioned documents (such as Naohisa Sugimoto, Protein
Expression and Purification, 2012,82,290-296；M.A.Lemos etc., Enzyme
And Microbial Technology, 2003,32:35-40；Markus Linder etc., The Journal
Of Biological Chemistry, 1996,271, No.35,21268-21272；Jantaporn
Thongekkaew etc., Enzyme and Microbial Technology, 2013,52:241-246；
Ahn J etc., Applied microbiology and biotechnology, 2004,64:833-839；
Thongekkaew J etc., Biochemical and Biophysical Research Communications,
2012,420:183-187；Ram ó n-Luing LA etc., Biotechnology letters, 2006,
28:301-7；Santiago-Hern á ndez J etc., Enzyme and microbial technology,
2006,40:172-6；Richins RD etc., Biotechnology and bioengineering, 2000,
69:Referred in 591-6) it is various for cellulase, lipase, chaperone, fructosidase or have
Machine phosphatase forms the CBD of fusion protein, herein includes the full content of above-mentioned document by reference
Herein.
The CBD of property as an example, the invention provides the NO of ID containing SEQ:CBD shown in 2 the 6th~108,
And the NO of ID containing SEQ:The CBD of the amino acid sequence coded by 10, or be made up of these amino acid sequences
CBD。
The fusion protein that CBD is formed with destination protein is typically suitable for fusion protein of the invention." purpose
Albumen " refers to herein it is intended that the various albumen for preparing and being separate, especially with each of specific function
Plant enzyme.In the present invention, destination protein includes but is not limited to cellulase, lipase, chaperone, fructose
Glycosides enzyme or organic phosphoric acid enzyme.
Lipase specifically preferred according to the invention.Lipase can be various lipase well known in the art, for example
TTL, CALB and RML.The example of property as an example, the nucleotide sequence and amino acid sequence of TTL are respectively such as
SEQ ID NO:Shown in 3 and 4；The nucleotide sequence of CALB such as SEQ ID NO:9 shown in 64-1014,
The nucleotide sequence of RML such as SEQ ID NO:Shown in 11.
It should be understood that in addition to CBD and destination protein, other compositions, such as CBD can be also contained in fusion protein
Joint sequence between destination protein.Docking header sequence is known in the art various suitable without special limitation
Share and can be used in the present invention to form the joint sequence of fusion protein in two kinds of albumen of connection.Certainly egg is merged
Other sequences beneficial to expression, screening, secretion and/or the purifying of fusion protein are may also include in white, these
Sequence is also all the sequence commonly used in by thalline fermenting and producing fusion protein known in the art.
Therefore, generally, fusion protein of the invention is by CBD, activated protein, optional joint sequence and appoints
The sequence of the expression, screening, secretion and/or the purifying that are conducive to fusion protein of choosing is constituted.
The technology of the expression vector of construction expression fusion protein of the invention is known in the art, including example
The coded sequence of the coded sequence of destination protein and CBD is connected by the method for overlap-PCR such as
Come, destination protein-CBD then is recombinated into coded sequence with ligase is connected on suitable carrier, extracts matter
The steps such as grain, sequencing.
After expression vector needed for building acquisition, the expression vector can be transformed into suitable host.Can root
Corresponding host microorganism is selected according to the destination protein of required expression.For example, it is preferable to from known in the art
Implement the present invention for the host microorganism of the Expression product destination protein.For example, for TTL,
Can select Pichia pastoris；For CALB and RML, aspergillus niger is can select.
After conversion, can using conventional fermentation medium and fermentation condition (for example fermentation time, temperature,
Inoculum density etc.) fermented.
It is a feature of the present invention that in fermentation process, specifically induced expression for a period of time after to fermentation ends,
Used again after the fusion protein in the zymotic fluid for thalline separate, separation and fermentation liquid and cellulose are adsorbed using cellulose
The zymotic fluid and/or separated thalline are fermented.
Therefore, the method for the present invention is generally included：
(1) adsorb
Induced expression for a period of time after, by thalline and separation of fermentative broth, obtain separate thalline and separate
Zymotic fluid, makes zymotic fluid be contacted with cellulose, to allow fusion protein therein to be adsorbed on cellulose,
And zymotic fluid is separated with the cellulose for having adsorbed fusion protein；
(2) continue to ferment
The zymotic fluid for by the thalline of foregoing separation and with cellulose separate is used to ferment；With it is optional
(3) the step of repeating foregoing (1) and (2).
Specifically, fermentation a period of time after, specially induced expression for a period of time after, thalline is made first
With separation of fermentative broth, zymotic fluid is then set to be contacted with cellulose, so that the fusion protein contained by it is adsorbed to fiber
On element, zymotic fluid is then set to be separated with cellulose.Can be so repeated multiple times, for example twice, three times, four times very
To more times, specific number of times can be determined according to total fermentation duration.Generally, the time between adsorbing twice
Interval is unsuitable too short, also unsuitable long, generally within the scope of 15~30 hours, preferably 20~26
Within the scope of hour.
The thalline of separation can be temporarily stored in such as 4 DEG C refrigerators, can also be applied directly to other ongoing hairs
In ferment operation, or can be applied directly to be fermented in fresh fermentation medium.For other fermentation process
When, can be used alone, or fermented after can mixing with new fresh thalli.
Zymotic fluid after being separated with cellulose can be used to ferment, also may be used with the separate thalline mixing, continuation
For the new fresh thalli that ferments.Whether with the separate thalline mixing, or for the fresh thalline that ferments,
Can be used alone the zymotic fluid of the separation, or after can it be mixed with fresh fermentation medium, then carry out
Fermentation.
In a specific embodiment, fermentation of the invention can be carried out with fed-batch mode.That is, the hair that will be flowed out
Zymotic fluid flow plus culture in the way of anti-adding again after being contacted with cellulose.Hair after being separated with cellulose
Zymotic fluid individually instead adds, and instead adds after can also mixing with fresh fermentation medium, or divides with fresh fermentation medium
Liu not add.
Preferably, after the zymotic fluid of outflow being contacted with cellulose again in the way of anti-adding to originally separating place
The thalline of reason flow plus culture.
In a specific embodiment, by thalline and separation of fermentative broth, process (contact) with cellulose and ferment
Liquid for a period of time and after zymotic fluid is separated with cellulose, then by separating obtained zymotic fluid with it is previous
The thalline mixing of separation, continues to ferment.Can be so repeated multiple times, for example twice, three times, four times even more
Repeatedly, specific number of times can be determined according to total fermentation duration.Generally, the time interval between adsorbing twice
It is unsuitable too short, it is also unsuitable long, generally within the scope of 15~30 hours, preferably at 20~26 hours
Within the scope of.
It should be understood that " in fermentation process " refers to after induced expression to fermentation ends herein.Generally, lure
Lead after expressing through that after a while, " a period of time " thalline and separation of fermentative broth should be about into 15~30 small
When, usually 18~28 hours, preferably 20~24 hours.
In other words, the method for the present invention may include：Induced expression 15~30 hours, preferably 18~28 hours,
More preferably 20~24 hours are afterwards, by thalline and separation of fermentative broth, zymotic fluid is contacted with cellulose, with
The fusion protein in zymotic fluid is set to be adsorbed on cellulose (" absorption " step), then again by zymotic fluid
Separated with cellulose, separating obtained zymotic fluid is mixed with the thalline for being previously separated acquisition and continue fermentation
(" continuing to ferment " step), then implemented described again every 15~30 hours, preferably 20~26 hours
Absorption and continuation fermentation step, until fermentation ends.
In another specific embodiment, continue in fermentation step, thalline can be fresh thalline, also may be used
Being the thalline of the separation, or its mixture；Fermentation medium can be fresh fermentation medium,
Can also be the zymotic fluid for cellulose separate, or both mixtures.It will be appreciated that,
It is at least a kind of comprising the bacterium from adsorption step in thalline and fermentation medium in the continuation fermentation step
Body and zymotic fluid.
Can be according to actual fermentation situation (such as fusion protein in fermentation time, fusion protein, zymotic fluid
Concentration etc.) zymotic fluid separated is contacted with a certain amount of cellulose.Absorption typically by zymotic fluid with
Cellulose sways 10~60min, e.g., from about 30 minutes at about 0~10 DEG C, such as 4 DEG C.
It should be understood that contacted with cellulose not necessarily adsorb the whole fusion proteins in zymotic fluid, only
The amount reduction of the fusion protein in zymotic fluid (for example, reducing at least 10%, is preferably decreased to few 20
%, at least 30%, at least 40%, at least 50%).Big portion preferably in zymotic fluid
Point fusion protein is adsorbed on cellulose, for example, at least 60%, at least 70%, at least 80%, extremely
Few 90% fusion protein is adsorbed on cellulose.Most preferably, almost all of fusion in zymotic fluid
Albumen is all adsorbed on cellulose.
Therefore, special limit is had no for the amount and both times of contact that add the cellulose in zymotic fluid
System, technical staff can easily be adjusted and be determined according to practical condition.Generally, cellulose plus
Enter amount for 0.1~2g celluloses/liter zymotic fluid, such as 0.3~1.0g celluloses/liter zymotic fluid.
After separating cellulose and zymotic fluid, cellulose can be isolated using conventional method as the case may be
The fusion protein of upper absorption.For example, if in order to detect sample, the Tris-Cl containing 8M urea can be used
The denatured by boiling wash-out of buffer solution.As referred to 100% concentration of method in document to obtain pure fusion protein
Ethylene glycol (analytica chimica acta 621 (2008) 193-199) and the cellobiose of 1% concentration are molten
Liquid (Biosci.Biotech.Biochem., 60 (7):1183-1185,1996.) elute.Or,
Albumen wash-out need not such as be got off from cellulose, the cellulose that also can will be directly adsorbed with fusion protein is answered
It is fit directly to regard immobilization product (being immobilized lipase in this case) to use.
Similarly, the fusion protein in fermentation ends after fermentation liquid can be separated using conventional method,
For example, taking the mode (removing water) for concentrating filtering fermentation liquor；Certainly, can also be inhaled using cellulose
Attached mode, makes to be contacted with cellulose in fermentation ends after fermentation liquid, so as to by the fusion protein contained by it
Separate.Then obtained fusion protein will be every time separated to merge, purify.Or also can be pure respectively
Change and separate obtained fusion protein every time, then remerge.
In the case where fusion protein is separated from cellulose, purifying can be carried out using conventional method and melted
Hop protein, including but not limited to gel permeation chromatography, ion-exchange cellulose chromatography, affinity chromatography etc..
Suitable for cellulose of the invention including absorbent cotton, microcrystalline cellulose and cotton etc..Reading this hair
After bright disclosure, those skilled in the art can select different fibers according to different fusion proteins
Element.For example, when fusion protein is CALB-CBD, preferably using microcrystalline cellulose；When fusion protein is
During RML-CBD, cotton is preferably used.
Hereafter the present invention will be illustrated in the way of specific embodiment.It should be understood that these embodiments are only example
Property, the scope being not intended to limit the present invention.The experimental technique of unreceipted actual conditions in the following example, leads to
Often according to the written Molecular Cloning of normal condition such as Sambrook＆Russell:A
Condition described in Laboratory Manual (the Molecular Cloning:A Laboratory guide third edition), or according to manufacture
Condition proposed by manufacturer.Unless otherwise defined, all specialties and scientific words and ability used in text
Meaning familiar to domain skilled person institute is identical.Additionally, any method similar to described content or impartial and
Material all can be applied in the present invention.Preferable implementation described in text only presents a demonstration with material and is used.This
If not being illustrated in invention, compound is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, is pure level.
Embodiment 1：The structure of recombinant yeast
It is classified as by optimization and through the CBD nucleotides sequences that Sangon Biotech (Shanghai) Co., Ltd. synthesizes
(containing Linker)：
CCGACGACGGGCTCGTGCGCCGTGACGTACACCGCGAACGGCTGGAGCGGTGGCTTCACGGCG
GCCGTCACGCTCACCAACACGGGCACGACGGCGCTGAGCGGCTGGACGCTCGGGTTCGCCTTCCCGT
CCGGGCAGACCCTGACCCAGGGCTGGAGCGCGCGGTGGGCGCAGTCCGGGTCGAGCGTCACGGCGAC
CAACGAGGCCTGGAACGCCGTCCTCGCCCCGGGGGCGAGCGTCGAGATCGGCTTCAGCGGCACGCAC
ACCGGCACCAACACGGCGCCGGCGACGTTCACGGTCGGCGGCGCGACCTGCACGACGCGCTGA(SEQ
ID NO:1, italicized item is Linker)；
CBD amino acid sequences are (containing Linker)：
PTTGSCAVTYTANGWSGGFTAAVTLTNTGTTALSGWTLGFAFPSGQTLTQGWSARWAQSGSSV
TATNEAWNAVLAPGASVEIGFSGTHTGTNTAPATFTVGGATCTTR(SEQ ID NO:2, italicized item
It is Linker)；
TTL nucleotides sequences are classified as：
GAAGTCTCTCAAGACTTGTTCAACCAGTTCAACTTGTTCGCTCAATACTCTGCCGCTGCCTAC
TGTGGTAAGAACAATGATGCTCCAGCTGGTACTAACATTACCTGTACTGGTAACGCTTGTCCAGAAG
TTGAGAAGGCTGATGCTACCTTCCTGTACTCCTTCGAAGACTCTGGAGTTGGAGATGTTACTGGTTT
CCTGGCCTTGGATAACACTAACAAGTTGATCGTTCTGTCCTTCAGAGGTTCCAGATCCATCGAGAAC
TGGATTGGTAACTTGAACTTTGACTTGAAGGAGATCAACGACATCTGTTCTGGATGTCGTGGTCACG
ATGGATTTACCTCCTCTTGGAGATCTGTTGCTGATACCTTGAGACAGAAGGTCGAAGATGCTGTCAG
AGAACATCCAGACTATAGAGTTGTCTTCACTGGTCACTCCTTGGGAGGTGCCTTGGCTACTGTTGCT
GGTGCTGACTTGCGTGGTAATGGTTATGACATTGATGTCTTCTCCTACGGTGCTCCAAGAGTTGGTA
ATCGTGCCTTCGCTGAGTTTCTGACCGTCCAAACTGGAGGTACTTTGTACAGAATTACCCATACTAA
CGACATTGTTCCAAGATTGCCACCACGTGAGTTCGGATACTCTCATTCCTCTCCAGAGTACTGGATC
AAGTCTGGAACCTTGGTTCCAGTCAGACGTAGAGACATCGTCAAGATTGAAGGTATTGATGCCACTG
GAGGTAACAATCAACCAAACATTCCAGACATTCCAGCTCACTTGTGGTACTTTGGTCTGATTGGTAC
TTGCTTGTAA(SEQ ID NO:3)；
TTL amino acid sequences are：
EVSQDLFNQFNLFAQYSAAAYCGKNNDAPAGTNITCTGNACPEVEKADATFLYSFEDSGVGDV
TGFLALDNTNKLIVLSFRGSRSIENWIGNLNFDLKEINDICSGCRGHDGFTSSWRSVADTLRQKVED
AVREHPDYRVVFTGHSLGGALATVAGADLRGNGYDIDVFSYGAPRVGNRAFAEFLTVQTGGTLYRIT
HTNDIVPRLPPREFGYSHSSPEYWIKSGTLVPVRRRDIVKIEGIDATGGNNQPNIPDIPAHLWYFGL
IGTCL(SEQ ID NO:4)。
Amplimer sequence used is：
TL-F AvrII(5’-3’)TTTTCCTAGGGAAGTCTCTCAAGACTTGTTCAACC(SEQ ID
NO:5)；
TL-R(5’-3’)CGAGCCCGTCGTCGGAAGCAAGTACCAATCAGAC(SEQ ID NO:6)；
CBD-F(5’-3’)ATTGGTACTTGCTTGCCGACGACGGGCTCGTGCG(SEQ ID NO:7)；
CBD-R-EcorI(5’-3’)TTTTGAATTCTCAGCGCGTCGTGCAGGTCGC(SEQ ID NO:8)。
TTL and CBD sequences are carried out respectively with primerstar exo+ polymerases (Takara companies)
PCR is expanded, the PCR primer with TTL and CBD as template, by the method for overlap-PCR by TTL
Together with CBD sequence tandem sequence repeats, there is naturally occurring Linker in CBD between TL and CBD sequences
Connection.Then TTL-CBD recombination sequences are connected to carrier pAOX1 with T4 ligases (Fermentas)
In.Extract plasmid, sequencing.It is red according to finishing after correct carrier SalI single endonuclease digestions linearisation will be sequenced
The standard transformation methods of yeast, electroporated Pichia pastoris competent cell, are applied to Selective agar medium MDS
Screening flat board, in 28 DEG C of overnight incubations.
Embodiment 2：Cellulose binding domain merges enzyme (TTL-CBD) expression
Picking embodiment 1 builds the yeast single bacterium colony for obtaining and is inoculated in 5mL YPD culture mediums, 28 DEG C,
200rpm incubated overnights.It is seeded in the BMGY culture mediums of 50mL, 28 DEG C, 220rpm cultures are collected
Thalline.With the resuspended washing thalline of sterilized water 2 times, then with the resuspended thalline of BMMY culture mediums.Trained to BMMY
Support the methyl alcohol of addition 2% in base, 28 DEG C, 220rpm induced expressions.Enzyme activity is measured by sampling every 24h, and
To adding 0.5mL methyl alcohol in 50mL culture mediums.After induction 3d, zymotic fluid concentration is collected.
Polyacrylamid gel electrophoresis (SDS-PAGE) detects sample, as a result as shown in Figure 1, it can be seen that,
Three transformants containing genes of interest by fermentation, expressed, and molecular weight is about 40kD by destination protein.
Embodiment 3：Absorbent cotton is directly added into culture medium to merging the influence of expression of enzymes
When the yeast that embodiment 1 is built acquisition goes to BMMY culture medium the step, 0.5g is added
Absorbent cotton, other steps are identical with the zymotechnique of embodiment 2.
After fermentation ends, absorbent cotton is denatured with 4mL 8M urea liquids, another to add 5 × Loading of 1mL
Buffer (is voluntarily prepared according to this formula：250mM Tris-HCl (pH6.8), 10% (W/V) SDS,
0.5% (W/V) Coomassie brilliant blue, 50% (V/V) glycerine, 5% (W/V) beta -mercaptoethanol) it is incubated altogether, boiling
Water boils 15min, and SDS-PAGE detects absorbent cotton denaturing sample, and remaining zymotic fluid and normal fermentation liquid are same
When sample preparation detection, as a result as shown in Figure 2.
From the figure, it can be seen that absorbent cotton is directly added into culture medium can influence yeast growth in itself, no matter cotton
Flower or culture medium are all not detected by destination protein in itself.
Embodiment 4：Absorbent cotton influence of the absorption to fusion expression of enzymes offline
As embodiment 2 is fermented, but after methanol induction 24h is added, 4000rpm, 4 DEG C of centrifugation 10min,
Thalline keeps in 4 DEG C of refrigerators, and bacterium solution is placed in new centrifuge tube, adds absorbent cotton 0.5g to sway absorption at 4 DEG C
30min, remaining zymotic fluid of the destination protein through absorbing continues to mix culture with thalline；The step is repeated during 48h.
Absorbent cotton is denatured with 4mL 8M urea liquids, another to add 5 × Loading of 1mL buffer (same
Embodiment 3) it is incubated altogether, boiling water boiling 15min, SDS-PAGE detection absorbent cotton denaturing sample, remnants fermentations
Liquid and the detection of sample preparation simultaneously of normal fermentation liquid, as a result as shown in Figure 3.
Be can see from Fig. 3 (a), a large amount of destination proteins, remaining zymotic fluid and control are detected on absorbent cotton
The destination protein content of sample is roughly equal.In Fig. 3 (b), swimming lane 1 is methanol induction 48h zymotic fluids
(before absorption), 2 is that 3 is fiber through the zymotic fluid (after absorption) of cellulose absorbed portion fusion protein
The fusion protein sample of wash-out is denatured on element.
Embodiment 5：Absorbent cotton absorption fusion enzyme total amount detection electrophoretogram
With the bovine serum albumin(BSA) (BSA) of 1mg/mL concentration as standard sample, 500,200 are diluted to,
100,50 μ g/mL, the common sample preparation of urea-denatured liquid of the absorbent cotton obtained with embodiment 4 and electrophoresis detection,
Result is as shown in Figure 4.
AlphaImager HP software analysis bands, the product with band area and density as abscissa, with dense
It is transverse and longitudinal mark to spend, and obtains calibration curve equation y=8 × 10-5*x-732.62 (R2=0.9856), and calculate institute
Obtain destination protein total amount on absorbent cotton and be about 2.4mg.
Embodiment 6：Lifting result of the absorbent cotton absorption fusion enzyme to total output
Using the same procedure of embodiment 4 and 5, destination protein content is determined in control fermentation liquid and through absorbent cotton
Destination protein content after absorption in remaining zymotic fluid is simultaneously mapped, as a result as shown in Figure 5.Can from Fig. 5
Arrive, due to the absorption of absorbent cotton, the destination protein total amount acquired in absorption fermented sample is about normal fermentation institute
Obtain 2 times of destination protein total amount.
Embodiment 7：The result of the nutrient solution for instead plus after being contacted with cellulose flowing out by different way
Cultivated using fed-batch mode, after adding methanol induction 24h, 4000rpm, 4 DEG C of centrifugation 10min,
Thalline keeps in 4 DEG C of refrigerators, and bacterium solution is placed in new centrifuge tube, adds microcrystalline cellulose or absorbent cotton 0.5g 4
Absorption 30min DEG C is swayed, remaining zymotic fluid of the destination protein through absorbing is again with anti-add mode (with fresh cultured
Liquid mixing after instead plus, or by its with fresh medium respectively flow plus) flow plus culture, wherein, 50% body
The outflow nutrient solution of product (25mL) instead adds after mixing with 50% volume (25mL) fresh medium, or not
Stream adds respectively for mixing.Remaining fermentation condition is with embodiment 2.After once adsorbed again after 48h and instead plus
Operation.
Method according to embodiment 4 and 5 calculates cellulosic substrates adsorbance and zymotic fluid destination protein
Amount.
Result is as shown in table 1 below.In table 1, " normal fermentation " is to be fermented by the method for the present embodiment,
But the fermentation for not using microcrystalline cellulose or absorbent cotton to be adsorbed.
Table 1：TTL-CBD adds obtained cellulose induced expression total amount (50mL systems) through different modes stream
Embodiment 8：The fermentation of CALB-CBD and RML-CBD
1st, CALB-CBD sequences and material
1.1 Host Strains and method for transformation
Aspergillus niger N8：PyrG defect bacterial strains；
Expression vector is transformed into by Host Strains using protoplasm body, selection markers are pyrG.
1.2 culture mediums
Fermentation medium：2% glucose, 6% maltose, 4% pancreas peptone soybean broth (TSB, purchase
From Chemical Reagent Co., Ltd., Sinopharm Group), 7% sodium citrate, 1.5% ammonium sulfate, 0.1% di(2-ethylhexyl)phosphate
Hydrogen sodium, 0.1% magnesium sulfate, 0.07%Tween 80, trace element.
1.3 CALB sequences：It is as follows using CALB original series
ATGATGGTCGCGTGGTGGTCTCTATTTCTGTACGGCCTTCAGGTCGCGGCACCTGCTTTGGCT
CTGCCTTCTGGCAGCGATCCTGCCTTCAGCCAGCCCAAGAGCGTGCTCGATGCTGGTCTGACCTGCC
AGGGTGCGTCGCCGTCCAGCGTCTCCAAGCCCATCCTTCTCGTCCCCGGAACTGGCACCACTGGTCC
GCAGAGCTTCGACAGCAACTGGATTCCCCTGTCTACGCAGTTGGGCTACACTCCCTGCTGGATCTCT
CCGCCTCCGTTCATGCTCAACGACACCCAGGTCAACACGGAATACATGGTCAACGCCATCACTGCGC
TCTACGCTGGCTCGGGCAATAACAAGTTGCCCGTGTTGACCTGGAGCCAGGGTGGACTGGTTGCTCA
GTGGGGTCTGACCTTCTTTCCCAGTATCCGCTCCAAGGTCGATCGACTTATGGCCTTTGCTCCCGAC
TACAAGGGCACCGTCCTCGCTGGCCCTCTCGATGCACTCGCTGTTAGTGCTCCGTCCGTATGGCAGC
AAACTACCGGTTCGGCACTGACTACCGCACTCCGTAACGCTGGAGGTCTGACCCAGATCGTGCCCAC
TACCAACCTGTACTCGGCGACCGACGAGATCGTTCAGCCTCAAGTGAGCAACTCGCCGCTCGACTCT
TCCTACCTGTTCAACGGAAAGAACGTTCAGGCACAGGCCGTGTGTGGGCCGCTGTTCGTCATTGACC
ACGCTGGCAGCCTCACCTCGCAGTTCTCCTACGTCGTTGGTCGATCTGCCCTACGCTCCACCACGGG
CCAGGCTCGTAGTGCTGACTATGGCATTACGGACTGCAACCCTCTTCCCGCCAATGATCTGACTCCC
GAGCAAAAGGTCGCCGCGGCTGCGCTCCTGGCTCCGGCAGCTGCAGCCATTGTGGCTGGTCCGAAGC
AGAACTGCGAGCCCGACCTGATGCCCTATGCCAGACCGTTTGCAGTTGGCAAGCGCACCTGCTCTGG
CATTGTCACTCCC(SEQ ID NO:9, underscore part is amylase signal peptide)
1.4 CBD sequences：It is as follows using CBD sequences
AGCACTGGTAACCCTAGCGGCGGTAACCCACCGGGTGGTAACCGTGGTACTACCACGACGCGT
CGCCCGGCTACTACCACCGGTTCTTCTCCGGGTCCGACCCAATCTCACTACGGTCAGTGTGGCGGCA
TCGGCTACTCCGGCCCGACCGTTTGTGCGTCCGGCACCACCTGCCAGGTGCTGAATCCGTACTATTC
CCAGTGCCTGTAA(SEQ ID NO:10)
2nd, RML-CBD sequences and material
2.1 Host Strains and method for transformation
With Host Strains used by TTL-CBD, convert and cultural method.
2.2 RML sequences：It is as follows using RML complete sequences
TCCATCGACGGAGGTATTAGAGCCGCTACTTCTCAGGAAATCAACGAACTTACTTACTATACA
ACTTTGTCAGCTAATTCTTACTGTAGAACTGTTATTCCTGGTGCTACTTGGGATTGCATACATTGTG
ACGCCACTGAAGATTTAAAGATAATTAAAACCTGGTCTACTTTGATTTACGACACTAACGCTATGGT
TGCTAGAGGAGATTCCGAGAAGACTATTTATATCGTGTTTAGAGGTTCTTCATCTATTCGTAATTGG
ATCGCTGATTTGACATTCGTTCCAGTCTCTTACCCTCCAGTTTCTGGTACTAAGGTTCACAAAGGAT
TTCTTGATTCTTATGGTGAAGTTCAAAACGAGTTGGTTGCTACTGTCTTGGATCAGTTTAAACAATA
CCCATCTTATAAGGTTGCTGTCACTGGTCACTCTTTGGGAGGTGCTACTGCCTTGCTGTGTGCTTTA
GGTTTATACCAGAGAGAGGAAGGATTGTCTTCAAGTAACCTATTCTTGTACACTCAAGGTCAGCCTA
GAGTTGGAGATCCAGCATTTGCTAATTATGTGGTTTCTACTGGTATTCCATATAGACGTACTGTTAA
CGAAAGAGACATAGTACCACACTTGCCTCCAGCTGCCTTCGGATTTCTGCATGCCGGTGAAGAGTAC
TGGATCACAGATAATTCTCCTGAAACCGTTCAAGTGTGTACATCTGATTTAGAGACTTCCGACTGCT
CTAACAGTATTGTTCCATTTACTTCAGTTCTTGATCATTTGTCTTATTTTGGAATTAACACCGGTTT
GTGTACTTAA(SEQ ID NO:11)
2.3 CBD sequences：Using CBD sequences with CBD sequences (SEQ ID NO used in TTL-CBD:2)
Other build, and screen and the same TTL-CBD of cultural method (embodiment 1 and 2)
3rd, CALB-CBD and RML-CBD is expressed
Polyacrylamid gel electrophoresis (SDS-PAGE) detects sample, as a result as shown in Figure 6.A and b distinguishes
It is the expression of CALB-CBD and RML-CBD, according to sequence and predicted molecular weight size, is at arrow
Destination protein (CALB-CBD is original fermentation liquor loading, and RML-CBD is 10 times of loadings of concentration).
4th, CALB-CBD and RML-CBD celluloses induced expression total amount
Fermented using method similar to Example 4, CALB-CBD and RML-CBD is fine with crystallite respectively
Dimension plain (Avicel PH101) and cotton repeat above-mentioned absorbent cotton induction step, cellulosic substrates adsorbance with
Zymotic fluid destination protein amount is determined with urea-denatured wash-out and destination protein electrophoresis in above-described embodiment 4 and 5 respectively
Amount method is determined, and numerical value is as shown in table 2 below.
Table 2：CALB-CBD and RML-CBD cellulose induced expressions total amount (50mL systems)
| Normal fermentation (μ g) | Microcrystalline cellulose (μ g) | Cotton (μ g) | |
| CALB-CBD | 1980.3±230.8 | 3433.5±240.5 | 3148.6±189.4 |
| RML-CBD | 921.7±98.6 | 1298.6±69.0 | 1539±249.2 |
As shown in Table 2, after cellulose adsorbing separation, CBD destination proteins total amount has growth in various degree.
Claims (10)
1. a kind of fermentation yield of the fusion protein for improving containing cellulose binding domain, ferment or prepare containing cellulose knot
The method for closing the fusion protein in domain or the fusion protein of separation and/or purifying containing cellulose binding domain, the side
Method includes the microorganism of expression vector of the fermentation containing the fusion protein that can express the containing cellulose binding domain, its
In, in fermentation process, methods described adsorbs the fusion egg in the zymotic fluid for thalline separate using cellulose
In vain, the step of being fermented with the zymotic fluid and/or separated thalline again after separation and fermentation liquid and cellulose.
2. the method for claim 1, it is characterised in that methods described includes：
(1) adsorb
Induced expression for a period of time after, by thalline and separation of fermentative broth, obtain separate thalline and separate
Zymotic fluid, makes zymotic fluid be contacted with cellulose, to allow fusion protein therein to be adsorbed on cellulose,
And zymotic fluid is separated with the cellulose for having adsorbed fusion protein；
(2) continue to ferment
The zymotic fluid for by the thalline of foregoing separation and/or with cellulose separate is used to ferment；With it is optional
(3) the step of repeating foregoing (1) and (2).
3. method as claimed in claim 2, it is characterised in that
In step (2), the thalline selected from fresh thalline, the thalline of the separation or its mixture is used
And the hair of the mixture selected from fresh fermentation medium, the zymotic fluid for cellulose separate or both
Ferment culture medium carries out continuation fermentation, and condition is, at least a kind of comprising from step in thalline and fermentation medium
Suddenly the thalline or zymotic fluid of (1).
4. method as claimed in claim 2, it is characterised in that the fermentation is carried out with fed-batch mode,
Flow plus culture in the way of anti-adding again after the zymotic fluid of outflow is contacted with cellulose, wherein, will be with fibre
Dimension element separate after zymotic fluid individually instead plus, or instead add after mix with fresh fermentation medium, or with it is fresh
Fermentation medium flows add respectively.
5. method as claimed in claim 2, it is characterised in that methods described includes：
(a) induced expression for a period of time after, by zymotic fluid with containing merging for the containing cellulose binding domain can be expressed
The thalline of the expression vector of albumen is separated；
B () makes the zymotic fluid that step (a) is obtained that a period of time is contacted with cellulose, then make cellulose with hair
Zymotic fluid is separated；With
C () mixes the thalline that step (b) gained zymotic fluid is obtained with step (a), continue to ferment.
6. the method as any one of claim 1-5, it is characterised in that methods described have with
Next or multiple features：
The fusion protein be cellulase, lipase, chaperone, fructosidase or organic phosphoric acid enzyme with
The fusion protein that cellulose binding domain is formed；
The cellulose is selected from absorbent cotton, microcrystalline cellulose and cotton；
The cellulose binding domain is the relative molecular mass in cellulase 0.4 × 104~2.0 × 104It
Between fragment；
Methods described includes, induced expression for a period of time after, make thalline and separation of fermentative broth, at described one section
Between be 15~30 hours, preferably 18~28 hours, more preferably 20~24 hours；
Cellulose is 10~60 minutes with the time of contact of zymotic fluid；
Cellulose is 4~15 DEG C with the Contact Temperature of zymotic fluid；
Methods described includes：Before fermentation ends, it is repeated several times described by thalline and separation of fermentative broth, by fiber
Element contacts, isolates cellulose and makes zymotic fluid and thalline to re-mix and continue the step of fermentation with zymotic fluid
Suddenly, time interval twice between the step is 15~30 hours, preferably 20~26 hours；With
The consumption of contact cellulose is 0.1~2.0g celluloses/liter zymotic fluid every time.
7. the method as any one of claim 1-6, it is characterised in that methods described have with
Next or multiple features：
Coded sequence ID containing the SEQ NO of the cellulose binding domain:1 the 16th~327, or such as SEQ ID
NO:Shown in 10；
The lipase is selected from：Dredge the fat of the thermophilic hyphomycete of cotton like (Thermomyces lanuginosus)
The lipase (RML) of fat enzyme mutant (TTL), rhizomucor miehei (Rhizomucor miehe i), and
The lipase B (CALB) of antarctic candida (Candida antarctica)；Preferably, the TTL
Amino acid sequence such as SEQ ID NO:Shown in 4, the amino acid sequence of the CALB is by SEQ ID NO:9
64-1014 it is nucleotide sequence coded, the amino acid sequence of the RML is by SEQ ID NO:11 compile
Code；
Methods described also includes the step of isolating fusion protein from the cellulose for adsorbed fusion protein；With
The cellulose for being adsorbed with fusion protein is directly used as immobilization product.
8. the method as any one of claim 1-7, it is characterised in that the fusion protein is
The fusion protein of lipase and cellulose binding domain, methods described merges egg using described in yeast fermenting and producing
In vain；Preferably, coded sequence ID containing the SEQ NO of the cellulose binding domain:1 the 16th~327, or
Such as SEQ ID NO:Shown in 10；And/or, the fusion protein is CALB-CBD, and the cellulose is crystallite
Cellulose；And/or, the fusion protein is RML-CBD, and the cellulose is cotton.
9. application of the cellulose in the fusion protein that fermentation prepares containing cellulose binding domain, wherein, it is described
The step of using including making the cellulose be contacted with the zymotic fluid separated during the fermentation.
10. application as claimed in claim 9, it is characterised in that the fusion protein be cellulase,
The fusion protein that lipase, chaperone, fructosidase or organic phosphoric acid enzyme and cellulose binding domain are formed；
The cellulose is selected from absorbent cotton, microcrystalline cellulose and cotton.
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| CN107365757A (en) * | 2017-08-21 | 2017-11-21 | 山东大学 | It is a kind of to separate the method for obtaining concentration β endoglucanases from mixture using specific adsorption |
| CN110776571A (en) * | 2019-11-04 | 2020-02-11 | 福建省中医药研究院(福建省青草药开发服务中心） | Metallothionein fusion protein construction, rapid preparation of immobilized carrier and application of metallothionein fusion protein construction and immobilized carrier in heavy metal ion removal |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| CN110776571B (en) * | 2019-11-04 | 2021-08-10 | 福建省中医药研究院(福建省青草药开发服务中心） | Metallothionein fusion protein construction, rapid preparation of immobilized carrier and application of metallothionein fusion protein construction and immobilized carrier in heavy metal ion removal |
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