CN106367409A - Method for simultaneous high-yield production of cellulase and [beta]-glucosidase - Google Patents
Method for simultaneous high-yield production of cellulase and [beta]-glucosidase Download PDFInfo
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Abstract
The invention discloses a method for simultaneous high-yield production of cellulase and [beta]-glucosidase. The operating method for simultaneous high-yield production of the [beta]-glucosidase and the cellulase is implemented by the following steps: expressing polypeptide, which has a cellobiase activity, in trichoderma reesei so as to obtain a recombinant trichoderma reesei strain, and conducting fed-batch fermentation by taking a soluble inducer of a glycose transglycosylation product as a carbon source. According to the method provided by the invention, cultivation is conducted in a fermentation tank by virtue of a fed-batch fermentation technology for 156h, so that the activity of the [beta]-glucosidase in a recombinant strain fermentation broth exceeds 300CBU/mL, which is more than 60 times above that of an original strain, and meanwhile, the activity of the cellulase exceeds 50FPU/mL. With the application of the method provided by the invention, shortcoming in the prior art that the [beta]-glucosidase of a trichoderma reesei cellulase system is too low in enzymatic activity is overcome, the compounding cost of the cellulase is reduced and the enzymolysis efficiency of lignocellulose is improved.
Description
Technical field
The present invention relates to genetic engineering and field of fermentation engineering are and in particular to one kind High Cellulase Production and β-Fructus Vitis viniferae simultaneously
The method of glycosidase, be more particularly a kind of in trichoderma reesei, using cheap solubility inducer fed-batch fermentation, with
When High Cellulase Production and beta-glucosidase method.
Background technology
Increasingly serious, the renewable money of the lignocellulose with straw as representative with energy crisis and environmental pollution
The bioconversion in source becomes production bio-fuel and biological-based chemicals, to mitigate the important selection of oil dependence.It is currently based on
The biotransformation efficiency of lignocellulose need to be improved, and wherein relatively costly to be that it is realized industrialized heavy for cellulase production
Want one of bottleneck.Therefore, the high efficiency, low cost of cellulase produces the attention of extremely various countries' researcher.
It is widely present the microorganism of eccrine fiber element enzyme, trichoderma reesei (trichoderma reesei) is in nature
One of the extracellular protein secretion capacity that has now been found that cellulase production bacterium the strongest.Trichoderma reesei cellulase enzyme system is mainly wrapped
Include exocellulase, endo cellulase and beta-glucosidase.Wherein exocellulase mainly acts on cellulose line
The end of shape molecule produces cellobiose；Endo cellulase acts on cellulose non-crystallized part, random hydrolysis β-Isosorbide-5-Nitrae-sugar
Glycosidic bond reduces cellulosic degree of polymerization thus generating new cellulose end, thus assisting the faster hydrolysis fiber of exocellulase
Element；And the product cellobiose that exocellulase generates has inhibitory action to exocellulase and endo cellulase,
After cellobiose reaches finite concentration, exocellulase and endo cellulase activity are suppressed thus affecting cellulose
The hydrolysis efficiency of enzyme；And beta-glucosidase can make outer with hydrolysis fiber disaccharide as glucose, thus releasing this inhibitory action
Enzyme cutting and restriction endonuclease keep continuous action, thus reaching efficient cellulose hydrolysis to produce glucose.
In trichoderma reesei cellulase system, exocellulase accounts for the 60%-80% of total extracellular protein, and 20%-36%'s is interior
Cut cellulase and 1% beta-glucosidase composition, so beta-glucosidase is fiber in trichoderma reesei cellulase
Rate-limiting enzyme in plain degradation process.Although m k Fuda draw etc. disclose by the extra method adding beta-glucosidase Lai
Optimize trichoderma reesei cellulase system (cn200880106102.4), but additionally add the increasing that commercial enzyme undoubtedly causes cost
Plus.And studies in China person discloses a kind of method expressing external source beta-glucosidase gene nfbgl3a by cbh1 promoter
To enrich trichoderma reesei cellulase enzyme system (cn201510795507.5), but the use of cbh1 promoter can reduce cbh1 base
The expression of cause, thus affect cellulase systems.For problem above, using suitable promoter, and optimize fermentation process,
Improve trichoderma reesei cellulase and beta-glucosidase to produce, be using cellulose series biomass resource produce bio-fuel and
The important need of biological-based chemicals.
Content of the invention
It is an object of the invention to provide a kind of method of High Cellulase Production simultaneously and beta-glucosidase it is therefore intended that gram
Take the too low problem of b- glucosidase content in trichoderma reesei cellulase system present in prior art, there is provided Yi Zhong
Strong promoter ppdc1 under glucose existence condition, overexpression external source beta-glucosidase gene aabgl1, thus to contain Portugal
The method of cheap solubility inducer High Cellulase Production and the b- glucosidase simultaneously of grape sugar.
The purpose of the present invention is achieved through the following technical solutions:
The present invention relates to a kind of method of utilization trichoderma reesei High Cellulase Production and beta-glucosidase simultaneously, described side
Method comprises the steps:
S1. in trichoderma reesei (trichoderma reesei), expression has the peptide coding of activity of beta-glucosidase
Nucleotide, obtain restructuring trichoderma reesei；
S2. with the sugar liquid containing b- disaccharide as carbon source with inducer, culture described restructuring trichoderma reesei produces cellulose simultaneously
Enzyme and beta-glucosidase；Described culture appending stuff in fermentation tank using the sugar liquid containing the β-disaccharide turning glycosides generation
Concentration of glucose in fermentation liquid.
Preferably, in step s1, the nucleotide of the described peptide coding with activity of beta-glucosidase be selected from funguses,
Antibacterial, plant or obtained by synthetic gene, or the nucleotide sequence after being optimized by protein engineering.More preferably
For beta-glucosidase gene.
Preferably, the nucleotide of the described peptide coding with activity of beta-glucosidase is selected from from microorganism Aspergillus aculeatus
The aabgl1 gene of (aspergillus aculeatus).
Preferably, in step s1, with the pcb301 containing hygromycin gene as skeleton plasmid, it has been sequentially connected nucleoside
Aabgl1 gene shown in pyruvic carboxylase promoter ppdc1, seq id no.2 as shown in seq id no.1 for the acid sequence
With the pdc1 terminator shown in seq id no.3, construction recombination plasmid.
Preferably, described structure comprises the steps:
A1. with trichoderma reesei genome dna as template, with the primer pair as shown in seq id no.4, seq id no.5
Pdc1 promoter is expanded, and is expanded with the primer pair pdc1 terminator as shown in seq id no.6, seq id no.7；
Using cla i single endonuclease digestion pcbhygb carrier, using being seamlessly connected enzyme, pdc1 promoter and terminator one step are connected to linearisation
Pcbhygb in, obtain carrier pcbhygb-pdc1；
A2. carried out for template with the cdna of microorganism Aspergillus aculeatus using the primer as shown in seq id no.8, seq id no.9
The clone of aabgl1 gene, is connected to carrier pcbhygb-pdc1 using being seamlessly connected enzyme by cloning the aabgl1 gene obtaining
In, obtain recombiant plasmid pcbhygb-pb.
Preferably, in step s2, the sugar liquid containing the b- disaccharide turning glycosides generation is the sugar using glucose or containing glucose
Slurry, turns what glycosides reaction was prepared by what beta -glycosidase was catalyzed.
Preferably, in step s2, described batch feeding is particularly as follows: be reduced to temperature when in fermentation liquid, glucose exhausts
25 DEG C, then start to flow plus the sugar liquid containing β-disaccharide；Stream according to the sugar liquid containing β-disaccharide for the concentration of glucose regulation and control in fermentation liquid
Acceleration so that in fermentation liquid concentration of glucose be 0.05g/l-0.30g/l.
Preferably, in step s2, in incubation, restructuring trichoderma reesei spore suspension is connect by fermentating liquid volume 10%
Plant in fermentation medium, cultivate for 28 DEG C and exhaust to glucose.
Preferably, in step s2, in the described sugar liquid containing β-disaccharide, β-disaccharide accounts for the 10%-20% of total sugar concentration.
The invention still further relates to a kind of trichoderma reesei (trichoderma reesei) pb3cgmcc no.12768.
The invention still further relates to the restructuring trichoderma reesei of a kind of High Cellulase Production simultaneously and beta-glucosidase, in Richter scale wood
Mould middle use ppdc1 promoter have expressed the beta-glucosidase gene aabgl1 of external source.
Preferably, described restructuring trichoderma reesei contains the beta-glucosidase base that have expressed external source using ppdc1 promoter
Recombiant plasmid because of aabgl1.
Preferably, the beta-glucosidase gene aabgl1 of described external source is selected from from microorganism Aspergillus aculeatus (aspergillus
Aculeatus aabgl1 gene).
In the present invention, cellulase production bacterial strain is derived from t.reesei, such as American type culture collection numbering
For56765tmAnd Chinese industrial Microbiological Culture Collection administrative center numbers and is13052 t.reesei
rut c30.
The present invention passes through to utilize pdc1 promoter overexpression aabgl1 gene in trichoderma reesei, obtains and is containing Fructus Vitis viniferae
Under the fermentation medium of sugar, recombinant conversion that beta-glucosidase enzyme activity is obviously improved.Compared with starting strain rut c30, β-Portugal
Polyglycoside enzyme enzyme activity improves 60 times.
In the present invention, described trichoderma reesei (trichoderma reesei) pb3 submits China on July 14th, 2016
Microbiological Culture Collection administration committee common micro-organisms center preservation (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Institute of Microorganism, Academia Sinica's postcode 100101), it is cgmcc no.12768 that culture presevation is numbered.
Compared with prior art, the invention has the following beneficial effects:
1st, the restructuring trichoderma reesei that the present invention builds excessive in the fermentation medium containing glucose can produce b- Portugal
Polyglycoside enzyme.
2nd, the restructuring trichoderma reesei that the present invention builds can be as inducer by the use of the Glucose Liquid containing β-disaccharide simultaneously
High Cellulase Production and beta-glucosidase.
3rd, the crude enzyme liquid of the present invention does not need additionally to add beta-glucosidase, reduces the cost of compound enzyme.
4th, the crude enzyme liquid of the high-cellulose enzymatic activity of the present invention is preferably is directly used in lignocellulose-like biomass biology
The hydrolysis of refining process cellulosic component.
5th, the high-cellulose enzyme of the present invention produces intensity and saves significantly on cellulase fermentations investment of production equipment and process energy
Consumption, reduces cellulase production cost.
Brief description
Fig. 1 expressing heterologous beta-glucosidase plasmid figure.A: containing promoter ppdc1 and pdc1 terminator expression vector matter
Grain figure；B:pdc expression cassette overexpression aabgl1 expression vector plasmid figure；
Fig. 2 contains the agrobacterium tumefaciens bacterium solution pcr checking electrophoretogram of expression vector；
Fig. 3 restructuring trichoderma reesei conversion subgenom dna checking aabgl1 gene electrophoretogram；
Fig. 4 produces beta-glucosidase and control strain comparison diagram with trichoderma reesei transformant shake flask fermentation of recombinating；
Fig. 5 utilizes trichoderma reesei transformant pb-3 to utilize cheap solubility inducer fed-batch fermentation in fermentation tank same
When high-yield beta-glucosidase and cellulase production curve chart.
Specific embodiment
With reference to embodiment, the present invention is described in detail.Following examples will be helpful to those skilled in the art
Further understand the present invention, but the invention is not limited in any way.It should be pointed out that to those of ordinary skill in the art
For, without departing from the inventive concept of the premise, can also make certain adjustments and improvements.These broadly fall into the guarantor of the present invention
Shield scope.
Embodiment 1, the recombiant plasmid pcbhygb-pb of construction expression external source beta-glucosidase gene aabgl1
With pan7-1 (sequence is shown in genbank:z32698.1) plasmid as template, hygbf (5 '-
Ggactagtccttgtatctctacacacaggctca-3 ' seq id no.10) and hygb (5 '-
Ggactagtccttaattaatcgagtggagatgtggagtgggcgc-3 ' seq id no.11) it is primer pair hygromycin resistance
Gene is expanded, using spe i restriction enzyme site by hygromycin expression cassette be connected to pcb301 (sequence is shown in genbank:
Af139061.1) in agrobacterium vector, the new support obtaining is named as pcbhygb.And (compiled with Li's Trichoderma strains rut c30
Number nrrl 11460) genome dna is template, with ppdc1f (5 '-
Cccccctcgaggtcgacggtaggacttccagggctacttg-3 ' seq id no.4) and ppdc1r (5 '-
Cggtcagacttcatgccgggcgtacggattgtgctgtagctgcgct-3 ' seq id no.5) start for primer pair pdc1
Sub (seq id no.1) is expanded, with tpdc1f (5 '-agcgcagctacagcacaatccgtacgcccggcatgaagtct
Gaccg-3 ' seq id no.6) and tpdc1r (5 '-aggaattcgatatcaagctttggacgcctcgatgtcttcc-3 '
Seq id no.7) expanded for primer pair pdc1 terminator (seq id no.3).Carried using cla i single endonuclease digestion pcbhygb
Body, using being seamlessly connected enzyme (work is given birth in Shanghai), pdc1 promoter and terminator one step is connected in linearizing pcbhygb, obtains
To carrier pcbhygb-pdc1 (as Fig. 1 a).Recycle primer pdcaabgl1f (agcgcagctacagcacaatcatgaagctc
Agttggcttga seq id no.8) and pdcaabgl1r (5 '-cggtcagacttcatgccgggtcattgcaccttcggga
Gcg-3 ' seq id no.9) aabgl1 gene is carried out for template with the cdna of aspergillus aculeatu no.f-50
Gene is directly connected in the carrier of above-mentioned structure also with being seamlessly connected enzyme, is named as by the clone of (seq id no.2)
Pcbhygb-pb (as Fig. 1 b), carrier is delivered to the errorless rear use of Shanghai biological engineering company limited sequencing.
Embodiment 2, using agrobacterium tumefaciens mediation method by aabgl1 expression casette import trichoderma reesei genome
Using electric method for transformation, pcbhygb-pb plasmid is converted to Agrobacterium agl-1, and coat containing 100mg/l profit
Lb selective medium (lb medium component, g/l:10nacl, 10 tryptones, 5 ferment of good fortune gentle 100mg/l kanamycin
Female extract) on flat board, after 28 DEG C of culture 2-4d, picking resistance single bacterium colony carries out activation and preserves.For determining whether to convert successfully,
Picking resistant clones, in the pcr reaction system preparing, carry out pcr reaction with pdcaabgl1f and tpdc1r for primer, it
After take 5 μ l amplified productions to carry out 1% agarose gel electrophoresiies detection, result is as shown in Fig. 2 obtain a treaty 3.5kb as expected
(3583bp) band.
Trichoderma reesei rut c30 is inoculated in product spore culture medium (3% fructus hordei germinatus leaching powder, 1.5% agar), 28 DEG C of culture 7d,
With the lower conidium of aseptic washing, six layers of filtered through gauze, add after 4000rpm centrifugation 4min after aseptic aqueous suspension spore again
4000rpm is centrifuged 4min, adds aseptic aqueous suspension spore blood cell plate to count, and adjustment concentration is every milliliter 107Individual conidium is put
Enter 4 DEG C to save backup.Conversion process takes 100 μ l to use im culture medium (g/l:k2hpo4, 1.45；kh2po4, 2.5；Nacl,
0.15；mgso4, 0.5；cacl2, 0.066；feso4, 0.0008；nh4so4, 0.5；Glucose, 1.8；40mmol/l 2-[n-
Morpholino] ethanesulfonic acid, ph5.3；0.5% glycerol (w/v)) activated proceeded to expression vector
Agrobacterium tumefaciens agl-1 and after 100 μ l trichoderma reesei rut c30 conidiospore suspension mix, even spread arrives in advance covering
On the cellophane in im culture medium (add 200 μm of acetosyringones and 5mm glucose), 25 DEG C of light culture 2d, by glass
(200g Rhizoma Solani tuber osi is cut into small pieces the pda culture medium that paper is transferred to containing 200 μ g/ml and 500 μ g/ml cephamycin hygromycin, adds water
Well-done, boil 20～30 minutes, with eight layers of filtered through gauze, heat, then plus 20g agar, continue heated and stirred and mix, treat that agar is molten
After having solved, add glucose, stir, after slightly cooling down, supply moisture again to 1000 milliliters, subpackage test tube or conical flask, plus
Fill in, wrap up, 20 minutes about the rear test tubes that take out of 115 DEG C of sterilizings are put inclined-planes or shaken up, and store standby after cooling.) on flat board, 28
DEG C culture 3-7d, will have trichoderma reesei recombinant clone to occur, choose in time, be transferred to new pda flat board, 20 plants of conversions of picking
Son, is named as pb-1, pb-2, pb-3 to pb-20, extract after the genome dna of 20 plants of recons using pdcaabgl1f and
Pdcaabgl1r primer carries out pcr checking, and result is as shown in Figure 3 it should obtain the band of a 2583bp it was demonstrated that aabgl1 base
Because being all successfully incorporated in trichoderma reesei genome.
Using turning, glycoside products shake flask fermentation screening high-yield beta-glucosidase trichoderma reesei conversion sub-step is as follows:
1st, the preparation of inducer: configuration 600g/l glucose solution, every gram of glucose adds 20cbu Xia Sheng industry group to have
The beta-glucosidase that limit company produces, is incubated 72h, then boiling sugar liquid 5min makes β-Fructus Vitis viniferae under the conditions of ph4.8 and 65 DEG C
Solubility inducer β-disaccharide and glucose mixture (mgd) can be prepared after glycosidase inactivation.Glucose is contained after measured in mgd
410.3g/l, 60.5g/l gentiobiose, 9.3g/l cellobiose and sophorose 13.7g/l.
2nd, slant culture: take a small amount of restructuring trichoderma reesei and host's trichoderma reesei rut c30 spore inoculating from -80 DEG C of refrigerators
In raw Spore cultivation base (Fructus Hordei Germinatus powder containing 30g/l and 15g/l agar), cultivate 7d at 28 DEG C, wash lower spore with aseptic, in
Standby at 4 DEG C.
3rd, seed culture: the spore under step 1 is washed, it is inoculated in seed culture medium (glucose containing 4g/l and 10g/l Semen Maydiss
Slurry) in, prepare spore suspension in 28 DEG C and 150rpm culture 24h, as seed liquor.
4th, shake flask fermentation culture the: be seeded to (liquid attractant containing glucose of fermentation medium containing 50ml by 10% inoculum concentration
Lead thing mgd 10g/l, peptone 1g/l, wheat bran 10g/l, carbamide 0.3g/l, (nh4)2so41.4g/l, kh2po42g/l,
mgso4·7h2O 0.3g/l, cacl20.4g/l, feso4·7h2O 5mg/l, mnso4·h2O 1.7mg/l, znso4·7h2o
1.4mg/l, cocl22mg/l, tween 80 0.2ml/l) 250ml triangular flask in, 28 DEG C culture 48h after sample 6000rpm
Supernatant is taken to carry out enzyme activity determination as crude enzyme liquid after centrifugation 5min, filter paper enzyme activity and beta-glucosidase enzyme activity determination method adopt
International theory and standard method (ghose, t.1987.measurement of cellulase that purely chemistry meeting iupac promulgates
, fermentation results as shown in Figure 4 activities.pure and applied chemistry59 (2): 257-268).Host Strains
The beta-glucosidase enzyme activity that trichoderma reesei rut c30 produces is 0.15cbu/ml, and trichoderma reesei enzyme activity of recombinating is from 0.45-
3.9cbu/ml, improves 3-25 times, result is as shown in Figure 4.The reason cause recon enzyme activity difference may be: 1. external source
The copy number that expression casette is incorporated in trichoderma reesei genome is different；2. the position being incorporated in genome is different；3. weight
After group son culture, part expression cassette is lost or base mutation.So those skilled in the art can obtain one plant of product by screening
Enzyme is higher and can stablize hereditary recon.
Embodiment 3, use restructuring trichoderma reesei High Cellulase Production and beta-glucosidase simultaneously
The method of the High Cellulase Production that fermentation mode is announced by Bai Fengwu etc. (201610309126.6) is as follows: (1-3
Step is identical with above-mentioned steps)
1. the preparation of inducer: configuration 600g/l glucose solution, every gram of glucose adds 20cbu Xia Sheng industry group to have
The beta-glucosidase that limit company produces, is incubated 72h, then boiling sugar liquid 5min makes β-Fructus Vitis viniferae under the conditions of ph 4.8 and 65 DEG C
Solubility inducer β-disaccharide and glucose mixture (mgd) can be prepared after glycosidase inactivation.Glucose is contained after measured in mgd
410.3g/l, 60.5g/l gentiobiose, 9.3g/l cellobiose and sophorose 13.7g/l.
2. slant culture: take a small amount of trichoderrna reesei pb3 cgmcc no.12768 (weight from -80 DEG C of refrigerators
Group trichoderma reesei, the method obtaining high enzyme activity recon according to disclosed in embodiment 2, it is heavy that those skilled in the art can obtain this
Group trichoderma reesei) spore inoculating, in raw Spore cultivation base (Fructus Hordei Germinatus powder containing 30g/l and 15g/l agar), cultivates 7d at 28 DEG C,
With the lower spore of aseptic washing, store for future use at 4 DEG C.
3. seed culture: the spore under step 1 is washed, it is inoculated in seed culture medium (glucose containing 4g/l and 10g/l Semen Maydiss
Slurry) in, under the conditions of 28 DEG C and 150rpm, culture 24h prepares spore suspension, as seed liquor.
4. fermentation culture: be seeded to (the liquid inducer containing glucose of fermentation medium containing 3l by 10% inoculum concentration
Mgd10g/l, peptone 1g/l, wheat bran 10g/l, carbamide 0.3g/l, (nh4)2so41.4g/l, kh2po42g/l, mgso4·
7h2O 0.3g/l, cacl20.4g/l, feso4·7h2O 5mg/l, mnso4·h2O 1.7mg/l, znso4·7h2o 1.4mg/
L, cocl22mg/l, tween 80 0.2ml/l) 7l stirred fermentor in, fermentation front 12-36h (in the present embodiment essence
True is 25h) it is thalli growth Main Stage (i.e. fast-growth), control fermentation temperature to be 28 DEG C, ph control utilizes Feeding ammonia water
Realize, supplement nitrogen source while controlling ph to be not less than 4.2.Concentration of glucose in monitoring fermentation liquid, treats glucose in fermentation liquid
Cool the temperature to 25 DEG C when exhausting, and start stream plus mgd, concentration of glucose in every 4h detection fermentation tank, and dense according to glucose
Degree regulates and controls mgd flow acceleration it is ensured that concentration of glucose is 0.05g/l-0.30g/l in fermentation tank, and wherein fermentation tank rotating speed is to reach
To for the purpose of fermentation liquid mix homogeneously.Filter paper enzyme activity and beta-glucosidase enzyme activity determination method adopt international theory and pureization
Standard method (ghose, t.1987.measurement of cellulase activities.pure that association iupac promulgates
And applied chemistry 59 (2): 257-268), fermentation results are as shown in Figure 5.Trichoderma during culture 156h
Reesei pb3 cgmcc no.12768 fermentation liquid cellulase unit alive reaches 55fpu/ml, cellulase production intensity
Reach 352fpu/l/h, beta-glucosidase enzyme activity is up to 300cbu/ml, compared to using original strain trichoderma reesei rut
C30, beta-glucosidase enzyme activity improves 60 times, has reached the excessive mesh producing cellulase and beta-glucosidase simultaneously
's.
Wherein the fermentative medium formula described in step (3) is: the liquid inducer mgd 10g/l containing glucose, egg
White peptone 1g/l, wheat bran 10g/l, carbamide 0.3g/l, (nh4)2so41.4g/l, kh2po42g/l, mgso4·7h2O 0.3g/l,
Cacl2 0.4g/l, feso4·7h2O 5mg/l, mnso4·h2O 1.7mg/l, znso4·7h2O 1.4mg/l, cocl2
2mg/l, tween 80 0.2ml/l.
In sum, the present invention is expanded from the genome dna of t.reesei rut c30 respectively by designing primer
The promoter of pdc1 and terminator, and clone aabgl1 base from the cdna of aspergillus aculeatu no.f-50
Cause, using the seamless Cloning Kit of instant (no.b632219, work is given birth in Shanghai), directly by promoter and termination sub-piece one step
Method is linked in the pcb301 containing Hygromycin resistance marker's gene (genbank:af139061.1) carrier, obtains containing pdc1 table
Reach the expression vector pcbhygb-pdc of box and between promoter and terminator added with bswi i restriction enzyme site, then utilize this limit
Property restriction endonuclease linearized vector processed, recycles seamless connection enzyme to be connected to beta-glucosidase gene nucleotide fragments
In pcbhygb-pdc, obtain expression vector pcbhygb-pb, using agrobacterium tumefaciens mediation method by overexpression aabgl1 base
Transformant is obtained in the genome of expression cassette importing t.reesei rut c30 of cause.Transformant is inoculated in product spore culture medium
(3% fructus hordei germinatus leaching powder, 1.5% agar), spore saves backup as at 4 DEG C, using the fermentation process of the announcements such as Bai Fengwu
(cn201610309126.6) fed-batch fermentation, High Cellulase Production and beta-glucosidase simultaneously are carried out in bioreactor
Enzyme.
Claims (10)
1. a kind of method of High Cellulase Production simultaneously and beta-glucosidase is it is characterised in that methods described includes walking as follows
Rapid:
S1. in trichoderma reesei (trichoderma reesei), expression has the core of the peptide coding of activity of beta-glucosidase
Thuja acid, obtains restructuring trichoderma reesei；
S2. with the sugar liquid containing β-disaccharide as carbon source with inducer, culture described restructuring trichoderma reesei produce simultaneously cellulase and
Beta-glucosidase；Described culture appending stuff fermentation in fermentation tank using the sugar liquid containing the β-disaccharide turning glycosides generation
Concentration of glucose in liquid.
2. the method for High Cellulase Production simultaneously according to claim 1 and beta-glucosidase is it is characterised in that step
In s1, the nucleotide of the described peptide coding with activity of beta-glucosidase is selected from funguses, antibacterial, plant or passes through people
Nucleotide sequence that work synthetic gene obtains or after being optimized by protein engineering.
3. the method for High Cellulase Production simultaneously according to claim 2 and beta-glucosidase is it is characterised in that described
The nucleotide with the peptide coding of activity of beta-glucosidase is selected from from microorganism Aspergillus aculeatus (aspergillus aculeatus)
Aabgl1 gene.
4. the method for High Cellulase Production simultaneously according to claim 3 and beta-glucosidase is it is characterised in that step
In s1, with the pcb301 containing hygromycin gene as skeleton plasmid, it has been sequentially connected nucleotide sequence such as seq id no.1
Shown in the shown aabgl1 gene shown in pyruvic carboxylase promoter ppdc1, seq id no.2 and seq id no.3
Pdc1 terminator, construction recombination plasmid.
5. the method for High Cellulase Production simultaneously according to claim 4 and beta-glucosidase is it is characterised in that described
Structure comprises the steps:
A1. with trichoderma reesei genome dna as template, with primer pair pdc1 as shown in seq id no.4, seq id no.5
Promoter is expanded, and is expanded with the primer pair pdc1 terminator as shown in seq id no.6, seq id no.7；Using
Pdc1 promoter and terminator one step are connected to linearizing by cla i single endonuclease digestion pcbhygb carrier using seamless connection enzyme
In pcbhygb, obtain carrier pcbhygb-pdc1；
A2. carried out for template with the cdna of microorganism Aspergillus aculeatus using the primer as shown in seq id no.8, seq id no.9
The clone of aabgl1 gene, is connected to carrier pcbhygb-pdc1 using being seamlessly connected enzyme by cloning the aabgl1 gene obtaining
In, obtain recombiant plasmid pcbhygb-pb.
6. the method for High Cellulase Production simultaneously according to claim 1 and beta-glucosidase is it is characterised in that step
In s2, the sugar liquid containing the β-disaccharide turning glycosides generation is the syrup using glucose or containing glucose, is catalyzed by beta -glycosidase
The glycosides reaction that turns be prepared.
7. the method for High Cellulase Production simultaneously according to claim 1 and beta-glucosidase is it is characterised in that step
In s2, described batch feeding, particularly as follows: temperature is reduced to 25 DEG C when glucose exhausts in fermentation liquid, starts stream plus contains β-two
The sugar liquid of sugar；Flow acceleration according to the sugar liquid containing β-disaccharide for the concentration of glucose regulation and control in fermentation liquid is so that Fructus Vitis viniferae in fermentation liquid
Sugared concentration is 0.05g/l-0.30g/l.
8. the restructuring trichoderma reesei of a kind of High Cellulase Production simultaneously and beta-glucosidase is it is characterised in that in trichoderma reesei
Middle use ppdc1 promoter have expressed the beta-glucosidase gene aabgl1 of external source.
9. restructuring trichoderma reesei according to claim 8 is it is characterised in that described restructuring trichoderma reesei contains use
Ppdc1 promoter have expressed the recombiant plasmid of the beta-glucosidase gene aabgl1 of external source.
10. a kind of trichoderma reesei (trichoderma reesei) pb3 cgmcc no.12768.
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| CN114703164A (en) * | 2022-03-03 | 2022-07-05 | 上海交通大学 | Efficient cellulase inducer and preparation and application methods thereof |
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