CN102071237A - Application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials - Google Patents
Application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials Download PDFInfo
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Abstract
The invention discloses application of LeEXP2 to improving efficiency of cellulase to degrade cellulosic materials. Experiments prove that LeEXP2 can cooperate with cellulase to improve the efficiency of cellulose degradation to generate reducing sugar. A lot of waste lignocelluloses exist in the nature, and by adopting LeEXP2 protein to cooperate with cellulase to degrade cellulose, the environmental pollution caused by the methods for pre-treating cellulose through high temperature and high pressure as well as acid and alkali hydrolysis in the prior art is avoided, having fundamental significance in cleanly and efficiently utilizing the waste celluloses to produce energy to solve current energy crisis.
Description
Technical field
The invention belongs to bioengineering field, relate to the purposes that a kind of LeEXP2 improves cellulose degraded cellulose materials efficient particularly.
Background technology
Mierocrystalline cellulose is the major ingredient of plant cell wall, is the renewable carbon source material of maximum on the earth.Utilize it to carry out bio-transformation and produce chemical such as alcohol fuel, the problems such as energy dilemma, food shortage, environmental pollution that solve for current mankind are extremely important.The special crystalline structure of Mierocrystalline cellulose makes Cellulase Molecules be difficult to the glycosidic link of close cellulosic molecule inside, and cellulase that enzymolysis consumption is a large amount of and time have become the bottleneck of Wood Adhesives from Biomass
[1]Modes such as utilization chemical reagent or High Temperature High Pressure change not only cost height and time-consuming of fibrous texture at present, and can bring environmental pollution.Therefore, it is particularly urgent to seek the method for change fibrous texture of gentle, efficient, environmental protection.
Expansins is a ubiquitous class protein in the cell walls of higher plant, can strengthen the extension ability of cell walls, thereby lax plant cell wall makes cell amplification, in growth and development of plant and reply and play an important role in coercing
[2- 3]Find that at present Expansins mainly contains four gene families, i.e. α-expansin (EXPA), β-expansin (EXPB), expansin-like A (EXLA) and expansin-like B (EXLB)
[4]The typical structure characteristics of Expansin are N-end signal peptide, and two structural domains of D1 (Domain1) and D2 (Domain2) are linked to each other by Linker.Recently nematode
[5]Mytilus edulis
[6], found that also some structurally are similar to the homologous protein of plant Expansin in the biologies such as minority fungi and bacterium
[7-10]Cell walls is the starting material of cellulose prods such as cotton, fiber crops and paper, if can utilize swollenin to improve or degraded cellulose, will produce chemicals to the cleaning degraded cellulose have important meaning.Not only the complex steps difficulty is big but also amount purifying is also comparatively limited but be purified into single Expansin albumen from the total protein of plant, and the plant swollenin is difficult to middle great expression and purifying on bacterium
[11]This has limited the application of Expansin.
There is LeEXP1 in the Expansins family of tomato, and LeEXP2, albumen such as LeEXP3, LeEXP2 are reported in expansion, the sophisticated tissue and express, and participates in the physiology role of aspects such as cell elongation
[12-13], but do not improve cellulosic degradation efficiency as yet at present with its collaborative cellulase.
Reference:
1?Merino?ST，Cherry?J.Progress?and?challenges?in?enzyme?development?forbiomass?utilization.Adv?Biochem?Engin/Biotechnol，2007，108：95-120.
2?Sampedro?J，Cosgrove?DJ.The?expansin?superfamily.Genome?Biology，2005，6(12)：1-11.
3?Cosgrove?DJ.Loosening?of?plant?cell?walls?by?expansins.NATURE，2000，407(21)：321-326.
4?Cosgrove?DJ，Bedinger?P，Durachko?DM.Group?I?allergens?of?grass?pollen?ascell?wall?loosening?agents.PNAS?USA，1997，94：6559-6564.
5?Qin?L，Kudla?U，Roze?EH，et?al.Plant?degradation：a?nematode?expansinacting?on?plants.Nature，2004，427(6969)：30.
6?Xu?BZ，Hellman?U，Ersson?B，et?al.Purification，characterization?and?amino-acid?sequence?analysis?of?a?thermostable，low?molecularmass?endo-b-(1，4)-glucanase?from?bluemussel，Mytilus?edulis.Eur?J?Biochem，2000，267(16)：4970-4977.
7?Kerffa?F，Amorosoa?A，Hermana?R，et?al.Crystal?structure?and?activity?ofBacillus?subtilis?YoaJ(EXLX1)，a?bacterial?expansin?that?promotes?rootcolonization.PNAS，2008，105(44)：16876-16881.
8?Yao?Q，Sun?TT，liu?WF，Chen?GJ.Gene?cloning?and?heterologous?expression?ofa?novel?endoglucanase，swollenin，from?Trichoderma?pseudokoningiiS38.BiosciBiotechnol?Biochem，2008，72(11)：2799-2805.
9?Lee?HJ，Lee?S，Ko?H，Kim?KH，Choi?I.An?Expansin-Like?Protein?from?Hahellachejuensis?Binds?Cellulose?and?Enhances?Cellulase?Activity.Mol.Cells，2010，29：379-385.
10?Saloheimo?M，Paloheimo?M，Hakola?S，et?al.Swollenin，a?Trichoderma?reeseiprotein?with?sequence?similarity?to?the?plant?expansins，exhibits?disruptionactivity?on?cellulosic?materials.Eur?J?Biochem，2002，269(17)：4202-4211.
11?Kim?ES，Lee?HJ，Bang?WG?et?al.Functional?Characterization?of?a?BacterialExpansin?From?Bacillus?subtilis?for?Enhanced?Enzymatic?Hydrolysis?of?Cellulose.Biotechnology?and?Bioengineering，2009.
12?Vogler?H，Caderas?D，Mandel?T，Kuhlemeie?C.Domains?of?expansin?geneexpression?define?growth?regions?in?the?shoot?apex?of?tomato.Plant?MolecularBiology.2003，53：267-272.
13?Reinhardt?D，Wittwer?F，Mandel?T，and?Kuhlemeier?C.Localized?Upregulationof?a?New?Expansin?Gene?Predicts?the?Site?of?Leaf?Formation?in?the?TomatoMeristem.The?Plant?Cell，1998：10，1427-1437.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of LeEXP2 to improve the purposes of cellulose degraded cellulose materials efficient.
Technical scheme of the present invention is summarized as follows:
LeEXP2 improves the purposes of cellulose degraded cellulose materials efficient, and the aminoacid sequence of LeEXP2 is shown in SEQ IDNO.12.
Prove that by experiment LeEXP2 can work in coordination with cellulase and improve the efficient that cellulose degradation produces reducing sugar.Occurring in nature has many waste lignocellulose; utilize the collaborative cellulose degraded Mierocrystalline cellulose of LeEXP2 albumen to avoid prior art method pollution on the environments such as High Temperature High Pressure and acid and alkali hydrolysis pretreatment of fiber element; this effectively utilizes depleted cellulose raw production capacity source to cleaning, and solves current energy dilemma and is extremely important.
Description of drawings
Fig. 1 is the PCR product gel electrophoresis of LeEXP2 gene.DNA standard molecular weight Marker shown in the swimming lane 1; Shown in the swimming lane 2 is the PCR product gel electrophoresis result of LeEXP2.
Fig. 2 is the construction strategy figure that contains the plasmid pGEM-LeEXP2 of tomato LeEXP2 gene.
Fig. 3 is the gel electrophoresis of pTLeEXP2 plasmid enzyme restriction.Swimming lane 1 is restriction enzyme EcoRI and the two pTLeEXP2 plasmids of cutting of XbaI among the figure; Swimming lane 2 is the contrast of pTLeEXP2 plasmid; M is DNA standard molecular weight Marker.
Fig. 4 is the gel electrophoresis of pPICZalpha A plasmid enzyme restriction.M is DNA standard molecular weight Marker among the figure, and swimming lane 1 is cut product swimming lanes 2 and is the contrast of pPICZalpha A plasmid for the EcoR I of pPICZalpha A and XbaI are two.
Fig. 5 is that LeEXP2 is gene constructed during to the pichia pastoris phaff expression vector, and the recombinant plasmid enzyme that obtains after the conversion is cut qualification result: swimming lane 1 is the plasmid contrast among the figure; Swimming lane 2 is that restriction enzyme EcoRI singly cuts the 4219bp fragment that plasmid produces; Swimming lane 3 is that XbaI singly cuts the 4219bp that plasmid produces; Swimming lane 4 is two 3530bp and two dna fragmentations of 689bp of cutting the plasmid generation of EcoRI and XbaI; Swimming lane M is DNA standard molecular weight Marker.
Fig. 6 is the gene constructed construction strategy figure to the pichia pastoris phaff expression vector of LeEXP2.
Fig. 7 cuts gel electrophoresis for plasmid LeEXP2-pPICZalpha A enzyme.Swimming lane 1 is the contrast of pPICZalpha A-LeEXP2 plasmid among the figure; Swimming lane 2 be plasmid pPICZalpha A-LeEXP2 singly cut the dna fragmentation of the 4219bp of generation through PmeI; Swimming lane M is Marker.
Fig. 8 identifies reorganization pichia pastoris phaff bacterial strain for PCR.M is Marker among the figure; Swimming lane 1 to 9 is corresponding L7 respectively, L9, L10, L11, L12, L13, L14, L15, the PCR result of bacterial strains such as L16; Swimming lane 10 is that swimming lane 11 is that water is the result of template as the PCR negative control with the fragment of the DNA 2.2kb that amplification produces as the PCR positive control of X-33.
Fig. 9 is different induction time recombinant bacterial strain L15 excretory total protein electrophorograms.Swimming lane 1 is albumen Marker, the LeEXP2 that swimming lane 2-9 expresses for the secretion in 1-8 days of reorganization bacterium, and swimming lane 10 is that control strain pichia pastoris phaff host bacterium X33 was at the 8th day excretory albumen.
Embodiment
The present invention is described further below in conjunction with the drawings and specific embodiments.
The clone of embodiment 1 tomato LeEXP2 gene
Extract total RNA of tomato with Trizol reagent according to the test kit explanation: get the total RNA of 2ug, with the reverse transcription test kit its reverse transcription is become cDNA (Promega company), with this cDNA is template, classify upstream primer as and downstream primer carries out PCR with nucleotides sequence shown in SEQ ID NO.1 in the sequence table and the SEQID NO.2, amplify the LeEXP2 gene (see figure 1) of the 744bp shown in the SEQ ID NO.3 in the sequence table.Reclaim the PCR product, be connected to TA carrier pGEM-T (Promega company) and obtain new plasmid, confirm that through order-checking this plasmid contains tomato LeEXP2 gene, plasmid called after pGEM-LeEXP2, i.e. acquisition contains the plasmid pGEM-LeEXP2 of tomato LeEXP2 gene.Construction strategy is seen Fig. 2.
With plasmid pGEM-LeEXP2 is template, classify upstream primer as with nucleotides sequence shown in the SEQ ID NO.4 in the sequence table, classify downstream primer as with nucleotides sequence shown in the SEQ ID NO.5 in the sequence table and carry out pcr amplification, obtain the 703bp fragment shown in the SEQ ID NO.6, described 703bp fragment is connected the transformant that transformed into escherichia coli TOP10F ' obtains with TA cloning vector pGEM-T, plasmid in identifying transformant is to be formed by connecting by LeEXP2 gene and pGEM-T, novel plasmid called after pTLeEXP2 with the transformant acquisition, and from above-mentioned transformant extraction plasmid pTLeEXP2, and from the intestinal bacteria that contain Yeast expression carrier pPICZalpha A plasmid, extract pPICZalpha A plasmid, with EcoRI and XbaI while digested plasmid pTLeEXP2 and pPICZalpha A; The agarose gel electrophoresis enzyme is cut product (Fig. 3 and Fig. 4), reclaims enzyme and cut the pTLeEXP2 plasmid and obtain the 689bp fragment (Fig. 3) shown in the SEQ ID NO.7 and enzyme and cut the 3530bp fragment (Fig. 4) shown in the SEQ ID NO.8 that pPICZalpha A plasmid obtains behind agarose gel electrophoresis.Be transformed in the competent cell of intestinal bacteria TOP10F ' after reclaiming the product connection, be coated with the LB flat board that contains Zeocin (available from Invitrogen company) 25 μ g/mL, be inverted for 37 ℃ and cultivate 12-16h, it is 1-2mm that bacterium colony grows to diameter; Select the mono-clonal on the flat board, by bacterium cracking PCR screening positive clone, the insertion of cutting foreign gene on the checking carrier with EcoRI and XhaI enzyme behind the extraction plasmid: singly cut plasmid and all produce fragment 4219bp, two plasmids of cutting all produce 3530bp and 689bp fragment (Fig. 5), the checking result is consistent with expection, after order-checking confirmed, called after pPICZalpha A-LeEXP2 promptly obtained containing the intestinal bacteria of pPICZalpha A-LeEXP2 plasmid.Extract pPICZalpha A-LeEXP2 plasmid in the intestinal bacteria, promptly obtain containing the recombinant vectors of tomato LeEXP2 gene.Construction strategy is seen Fig. 6.
Get 10-15 μ g pPICZalpha A-LeEXP2 plasmid, be cut into the 4219bp fragment shown in the linear SEQ ID NO.9 with the PmeI enzyme, electrophoresis detection produces the 4219bp fragment as shown in Figure 7, after phenol/chloroform extracting, linear DNA fragment shown in the linear SEQ ID of the ethanol sedimentation NO.9, be dissolved in sterilized water after the drying, making DNA concentration is the linear dna solution of 0.5-1 μ g/ μ L;
Transform the competent cell that preparation yeast competent cell method prepares pichia pastoris phaff host bacterium X-33 with reference to the electroporation of fine works molecular biology experiment guide (the 4th edition) in (P512-513), behind the linear dna solution mixing of 0.5-1 μ g/ μ L with the competent cell of 80 μ L pichia pastoris phaff host bacterium X33 and 10 μ L, transfer in the pole cup of 4 ℃ of precoolings, ice bath 5min, voltage 1500V, electroporation with eppendorf company shocks by electricity, electric shock 5ms, add the ice-cold 1M sorbitol aqueous solution of 1mL after the electric shock immediately in electric revolving cup, mixing, mixed solution is transferred in the 15mL centrifuge tube, 28-30 ℃ leaves standstill cultivation 1h, at the mixed solution that is coated with on the YPD flat board of the Zeocin that contains 100 μ g/mL (sale of Invitrogen company) after 100 μ L cultivate, be inverted for 30 ℃ and cultivate until the appearance of reorganization bacterium.
The YPD substratum is yeast extract 10g/L, peptone 20g/L, glucose 20g/L, agar 18g/L.
The temperature of the pole cup of precooling also can be any value in 0-4 ℃; 28-30 ℃ of time of leaving standstill cultivation also can be any value among the 1-2h; The volume that is coated with the mixed solution after the cultivation on flat board also can be any value among the 50 μ L-200 μ L.More than experiment all can obtain to contain the reorganization pichia pastoris phaff bacterium of LeEXP2 gene.
The PCR of embodiment 4 LeEXP2 pichia pastoris phaff transformants identifies
Extract the genomic dna of the reorganization bacterium that is obtained among the embodiment 3, genomic dna with the reorganization bacterium is a template, classify the upstream and downstream primer as with nucleotides sequence shown in SEQ ID NO.10 and the SEQ ID NO.11 and carry out PCR, to detect the insertion of foreign DNA, the chromogene group DNA of pichia pastoris phaff host bacterium X33 be the PCR of template as positive control, the PCR that with water is template is as negative control.The pcr amplification condition is 95 ℃ of sex change 30s, 54 ℃ of annealing 30s, and 72 ℃ are extended 3min.PCR product electrophoresis, L7 as shown in Figure 8, L9, L10, L11, L12, L13, L14, L15, bacterial strains such as L16 all can amplify the fragment that is inserted into the 1.215kb that comprises the LeEXP2 gene on the pichia pastoris phaff host bacterium X33 karyomit(e), and the 2.2kb gene fragment of the AOX1 gene of pichia pastoris phaff host bacterium X33, illustrate that above-mentioned reorganization bacterium all is integrated with the LeEXP2 gene.
The LeEXP2 genetic expression of embodiment 5 methanol induction pichia pastoris phaff transformants
1. inoculate the reorganization pichia pastoris phaff bacterium L7 that contains the LeEXP2 gene that identifies through embodiment 4, L9, L10, L11, L12, L13, L14, L15, L16 and pichia pastoris phaff host bacterium X33 are switched to 2ml, and the BMGY substratum of pH=4.8 is cultivated in advance, and 220 rev/mins, 29 ℃ are cultured to OD
600=4;
2. by 1: 100 volume ratio respectively with step being forwarded in the 25ml BMGY substratum 1. through pre-incubated bacterium liquid, 220 rev/mins, 29 ℃ are cultured to OD
600=4;
3. the bacterium liquid of 2. step being cultivated is transferred respectively and is added to the containing in the BMMY substratum that volumetric concentration is 0.5% methyl alcohol of 50ml pH=4.8, makes the concentration OD of bacterium liquid
600=1, at 220 rev/mins, cultivate in 29 ℃ of shaking tables, every 24h mends methyl alcohol to 0.5%, induces reorganization bacterium secreting, expressing LeEXP2.For detecting that LeEXP2 can express effectively and target protein being secreted into the extracellular of reorganization pichia pastoris phaff bacterium, carry out protein electrophoresis from the substratum of inducing the back to get the reorganization bacteria growing in the 1st day to 8 days.The sampling and the electrophoresis mode as follows: get 100 μ l substratum, centrifugal after, get supernatant 16 μ l and mix with the sample-loading buffer of 4 μ l, after boiling water boils 5 minutes, get the 12ul electrophoresis.As shown in Figure 9, increase along with induction time, target protein LeEXP2 excretory many more (aminoacid sequence of excretory target protein LeEXP2 is shown in SEQ ID NO.12), control strain pichia pastoris phaff host bacterium X33 does not then have the secretion (Fig. 9 is different induction time recombinant bacterial strain L15 excretory total protein electrophorograms) of target protein LeEXP2, the amount of the LeEXP2 of each bacterial strain secreting, expressing is difference to some extent also, the amount of bacterium L15 excretory recombinant protein LeEXP2 of wherein recombinating is bigger, therefore, pichia pastoris phaff bacterium (Pichia pastoris) the L15 CGMCC NO.4123 that will recombinate carries out preservation, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date: on August 27th, 2010, (seeing preservation survival proof).
The rotating speed that step is cultivated in 1. and 2. also can be any value in 200-250 rev/min; Culture temperature also can be any value in 28 ℃-30 ℃; Cultivate the concentration OD of bacterium
600It also can be any value among the 2-6.The concentration that contains methyl alcohol in the 3. middle BMMY substratum of step also can be any value among the 0.5%-1%; The concentration OD of thalline
600It also can be any value in the 0.8-1.5 scope; Rotating speed also can be any value in 200-250 rev/min; Culture temperature also can be any value in 28 ℃-30 ℃, and the concentration of every 24h benefit methyl alcohol also can be any value among the 0.5%-1%.Prove by electrophoresis and also can finish the LeEXP2 genetic expression of inducing the pichia pastoris phaff transformant under these conditions.
The BMGY substratum is 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 100mM potassiumphosphate, 4x10
-5The % vitamin H, 1% glycerine, surplus is a water.
The BMMY substratum is 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 100mM potassiumphosphate, 4x10
-5The % vitamin H, 0.5%-1% methyl alcohol, surplus is a water.
Sample-loading buffer is the Tris of 15.1g/L, the 94g/L glycine; 5.0g/L sodium lauryl sulphate (SDS), surplus are water.
Embodiment 6 LeEXP2 improve the efficient of cellulose degraded filter paper
Induce pichia pastoris phaff host bacterium X33 and reorganization pichia pastoris phaff bacterium (Pichiapastoris) L15 CGMCC NO.4123 (being called for short reorganization bacterium L15) with the method for embodiment 5, when inducing the 6th day, get 2 milliliters of the substratum of pichia pastoris phaff host bacterium X33 and reorganization bacterium L15 secreting, expressing respectively, 12000 rev/mins after centrifugal 1 minute, Xinhua's filter paper bar of getting 1.5ml supernatant and 50mg in 25ml tool plug test tube under the room temperature temperature bathe 48h, 7U/L cellulase (the GC220 that adds 0.5ml again, company of outstanding person's energy section) liquid reacts, behind 50 ℃ of water-bath 1hr, every pipe adds 3mlDNS solution, boiling water bath heating 5min, be settled to 25ml after being cooled to room temperature, measure OD
540Value, determine the amount of the glucose that discharges (measuring method of glucose typical curve is as follows: be 0,0.5,1 with 2 ml concns respectively according to the typical curve of glucose, 1.5,2.0,2.5,3.0.3.5,4.0mg/ml glucose solution and 3 milliliters of DNS mixings, boiling water bath heating 5min is settled to 25ml after being cooled to room temperature, measures OD
540Value, be X-coordinate with concentration, with each OD
540Value be ordinate zou drawing standard curve).Contain LeEXP2 albumen in the substratum of reorganization bacterium L15, the reducing sugar that itself and cellulase effect filter paper produce is 1.34mg; And the LeEXP2 secretion is not arranged in the substratum of contrast pichia pastoris phaff host bacterium X33, the reducing sugar that produces with cellulase effect filter paper is 0.88mg.It is 1.52 times that cellulase acts on separately that the LeEXP2 of reorganization bacterium L15 abduction delivering and cellulase synergistic effect filter paper produce sugar.Described DNS reagent composition is as follows: contain 3 of 10g in 1 liter of DNS solution, 5-dinitrosalicylic acid, 10g NaOH, the Seignette salt of 200g, the phenol of 2g, the NaHSO of 5g
3, surplus is a water.
The bacterium L7 that will recombinate, L9, L10, L11, L12, L13, L14 or L16 adopt the method for present embodiment to carry out above-mentioned experiment, and the collaborative cellulose degraded filter paper fibre element of proof LeEXP2 is stronger than the independent effect of cellulase.This test has illustrated that LeEXP2 can improve the efficient of cellulose degraded cellulose materials-filter paper.
Induce pichia pastoris phaff host bacterium X33 and reorganization bacterium L15 with the method for embodiment 5, when inducing the 6th day, get 2 milliliters of the substratum of pichia spp host bacterium and reorganization bacterium L15 secreting, expressing respectively, 12000 rev/mins after centrifugal 1 minute, the crystalline fibers (Sigma company) of getting 1.5ml supernatant and 50mg in 25ml tool plug test tube under the room temperature temperature bathe 48h, 7U/L cellulase (the GC220 that adds 0.5ml again, company of outstanding person's energy section) liquid reacts, behind 50 ℃ of water-bath 1hr, every pipe adds 3mlDNS solution, boiling water bath heating 5min is settled to 25ml after being cooled to room temperature, by surveying OD
540Value determine the amount of the glucose that discharges.Contain LeEXP2 albumen in the substratum of reorganization bacterium L15, the reducing sugar that itself and cellulase effect crystalline fibers produce is 8.99mg; And the LeEXP2 secretion is not arranged in the substratum of contrast pichia spp host bacterium X33, the reducing sugar that produces with cellulase effect crystalline fibers is 6.34mg.It is 1.42 times that cellulase acts on separately that LeEXP2 and cellulase synergistic effect crystalline fibers are produced sugar.
The bacterium L7 that will recombinate, L9, L10, L11, L12, L13, L14 or L16 adopt the method for present embodiment to carry out above-mentioned experiment, and the collaborative cellulose degraded crystalline fibers of proof LeEXP2 is stronger than the independent effect of cellulase.This test has illustrated that LeEXP2 can improve the efficient of cellulose degraded cellulose materials-crystalline fibers.
Embodiment 8 LeEXP2 improve the efficient of cellulose degraded straw
Induce pichia pastoris phaff host bacterium X33 and reorganization bacterium L15 with the method for embodiment 5, when inducing the 6th day, get 2 milliliters of the substratum of pichia spp host bacterium X33 and reorganization bacterium L15 secreting, expressing respectively, 12000 rev/mins after centrifugal 1 minute, the straw powder of getting 1.5ml supernatant and 50mg in 25ml tool plug test tube under the room temperature temperature bathe 48h, 7U/L cellulase (the GC220 that adds 0.5ml again, company of outstanding person's energy section) liquid reacts, behind 50 ℃ of water-bath 1hr, every pipe adds 3mlDNS solution, boiling water bath heating 5min is settled to 25ml after being cooled to room temperature, by surveying OD
540Value determine the amount of the glucose that discharges.Contain LeEXP2 albumen in the substratum of reorganization bacterium L15, the reducing sugar that itself and cellulase effect straw produce is 8.10mg; And the LeEXP2 secretion is not arranged in the substratum of contrast pichia spp host bacterium X33, the reducing sugar that produces with cellulase effect straw is 6.10mg.It is 1.32 times that cellulase acts on separately that LeEXP2 and cellulase synergistic effect straw produce sugar.
Bacterium L7 will recombinate, L9, L10, L11, L12, L13, L14 or L16 adopt the method for present embodiment to carry out above-mentioned experiment, the result shows that the collaborative cellulose degraded cellulose materials-straw of LeEXP2 is stronger than the independent effect of cellulase, and promptly LeEXP2 can improve the efficient of cellulose degraded cellulose materials-straw.
Claims (1)
1.LeEXP2 improve the purposes of cellulose degraded cellulose materials efficient.
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| WO2013155866A1 (en) * | 2012-04-16 | 2013-10-24 | 山东七河生物科技股份有限公司 | Liquid fermentation method for increasing yield of cordyceps polysaccharide by expansin |
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