CN1335395A - High-activity cellulase composition and its prepn - Google Patents
High-activity cellulase composition and its prepn Download PDFInfo
- Publication number
- CN1335395A CN1335395A CN 01128578 CN01128578A CN1335395A CN 1335395 A CN1335395 A CN 1335395A CN 01128578 CN01128578 CN 01128578 CN 01128578 A CN01128578 A CN 01128578A CN 1335395 A CN1335395 A CN 1335395A
- Authority
- CN
- China
- Prior art keywords
- gene
- cellulase
- conversion
- strain
- cbhi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The high activity cellulase composition consists of the cellulases including: CBHI 30-40 wt%, CBHII 10-25 wt%, EGI 10-15 wt%, EGII 6-10 wt%, EGIII 1-4 wt%, EGIV 0.5-2 wt%, EGV 0.5-1.0 wt% and CeIB 10-30 wt%, where the CeIB gene is the promoter and terminal signal of CBHI gene, and the secretion signal is the front 28 amino acids coded by CBHI. It is prepared through DNA recombinant technological process including plasmid construction, Trichoderma conversion, screen of high yield strain, spawn fermenting expression, etc. The present invention has the advantages of high cellulase activity, low cost, stable product quality, and excellent application effect in various industry.
Description
Technical field
The utility model relates to a kind of enzyme, the cellulase composition that especially utilizes gene engineering to obtain.
Background technology
Cellulase is used for industries such as weaving, wine brewing, feed, traditional Chinese medicine extraction and fruit juice production in a large number.Wood etc. are at " Enzymology method " (Methodsin Enzymology), 160,25, disclose complete fungin enzyme system in 234 pages (1986) and comprised several different enzyme classes, comprise that those are accredited as exocellobiohydrolase (EC3.2.1.91) (" CBH "), the enzyme of endoglucanase (EC3.2.1.4) (" EG ") and beta-glucosidase enzyme (EC3.2.1.21) (" BG ").CBH, EG and BG fungin enzyme classification can further expand to the multiple composition among comprising every type, have been separated to CBH and EG from multiple fungus resource.The bacterial classification of existing production of cellulose enzyme mostly is trichoderma viride, these bacterial classifications screen then by chemistry and physical mutagenesis and obtain, though trichoderma viride can be secreted 10-20 grams per liter protein when producing for big jar, and major part is a cellulase, but because the enzyme specific activity that viride itself produces is lower, generally at 200,000 international unit/liter (Yu Xiaobin, 2000, ChinaEnzyme).Anaerobic fungi is that recent two decades comes the novel microorganism bacterial classification separated from the phytophagous animal gi tract, major part to have the enzymic activity of secretion plant cell wall degraded, and the specific activity of these enzymes high especially (Wood et al.1986).The enzyme of unit weight is to high times of the enzyme (Gilbert et al.1992) of the energy force rate viride product of cellulose degradation, but the anaerobic fungi speed of growth is slow, is not suitable for suitability for industrialized production.A kind of effective means changes viride over to for the gene of cellulase that anaerobic fungi is encoded, can secrete a large amount of enzymes like this, and these enzymes is active high, Cellulase B (the CelB) (Lietal of anaerobic fungi Orpinomyces for example, 1997) specific enzymes activity is 220,000 international unit/gram exceeds more than ten times than the specific activity of the enzyme of viride.How changing the CelB gene of the anaerobic fungi of high specific activity over to viride produces in the bacterium, thereby improve the output of cellulase element, reach 400,000-1200,000 international unit/liter, and 1,4 beta-glucanase activity can reach 1000,000-3000,000 international unit is the technical barrier that this area need solve.Can the production of enzyme preparation gordian technique be the activity unit of every liter of bulk fermentation liquid, and the activity level that unit volume is produced directly restricts production cost, energy consumption and be used for the various fields of industrial or agricultural.
Summary of the invention
Purpose of the present invention is produced the product that a kind of activity is high, can be widely used in above-mentioned industry for improving the active yield of cellulase.Its principle is that the cellulase Cellulase B gene of the high specific activity of coding is implanted the existing bacterium pearl HypocreaiecorinaMGG80 that produces with transgenic method, pass through recombinant DNA technology simultaneously, to produce the bacterium pearl is subjected to inductive promotor (Promoter) and adds this gene locus under working condition, implant efficiently expressing of gene thereby instruct, obtain a kind of new cellulase composition.
The present invention adopts gene recombination technology to produce a kind of high-activity cellulase composition, it is characterized in that: it is that cellulase by following weight per-cent constitutes: the cellulase CBH I 30～40% that is produced by trichoderma strain, CBH II 10～25%, EG I 10～15%, EG II 6～10%, EG III 1～4%, EG IV 0.5～2%, EG V 0.5～1.0%, cellulase CelB10～30% with the anaerobic fungi generation, wherein, the CelB gene is the promotor and the termination signal (Terminater) of CBH I gene, and secretion signal (Secretion Signal) is preceding 28 amino acid coding of CBHI coding.
Preceding 28 amino acid of described CBHI coding are encoded to MYRKLAVISAFLATARAQSACTLQSETH in detail.
The preparation method of high-activity cellulase composition is to adopt the DNA recombinant technology, comprises that structure, the conversion of Li's Trichoderma, the screening of superior strain, the strain fermentation of plasmid expressed, and it is characterized in that:
(1) structure of plasmid
The DNA of Trichodermareesei extracts with Easy-DNA reagent, with the HindIII restriction enzyme as partial hydrolysis, pack into then with in the PUC18 plasmid after the same enzymic hydrolysis, the dna segment that connects Trichodermareesei (Treesei) and PUC18 with T4 ligase enzyme (T4 ligase), sample after the connection is used for transformed into escherichia coli (Ecoli), and the sample after the conversion is seeded in equably and carries out the bacterium colony screening on the solid LB substratum that contains 50 microgram/grams; By screening, 12 bacterium colonies that contain the CBHI gene are selected, and their contained plasmid DNA are separated, and carry out determined dna sequence then; CBH I gene 5 ' 1500 base pairs of end, with 3 ' 1000 base pairs with polymerase chain reaction (Polymerase Chain Reactions (PCR)] amplify, reload in the PUC18 plasmid, 5 ' dististyle is disconnected except that containing the promotor part, preceding 28 the amino acid whose sequences that also contain the CBHI that encodes, and 5 ' end and 3 ' joint add several restriction endonuclease sites, the CelB gene of anaerobic fungi also is to amplify by PCR, include only coding to the amino acid whose interval of the activated middle portion of Walocel MT 20.000PV CMC, the site that has added restriction enzyme simultaneously is so that embed the joint of CBH I gene 5 ' and 3 ' end smoothly;
(2) conversion of Trichodermareesei
The method of Penttila is adopted in the conversion of Trichodermareesei, on double-deck agarose substratum, obtain about 120 and transform bacterium colony, transformed bacteria dropped into to have gone select and non-selection cultivation is alternately cultivated three times, from each elementary conversion bacterium colony, separate two single bacterium colonies more arbitrarily, be inoculated on the potato nutrient agar,, write down every strain growth speed in 28 ℃ of cultivations 5 days;
(3) screening of superior strain
In above-mentioned bacterial strains, select approaching bacterium pearl before fast growth, form and the conversion, under aseptic condition, take out about one square centimeter mycelium, the cellulase that is used to inoculate 50 milliliters is induced liquid nutrient medium, culturing bottle seals with tampon, cultivated 5 days in 28 ℃ shaking table the inoculation back, after cultivating end, mycelium and residual Mierocrystalline cellulose are removed with centrifugal, get supernatant liquor, get supernatant liquor and survey enzymic activity, the bacterial strain that unconverted bacterial strain under the supernatant liquor specific activity similarity condition is exceeded 1.5 times carries out further separation and Culture;
(4) strain fermentation is expressed
By said gene high-yield strains that engineering obtains, with Microcrystalline Cellulose as main carbon source with induce substrate, in 28 ℃, PH4.6-4.8, dissolved oxygen 20% above condition three grade fermemtation, can obtain the fermented liquid of high-activity cellulase composition, its output can reach 300,000-500,000 international unit/liter.
The high active cellulase fermented liquid that obtains, through Plate Filtration, get clear and bright filtrate, via hole diameter is 10000 dalton's ultra-filtration membrane ultrafiltration and concentration 3-8 times again after the 0.4um mocromembrane filters, after getting different activities unit's concentrated solution, in the ratio of sucrose 20%, Sodium Benzoate 0.2%, potassium sorbate 0.2%, detect qualified back and get final product by the specification packing.
Under equal conditions fermentation, cellulase activity of the present invention improves 1-5 doubly, reaches 400,000-1200,000 international unit/liter, the external import of product price descends more than 33% with veriety, and constant product quality, and result of use is good, the product that obtains by this kind method is because of its super quality and competitive price, can be widely used in weaving, wine brewing, food, beverage, medicine, oil production and feedstuff industry, produce huge economical, societal benefits, promote the level of industry of these industries.
Description of drawings
Fig. 1 is a recombination design diagram of the present invention.
The promotor of 1-CBHI (CBHI Promoter)
The CelB gene of 2-anaerobic fungi (0rpinomyces CelB Gene)
The termination signal of 3-CBHI (CBHI Terminater)
4-plasmid sequence (Plasmid Sequence)
The secretion signal of 5-CBHI (CBHI Secretion Signal)
The specific embodiment
Embodiment 1: as shown in Figure 1, contain the plasmid of Fig. 1 by the conversion to trichoderma reesei (Transformation) and screening, obtain the bacterial strain with hereditary information shown in Figure 1, right Afterwards for the production of keep in the products obtained therefrom that the original production bacterial strain produces with add new source gene institute Several cellulases that produce, the product that reconfigures is complex enzyme, concrete component is: CBHI36%, the CBHII20% that H.iecorina produces, EGI12%, EGII8%, EGIII2%, The CelB20% that EGIV1%, EGV1% and anaerobic fungi produce has collaborative the promotion between each component Effect is promoter and the termination signal of CBHI gene in the new CelB gene that adds, and divides Pil signal is front 28 amino acid coding of CBHI coding.
Its preparation method is to adopt the DNA recombinant technique, comprises structure, the trichoderma reesei of plasmid The steps such as the conversion of bacterium, the screening of superior strain, strain fermentation expression and product extraction. Specifically Be respectively:
(1) structure of plasmid: the DNA of trichoderma reesei QM9414 American I nvitrogen The Easy-DNA reagent of company extracts, and uses the HindIII restriction enzyme as partial hydrolysis, Pack into then with in the PUC18 plasmid after the same enzyme hydrolysis, connect with T4Ligase The dna segment of Treesei and PUC18, the sample after the connection are used for transformed into escherichia coli (Ecoli), the sample after the conversion is seeded in the solid LB training that contains 50 microgram/grams equably Support and carry out the bacterium colony screening on the base; The probe that is used for screening is the dna segment (Shoe of T.reesei Maker et al.1983). By screening, 12 bacterium colonies that contain the CBHI gene are selected, it Contained DNA be separated, carry out then determined dna sequence, altogether survey Decided about 600 base-pairs, and the sequence of positive anti-chain is measured all; CBH I base Because of 5 ' 1500 base-pairs of end and 3 ' 1000 base-pair Polymerase Chain Reactions (PCR) amplifies, and reloads in the PUC18 plasmid, and 5 ' dististyle is disconnected to be opened except containing Outside the mover part, also contain front 28 amino acid whose sequences of the CBHI that encodes, and 5 ' end Add several restriction endonuclease sites with 3 ' joint. Two kinds of segments are packed into behind the PUC18, Carry out dna sequencing, to get rid of the possibility of when PCR amplifies, introducing sudden change. Anaerobic fungi The CelB gene also is to amplify by PCR, includes only coding to the activated centre of CMC The interval of partial amino-acid has added the site of restriction enzyme simultaneously, in order to embed smoothly The joint of CBH I gene 5 ' and 3 ' end. Plasmid behind the embedding CelB gene is also surveyed Order.
(2) conversion of trichoderma reesei: (1987) such as Penttila are adopted in the conversion of trichoderma reesei Method. For the ease of having selected the bacterium colony of PUC18 segment chimeric, contain Aspergillus The PUC18 of the acetamidase of nidulans (acetamidase) gene is as a kind of The plasmid of co-transfomation (Penttila etc., 1987). The method that transforms please refer to Penttila etc. (1987). On double-deck agarose culture medium, obtain about 120 conversions Bacterium colony is dropped into to have gone to transformed bacteria and is selected and non-selection cultivation alternate culture three times, again from each Separate arbitrarily two single bacterium colonies in the elementary conversion bacterium colony, be inoculated on the potato agar medium, In 28 ℃ of cultivations 5 days, record every strain growth speed;
(3) screening of superior strain: in above-mentioned bacterial strains, select fast growth, form and turn to The bacterium pearl that approaches before changing is taken out about one square centimeter mycelium under aseptic condition, be used for Inoculate 50 milliliters cellulase induction fluid nutrient medium, blake bottle is 250 milliliters of triangular flasks, With the tampon sealing, the shaking table in 28 ℃ after the inoculation was cultivated 5 days, after cultivation finishes, with mycelia Body and residual cellulose are got supernatant and are surveyed enzymatic activity with centrifugal remove (5000rpm, 15min) (1%CMC, PH4.8), exceeds unconverted bacterial strain under the supernatant specific activity similarity condition by 50 ℃ 1.5 bacterial strain doubly further separates cultivation;
(4) strain fermentation is expressed: by said gene high-yield strains that engineering obtains, with the crystallite fibre Dimension is plain as main carbon source with induce substrate, in 28 ℃, PH4.6-4.8, dissolved oxygen more than 20% The condition three grade fermemtation can obtain the zymotic fluid of high-activity cellulase composition, and its output can Reach 300,000-500,000 international unit/liter.
(5) product extracts: get above-mentioned zymotic fluid, through plate-frame filtering, get clear and bright filtrate, through 0.4um After mocromembrane filters again via hole diameter be 10000 dalton's milipore filter ultrafiltration concentration 3-8 doubly, no After active unit's concentrate, press sucrose 20%, Sodium Benzoate 0.2%, potassium sorbate 0.2% Ratio, detect and get final product by the specification packing after qualified.
Claims (3)
1, a kind of high-activity cellulase composition, it is characterized in that: it is that cellulase by following weight per-cent constitutes: the cellulase CBH I 30～40% that is produced by trichoderma strain, CBH II 10～25%, EG I 10～15%, EG II 6～10%, EG III 1～4%, EGIV0.5～2%, EGV0.5～1.0%, cellulase CelB10～30% with the anaerobic fungi generation, wherein, the CelB gene is the promotor and the termination signal (Terminater) of CBH I gene, and secretion signal (Secretion Signal) is preceding 28 amino acid coding of CBHI coding.
2, the preparation method of the cellulase composition of claim 1 is to adopt the DNA recombinant technology, comprises that structure, the conversion of Li's Trichoderma, the screening of superior strain, the strain fermentation of plasmid expressed, and it is characterized in that:
(1) structure of plasmid
The DNA of Trichodermareesei extracts with Easy-DNA reagent, do partial hydrolysis with the HindIII restriction enzyme, pack into then with in the PUC18 plasmid after the same enzymic hydrolysis, the dna segment that connects Treesei and PUC18 with T4Ligase, sample after the connection is used for transformed into escherichia coli (Ecoli), and the sample after the conversion is seeded in equably and carries out the bacterium colony screening on the solid LB substratum that contains 50 microgram/grams; By screening, 12 bacterium colonies that contain the CBHI gene are selected, and their contained plasmid DNA are separated, and carry out determined dna sequence then; CBH I gene 5 ' 1500 base pairs of end, with 3 ' 1000 base pairs amplify with polymerase chain reaction Polymerase Chain Reactions (PCR), reload in the PUC18 plasmid, 5 ' dististyle is disconnected except that containing the promotor part, preceding 28 the amino acid whose sequences that also contain the CBHI that encodes, and 5 ' end and 3 ' joint add several restriction endonuclease sites, the CelB gene of anaerobic fungi also is to amplify by PCR, include only coding to the amino acid whose interval of the activated middle portion of CMC, the site that has added restriction enzyme simultaneously is so that embed the joint of CBH I gene 5 ' and 3 ' end smoothly;
(2) conversion of Trichodermareesei
The method of Penttila is adopted in the conversion of Trichodermareesei, on double-deck agarose substratum, obtain about 120 and transform bacterium colony, transformed bacteria dropped into to have gone select and non-selection cultivation is alternately cultivated three times, from each elementary conversion bacterium colony, separate two single bacterium colonies more arbitrarily, be inoculated on the potato nutrient agar,, write down every strain growth speed in 28 ℃ of cultivations 5 days;
(3) screening of superior strain
In above-mentioned bacterial strains, select approaching bacterium pearl before fast growth, form and the conversion, under aseptic condition, take out about one square centimeter mycelium, the cellulase that is used to inoculate 50 milliliters is induced liquid nutrient medium, culturing bottle seals with tampon, cultivated 5 days in 28 ℃ shaking table the inoculation back, after cultivating end, mycelium and residual Mierocrystalline cellulose are removed with centrifugal, get supernatant liquor and survey enzymic activity, the bacterial strain that unconverted bacterial strain under the supernatant liquor specific activity similarity condition is exceeded 1.5 times carries out further separation and Culture;
(4) strain fermentation is expressed
By said gene high-yield strains that engineering obtains, with Microcrystalline Cellulose as main carbon source with induce substrate, in 28 ℃, PH4.6-4.8, dissolved oxygen 20% above condition three grade fermemtation, can obtain the fermented liquid of high-activity cellulase composition, its output can reach 300,000-500,000 international unit/liter.
3, the preparation method of cellulase composition according to claim 2, it is characterized in that: get fermented liquid, through Plate Filtration, get clear and bright filtrate, via hole diameter is 10000 dalton's ultra-filtration membrane ultrafiltration and concentration 3-8 times again after the 0.4um mocromembrane filters, after different activities unit's concentrated solution, successively add sucrose, Sodium Benzoate, potassium sorbate in the ratio of sucrose 20%, Sodium Benzoate 0.2%, potassium sorbate 0.2%, detects qualified back and presses the specification packing and get final product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128578 CN1335395A (en) | 2001-09-04 | 2001-09-04 | High-activity cellulase composition and its prepn |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 01128578 CN1335395A (en) | 2001-09-04 | 2001-09-04 | High-activity cellulase composition and its prepn |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1335395A true CN1335395A (en) | 2002-02-13 |
Family
ID=4668421
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 01128578 Pending CN1335395A (en) | 2001-09-04 | 2001-09-04 | High-activity cellulase composition and its prepn |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1335395A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102414315A (en) * | 2009-05-19 | 2012-04-11 | 森迪奈尔Ch有限责任公司 | A dried and stabilized ready-to-use composition containing nucleic acid polymerization enzymes for molecular biology applications |
| CN101809151B (en) * | 2007-08-30 | 2013-04-24 | 埃欧金能源公司 | Method for cellulase production |
| CN101827939B (en) * | 2007-08-30 | 2013-08-07 | 埃欧金能源公司 | Enzymatic hydrolysis of lignocellulosic feedstocks using accessory enzymes |
| CN109234301A (en) * | 2018-09-11 | 2019-01-18 | 中国农业科学院饲料研究所 | For quickly improving recombinant expression carrier and its application of trichoderma reesei Cellulase enzyme activity |
-
2001
- 2001-09-04 CN CN 01128578 patent/CN1335395A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101809151B (en) * | 2007-08-30 | 2013-04-24 | 埃欧金能源公司 | Method for cellulase production |
| CN101827939B (en) * | 2007-08-30 | 2013-08-07 | 埃欧金能源公司 | Enzymatic hydrolysis of lignocellulosic feedstocks using accessory enzymes |
| CN102414315A (en) * | 2009-05-19 | 2012-04-11 | 森迪奈尔Ch有限责任公司 | A dried and stabilized ready-to-use composition containing nucleic acid polymerization enzymes for molecular biology applications |
| CN109234301A (en) * | 2018-09-11 | 2019-01-18 | 中国农业科学院饲料研究所 | For quickly improving recombinant expression carrier and its application of trichoderma reesei Cellulase enzyme activity |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102325872B (en) | Fermentation broth formulations | |
| EP2150615B1 (en) | Method for producing cellulase and hemicellulase having high hydrolytic activity | |
| CN102149819B (en) | Beta-glucosidase variants having improved activity, and uses thereof | |
| US20090035811A1 (en) | Artificial scaffolding material for protein retention and use of the same | |
| CN102762727A (en) | Host cell capable of producing enzymes useful for degradation of lignocellulosic material | |
| RU2361918C1 (en) | Strain of mycelial mushroom penicillium verruculosum - producer of complex of cellulase, xylanase and xyloglucanase and way of reception of fermental preparation of complex of cellulase, xylanase and xyloglucanase for hydrolysis of cellulose and hemicellulose | |
| KR101150280B1 (en) | Cellulase producing Nectria cinnabarina and its use for saccharification | |
| Jang et al. | Thermostable cellulases from Streptomyces sp.: scale-up production in a 50-l fermenter | |
| CN102234639A (en) | Method for the production of cellulases based on the regulation of the oscillation of the pression of the oxygen disolved in the culture medium | |
| CN104498365A (en) | Bacterial strain capable of producing chitin deacetylase and application of bacterial strain in production of chitin deacetylase through fermentation | |
| CN101855973B (en) | Fungus strain irpex iacteus for producing laccase, and culturing method and application thereof | |
| Ghoshal et al. | Solid state fermentation in food processing | |
| CN1335395A (en) | High-activity cellulase composition and its prepn | |
| CN105219661A (en) | The special strain therefore of synthesis of oligonucleotides semi-lactosi and the method with its synthesis of oligonucleotides semi-lactosi | |
| CN1176205C (en) | Acetic acid leakage type high-yield pyruvate bacterium and its breeding method and producing pyruvic acid by said bacterium via fermentation process | |
| Joshi et al. | Fermentation technology: current status and future prospects | |
| Gunathilake et al. | Evaluation of cellulose degrading efficiency of some fungi and bacteria and their biofilms | |
| CN1060518C (en) | Method for producing dictyophora indusiata nutritive mycelium using bamboo fungus submerged culture fermentation technology | |
| Zoppas et al. | Alternatives for cellulase production in submerged fermentation with agroindustrial wastes | |
| Nishise et al. | Production of raw cassava starch-digestive glucoamylase by Rhizopus sp. in liquid culture | |
| CN108424896B (en) | Method for producing cellulase by mixed fermentation of corn straw furfural residues | |
| CN1683520A (en) | A strain for producing composite enzyme and process for producing compoiste emzyme using said strain fermentation | |
| KR101255090B1 (en) | Efficient production of lactic acid by the simulataneous saccharification and fermentation of algal biomass | |
| CN102212482B (en) | Method for aspergillus niger and solid starters for fermenting and producing feed | |
| US8518679B2 (en) | Complementation of the Trichoderma reesei secretome limiting microbiological contaminations within the context of industrial processes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |