CN1049684C

CN1049684C - Preparing method of high active cellulase

Info

Publication number: CN1049684C
Application number: CN96116049A
Authority: CN
Inventors: 朱明田; 李波; 徐海; 钱卫; 曲音波; 高培基
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 1996-11-13
Filing date: 1996-11-13
Publication date: 2000-02-23
Anticipated expiration: 2016-11-13
Also published as: CN1157853A

Abstract

The present invention relates to a preparing method of high-activity cellulase, which belongs to the field of microorganism preparation. The present invention is characterized in that natural cellulose substances are pulverized and blasted at high pressure, and the natural cellulose substances are used as carbon sources; a penicillium decumbiens thom AS3.524 strain is used for deep liquid fermentation, and cellulase powder is produced through filtration, concentration, spray drying, etc. The method has the advantages of wide source of cellulase raw materials, low cost and high stability, and is suitable for mass industrial production. Fermented waste residues can be prepared into fodder, and the problem of environmental pollution is solved. The activity of the cellulase powder prepared by the method is 8000 u/g; compared with the activity of the product prepared by a national solid fermentation method, the activity of the cellulase powder is increased by 20%. The cellulase is widely used in industry of textile brewing, etc.

Description

The manufacture method of high active cellulase

The invention belongs to microorganisms producing and make the field.

Cellulase is used for light textile, feed and brewages and fermentation industry.Cellulase production adopts solid and two kinds of zymotechniques of liquid at present, the former one-time investment is little than the latter, but the processing requirement floor space is huge, easy pollution microbes, and production stability is poor, technology is difficult for grasping, and is difficult to carry out suitability for industrialized production, and continuous continent, Shenyang, Liaoning cellulase factory promptly uses this technology.Although liquid fermenting has the production stability height, technology is easily grasped, can large-scale industrial production etc. characteristics, combine between Mierocrystalline cellulose in but inductive substance---the natural cellulose class material (corn tangerine, jowar tangerine, wheat tangerine, straw etc.) and xylogen and the Mierocrystalline cellulose and closely be difficult to decompose utilize because of cellulase, so it is raw material that manufacturing process need adopt expensive import cellulose powder, therefore the production cost of cellulase is quite high, influenced applying of this technology, the present domestic sophisticated liquid fermentation producing technology that still do not have.

The present invention is intended to overcome the deficiency of prior art, utilizes natural cellulose class material to produce high active cellulase for raw material by the deep liquid fermentation process manufacturing.

The present invention realizes as follows, select for use natural cellulose class material such as corn tangerine, wheat tangerine, straw, jowar tangerine etc. to make raw material, be crushed to 20 orders with pulverizer and make particle, particle adds 50% water and mixes thoroughly, the high pressure steam of packing into is sprayed and is forced into the 1.2-1.5 MPa in the quick-fried machine, keeps 5-15 minute, reduces to normal barometric pressure suddenly, make particle spray the bulk powdery of quick-fried one-tenth, with this inductive substance---carbon source as cellulase; Use Penicillium penicillium decumbens (Penicillium decumbens AS3.524) [Beijing Microbe Inst., Chinese Academy of Sciences preservation bacterial classification] to make bacterial classification,, go out liquid seeds through the triangular flask shake-flask culture again through the test tube slant bacterial classification.

Substratum: carbon source 2.5-5%

Auxiliary material: soybean cake powder or wheat bran 0.5-0.8% KH ₂PO ₄0.35%

(MH ₄) ₂SO 0.2％ CaCO ₃ 0.5％

Above substratum sized mixing pack in three general fermentor tanks, coefficient 70%, the accent pH value is 5.0-6.0, respectively as first class seed pot, secondary seed jar and fermentor tank, after sterilizing, in first class seed pot, insert seed liquor in the triangular flask by 10% inoculum size, 28 ℃ of-31 ℃ of following ventilations is 1: 0.8-1.0, the fermented liquid that deep liquid was cultivated after 36 hours is access in the secondary seed jar after the sterilising treatment with 10% inoculum size again, deep liquid was cultivated 48 hours under the condition identical with first class seed pot, the nutrient solution that generates is linked in the fermentor tank after the sterilising treatment with 10% inoculum size again, liquid submerged fermentation was finished fermentation in 56 hours under identical condition, remove residue to make feed through Plate Filtration or whizzer separation, be concentrated into 1/10 of original volume behind the filtrate collection, then with the spray-dried cellulase powder of making of concentrated solution.

The measuring method of cellulase activity: with 20mg Xinhua filter paper is substrate, the acetate buffer solution 0.9ml that adds PH4.8,0.1M, the enzyme liquid 0.1ml that adds suitable dilution again, 50 ℃ of reactions 60 minutes, measure the reducing sugar that generates with 3.5-dinitrosalicylic acid method (DNS method), with glucose as a standard sugared, i.e. per minute generation 1ug glucose is an enzyme activity unit.

The inventive method is expected tangerine owing to adopt high pressure steam to spray quick-fried operation to natural cellulose class material such as jade, the wheat tangerine, straw etc. are handled, make that Mierocrystalline cellulose in this type of material and the imporosity between xylogen and the Mierocrystalline cellulose are destroyed, thereby help the decomposition utilization of microorganism, can induce a large amount of cellulases synthetic, the utilization of natural cellulose class material replaces the cellulose powder that involves great expense and makes raw material, can significantly reduce the production cost of cellulase, this process using Penicillium penicillium decumbens AS3.524 (Penicillium decumbens AS3.524) liquid submerged fermentation, the production stability height, pollution microbes not, be convenient to large-scale industrial production, the residue of fermenting process filtering can be used to produce feed again, has overcome the environmental pollution that brings thus.Utilize the enzyme powder of this explained hereafter to be 8000U/g, exceed 20%, produce enzyme powder enzyme with Denmark NOVO company and live close than the enzyme work of domestic solid fermentation through activation.

Fig. 1 is a process flow sheet of the present invention, and wherein 1 is natural cellulose class material, the 2nd, pulverize, the 3rd, the high pressure steam spray is quick-fried, and the 4th, slant strains, the 5th, shake a bottle bacterial classification, the 6th, auxiliary material, the 7th, first class seed pot, the 8th, secondary seed jar, the 9th, fermentor tank, the 10th, strainer, the 11st, filter residue, the 12nd, filtrate, the 13rd, concentrated solution, the 14th, spraying drying, the 15th, cellulase powder.

Known that by figure native cellulose class material 1 is processed into 20 purpose particles and is admitted to high steam spray through pulverizing 2 operations In the quick-fried machine, spray quick-fried 3 operations through high steam and be processed into powder and form culture mediums as carbon source and auxiliary material 6 and add respectively Enter in first class seed pot 7, secondary seed tank 8 and the fermentation tank 9 to carry out sterilization treatment, with penicillium decumbens AS3.524 bacterial classification Carry out test tube slant bacterial classification 4 and triangular flask shaking flask bacterial classification 5 is turned out liquid seeds, insert in the first class seed pot 7 and carry out Submerged fermentation is cultivated, and nutrient solution is inserted carry out the submerged fermentation cultivation in the secondary seed tank 8, takes out then nutrient solution Insert and carry out submerged fermentation in the fermentation tank 9, fermenting completely removes by filter filter residue 11 by filter 10 and is processed to Feed, filtrate 12 is condensed to concentrate 13, and spray-dried 14 operations are made cellulase powder 15 again.

The embodiment of the invention is as follows:

1, high pressure spray quick-fried (corn tangerine particle): vapour pressure 1.2 MPas, 15 minutes time.Substratum: carbon source 5%

Auxiliary material: soybean cake powder 0.5% KH ₂PO ₄0.35%

(NH ₄) ₂SO ₄ 0.2％ CaCO ₃ 0.5％

Transferring pH value after the tinning is 5.0,28 ℃, ventilation 1: 0.8, and inoculation fermentation step by step after the sterilising treatment, fermentation time:

First class seed pot 36 hours

Secondary seed jar 48 hours

After fermentor tank 56 hours fermentation is finished after filtration, concentrate, operation such as spraying drying makes the cellulase powder.

2, high pressure spray quick-fried (straw tangerine particle): vapour pressure 1.4 MPas, 9 minutes time.

Substratum: carbon source 4%, wheat bran 0.6% adds other auxiliary materials with embodiment 1, transfers 5.5,30 ℃ of pH values after the tinning, ventilation 1: 0.9, other technological processs are with embodiment 1.

3, high pressure spray quick-fried (jowar tangerine particle): vapour pressure 1.5 MPas, 5 minutes time.

Substratum: carbon source 5% wheat bran 0.8%

Other auxiliary materials are with embodiment 1.It is 6.0,31 ℃ that pH value is transferred in tinning, and 1: 1 other process of ventilation is with embodiment 1.

Claims

1, a kind of manufacture method of high active cellulase is made raw material with natural cellulose class material, adds soybean cake powder or wheat bran, KH ₂PO ₄, (MH ₄) ₂SO ₄, CaCo ₃For auxiliary material is made substratum, inoculation is carried out secondary seed cultivation and fermentation through the liquid spawn of test tube and triangular flask shake-flask culture after sterilising treatment, it is characterized in that the 20 order natural cellulose class material particles that will pulverize earlier spray quick-fried powdering as carbon source through high pressure steam, spray quick-fried condition: vapour pressure is: 1.2-1.5 MPa, hold-time: 5-15 minute; Use bacterial classification to be Penicillium Penicillium decumbens AS3.524, secondary seed is cultivated and fermentation is all carried out liquid submerged fermentation in three general fermentor tanks of above-mentioned substratum are housed, and the fermentor tank coefficient is 70%, the substratum proportioning:

Carbon source: 2.5-5%

Soybean cake powder or wheat bran: 0.5-0.8% KH ₂O ₄0.35%

(MH ₄) ₂SO ₄0.2% CaCO ₃PH value is 5.0-6.0 in 0.5% 3 general fermentor tank, 28 ℃-31 ℃ of temperature, ventilation 1: 0.8-1.0, first class seed pot fermentation time: 36 hours, secondary seed jar fermentation time: 48 hours, the ferment tank time: 56 hours, after fermentation finishes after filtration, concentrate, the spraying drying operation makes the high active cellulase powder.