CN1665924A

CN1665924A - Hydrolysed n-source

Info

Publication number: CN1665924A
Application number: CN038155850A
Authority: CN
Inventors: 莫根斯·沃普尔曼; 尼尔斯·班克; 索伦·迈克尔森
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 2002-07-01
Filing date: 2003-06-26
Publication date: 2005-09-07
Also published as: AU2003236810A1; WO2004003216A2; AU2003236810A8; EP1520011A2; WO2004003216A3

Abstract

A method for the production of an enzyme of interest, on an industrial scale, comprising a) fermentation of a microbial strain producing an enzyme of interest in a fermentation medium comprising one or more partially prehydrolysed complex N-source(s), wherein said partially prehydrolysed N-source(s) are sterilised separately from any other source containing carbohydrates, the prehydrolysis being achieved by addition of an acid and/or a hydrolytic enzyme; and b) recovery of the enzyme of interest from the fermentation broth.

Description

Hydrolysed n-source

Technical field

The present invention is by add the method for one or more part prehydrolysis compound nitrogen sources in fermention medium, with the more economical required enzyme of mode fermentative production.

Background technology

Be used for industrial fermentation and produce the substratum of useful compound, wherein contain usually just like traditional nitrogenous sources such as soybean, corn steep liquor, yeast extracts.But use these traditional nitrogenous sources that many shortcomings are arranged, as viscosity height when the heat sterilization, starting material change, enzyme is difficult to renaturation and reclaims, produces coloured by product etc.And the cost costliness of these traditional nitrogenous sources, or consume too fastly.

Can replace traditional nitrogenous source by minimal medium, for example method described in the WO 98/37179 is exactly an example, but the shortcoming of this method to be thalli growth slow and production of enzyme is low.

Produce tetanus toxin and described in WO 01/05997 with the substratum that contains hydrolytic soya bean protein, the inventor will help the growth of thalline and the generation of toxin with glucose with remaining substratum autoclaving described in 67 pages.

Brief summary of the invention

The inventor finds, is beneficial to the quick growth of microorganism strains and/or guarantees the high yield of required product in order to ensure amino acid/peptide, just need add the compound nitrogen source of part prehydrolysis in fermention medium, so we wish to protect:

A kind of method of suitability for industrialized production enzyme comprises:

When a) utilizing microbial fermentation production relevant enzyme, the compound nitrogen source that contains one or more prehydrolysis in the fermention medium, wherein the nitrogenous source of these part prehydrolysis should separate sterilization with any other carbohydrate source, and the prehydrolysis of compound nitrogen source can be by reaching to its mode that adds acid and/or lytic enzyme.And

B) from fermention medium, reclaim required enzyme.

Detailed Description Of The Invention

Microbial strains

Used microbial strains can be the microorganism of any kind in this invention.

Preferably can come the production relevant enzyme with bacterium or fungi.

For example, can use some gram-positive microorganism, as genus bacillus, as bacillus alcalophilus, as bacillus amyloliquefaciens, bacillus brevis, bacillus circulans, bacillus coagulans, slow bacillus, Bacillus licheniformis, Bacillus megatherium, bacillus stearothermophilus, subtilis, bacillus thuringiensis; Or streptomyces, as shallow Streptomyces glaucoviolaceus, mouse ash streptomycete etc.; Also can utilize in addition such as Gram-negative bacterias such as intestinal bacteria, pseudomonass.

Equally also can obtain relevant enzyme by fungi, belong to as mycocandida, saccharomyces carlsbergensis (Kluyveromyces) genus, Pichia, saccharomyces, Schizosaccharomyces, Yarrowia, for example saccharomyces carlsbergensis, cereuisiae fermentum, saccharomyces diastaticus, Saccharomyces douglasii, Crewe Vickers yeast, promise ground yeast or Saccharomyces oviformis etc.

Utilize some filamentous funguss also can produce needed enzyme.These filamentous funguss comprise: the mould genus of top spore (Acremonium), and Eurotium, mahogany belongs to (Aureobasidium), genera cryptococcus, Fusarium, Humicola, Magnaporthe, mucor, myceliophthora, Neocallimastix, Neurospora, the Paecilomyces varioti Pseudomonas, Penicillium, cud fungi (Piromyces), Schizophyllum, the Talaromyce thermophilic ascomycete belongs to, Thielavia, Tolypocladium belongs to, Trichoderma etc., particularly, enzyme can derive from microorganism Aspergillus aculeatus, Aspergillus awamori, smelly aspergillus, aspergillus japonicus, Aspergillus nidulans, aspergillus niger, aspergillus oryzae, broomcorn millet sickle-like bacteria, fusarium culmorum, Fusarium graminearum, the withered sickle-like bacteria of fringe, Fusarium oxysporum, Fusarlum roseum, fusarium sambucinum, fusarium sulphureum, the banana sickle-like bacteria, sickle spore bacterium, neurospora, Rhizomucor miehei, the thermophilic silk mould (Myceliophthora thermophila) of ruining, Neuraspora crassa, penicillium purpurogenum, lignin Trichoderma, healthy and free from worry Trichoderma, the long shoot Trichoderma, Li's Trichoderma, trichoderma viride etc.

More than all related bacterial classifications can obtain at common microorganism-collecting center, for example American type culture collection (ATCC), German microorganism and cell are cultivated center (DSM), center (CBS) collected by Dutch fungi yeast and the farming research service is cultivated and the collection center, northern territory research service centre (NRRL) of american agriculture research DSMZ.

In a word, produce required enzyme in order to use this invention, used here term " from ... obtain " be be associated to the source, this just represents that relevant enzyme is that the cell that has inserted the gene in source produces.

Relevant enzyme

Involved enzyme may be certain peptide section or certain enzyme.

First-selection contains 5 to 100 amino acid whose peptide sections (peptide) concerning this paper; It is better successively to contain 10 to 80 amino acid, 15 to 60 amino acid, 15 to 40 amino acid whose peptide Duan Ze.

Specifically, this method need be applied to enzyme, particularly lytic enzyme (enzyme nomenclature is the EC3 class; Recommend according to NK of international biological chemistry alliance).

More specifically recommend following lytic enzyme:

Proteolytic enzyme: the proteolytic enzyme that is fit to derives from and comprises animal, plant or microorganism.Microbe-derived proteolytic enzyme is better, comprises through chemically modified crossing or through the proteolytic enzyme of protein engineering sudden change.Proteolytic enzyme can be aspartic protease, serine protease or metalloenzyme, preferably microbe-derived Sumizyme MP or trypsin-like proteolytic enzyme (tryptase trypsin-like protease).

Proteolytic enzyme and peptase respectively according to enzymic activity in cell＞=or＜10% be defined as self-destructiveness and nondestructive, cell cultures is in acellular broth culture, incubation time is 24 hours, the selection of acellular broth culture potential of hydrogen and temperature value is according to certain numerical value in the fermenting process, and these values are from gathering in the crops preceding 24 hours representational potential of hydrogen during to results and temperature range in the fermenting process.

Acellular broth culture is separated and is obtained by the nonsolute in the meat soup solution (comprising cell) through filtration, centrifugal or similar program by meat soup.

The example of Sumizyme MP is a subtilisin, particularly those originate from the enzyme of gemma rod bacterium, subtilisin Novo for example, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (in WO 89/06279, addressing).The trypsin-like examples of proteases has trypsin for example pig or Niu Qiyuan) and the reaping hook fungi protease, in WO 89/06270 and WO 94/25583, address.

The available examples of proteases is the variant of addressing among WO 92/19729, WO 98/20115, WO 98/20116 and the WO 98/34946, one or more sites below especially having replaced: 27,36,57,76,87,97,101,104,120,123,167,170,194,206,218,222,224,235 and 274.

Available commercially available protein enzyme preferably comprises ALCALASE ^TM, SAVINASE ^TM, PRIMASE ^TM, DURALASE ^TM, ESPERASE ^TM, RELASE ^TMAnd KANNASE ^TM(the letter A/S of Novi), MAXATASE ^TM, MAXACAL ^TM, MAXAPEM ^TM, PROPERASE ^TM, PURAFECT ^TM, PURAFECTOXP ^TM, FN2 ^TMAnd FN3 ^TM(Genencor international corporation).

Peptase: suitable peptase is FLAVOURZYME for example ^TM(the letter A/S of Novi).

Lipase: lipase comprises and derives from bacterium or fungi, also comprises deriving from through the mutant of chemically modified and the mutant of transforming through protein engineering.Lipase can originate from detritus Pseudomonas (have another name called thermophilic fungus belong to) as thermophilic hyphomycete of cotton shape or humicola lanuginosa (EP 258 068 and EP 305 216); Pseudomonas as: produce alkali pseudomonas (WO 96/13580); Class produce the alkali pseudomonas as: onion pseudomonas (EP331 376), (GB 1 for the Si Shi pseudomonas, 372,034), fluorescent pseudomonas, Pseudomonas (WO95/06720 and WO 96/27002) and bacillus are as subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131,253-360), bacstearothermophilus (JP64/744992) or bacillus pumilus (WO 91/16422) etc.

Other example is lipase variation thing, for example described in WO 92/05249, WO 94/01541, EP 407225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Available commodity lipase preferably has LIPOLASE ^TM, LIPOLASE ULTRA ^TMAnd LIPEX ^TM(the letter A/S of Novi).

Amylase: the amylase (α and/or β) that is fit to comprises and derives from bacterium or fungi.Also comprise through chemically modified and crossing or through protein engineering sudden change.For example amylase has the α-Dian Fenmei that obtains from genus bacillus, also has a kind of Bacillus licheniformis of particular variety, sees GB 1,296 for details, 839.

The diastatic example of available sees WO 94/02597, WO 94/18314, WO 96/23873 and WO 97/43424 described variant, one or more sites below especially those have been replaced: 15,23,105,106,124,128,133,154,156,181,188,190,197,202,208,209,243,264,304,305,391,408 and 444.

Available commodity starch enzyme comprises DURAMYL ^TM, TERMAMYL ^TM, FUNGAMYL ^TM, NATALASE ^TM, TERMAMYLLC ^TM, TERMAMYLSC ^TM, LIQUIZYME-X ^TM, BAN ^TM(originating from letter A/S company of Novi), RAPIDASE ^TMAnd PURASTAR ^TM(originating from Genencor international corporation).

Cellulase: the cellulase that is applicable to this purposes comprises bacterium or originated from fungus, comprise wild-type and through chemomorphosis or the prominent body of protein engineering, as bacterium, pseudomonas, humicola lanuginosa (Humicola), sickle-like bacteria, shuttle spore bacterium (Thielavia), straight branch top spore mould etc.US4 for example, 435,307, US 5,648,263, US 5,691,178, US 5,776,757 and Humicola insolens, the Myceliophthora thermophila of WO 89/09259, the cellulase of Fusarium oxysporum preparation.

Particularly the plain enzyme of useful fiber is the alkalescence or the neutral cellulase of those colour developing advantages, comprises that the patent No. is the cellulase of EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940.Other comprises that cellulase variants such as WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299 are also very suitable.

The plain enzyme of available commercial fibre has comprised the CELLUZYME of letter A/S company of Novi ^TMAnd CAREZYME ^TM, the CLAZINASE of Genencor international corporation ^TMAnd PURADAXHA ^TMAnd the KAC-500 of Kao company (B) ^TMDeng product.

Oxydo-reductase:

Available oxydo-reductase of the present invention comprises peroxidase, and oxydase is as laccase, catalase.

Other preferred lytic enzyme is the carbohydrate inversion enzyme, comprises the MANNAWAY of letter A/S company of Novi ^TMAnd pectate lyase (for example BIOPREPARATION 3000 ^TM).Shou Xuan enzyme also has transferring enzyme, lyase, isomerase, ligase enzyme in addition.

Compound nitrogen source:

According to requirement of the present invention, suitable compound nitrogen source is the protein of plant and animal material, and especially those carbohydrate contents are 10% with interior protein, if carbohydrate content 5% or even 3% with interior will be better.

The benefit that carbohydrate content is low just is to avoid plum La Deshi reaction (Maillardreactions).Usually to the substratum that contains primary amine groups compound and reductibility carbohydrate during through the row heat sterilization, the formation of color (plum La Deshi reaction) extremely is unfavorable for reclaiming and/or may suppressing growing.Therefore when heat sterilization, plum La Deshi reaction " partner " separately is controlled at and seem very important in certain scope.It is necessary will separating sterilization with compound nitrogen source such as simple carbohydrate such as glucose, sucrose in other words, and the compound nitrogen source lower protein of those carbohydrate contents preferably, as potato protein, fresh kidney beans protein, hematoglobin protein, fish protein and other animal protein.

Yet the people who is proficient in this field must know that when compound nitrogen source is sterilized the small amount of carbon hydrate does not have big influence to the recovery and the growth of enzyme.Therefore, when carbohydrate and compound nitrogen source are separately sterilized, in compound nitrogen source, can add and be less than 10% carbohydrate.

The people that are familiar with this field know that heat sterilization is different with scale to the influence of the reductibility carbohydrate quantity in the substratum that plum La Deshi reaction might take place.So, whether selected suitable compound nitrogen source and suitable nitrogenous source prehydrolysis condition to estimate according to industrial scale.

Usually the compound nitrogen source that adds prehydrolysis in the fermention medium should be 5% (mass ratio) of Kjeldahl determination (N-Kjeldahl) nitrogen amount at least, specifically should be 10-75% (mass ratio).

Prehydrolysis

The enzyme prehydrolysis of compound nitrogen source is first-selected technology, but this invention also can be finished with the such technology of acid hydrolysis.

Illustrate the performance of the preferred embodiment of prehydrolysis process.

The desirability of prehydrolysis can be as much as possible by correctly adjusting the hydrolytic action temperature, the proteolytic enzyme that adds and/or the quantity of peptase, consider the time that prehydrolysis takes place, selected when the selection of lytic enzyme and prehydrolysis take place in the prehydrolysis process to select proper pH value to realize at interval under the prerequisite of lytic enzyme.

The prehydrolysis degree that needs will depend on Several Factors:

According to from obtaining the viewpoint of high density and high volume production throughput, highly spissated charging substratum has the potential advantage.Therefore, for the formation of stimulating organism amount and/or the formation of product, can prepare by the compound N-source that is not easy to obtain in the ready-made substratum that is present in before the inoculation in the fermentor tank if the compound nitrogen source of the easy utilization of sufficient amount is arranged---gradually between whole yeast phase---, avoid adding through difference disinfectant compound nitrogen source in this charging substratum.Reaching the lasting availability of easily utilizing compound nitrogen source is the target of carrying out prehydrolysis, its can according to the hydrolysis degree that reaches and in culturing process proteolytic enzyme that bacterial strain produces and/or peptase quantity regulate.

According to the throughput that obtains high specific product---promptly, the high product production rate of one viable cell also can be discussed like the application class.

From obtaining the throughput of high efficiency products, when product is a kind of enzyme that makes own inactivation in unit molecule or dimolecular reaction, add medium component in order to avoid to make product self inactivation be very favorable.When add such medium component in fermenting broth the time compound nitrogen source can be such medium component.Therefore, can find that adding such medium component is very favorable in substratum---especially arrive to a certain degree, can pump, when still keeping high degree of protection simultaneously with mammoth pump when this medium component hydrolysis.

It is the suspended state that is used for describing certain solid particulate that term " can be pumped with pump ".It is block that these solid particulates but seldom become in pump, valve and tubing system---and the existence of agglomerate can change feeding speed and surpass 5%.

If involved enzyme is a kind of amylase, a kind of cellulase, lipase, a kind of oxydo-reductase, a kind of carbon lytic enzyme or a kind of nondestructive proteolytic enzyme or peptase, prehydrolysis preferably causes the destruction of peptide bond 10-70%, preferred is between the 15-40% of peptide bond.

If involved enzyme is a kind of proteolytic enzyme or peptase with self-destructiveness, prehydrolysis preferably destroys the 1-20% of peptide linkage content, and more that destructible is the 2-10% of peptide bond.

If involved enzyme is a kind of proteolytic enzyme or peptase with self-destructiveness, as a kind of compound nitrogen source, it is utilizing height protolysate and is only slightly having peculiar advantage during the mixture of protolysate.The degree of prehydrolysis is to producing involved enzyme, and the quantity that adds compound nitrogen source is all explained above, can calculate according to following formula:

[DPH (height hydrolysis) * W (height hydrolysis)+DPH (slight hydrolysis) * W (slight hydrolysis)]/[W (height hydrolysis)+W (slight hydrolysis)];

Wherein

DPH (height hydrolysis) is meant the degree of the prehydrolysis of height protolysate

DPH (slight hydrolysis) is meant the prehydrolysis degree of slight protolysate

W (slight hydrolysis) is meant the weight of slight protolysate in medium

W (height hydrolysis) is meant the weight at medium camber protolysate

Fermentation

The present invention can be used for any fermenting process that meets the industrial production standard.For example can satisfy the fermention medium volume and be at least the different requirements of 50 liters, 100 liters, 500 liters, 1000 liters or even 5000 liters.

These microbial strainss can be fermented according to existing any method.Fermention medium can be the substratum that is mixed by compound nitrogen source and compounded carbons.Fermenting process can be that the bonus point batch fermentation is fermented, flowed to batch-type fermentation, repeated batch formula, repetitive stream adds formula fermentation or continous way fermenting process.

In stream bonus point batch fermentation process, in substratum, do not add usually before the fermentation or only part add those and comprise structure and/or catalysis element.In the process of fermentation reaction, just add all or other comprise structure and/or catalysis element.Select the compound of charging to add together or separately.

In repetitive stream bonus point batch fermentation or continous way fermenting process, add whole initial medium components during the fermentation in addition.Initial substratum can be with the structural unit charging or is separately added.Different is in repetitive stream bonus point batch fermentation process, at set intervals, to remove the culture that contains biomass termly.And in the continous way fermenting process, when bio-reactor added fresh culture continuously, the culture that contains biomass also flowed out from reactor continuously with identical speed with certain speed.This dual mode all is by therefrom taking out the culture of isodose when adding the certain quantity of fresh substratum, keeping the constant of volume of culture.

In the present invention, it is preferred can adopting stream bonus point batch fermentation, repetitive stream bonus point batch fermentation or three kinds of modes of continous way fermentation.

The recovery of useful compound

This invention further relates to the processing of fermenting broth.After fermentation reaction finished, relevant enzyme should adopt different standard techniques to reclaim from fermenting broth according to different separately characteristics.

Below be the example that several products reclaim, but this invention is not limited only to following given example.

Example one

The hydrolysis of Rhizoma Solani tuber osi protein: OPA=51%

In 3.2 kilograms of potatos, add tap water to 12.5 liter, stir Rhizoma Solani tuber osi protein is fully suspended.

In mixing it is heated, temperature is set in 54 degrees centigrade.

When temperature reaches 45 degrees centigrade, the pH value is transferred to 6.0 with the sodium hydroxide solution of 4N.

When temperature arrives 50 degrees centigrade, to the ALCALASE that wherein adds 80 milliliters ^TM2.4L FG (available from letter A/S company of Novi), but must guarantee before this pH value to be maintained 6.0 with the NaOH of 4N.

After the about 5 minutes kinds, temperature rises to 54 degrees centigrade.

Adding ALCALASE ^TMSet the pH value after the about 10 minutes kinds by 6.0 to 8.0.At ALCALASE ^TMAfter adding again 26 fens kinds, stop to drip the sodium hydroxide solution of 4N, add 1.6 kilograms Rhizoma Solani tuber osi protein to it.

Abundant mixing with the Rhizoma Solani tuber osi protein 3 fens kinds that fully suspend, adds 150 milliliters of FLAVOURZYME ^TM(available from letter A/S company of Novi).

Adding ALCALASE after 20 hours, adding water to cumulative volume is 16 liters, contain the proteic suspension of prehydrolysis with these 16 liters and be packed as part, and 4 liters every part, be positioned over-18 degrees centigrade of preservations immediately, albumen prehydrolysis this moment process stops.

How defining albumen prehydrolysis degree (OPA) will explain in example four.Dry matter content is 93% in this example assumes potato protein, and wherein proteinic % content is 80% of dry-matter.

Example two

The hydrolysis of potato protein (OPA=2.9%)

Take by weighing 2.09 kilograms of Rhizoma Solani tuber osi proteins and add tap water to 10.5 liter, Rhizoma Solani tuber osi protein can fully be suspended its stirring.

In the time of mixing to its heating (temperature is set in 55 degrees centigrade).

When temperature arrives 30 degrees centigrade, the pH value is transferred to 6.2 to its sodium hydroxide solution that adds 4N.

When temperature reaches 55 degrees centigrade, add 58.5 milliliters of ALCALASE to it ^TM2.4LFG, and constant by the sodium hydroxide solution maintenance pH value that adds 4N 6.2.

During from adding ALCALASE, its pH set(ting)value becomes 8.0 by 6.2 after 5 fens kinds.

In the kind pH value was reduced to 5.6 by 8.0 at 5 minutes through adding 15% phosphoric acid solution (H3PO4) after 30 fens kinds again, at the Rhizoma Solani tuber osi protein of 1.575 kilograms of addings.

Add tap water to 15 liter then immediately, by every part of 2 liters of packing, it is stand-by to be stored in-18 degrees centigrade of preservations with these suspension that contain protolysate, and this moment, hydrolytic process stopped.

Albumen prehydrolysis degree (OPA) will be explained in example four.Suppose that dry matter content is 93% in the potato protein, wherein proteinic % content is 80% of dry-matter.

Example three

The hydrolysis of potato protein (OPA=19.5%)

Take by weighing 1.2 kilograms of Ma Lingzhu albumen and add tap water to 13 liter, Rhizoma Solani tuber osi protein can fully be suspended its mixing.

In the time of mixing to its heating (setting 55 degrees centigrade).

When temperature arrives 55 degrees centigrade,, add 116.6 gram ALCALASE by making its pH value transfer to 7.0 to its sodium hydroxide solution that adds 4N ^TM2.4L FG also constantly keeps the pH value constant 7.0 to its sodium hydroxide solution that drips 4N.

Count when adding ALCALASE, add tap water to 16 liter after 4 hours, these are contained suspension with protolysate by every part of 4 liters of packing, it is stand-by to be stored in-18 degrees centigrade of preservation, and hydrolytic process stops at this moment.

Proteoclastic degree (OPA) is how to define to explain in example four.Suppose that dry matter content is 93% in the potato protein, wherein proteinic % content is 80% of dry-matter.

Example four

The parsing of OPA, the proteolysis degree

About 1 gram sample (example weight W1) is mixed with the sodium hydroxide solution of 4 milliliters of 0.1N.

Centrifugal mixture to supernatant liquor is clarified, and the deionized water dilution supernatant liquor of using appropriate amount is to the V1 milliliter.

Start time adds 3 milliliters of OPA reaction reagents (as follows), violent mixing.After two minutes (this time is quite accurate), survey the light absorption value (1cm cuvette) under 340 nanometers.

Each sample is all pressed twice of same mode parallel processing.

Average light absorption value should contrast between light absorption value and the standard light absorption value, otherwise will be according to the result again with diluted sample.

Contrast light absorption value (blind): the light absorption value of deionized water

Standard light absorption value: the light absorption value after 50 milligrams of L-leucines add 500 ml deionized water

The OPA reaction reagent:

Take by weighing 7.62 gram disodium tetraborates, 200 milligrams of sodium laurylsulfonates add deionized water approximately to 175 milliliters.In 4 milliliter of 96% ethanol, add 160 milligrams of OPA, and OPA is fully dissolved.Then above-mentioned two kinds of solution are mixed, add 176 milligrams dithiothreitol (DTT) (99%) again and add water final volume is transferred to 200 milliliters.Place and after 4 hours the OPA reaction reagent is discarded.

OPA (proteolysis degree) is calculated by following formula and gets:

((A * (ODav., sample-ODav., contrast)/(ODav., standard-ODav., contrast) *

(V1(ml)×100)/(W1(mg)×P))-B)×100％/(C×D)

A=0.9516=leucine concentration

ODav., sample, the mean value of sample light absorption value under 340 nanometers.

ODav., standard, the mean value of leucine standard light absorption value under 340 nanometers

ODav., contrast, the mean value of deionized water light absorption value under 340 nanometers.

V1 (ml)=dilution volume, unit are milliliter

W1 (mg)=example weight, unit are milligram

Potato protein content percentage ratio in the P=hydrolyzation sample

B=0.4, for potato protein, this numerical value is constant

C=1.0, for potato protein, this numerical value is constant

D=9.1 is for potato protein, and this numerical value is constant

B, C, D are corresponding to the different numerical value of different proteins

Protein	????B	????C	????D
Protein	????B	????C	????D	Soybean	????0.342	????0.97	????7.8
Seitan	????0.4	????1.0	????8.3	Soybean	????0.342	????0.97	????7.8
Seitan	????0.4	????1.0	????8.3	Casein	????0.383	????1.039	????8.2
Meat	????0.4	????1.0	????7.6	Casein	????0.383	????1.039	????8.2
Meat	????0.4	????1.0	????7.6	Fish	????0.4	????1.0	????8.6
Other	????0.39	????1.0	????8.5	Fish	????0.4	????1.0	????8.6

Therefore, the OPA value can well be reacted in the analyzed sample percentage composition of hydrolysising peptide key.

Example five

Microbial strains

The Af50-34 that is used to make proteolytic enzyme in example six, example seven and example eight separates from NCIB 10309 to obtain, and carries out the improved bacterial classification of genetics according to method described in EP 0 506 780 B1.The SJ5262 that is used to make αDian Fenmei in example six, example nine and example ten derives from SJ4671, derives from US 6,100,063.At first, select the spontaneous mutation bacterial classification of anti-Rifampin, wherein the amino acid of RpoB the proteic the 478th has replaced original L-Ala by Xie Ansuan, and this bacterial classification is SJ4671rif10, and has applied for patent in Denmark, and PA 2,001 01972.Then, by the method for the two homologous recombination described in the WO 02/00907, with the coding extracellular protease gene (its protein sequence and gene order are at the AAE00011 that is numbered of GeneSeqP; WO 01/16285; EP482 879) disappearance.

Example six

The propagation program

The Af50-34 bacterial strain:

The B3-nutrient agar

Peptone 6 grams

Stomach en-4 grams

Yeast extract 3 grams

Meat extract 1.5 grams

One DEXTROSE MONOHYDRATE, 1 gram

Agar 20 grams

Add deionized water to a liter, with sodium hydroxide solution or hydrogen chloride solution the pH value transferred to 7.35 then,

121 celsius temperatures sterilization 40 minutes is stand-by.

Finish Deng sterilization, temperature is reduced to 40-50 degree centigrade,

Add 1 mole of every liter of sodium hydrogen carbonate solution (pH value is 9, and volume ratio is 10%) of sterilization after filtration and through the skim-milk (mass volume ratio is 10%) that disposes by deionized water of 121 degrees centigrade of 40 minutes high-temperature sterilizations.

M9-buffered soln

Two hypophosphite monohydrate disodium hydrogens, 8.8 grams

Potassium primary phosphate 3 grams

Sodium chloride 4 grams

Bitter salt 0.2 gram

Add deionized water to 1 liter

121 celsius temperatures sterilization 20 minutes

Seed shake-flask culture base:

PRK-1：

Soybean 50 grams

Two hypophosphite monohydrate disodium hydrogens, 20 grams

Add deionized water to 1 liter, with sodium hydroxide solution or hydrogen chloride solution the pH value is transferred to 9.0 then, it is installed in 500 milliliters of Erlenmeyer flasks with two baffle plates by every part of 100 milliliters of branches.

121 celsius temperatures sterilization 20 minutes is stand-by.

With the Af50-34 bacterial classification on the B3 nutrient agar 37 degrees centigrade cultivate 24 hours after.

With fully suspend thalline and measure light absorption value under 650 nanometers of M9 damping fluid with extinction photometer, get y milliliter (the y value is calculated by OD (650nm) * y=0.1) cell suspending liquid and be inoculated into PRK-1 and shake in the bottle, with substratum be positioned over the rotary shaking table of HT Infors Unitson with 37 degrees centigrade 300 rpms speed cultivate 22 hours stand-by.

Inoculation 80 ml shake flask nutrient solutions can carry out fermentation reaction in each fermentor tank.

SJ 5262 bacterial classifications:

The LB nutrient agar

Peptone (from casein) 10 grams

Yeast extract 5 grams

Sodium-chlor 10 grams

Agar 12 grams

Add deionized water to 1 liter, and the pH value is transferred to 7 (+/-0.2) with sodium hydroxide or hydrochloric acid soln.

121 celsius temperatures sterilization 20 minutes

M9-buffered soln

Two hypophosphite monohydrate disodium hydrogens, 8.8 grams

Potassium primary phosphate 3 grams

Sodium chloride 4 grams

Bitter salt 0.2 gram

Add deionized water to 1 liter

121 celsius temperatures sterilization 20 minutes

Seed shake-flask culture base:

PRK-50：

Soybean sheet (soy flakes) 44 grams

Two hypophosphite monohydrate disodium hydrogens, 2 grams

Add 1 liter of tap water, with sodium hydroxide solution or hydrogen chloride solution the pH value is transferred to 8.0 then, it is installed in 500 milliliters of Erlenmeyer flasks with two baffle plates by every part of 100 milliliters of branches.

121 celsius temperatures sterilization 60 minutes is stand-by.

With SJ 5262 bacterial classifications on the LB agar slant 37 degrees centigrade cultivate 24 hours after.

Measure light absorption value under 650 nanometers with M9 damping fluid suspension gained thalline and with extinction photometer, get y milliliter (the y value is calculated by OD (650nm) * y=0.1) cell suspending liquid and be inoculated in the PRK-50 substratum, with substratum be positioned over the rotary shaking table of HT Infors Unitson with 37 degrees centigrade 300 rpms speed cultivate 20 hours stand-by.

Inoculation is 80 milliliters in each fermentor tank, and just cultured thalline can carry out fermentation reaction.

Example seven

The fermentation reaction of Rhizoma Solani tuber osi protein in substratum with Af50-34 bacterial classification and OPA=2.9%

Fermentation reaction is to carry out in 2 liters the fermentor tank in that 4 baffle plate capacity are arranged, and it is about more than 20% to guarantee that enough ventilation rates make in the whole fermentor tank that the concentration of dissolved oxygen remains on, but ventilation rate can not surpass 2l/l/min.

Temperature is controlled at 37 degrees centigrade, produces in order to prevent foamy, will add the anti-foam oil of q.s at the beginning in substratum.Keep pH value of reactants between 8.0 and 7.7 by the phosphoric acid solution of continuous interpolation 15% or 10% ammoniacal liquor.

Growth medium promptly was added in inoculation in back 0.1 hour, and the speed below keeping.

Apart from time that charging begins (hour): 0 10 200

Substratum adds speed (grams per minute) 0 0.2 0.2

Form (make-up) substratum:

The Rhizoma Solani tuber osi protein of hydrolysis (OPA=2.9%) 100 grams

Potassium primary phosphate 5 grams

Two hypophosphite monohydrate disodium hydrogens, 5 grams

Bitter salt 2.5 grams

Manganous sulfate monohydrate 0.02 gram

Ferrous sulfate 0.08 gram

Salzburg vitriol 0.008 gram

Zinc chloride 0.008 gram

Citric acid 0.39 gram

Sulfur subchloride ammonium 0.05 gram

Riboflavin 0.004 gram

Nicotinic acid 0.03 gram

Pantothenate calcium 0.04 gram

Pyridoxal hydrochloride 0.008 gram

Vitamin H 0.0015 gram

Folic acid 0.004 gram

After regulating pH value to 8.0 with phosphoric acid solution or ammoniacal liquor, add tap water to 1 and liter in each fermentor tank, add 720 milliliters, 121 celsius temperatures sterilization 1 hour.

The charging substratum

The Rhizoma Solani tuber osi protein of hydrolysis (OPA=2.9%) 135 grams

Sucrose 300 grams

Add tap water to 1 liter 121 celsius temperatures sterilization 1 hour

Reactant from fermentor tank, took a morsel in back 49 hours, 71 hours according to the methods analyst of example 11 protease activities wherein in inoculation respectively.

Example eight

With Af50-34 bacterial classification and OPA=51% the fermentation reaction of Rhizoma Solani tuber osi protein in growth medium.

This fermentation reaction, what use in the charging substratum is that degree of hydrolysis is 51% the Rhizoma Solani tuber osi protein, in full accord in all the other and the example seven, when based on deriving from Rhizoma Solani tuber osi protein dry-matter in the hydrolyzate (110g hydrolyzate/l), the amount of used proteolysate among the corresponding embodiment 7 of this quantity.

Example nine

With SJ 5262 bacterial classifications and degree of hydrolysis 19.5% the fermentation reaction of Rhizoma Solani tuber osi protein in fermention medium

Fermentation reaction is to carry out in 2 liters the fermentor tank in that 4 baffle plate capacity are arranged, rotating speed and ventilate must guarantee that enough ventilation rates make that the concentration of dissolved oxygen remains on more than about 20% saturation ratio in the whole fermentor tank, but whenever ventilation can not surpass 2l/l/min.

Temperature is controlled at 37 degrees centigrade, produces in order to prevent foamy, will add the anti-foam oil of q.s at the beginning in substratum.Keep pH value of reactants between 7.5 and 7.0 by the phosphoric acid solution of continuous interpolation 15% or 10% ammoniacal liquor.

Growth medium promptly was added in inoculation in back 0.1 hour, and speed remains unchanged subsequently.

The time of the initial cultivation of distance (hour): 05 200

Substratum adds speed (grams per minute) 0 0.15 0.15

Form substratum:

Rhizoma Solani tuber osi protein hydrolyzate (OPA=19.5%) 187.5 grams

Potassium primary phosphate 5 grams

Dipotassium hydrogen phosphate 5 grams

Two hypophosphite monohydrate disodium hydrogens, 2.5 grams

Bitter salt 2.5 grams

Ammonium sulfate 2.5 grams

Manganous sulfate monohydrate 0.02 gram

Ferrous sulfate 0.08 gram

Salzburg vitriol 0.008 gram

Zinc chloride 0.008 gram

Citric acid 0.39 gram

Add tap water to 1 liter,

In each fermentor tank, add 720 milliliters of fermention mediums that prepare of power, 121 celsius temperatures sterilization 1 hour.

The charging substratum:

1 DEXTROSE MONOHYDRATE, 400 grams

Add tap water to 1 liter 121 celsius temperatures sterilization 1 hour

Reactant from fermentor tank, took a morsel in back 95 hours, 116 hours according to the methods analyst of example 11 protease activities wherein in fermentation respectively.

Example ten

With the fermentation reaction of SJ 5262 bacterial classifications, in forming substratum, use the Rhizoma Solani tuber osi protein of non-hydrolysis

This fermentation reaction is the Rhizoma Solani tuber osi protein of non-hydrolysis except what use in forming substratum, and is in full accord in all the other and the example nine., when based on deriving from Rhizoma Solani tuber osi protein dry-matter in the hydrolyzate of hydrolyzate/not (15g hydrolyzate/l), the amount of used proteolysate among the corresponding embodiment 9 of this quantity.

Example 11

Draw the activity of enzyme in the fermented liquid by analytical calculation

What the mensuration of proteolytic enzyme titre (example seven and example eight) adopted is method the most frequently used in the zymology, i.e. the vigor of enzyme in the assaying reaction product, and the 29-31 page or leaf is described among the WO 89/06279 just can be used for measuring protease activities.

What the mensuration of αDian Fenmei titre (example nine and example ten) also adopted is method the most frequently used in the zymology, i.e. the vigor of enzyme in the assaying reaction product, the described activity that just can be used for measuring αDian Fenmei of 9-10 page or leaf among the WO 95/26397.

Example 12

Enzymic activity in comparative analysis example seven examples eight examples nine examples ten:

Af50-34 bacterial classification/proteolytic enzyme

The Rhizoma Solani tuber osi protein hydrolysate; OPA=2.9% in the charging (example seven)

49,71 hours relevant titres of sampling: 139,130

The Rhizoma Solani tuber osi protein hydrolysate; OPA=51% in the charging (example eight)

The relative titre at 49,71 hours: 100,68

(all titre values all with the titre value of 49 hours samples in the example eight as standard.)

SJ 5262/ αDian Fenmei

The Rhizoma Solani tuber osi protein hydrolysate; OPA=19.5% in the composition (example nine)

The relative titre of taking a sample at 95,116 hours: 111,130

The non-hydrolysate of Rhizoma Solani tuber osi protein (example ten)

The relative titre of taking a sample at 95,116 hours: 100,117

(all titre values all with the titre value of 95 hours samples in the example ten as standard)

Can draw according to above-mentioned analysis: when the compound nitrogen source (potato) with prehydrolysis came the fermentation production of protein enzyme, lower prehydrolysis degree helped the formation of proteolytic enzyme.When the compound nitrogen source (potato) with prehydrolysis came the fermentative production αDian Fenmei, fully the compound nitrogen source of prehydrolysis also helped the increase of alpha-amylase activity very much, and its major cause is that the compound nitrogen source degree of prehydrolysis can better be absorbed by microorganism.

Claims

1. method with the industrial-scale production enzyme comprises:

A) produce needed enzyme with the fermention medium that contains one or more prehydrolysis compound nitrogen source by the mode of microbial fermentation, these compound nitrogen sources will separate sterilization with other nutrition sources of carbohydrate containing.In compound nitrogen source, add acid or lytic enzyme to reach the purpose of described prehydrolysis;

B) from fermentation culture, reclaim required enzyme.

2. method described in the claim 1, enzyme wherein comprises amylase, cellulase, lipase, oxydo-reductase, carbohydrolase and nondestructive proteolytic enzyme or peptase.

3. method described in the claim 1, enzyme wherein are can self-destructive proteolytic enzyme or peptase.

4. method described in the claim 1, microorganism wherein can be selected bacterium or fungi for use.

5. method described in the claim 4, bacterium wherein is a genus bacillus.

6. method described in the claim 1, compound nitrogen source wherein is to contain the plant origin albumen that is less than 10% carbohydrate.

7. method described in the claim 1, compound nitrogen source wherein is selected from Rhizoma Solani tuber osi protein or Semen Pisi sativi protein.

8. method described in the claim 1, compound nitrogen source wherein is to contain the protein for animal that is less than 10% carbohydrate.

9. method described in the claim 1, compound nitrogen source wherein is selected from blood protein, fish mytolin and animal muscle albumen.

10. method described in the claim 2, compound nitrogen source prehydrolysis wherein destroys the peptide bond of 10%-70%.

11. method described in the claim 3, compound nitrogen source prehydrolysis wherein destroys the peptide bond of 1-20%.

12. calculating by weight, method described in the claim 1, the content of prehydrolysis compound nitrogen source wherein reach at least 5% of whole kjeldahl nitrogen total amounts of adding substratum.

13. method described in the claim 1, the amount of fermention medium wherein are 50 liters at least.

14. method described in the claim 1, fermentation wherein are that the fermentation of repeated batch formula, stream add the formula fermentation, repetitive stream adds formula fermentation or continous way fermentation reaction.