CN1252438A - Inulinase generating saccharomycetes strain and its application in producing high fructure syrup - Google Patents

Abstract

The present invention provides an inulinase-generating saccharomycetes strain, the inulinase generated by the saccharomycetes strain and the production process of high fructose syrup by utilizing this kind of saccharomycetes strain to and the inulinase secreted by the saccharomycetes to degrade inulin. The present invention can raise yield of inulinase, shorten the fermentation peak period, and reduce the production cost of inulinase.

Description

A kind of yeast strain and application in making high fructose syrup thereof that produces inulinase

The present invention relates to a kind of yeast strain that produces inulinase; Inulinase by the yeast generation; By the method for yeast strain high density fermentation production inulinase, and the method for producing high fructose syrup by described inulinase glycosylated degradation inulin.

, flavor inexpensive because of it by the sucrose that glucose and fructose are formed is sweet widely to be used in food, beverage, pharmaceutical industries as sweeting agent, in still less-developed epoch of human material conditions and area, sucrose with its high-energy and high sugariness at additional body energy and satisfy the seasoning demand side and played the crucial effect that also still plays.

Along with The development in society and economy progress, human living standard's raising, the nutrition drawback of sucrose, glucose displays day by day.A large amount of nutrition and medical research show: long-term edible sucrose causes obesity, diabetes, carious tooth, hypertension and cardiovascular disorder etc. easily.Some national nutritionist even the suggestion that has proposed to limit the use of or forbid sucrose.Therefore, research, the new sweeting agent substitute of searching are heat subjects always.

Fructose is the sweetest natural sweeteners of nature, and its sweetness ratio sucrose is high by 80%.Single from function as sweeteners and economic benefit, fructose obviously is better than sucrose.Also the sugariness than the special-purpose sweeting agent sorbyl alcohol of present widely used diabetes food also exceeds 50%.

Fructose also has than glucose, obvious superior health care and the nutritive effect of sucrose.The phosphorylation of fructose in the human liver do not resemble the control that is subjected to Regular Insulin the glucose, directly enters the glycolysis-tricarboxylic acid cycle after most fructose phosphorylations, and fructose is not only the active and effective carbon nutrition of diabetic subject source; Edible fructose can also avoid bringing out diabetes, and blood sugar peak and hypoglycemia are avoided producing lactic acid and caused sore muscle and tired sense and childhood caries tooth disease and obesity after meal.

In addition, fructose has better performance of keeping humidity and better solubility property because of its osmotic pressure that is higher than 1 times of sucrose, and this can bring the advantage of producing with application facet.

Therefore, the research and development of fructose resource are significant.High fructose syrup is the especially main products of the edible and medical fructose of developing country of majority state always.A generation, two generations and three generation products have been gone through in the production of high fructose syrup, be respectively high fructose syrup (FGS), rich high fructose syrup (RFS) and high fructose syrup (HFS), the core mass index sign of three generation products is that fructose content reaches respectively more than 42%, 55% and 90% in the product, and its corresponding production technology feature division is as back:

1) FGS: starch → glucose → glucose+fructose, from the substrate to the product, all finish by a series of enzymic catalytic reaction, product fructose contains theoretical value=＜50% of rate, and this index is generally 42% in the production practice, and this approach at home and abroad widely adopts;

2) RFS: starch → glucose → glucose+fructose → fractionation by adsorption goes out fructose → remaining glucose → glucose+fructose, and the end product fructose content can reach 55%; Because the RFS products production need not newly to add any equipment, there is a lot of high fructose syrup factory newly to go up s-generation high fructose syrup product RFS successively in recent years both at home and abroad, with generation product co-production.

3) HFS: polyfructosan → oligofructose → fructose, end product fructose content＞90%, only contain glucose, in the piece root of polyfructosan substrate from the feverfew of being rich in polyfructosan such as jerusalem artichoke (Jerusalem artichokd), witloof (Chicory) less than 5%.The biochemical basis of HFS production approach is obviously different with preceding two kinds of fructose products with corresponding technology, more simple comparatively speaking, preceding two kinds of fructose FGS need starch degrading enzyme more than three kinds, 6 carbon sugar isomerase and corresponding different technical process section at least with RFS production, and HFS only needs a kind of inulinase, is finished by a step enzymic catalytic reaction from the substrate to the product; Since there is not the scale production of polyfructosan degradation enzyme system at present, the necessary self-produced inulinase of manufacturer, corresponding product enzyme and two process sections of enzymolysis of just having required.Because raw material is cheap and easy to get, technology is simplified relatively, the product fructose content than be higher than far away one, two generation product, improve nearly 1 times, so this is an approach producing the most promising and magnetism of fructose.But the bacterium producing multi enzyme preparation inulinase yields poorly in the prior art, produces enzyme cost height.

Result of study about the inulinase zymologic property shows: the mode of cutting polyfructosan according to the inulinase enzyme is divided into circumscribed and endoinulase, obtain fructose and fructose and oligosaccharide mixture after the effect respectively, known a kind of molecular weight 53KDa from the thick reagent of aspergillus (Aspergillus) endoinulase commercial enzyme, Km=19mM, and with a kind of circumscribed inulinase molecular weight 81KDa of source, Km=60mM, restriction endonuclease and excision enzyme Km reduce to 6 and 10mM respectively behind the purifying; Fe ³⁺Can make the endoinulase activity of purifying improve 20 times, Mn ⁺And Mg ⁺The activity that then can partly suppress these two kinds of enzymes; A lot of inulinases have invertase activity nearly all simultaneously, the inulinase of endo-type enzyme compares I/S=1/0.2 with invertase activity, the I/S=1/8 of excision enzyme (Rosa Azhari, Alada M.Szlak, EhudIlan, Samuel Sideman, and Noah Lotan., Purification andcharacterization of endo-and exo-inulinase Biotechnology andApplied Biochemistry, 1989,11:105-117.); In the time of more earlier, it is 60 ℃ that report aspergillus inulinase optimal reactive temperature is just arranged, and optimal reaction pH value is 4.4 (Starch.1981,33:373-377, Enzymatic hydrolysis of inulin-analternative way to fructose production, L.Zittan); According to another report, a kind of aspergillus niger of inulinase that produces is under the shaking table level, 30 ℃ of * 140rpm, reach 27U/ml to the work of the 5th day (120h) enzyme, the work of the 7th day (168h) enzyme peaks and is 48.4U/ml, biomass reaches 28.8g/l (Kazuyoshi Ohta, Shigeyuki Hamada, and Toyohiko Nakamura.Production of high concentrations of ethanol from inulin bysimultaneous Saccharification and fermentation using Aspergillusniger and Saccharomyces cerevisiae.Applied and EnvironmentalMicrobiology, 1993,59:729-733).Report is thought recently: a kind of aspergillus niger that produces inulinase is shaking under bottle level, 28 ℃ of * 200rpm, inulinase rises to 48U/ml from 0 straight line between 0-120h, then keep stable afterwards, reach the peak value of 50U/ml to the 7th day (168h),＞55 ℃ temperature tolerance is relatively poor, under 60 ℃ of * 1h, residual activity only under 78%, 70 ℃ of * 1h enzyme work to be almost inulinase transformation period of 0,60 ℃ be 71min; Optimal reaction pH value is 5.0, and inulinase stability is fine in the pH5.0-5.8 scope; Na ⁺Can make enzymic activity improve 15%, Ca ²⁺, Zn ²⁺, Cu ²⁺, Fe ³⁺There is not obviously influence, Ba ²⁺, Mn ²⁺, Pb ²⁺, Ag ¹⁺Make enzymic activity (the Gaye Ongen-Baysal that significantly descends, S.Suha Sukan, Nikolay Vassilev., Productionand properties of inulinase from Aspergillus niger.BiotechnologyLetters, 1994,16:275-280).

Another focus bacterial classification that produces inulinase is exactly that kluyveromyces Kluyveromyces. it is reported that Kluyveromyces marxianus CBS6556 can secrete inulinase, the inulinase molecular weight 64KDa that produces, degree of glycosylation reaches 26-37%, the inulinase of secreting to the substratum is a dimer, with the inulinase of cell wall-bound be the tetramer (Rouwenhorst R.J.et al.Structure andproperties of the extracellular inulinase of Kluyveromycesmarxianus CBS6556, Applied and Environmental Microbiology 1990,56:3337-3345.), the suitableeest growth of this bacterial strain is 40-45 ℃ with producing the enzyme temperature, in the fermentor tank of 1L, adopt inorganic medium to carry out cultured continuously, the culture medium C source is restricted to 0.25% sucrose, and fermentation parameter is pO ₂Be controlled at 50-70%, by 1MKOH and 0.5MH ₂SO ₄Control pH4.5, under the dilution rate of 0.1/h, biomass is up to 1.05mg (DW)/ml, the highest extracellular enzyme work reaches 32U/ml, I/S=1/15, extracellular enzyme and intracellular enzyme ratio are roughly according to incubation time: 50-65%/50-35%, the optimal reactive temperature of enzyme are 50 ℃, and optimal reaction pH value is 4.5-5.0; Remaining sucrose concentration and production of enzyme are negative correlation (Rouwenhorst R.J.et al., 1988) in the substratum.Another kind of Kluyveromyces marxianus NCYC587 is in the shaking table condition, 28 ℃ of * 200rpm, the inulinase activity reaches 34.5U/ml when cultivating 120h, I/S=1/0.6 (Ongen-Baysal G and Sukan S S.Production of inulinase nymixed culture of Aspergillius niger and Kluyveromyces marxianus.Biotechnology Lett.1996,18:1431-1434).The Kluyveromyces bacterial strain that latest domestic report is a kind of to produce inulinase is by extractum carnis, corn steep liquor, on organic substratum that urea and jerusalem artichoke juice are formed through shaking a bottle condition of enzyme production orthogonal test gained result: the highest inulinase extracellular enzyme is active to be 15U/ml, then test gained the highest extracellular enzyme output is respectively 17.63 and 18.7U/ml on 15L and the homemade fermentor tank of 1000L, the distribution proportion of intracellular enzyme and extracellular enzyme is 0.2561: 0.7439 (Wei Wenling, Zheng Zhonghui, Zheng Zhicheng etc., the research of the synthetic inulinase optimum condition of kluyveromyces Y-85, the microorganism journal, 1998,38:208-212; The screening of Zheng Zhonghui, Liu Yueying, Cai Wenzheng, Wei Wenling, Zheng Zhicheng, inulinase high dynamic strain and the research of fermentation condition, Xiamen University's journal 1993,32:360-364.).Do not measure because of the inulin solvability is relatively poor about kluyveromyces inulinase Km value is many, have only NegoroH report Km measured value be 7.4mM (Purification andcharacterization of inulinase from Kluveromyces fraglis.J.Ferment.Techno1978,1.56:102-107).Reports such as Wei Wenling are about 14.0-15.7mM.

Molecular biology research about the polyfructosan degradation enzyme system starts from the nineties, alantin excision enzyme (EC 3.2.1.7) gene inul and order-checking were cloned into first in Belgian Vandenhaute group from Crewe Wei Makesi yeast Kluyveromyces marxianus in 1991, by 555 AA of 1.668Kb coding, reach 67% with the saccharase homology; Endo-inulinase (EC 3.2.1.7) gene inuA and order-checking are cloned into first in the Onodera group of Japan in 1996 from mould (Penicillium purprogenum), the structure gene of finding this enzyme contains 1548bp, 515 amino acid of encoding, wherein 25AA is a signal peptide, 490AA is a maturing enzyme albumen, and molecular weight is 54KDa.With E.coli is that the expression preliminary study that the host bacterium is done shows, the zymoprotein output that inuA expresses is very low, almost detect less than; Find by the comparative analysis dna nucleotide sequence: mould (Penicilliumpurprogenum) endo-inulinase gene and Crewe Wei Makesi yeast (Kluyveromycesmarxianus) alantin excision enzyme gene, be respectively 27%, 29% and 39% with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) invertase gene with the homology of subtilis (Bacillius subtilis) levanase.Illustrate that endo-inulinase gene and above-mentioned enzyme dna homolog are all lower.In a word, structure inulinase high-yielding engineering bacterial strain and the further good natural bacterial strain of inulinase high yield of screenability all are very necessary.

The applied research for preparing HFS about inulinase degraded inulin is more, it is reported, the aspergillus inulinase carries out DeR with 2U/g (inulin) under 60 ℃, the 24h degradation rate reaches 86.7%, 48h reaches 99.2%, Shandong dimension yeast inulinase carries out DeR with 6U/g (inulin) under 55 ℃, the 1h degradation rate reaches 30.6%, 24h reaches 82.2%, 48h reaches 89.6% (Starch.1981,33:373-377, Enzymatic hydrolysis of inulin-an alternative way tofructose production, L.Zittan), the aspergillus niger inulinase is stabilized in 12% at jerusalem artichoke juice degradation rate between 1-6h of 50 ℃ of sugar degrees 16% of degrading down according to another report, degraded product is the mixture of monose and oligofructose, and behind the kluyveromyces inulinase degraded 4h, degradation rate reaches 43%, and product is based on fructose (Gaye Ongen-Baysal, S.Suha Sukan, 1996).Restriction endonuclease degraded 60 behind the aspergillus niger commodity inulinase crude preparation by using purifying, 120, degradation rate reaches 36.7 respectively behind 240mM (glycosidic link) the inulin 4h, 28.3 with 21.7%, degradation rate reaches more than 90% behind the 24h, GF4-GF6 accounts for more than 80% in the product, and G, F and sucrose account for 8%, and synanthrin accounts for 8%, and excision enzyme is used to degrade 60,120 and 240mM (glycosidic link) inulin 4h after degradation rate reach 20 respectively, 12.5 with 10.4%, degradation rate reaches more than 90% behind the 24h, oligofructose 6% in the product, F accounts for more than 85%, synanthrin accounts for 8% (Rosa Azhari, Alada M.Szlak, Ehud Ilan, SamuelSideman, and Noah Lotan.Biotechnology and AppliedBiochemistry, 1989,11:105-117.Purification and characterizationof endo-and exo-inulinase).According to another report, mould (Penicillium purprogenum) endoinulase, degraded inulin product is with F3, F4 and F5 oligofructose are main, degradation rate is 32%, the endoinulase of another kind of mould be used to degrade inulin before 24h product with GF2, GF3, GF4 is main, degradation rate reaches 50%, reaches 70% to the 72h degradation rate, and product is based on F3 (NakamuraT, Shitara A, Matsuda S., et al., 1997, Production, purification and properties of an endoinulinase of Penicilliumsp.TN-88 that liberates inulotriose.J.Ferment.Bioeng.84:313-318).In a word, use in the prior art inulinase degraded jerusalem artichoke produce HFS comparatively the ideal condition be: 10-15% inulin concentration, 2-5U INU (pure inulinase)/g (inulin), 50 ℃, 15-20h.The endo-inulinase degraded product is based on oligofructose, and the excision enzyme degraded product is based on fructose.

At present about many than the yeast inulinase of the research of aspergillus inulinase, and the aspergillus inulinase rate ratio zymic of having reported is also high.But it is considered herein that,, especially be applied to be inferior to kluyveromyces when fermentation industry is produced greatly at aspergillus aspect the industrialization no matter bypass concrete enzyme.

At first, aspergillus carries out suitability for industrialized production by liquid fermenting has two problems effectively to solve, and the one, thalline and enzyme liquid separation difficulty; The 2nd, the product enzyme cycle is oversize, and enzyme is lived the peak period appearance in 5-8 days, and this is very uneconomic on the fermentation industry cost; Second, the the nourishing and growing of mould is what the elongation growth by the mycelium tip realized, this growth characteristics especially are fit to solid culture, and and be not suitable for the liquid state fermentation that modern zymotechnique is used always, generally speaking the solid fermentation work efficiency is low, cycle is long, the cost height, and the liquid state fermentation that the cost that obtains the bulk fermentation product is compared will increase a lot.By contrast, the very suitable liquid state fermentation of yeast cell reproductive characteristic; The 3rd, the mould nourishing body needs to provide enough complicated carbon nitrogen organic nutrients during growing, this also can increase fermentation costs, growing of kluyveromyces nourishing body can utilize cheap simple nutrient inorganic salt, sucrose and ammoniacal liquor etc., and the nourishing body kluyveromyces that obtains isodose is wanted considerably cheaper than the nutrient that mould consumes.Zymic high-cell density, low-cost fermentation process set up (Siegel R.S., Biotechnol.Bioeng, 1989,34:403-404), employed carbon source, nitrogenous source, salt, trace element and VITAMIN etc. are all very cheap in the fermention medium; The 4th, the distinctive musty of mould can influence the local flavor of inulinase and degraded product thereof, and the kluyveromyces fermentation does not have this drawback; The 5th, kluyveromyces contains the organic compound that multiple a large amount of promotion is grown, and as oligose, Nucleotide, each seed amino acid, small peptide etc., has many-sided DEVELOPMENT PROSPECT.These advantages all be mould less than or do not have.

For improving inulinase output and shortening and produce the enzymic fermentation peak period, solve the problem that inulinase yields poorly, cost is high in the HFS production practice, an object of the present invention is to screen a kind of new kluyveromyces yeast strains.

The inventor has screened a kind of kluyveromyces marxianus bacterium (Kluyveromycesmarxinus) bacterial strain, IW9801, and in China Committee for Culture Collection of Microorganisms common micro-organisms center (China, Beijing, the Zhong Guan-cun, 100080) carried out preservation, preserving number is: CGMCCNO.0360, preservation date is: on September 23rd, 1998.

Another object of the present invention provides a kind of inulinase that is produced by described yeast strain.It is 13.3mM that inulinase of the present invention has following feature: Km (synanthrin), Km (sucrose) 62.6mM, I/S=1/24.72; Optimum pH is 4.4, and has all kept the residual enzyme activity more than 90% in the pH3.8-6.4 scope; Optimal reactive temperature is 55 ℃, and higher inulinase reactive behavior is all arranged in 50-57.5 ℃ of scope, and the transformation period under 50 ℃ is 16 hours; Magnesium ion can improve inulinase activity 11.28%.Inulinase of the present invention is present in the fermented liquid after producing the enzyme yeast fermentation, with fermented liquid and the enzymolysis that can be directly used in inulin after the yeast thalline separates, if desired, also can as column chromatography, separate from fermented liquid with inulinase by ordinary method.

A further object of the present invention provides a kind of method (unless stated otherwise, the used per-cent of the present invention all is weight percentage) of producing inulinase by the yeast strain high density fermentation, comprising:

(1) be nitrogenous source with inoculation to containing 0.6-1.4% (weight) nitrogen compound, 0.8-1.2% organic carbon compound is in the inorganic salt basic medium of carbon source, at 25-40 ℃ of following aerated culture, makes pO ₂＞25%, pH4-5;

(2) be cultured to 4-7 hour, work as pO ₂Value begins stream and adds and replenish material when rising suddenly, it is identical with basic medium to replenish material, and just wherein macroelement content increases 5-10 doubly, and micronutrient levels increases 10-30 doubly, and carbon source content increases 30-50 times;

(3) stream adds in the additional material process, makes pO all the time ₂＞30%, pH4-5, a preceding 5-30 hour accelerating flow adds, latter stage 30-70 hour at the uniform velocity stream add, final fermentating liquid volume increases 40-80%, thalline biomass weight in wet base reaches the 150-300g/ liter;

(4) enzyme liquid is separated from thalline.

In above-mentioned fermentation process, preferably use sucrose or raffinose or glucose or jerusalem artichoke juice to be sole carbon source, use inorganic nitrogen compound or urea to be culture media nitrogen source.And preferred carbon source is a sucrose, and nitrogenous source is an ammonium sulfate.

In above-mentioned fermentation process, basic medium and additional material composition main reference Shau L.K and G.H.Wegner method (J.Dairy Sci., 1986,69:676～683), and improved.The one, substratum concentration is increased by 1 times, the 2nd, increase vitamins nutrition and EDTA such as vitamin H, nicotinic acid and calcium pantothenate, the 3rd, change the carbon source whey into sucrose.C source concentration is 0.8-1.2% in the preferred basic medium; Stream adds and replenishes that C source concentration is 35-60% in the material, and adds at stream and to keep in the process that C source concentration is 1.5-3.0% in the fermented liquid.Wherein more preferably in the basic medium C source concentration be 1.0%; Stream adds and replenishes that C source concentration is 50% in the material, and adds at stream and to keep in the process that C source concentration is 2.5% in the fermented liquid.

In this fermentation process, the range of choice of carbon source and nitrogenous source is mainly based on producing enzyme effect and this two aspects factor of raw materials cost.If carbon source concentration is lower than 0.8%, then be not enough to satisfy the demand of bacterial strain early growth breeding, delay logarithmic phase, if be higher than 1.2%, then can produce tangible product enzyme repression effect, promptly be unfavorable for producing enzyme, also cause the waste of C source.

In above-mentioned fermentation process, the preferred culture medium loading amount is the 40-60% of jar effective cumulative volume; Stream adds to replenish expects that volume is the 40-80% of basestocks volume.More preferably the substratum loading amount is 50% of jar effective cumulative volume; Stream adds to replenish expects that volume is 70% of a basestocks volume.

In above-mentioned fermentation process, in the preferred fermenting process pH is controlled at 4.5, with ammoniacal liquor control pH value.

In above-mentioned fermentation process, described minimal medium can use and be generally used for yeast-leavened minimal medium, but preferred is nitrogenous source except that containing 0.6-1.4% (weight) nitrogen compound, 0.8-1.2% organic carbon compound is beyond the carbon source, preferably at least also contain 0.4-0.7% phosphoric acid salt, the 0.8-1.2% magnesium salts, copper, zinc, cobalt and the salt compounded of iodine of the iron of 1.5-4.0ppm, manganese salt, borate, vitamin substances and 0.2-1.10ppm.

In above-mentioned fermentation process, preferably with the pO in the fermenting process ₂Value is controlled at 25-50%, is preferably 30-40%, and rotating speed is 450-1000rpm.

In above-mentioned fermentation process, preferably the thalline weight in wet base is controlled at 180-300g/L, be preferably 230g/L.

In above-mentioned fermentation process, preferably make in the fermented liquid the outer inulinase activity of the highest born of the same parents reach 45-75U/ml, and leavening temperature is preferably 38 ℃, the outer inulinase activity of the highest born of the same parents reaches 60U/ml in the fermented liquid.

In fermentation process of the present invention, the apparent foundation of make-up stream acceleration is pO ₂Value＞30%, the essence foundation is thalline biomass and fermented liquid air flow, early stage in fermentation, when air flow one timing, replenish material thalline biomass and the proportional example of flow acceleration,, fermenting the later stage when the biomass weight in wet base reaches 120-180g/L so take accelerating flow to add measure, biomass increases along with the increase of stream dosage and reduces, and stream adds measure so take at the uniform velocity.At fermentor tank rotating speed one regularly, pO ₂If be worth too high (＞50%), substratum nutrient deficiency is described, should strengthen the make-up stream acceleration, otherwise, pO ₂If be worth too low (＜30%), illustrate that the prosperous excessively thalline of growth and breeding is too much with oxygen depletion in the substratum, thereby force thalline when oxygen is not enough, to change anaerobic fermentation over to, carbohydrate is converted into alcohol, enzyme is unfavorable in producing, and also unfavorable in the thalline survival, should reduce the make-up stream acceleration this moment.What of make-up stream dosage are also relevant with leavening temperature, in the time of 25-32 ℃, always flow the 35-45% that dosage is equivalent to the basestocks volume, in the time of 35-40 ℃, always flow the 55-80% that dosage is equivalent to the basestocks volume.

The fermentation medium speed is actually high more good more, therefore should take all factors into consideration the rotating speed that factors such as equipment performance, energy consumption efficiency, yield of enzyme and product enzyme efficient preferably determine the air flow size.

By ammoniacal liquor control pH4.5, under the normal fermentation condition of this bacterial strain, secrete more organic acid and make medium pH value＜3.0 in the fermenting process, this at first is unfavorable for the generation of inulinase, also is unfavorable for the thalline breeding.Ammoniacal liquor is easy to control pH4.5 on the one hand, can also replenish an amount of N source on the other hand.

Usually, unit fermentation broth enzyme output and thalline weight in wet base are proportional, but the inulinase ultimate production in the unit fermentor tank and the relation of thalline weight in wet base are complicated, in 0-250g/L thalline weight in wet base scope, and the two proportional example relation,＞300g/L thalline weight in wet base, total production of enzyme increases and reduces, and until zero growth rate even negative growth occurring, causes the unit thalline to produce enzyme and reaches to greatest extent, fermentation raw material produces the enzyme decrease in efficiency, and producing the enzyme cost increases.Therefore, the fermentation process in high density among the present invention shows: based on the purpose of total inulinase output that obtains maximum and best product enzyme economic benefit, the preferable range of cell density is 180-300g/L, the suitableeest 230g/L.

Fermentation process in high density of the present invention not only can be used for yeast strain CGMCC0360 IW9801 of the present invention, also can be used for yeast strain.

Another purpose of the present invention provides the method that a kind of inulinase degraded inulin that utilizes method of the present invention to produce is produced high fructose syrup, comprise: thalline is with after enzyme liquid separates, inulinase liquid is mixed according to 1: 8～17 volume ratio with the inulin solution of sugar degree 8-17%, at 45-55 ℃ of following enzymatic saccharification 30-40 hour, obtain high fructose syrup.

In aforesaid method, the optimum condition of inulinase degraded inulin is: synanthrin concentration 15%, and enzyme liquid and 8-17% inulin liquid blending ratio are 1: 15, and hydrolysis temperature is 50 ℃, and enzymolysis time is 36 hours.

During the glycosylated degradation inulin, inulin concentration＞17%, having surpassed the inulin maxima solubility causes saccharifying tank to have the inulin precipitation to separate out, naturally thoroughly saccharification, if inulin concentration＜8%, it is no doubt good and fast to dissolve fully with saccharification in inulin, but certainly will increase the saccharifying tank volume like this, increase concentrated cost, finally raise product cost.Therefore the preferred saccharification inulin of the present invention concentration is 15%.

Bacterial strain of the present invention reaches 64.43U/ml in the highest inulinase output of 6.6L fermentor tank.The aspergillus inulinase production peak that the inulinase rate ratio is reported the nineties in the world is that 50U/ml (Ongen-Baysal et al.1996) is high by 29%, and is higher by 74% than kluyveromyces inulinase production peak 37U/ml (GrootWassnik et al.1980).

Can shorten the time of fermentation period and the appearance of inulinase output peak with yeast strain of the present invention, the present invention adopts the yeast fermentation process in high density, in fact the strain enzyme-producing peak is promptly occurred at 60h, with this time of aspergillus comparison in advance or shortened 60-120h, compare with the kluyveromyces shake flask fermentation in advance or shortened 48h (table 1).

Can reduce fermentation costs with method of the present invention.Because the preferred inorganic medium that adopts of strain enzyme-producing fermentation of the present invention, so fermentation costs is than organic substratum low about 30%, moreover, adopt inorganic medium to also have two other advantage: the one, be difficult for producing foam, reduce froth breaking cost and difficulty, help ventilation, the 2nd, be difficult for pollution microbes.Add because fermentation period shortens dramatically, so product cost is lower at least more than 1 times than the best level of the research nineties.

Bacterial strain inulinase optimal reactive temperature of the present invention is 55 ℃, and is good at 50-57.5 ℃ of scope internal stability, can keep greater activity (Fig. 7).。The temperature tolerance of inulinase of the present invention under 50 ℃ still can, the transformation period of 50 ℃ of following enzymes is about 16h, the residual enzyme activity has 83.02% behind the 6h, still has 53.74% behind the 12h, still keeps 32.09% (Fig. 9) to 36h.Mg ²⁺Improving active 11.28% (table 3) finds first for bacterial strain inulinase of the present invention.As a kind of measure that improves existing enzymic activity is with practical value.

Table 1. the present invention and inulinase superior strain of the prior art compare with product enzyme result

Bacterial classification	The C source	The N source	Training method	Peak times (h)	Extracellular enzyme activity (U/ml)	Document
							Aspergillus niger Aspergillus niger Aspergillus phoenicis Kluyveromyces fragilis Kluyveromyces marxianus Kluyveromyces marxianus Kluyveromyces Y-85 Kluyveromyces IW9801	Sucrose corn steep liquor jerusalem artichoke inulin inulin jerusalem artichoke powder inulin corn steep liquor, jerusalem artichoke juice sucrose	Peptone+inorganic nitrogen yeast culture+inorganic nitrogen yeast culture+inorganic nitrogen yeast culture inorganic nitrogen inorganic nitrogen beef extract, the urea inorganic nitrogen	Shaking flask shaking flask shaking flask 5L fermentation tank shaking flask 1L fermentation tank 1000L fermentation tank 6.6L fermentation tank	168 120 192 chemostats, 120 chemostats 30 72	48.4 50 36 37 34.5 32 18.7 62.43	Ohta et al.1993 Ongen-Baysal et al.1994 Peters et al.1983 Groot Wassnik et al.1980 Ongen-Baysal et al.1996 Rouwenhorst et al.1988 Wei Wen bell etc.; 1998 the present invention, 1998

Four, Brief Description Of Drawings:

Fig. 1 .Kluyveromyces IW9801 time curve that inulinase and biomass increase in shake flask fermentation

Fig. 2 .Kluyveromyces IW9801 is inulinase and protein secreting amount in 32 ℃ of fed-batch fermentations, the time curve that biomass increases

Fig. 3 .Kluyveromyces IW9801 is inulinase and protein secreting amount in 38 ℃ of fed-batch fermentations, the time curve that biomass increases

Fig. 4. the relation between synanthrin concentration and the Kluyveromyces IW9801 inulinase speed of response

Fig. 5. the relation between sucrose concentration and the Kluyveromyces IW9801 inulinase speed of response

The pH of Fig. 6 .Kluyveromyces IW9801 inulinase fits the property scope

The temperature of Fig. 7 .Kluyveromyces IW9801 inulinase is fitted the property scope

The temperature tolerance of Fig. 8 .Kluyveromyces IW9801 inulinase is observed

The stability observing of Fig. 9 .Kluyveromyces IW9801 liquid inulinase under 50 ℃

The result of Figure 10 (A) .Kluyveromyces IW9801 inulinase degraded inulin observes

The result of Figure 10 (B) .Kluyveromyces IW9801 inulinase degraded inulin observes

Figure 11. the HPLCl collection of illustrative plates of inulinase degraded inulin products therefrom.Wherein, on, in, figure below is respectively standard model, the collection of illustrative plates of 10% inulin+40UINU/g (inulin) and 11% inulin+22UINU/g (inulin) degraded product

Figure 12. the technical process experiment that the inventor carries out in finishing process of the present invention that inulinase fermentative production, degraded inulin prepare HFS: bacterial strain, substratum, definition of inulinase activity unit and mensuration

1. bacterial strain

Kluyveromyces IW9801 (Kluyveromyces) is for the inventor separates from kluyveromyces yeast strains and Hubei distiller's yeast are preserved in the laboratory, screening, mutagenesis, stablize gained.

2. substratum:

(1) bacterial strain keeps slant medium (1L): peptone 20g, yeast extract 10g, sucrose 20g, agar 20g;

(2) screening culture medium (1L): the enzyme inorganic medium is identical with producing, and just wherein sucrose changes synanthrin into:

(3) shake bottle and produce the organic substratum YIP of enzyme (1L): peptone 20g, yeast extract 10g, synanthrin 5g;

(4) shake bottle and produce enzyme inorganic medium MG (1L): (NH ₄) ₂SO ₄5g, KH ₂PO ₄3g, MgSO ₄7H ₂O0.5g, EDTA15mg, ZnSO ₄7H ₂O0.45mg, FeSO ₄7H ₂O1.5mg, CuSO ₄5H ₂O0.3mg, CaCl ₂2H ₂O 0.45mg, MnCl ₂4H ₂O 1.0mg, CoCl ₂6H ₂O 0.3mg, H ₃BO ₃1.0mg, KI 0.1mg, NaMoO ₄2H ₂O 0.04mg, calcium pantothenate 1.0mg, nicotinic acid 1.0mg, vitamin H 0.4mg, glucose 2.5g;

(5) fermentor tank produces the seed liquor substratum (1L) of enzyme substratum: the organic substratum composition of bottle product enzyme is identical with shaking;

(6) fermentor tank produces enzyme inorganic medium basestocks (1L): (NH ₄) ₂SO ₄10g, KH ₂PO ₄6g, MgSO ₄7H ₂O 1.0g, EDTA 30mg, ZnSO ₄7H ₂O 0.9mg, FeSO ₄7H ₂O 3.0mg, CuSO ₄5H ₂O 0.6mg, CaCl ₂2H ₂O 0.9mg, MnCl ₂4H ₂O 2.0mg, CoCl ₂6H ₂O0.6mg, H ₃BO ₃2.0mg, KI 0.2mg, NaMoO ₄2H ₂O 0.08mg, calcium pantothenate 2.0mg, nicotinic acid 2.0mg, vitamin H 0.8mg, each 0.075ml of defoamer GE and GPE, sucrose 10g;

(7) fermentor tank produces the enzyme inorganic medium and replenishes material (1L): (NH ₄) ₂SO ₄10g, KH ₂PO ₄20g, MgSO ₄7H ₂O 5.0g, EDTA 0.50g, ZnSO ₄7H ₂O 0.225g, FeSO ₄7H ₂O 0.15mg, CuSO ₄5H ₂O 0.015g, CaCl ₂2H ₂O 0.225g, MnCl ₂4H ₂O 0.05g, CoCl ₂6H ₂O0.015g, H ₃BO ₃0.05mg, KI 5.0mg, NaMoO ₄2H ₂O 0.02g, calcium pantothenate 0.10g, nicotinic acid 0.10g, vitamin H 0.8mg, each 0.075ml of defoamer GP and GPE, sucrose 500.0g.

All organic substratum are all through 121 ℃ of sterilization 40min, and inorganic medium is through 121 ℃ of sterilization 30min, and sugar soln is through 105 ℃ of sterilization 20min, VITAMIN and micro-filtration sterilization.

Enzyme live definition with measure: be defined as in the reaction system every milliliter of per minute according to most conventions inulinase of the present invention activity unit in the world and produce the required enzyme amount of 1 μ M hexose and be 1 unit of enzyme activity.

Inulinase reaction conditions of the present invention is: enzyme liquid 0.50ml and 2.5-3.0% synanthrin (Shanghai chemical reagent two factories) through suitably dilution react 10min at 55 ℃; reaction system is 1ml; pH value of reaction system is 4.5, is provided by 0.02mM acetate-sodium acetate buffer.After boiling water 5min stops enzyme reaction, add fehling reagent A, B mixes colour developing liquid 4ml, puts boiling water colour developing 15min, and the centrifugal 5000rpm*6min in cooling back gets supernatant and measures light absorption value in 590nm.Obtain reducing sugar content and enzymic activity in the enzymolysis product according to typical curve.Mutagenesis and screening that experiment 1. produces the natural bacterial strain of inulinase

The kluyveromyces yeast strains of preserving from this laboratory produces the inulinase bacterial strain with spilling the song screening and separating, and the diameter of the transparent circle that forms with inulinase degraded inulin is a foundation, carries out plate isolation and primary dcreening operation.

The bacterium colony cell of the elected bacterial strain of primary dcreening operation is directly in the primary dcreening operation plate that opens wide, accept the UV-light mutagenesis 10-20min of 20W*39CM, the mutagenesis bacterium colony carries out the mutagenesis primary dcreening operation again on the primary dcreening operation flat board, elected bacterial strain further shakes the multiple sieve of bottle, and it is active big or small to measure inulinase, the tentative Kluyveromyces IW9801 by name of then selected superior strain one strain, this bacterial strain subculture is more than 20 generations, and it is stable to produce inulinase performance and other main biological characteristics.Test the condition of the outer inulinase of 2. Crewes dimension IW9801 bacterial strain production born of the same parents

1, shake bottle and produce the enzyme time curve

(1) the product enzyme time curve on inorganic salt-dextrose culture-medium

Product enzymic fermentation condition on inorganic salt-dextrose culture-medium is: 32 ℃ of * 200rpm, 250ml triangular flask substratum loading amount 60ml.As can be seen from Figure 1: shake bottle and produce an enzyme and promptly peak at 30h, production of enzyme is 10.4U/ml, after this descends and keeps low-level about 4.0U/ml at 100h.Correspondingly biomass does not all surpass 1.3mg (DW)/ml all the time.

(2) the product enzyme time curve on organic substratum

Identical on the condition of enzyme production on organic substratum and inorganic salt-dextrose culture-medium.The result shows: the secretory volume straight line of enzyme increases to 18.12U/ml in the substratum in 0-42h, and keeps this straight horizontal of the 18U/ml left and right sides to 88h always; Correspondingly biomass rises to 30h from the 0h straight line, reaches 14.25mg/ml, is stabilized in ever since about 12mg (DW)/ml (Fig. 1).Demonstrate: thalline biomass and inulinase secretory volume have close proportional relationship.

2, fed-batch fermentation produces the time curve of enzyme in the pattern fermentor tank

(1) 32 ℃ of following inorganic medium fed-batch fermentation produces the enzyme time curve

Loading amount and fermentation condition: fed-batch fermentation carries out in the 6.6LBraun automatically controlled fermentor, inorganic salt are at 121 ℃ of 30min that sterilize down in the basestocks, sucrose is at 105 ℃ of 20min that sterilize down, trace element and VITAMIN filtration sterilization, just dress substratum total amount is 2.2L, inoculum size is 7.5%, and seed liquor adopted organic culture media shaking vase to cultivate in 1 day in advance under 32 ℃ of * 200rpm.Proofread and correct pH and pO before the charging ₂Electrode, 32 ℃ of leavening temperatures, rotating speed 1000rpm, air flow 9L/min is by rotating speed with add materials flow speed control system pO ₂＞30%, by 2M HCl and ammoniacal liquor control pH value 4.5.Be cultured to that the C source is exhausted in the 5h basestocks, the beginning feed supplement, the feed supplement flow velocity at the uniform velocity increases from 5h to 36h, initial flow rate is 0.56ml/l/min, it is 11.19ml/l/min that 36-60h stablizes flow velocity, fermentating liquid volume increases 433ml for every liter during fermentation ends, increases by 43.3%, and total fermentating liquid volume is about 2.80L.

As shown in Figure 2, the active straight line of inulinase rises between 0-18h, reaches 33.54U/ml to 18h, and 18-52h remains between the 37-40U/ml, rises to 46-47U/ml afterwards.Correspondingly, the thalline biomass, between 0-30h, almost rise to 53mg/ml (DW) from 0 straight line, reach 61mg/ml (DW) to 42h, basically keep this level to 60h, similarly, have obvious proportional relationship between the outer inulinase output of thalline biomass and born of the same parents, this point can visually see from Fig. 2.As for the specific activity of inulinase, keep the higher level 150-180U/mg protein except before the 12h, be stabilized in ever since about 27-31U/mg protein.

(2) 38 ℃ of following inorganic medium fed-batch fermentations produce the enzyme time curve

38 ℃ down other fermentation condition with 32 ℃ under basic identical, 38 ℃ of leavening temperatures, air flow 9L/min, rotating speed 0-10h are 900rpm, after this are 1000rpm, by rotating speed with add materials flow speed control system pO ₂＞30%, by 2MHCl and ammoniacal liquor control pH value 4.5.Be cultured to that the C source is exhausted in the 4.5h basestocks, the beginning feed supplement, the feed supplement flow velocity at the uniform velocity increases from 4.5-11h, and initial flow rate is 1.40ml/l/min, reach 12.87ml/l/min to 11h, 11-48h still keeps this flow velocity, and 48-60h keeps 10.3ml/l/min, and 60-72h keeps 7.38ml/l/min, during fermentation ends, fermentating liquid volume increases 723.65ml for every liter, increases by 72.4%, and total fermentating liquid volume is about 3.40L.Actual observation shows: pO ₂Value keeps 80-90% during 4.5-11h, remain on about 40% substantially at 11-72h.

As shown in Figure 3, the active straight line of inulinase rises between 6-30h, rise to 53.45U/ml from 7.11, to the 54h kept stable, further slowly rise to 64.43U/ml to 72h afterwards, as for 3h enzymic activity 11.43U/ml on the contrary 6h exceed approximately 40%, then be because before the feed supplement relatively due to the inductive effect of C hunger.As for the specific activity of inulinase, except 3-6h keeps higher level 142-155U/mg protein, after this slowly drop to the 15.4U/mg protein of 60h from the 5.54U/mg protein of 12h always; In fact single from the total secretory volume of protein, the dependency that increases with biomass is higher, from the 50mg/ml of 8h almost straight line rise to the 31.83mg/ml of 56h, keep the high level of 37-40mg/ml afterwards substantially at 58-72h.Correspondingly, the thalline biomass all is that straight line rises between whole yeast phase, 12,24,36,48,60 and the 72h biomass reach 12.5,27.0,36.5 respectively, 55.0,61.5 and 65mg (DW)/ml, have very high positive correlation between the outer inulinase output of thalline biomass and born of the same parents, this point can visually see from Fig. 3.

In a word, the total secretory volume of thalline inulinase and protein, biomass all have positive correlation preferably each other.Especially, the inulinase secretory volume has promptly reached higher level＞50U/ml at 28h, and highest level reaches 64.43U/ml.Test the outer inulinase zymologic property of 3. born of the same parents

1, the relation of the substrate specificity of inulinase and speed of response thereof and concentration of substrate

(1) substrate specificity of inulinase

The substrate specificity of table 2. inulinase

Substrate and concentration thereof	Enzymic activity (U/ml)	Relative activity (%)	????I/S
				Synanthrin, 5.0mM sucrose, 73.04mM	????28.20 ????696.97	????100.00 ????2471.52	????1/24.72

By table 2 data as seen, this bacterial strain excretory inulinase can act on synanthrin and sucrose simultaneously, and the I/S value is 1/24.72.

Inulinase Km value when (2) substrate is synanthrin

Being generally 30 according to the synanthrin polymerization degree can calculate the synanthrin molecular weight and be about 5000, therefore the synanthrin solution of preparing the 0-30mM concentration gradient is measured inulinase catalyzed reaction gained monose efficiency of pcr product simultaneously, set up equilateral hyperbola equation gained Vmax=24.4mg/ml/min, Km=13.3mM (Fig. 4) according to measured value.

Km value when (3) substrate is sucrose

The sucrose solution of 0-600mM concentration gradient is set, and experiment records Vmax=234.6mg/ml/min, Km=62.6mM (Fig. 5).

The present invention is two kinds of substrate gained Km values relatively, and the suitableeest substrate that can affirm this enzyme effect is synanthrin rather than sucrose, although I/S is 1/24.72, therefore judge that this enzyme is inulinase rather than saccharase.

2, the inulinase pH value in reaction is fitted the property scope

As shown in Figure 6, inulinase optimal reaction pH value is 4.4, but has all kept higher activity in the scope of pH3.8-6.4, is equivalent under the optimum pH active 90%.

3, inulinase optimal reactive temperature, the suitable property scope of temperature and heat resistance

(1) inulinase optimal reactive temperature

Bacterial strain inulinase optimal reactive temperature of the present invention is 55 ℃, and is good at 50-57.5 ℃ of scope internal stability, can keep greater activity (Fig. 7).

(2) suitable property scope of inulinase temperature and heat resistance

The heat resistance research (Fig. 8) of bacterial strain inulinase of the present invention: storage liquid inulinase 1h under the differing temps environment, the back is measured enzymic activity down at 55 ℃.The result shows: temperature tolerance raises with temperature and descends, and the temperature tolerance under 25,35 and 45 ℃ differs very little, and 55 ℃ of * 1h residual enzyme activity have only 67.91% (5 ℃ of following enzymic activitys are 100%), and 65*1h residual enzyme activity more is low to moderate 26.11%.In a word, the temperature tolerance of inulinase＞50 ℃ is poor.

As can be seen from Figure 9: the temperature tolerance of inulinase of the present invention under 50 ℃ still can, the transformation period of 50 ℃ of following enzymes is about 16h, the residual enzyme activity has 83.02% behind the 6h, still has 53.74% behind the 12h, still keeps 32.09% to 36h.

4, metal ion is to the active influence of inulinase

From table 3 data as can be seen, metal ion has a significant effect to inulinase activity of the present invention, wherein removes Mg ²⁺Improve outside active 11.28% Ca ²⁺, Mn ²⁺, Fe ³⁺Then make enzymic activity reduce by 15.72,22.41 and 27.60% respectively.

Table 3. metal ion is to the active influence of inulinase

Metal ion and concentration thereof (mM)	Enzymic activity (U/ml)	Relative activity (%)
			Contrast MgCl 2，25mM ????CaCl 2，25mM ????MnCl 2，25mM ????FeCl 3，25mM	????58.59 ????65.20 ????49.38 ????45.46 ????42.42	????100 ????111.28 ????84.28 ????77.59 ????72.40

The experiment 5. usefulness inulinases inulin of degrading

Degradation condition is 50 ℃, carries out in sealing triangular flask.

Figure 10 (A) shows that 6.0% synanthrin 5h under the inulinase effect of 200U/g (S) can degrade fully; 10.0% synanthrin 24h under the inulinase effect of 40U/g (S) can degrade fully; 11.0% synanthrin 40h under the inulinase effect of 22U/g (S) can degrade fully.

Figure 10 (B) shows that 2.5%, 5.0% and 7.5% synanthrin is respectively at 400U/g (S), and under the inulinase effect of 200U/g (S) and 133U/g (S), the 6h degradation rate is all above 100%.10%, 12.5% and 15% synanthrin is respectively at 100U/g (S), and to surpass time of 100% corresponding be 24h to degradation rate under the inulinase effect of 80U/g (S) and 67U/g (S), 24h and 36h.

From cost-effective angle, comprehensive Figure 10 (suitable degradation condition is for A, B) result: synanthrin concentration 15.0%, enzyme effect dosage 22U/g, action time 30-36h, degradation of substrates rate about 95%.

The concrete practice is: get the enzyme liquid (50U/ml) that produces enzymic fermentation 30-36h and mix with 15% synanthrin, substrate is sterilized in advance, carries out the saccharification enzymolysis under 50 ℃ of * 30-36h conditions, and enzyme liquid and synanthrin blending ratio are: 1/15.Be equivalent in the whole enzymatic hydrolysis system the active 3.0U/ml of inulinase, synanthrin concentration 14.1%, efficiency of pcr product 95%.Test 6. inulin degraded product qualitative and quantitative analyses

Table 4 and Figure 11 data are the qualitative and quantitative analytical resultss of inulinase degraded inulin.Figure 11 is the efficient liquid phase chromatographic analysis collection of illustrative plates of inulinase degraded inulin products therefrom.The HPLC analysis condition: adopt nh 2 column and Waters401 differential detector, second is fine in the moving phase: water=70: 30, flow rate of mobile phase are 1.0ml/min, and column temperature is 25 ℃.The result that several different methods such as HPLC and glucose oxidase method is measured clearlys show: fructose accounts for more than 95% in the degraded product, and glucose accounts for below 5%, does not contain other sugar.The reason that the degraded product total sugar content is higher than substrate content is: monose of every generation was just in conjunction with a water molecules when polyfructosan was degraded to monose, and the result makes the product amount increase about 10% than amount of substrate.

The efficient liquid phase chromatographic analysis result of table 4. inulinase degraded inulin products therefrom (%, W/V) ^*

Inulin concentration (%)	Enzyme dosage (U/g inulin)	Fructose content	Glucose content **	Sucrose content	Total sugar content	Degradation rate (%)	Fructose rate (%)
								????10 ????11	????40 ????22	??10.98 ??11.80	?0.16(0.545) ?0.22(0.530)	????0 ????0	??11.14(11.53) ??12.02(12.33)	??111.4 ??109.3	??98.56(95.27) ??98.17(95.70)

^*The substrate synanthrin is with the 0.02M acetate buffer heat of solution of pH4.5, and the inulinase liquid sealing with filtration sterilization is incubated through 115 ℃ of * 15min sterilization backs again, and degradation condition is 50 ℃ of * 36h. ^*Expand the corresponding data that the data system in the arc is calculated according to the sample glucose content result of determination of glucose oxidase.

Degraded product explanation inulinase of the present invention belongs to excision enzyme.Test 7. inulinase fermentative production, the degraded inulin prepare HFS

The technical process that bacterial strain inulinase fermentative production of the present invention, degraded inulin prepare HFS as shown in figure 12.Fs: seed liquor cultivation, inoculation and basestocks preparation and sterilization

Put forward the day before yesterday of shake-flask culture seed liquor on organic substratum, under 32 ℃ of * 200rpm, cultivated 24 hours, second day preparation basic medium also sterilized 0.5-1.0 hour under 121 ℃, wherein edible sucrose reaches 1% prewired back 105 ℃ of following sterilizations 0.5-1.0 hour according to final concentration, the cooling back adds in the basestocks, add in the basestocks after the VITAMIN filtration sterilization, the seed liquor inoculum size is 10% of a basestocks.Subordinate phase: enzymatic production

1, rotating speed 800-1000rpm, air flow 9L/min is by rotating speed and feed supplement flow rate control pO ₂＞=30%, keep 4.5 by ammoniacal liquor control fermented liquid pH value, temperature 32-38 ℃, time 4-6 hour.

2, supplemented medium preparation, sterilization and flow feeding

Under (1) 32 ℃, be cultured to that the C source is exhausted in the 5h basestocks, the beginning feed supplement, the feed supplement flow velocity at the uniform velocity increases from 5h to 36h, it is 11.19ml/l/min that 36-60h stablizes flow velocity, and fermentating liquid volume increases 433ml for every liter during fermentation ends, and total fermentating liquid volume is about 2.80L.The active straight line of inulinase rises between 0-18h, reaches 33.54U/ml to 18h, and 18-52h remains between the 37-40U/ml, rises to 46-47U/ml afterwards.The thalline biomass, between 0-30h, almost rise to 53mg/ml (DW) from 0 straight line, reach 61mg/ml (DW) to 42h, basically keep this level to 60h, specific activity as for inulinase, keep the higher level 150-180U/mg protein except before the 12h, be stabilized in ever since about 27-31U/mg protein.

Under (2) 38 ℃: basic identical except that 38 ℃ of temperature under other fermentation condition and 32 ℃.Be cultured to that the C source is exhausted in the 4.5h basestocks, the beginning feed supplement, the feed supplement flow velocity at the uniform velocity increases from 4.5-11h, reach 12.87ml/l/min to 11h, 11-48h still keeps this flow velocity, and 48-60h keeps 10.3ml/l/min, 60-72h keeps 7.38ml/l/min, during fermentation ends, fermentating liquid volume increases 723.65ml for every liter, and total fermentation volume is about 3.40L.The active straight line of inulinase rises between 6-30h, rises to 53.45U/ml from 7.11, to the 54h kept stable, further slowly rises to 64.43U/ml to 72h afterwards.The specific activity of inulinase except 3-6h keeps higher level 142-155U/mg protein, after this slowly drops to the 15.4U/mg protein of 60h always from the 5.54U/mg protein of 12h; The thalline biomass is that straight line rises between whole yeast phase, 12,24,36,48,60 and the 72h biomass reach 12.5,27.0,36.5,55.0 respectively, 61.5 and 65mg (DW)/ml.Phase III: yeast thalline and the centrifugation of enzyme liquid

At 10 ℃, 3000rpm, centrifugal 15 minutes, supernatant was an enzyme liquid, was precipitated as pure wet thallus cell.The quadravalence section: inulinase degraded inulin prepares high fructose syrup

15% synanthrin (15 parts) of inulinase liquid (1 part)+heat of solution, degradation condition: 50 ℃, 30-36h, degradation rate reach more than 95%, and product fructose rate reaches more than 95%.Make fructose absolute content＞90% among the HFS after 4 times of evaporation concentration.

Claims (23)

1, a kind of kluyveromyces yeast strains that produces inulinase, its preserving number is: CGMCC NO.0360.

2, a kind of inulinase that produces by the described yeast strain of claim 1.

3, according to the described inulinase of claim 1, it is characterized in that, its Km (synanthrin) is 13.3mM, Km (sucrose) is 62.6mM, I/S=1/24.72, and optimum pH is 4.4, and in the pH3.8-6.4 scope, all kept the residual enzyme activity more than 90%, optimal reactive temperature is 55 ℃, and the transformation period under 50 ℃ is 16 hours, and magnesium ion can improve inulinase activity 11.28%.

4, a kind of method of producing inulinase by the yeast strain high density fermentation comprises:

(1) be nitrogenous source with inoculation to containing 0.6-1.4% (weight) nitrogen compound, 0.8-1.2% (weight) organic carbon compound is in the inorganic salt basic medium of carbon source, at 25-40 ℃ of following aerated culture, makes pO ₂＞25%, pH4-5;

(2) be cultured to 4-7 hour, work as pO ₂Value begins stream and adds and replenish material when rising suddenly, it is identical with basic medium to replenish material, and just wherein macroelement content increases 5-10 doubly, and micronutrient levels increases 10-30 doubly, and carbon source content increases 30-50 times;

(3) stream adds in the additional material process, makes pO all the time ₂＞30%, pH4-5, a preceding 5-30 hour accelerating flow adds, latter stage 30-70 hour at the uniform velocity stream add, final fermentating liquid volume increases 40-80%, thalline biomass weight in wet base reaches the 150-300g/ liter;

(4) from culture, isolate enzyme liquid.

5, in accordance with the method for claim 4, it is characterized in that, use sucrose or raffinose or glucose or jerusalem artichoke juice to be sole carbon source, use inorganic nitrogen compound or urea to be culture media nitrogen source.

6, in accordance with the method for claim 5, it is characterized in that described carbon source is a sucrose, described inorganic nitrogen compound is an ammonium sulfate.

7, in accordance with the method for claim 4, it is characterized in that C source concentration is 0.9-1.1% in the basic medium; Stream adds and replenishes that C source concentration is 35-60% (weight) in the material, and adds at stream and to keep in the process that C source concentration is 1.5-3.0% (weight) in the fermented liquid.

8, in accordance with the method for claim 7, it is characterized in that C source concentration is 1.0% (weight) in the basic medium; Stream adds and replenishes that C source concentration is 50% (weight) in the material, and adds at stream and to keep in the process that C source concentration is 2.5% (weight) in the fermented liquid.

9, in accordance with the method for claim 4, it is characterized in that the substratum loading amount is the 40-60% of jar effective cumulative volume; Stream adds to replenish expects that volume is the 40-80% of basestocks volume.

10, in accordance with the method for claim 9, it is characterized in that the substratum loading amount is 50% of jar effective cumulative volume; Stream adds to replenish expects that volume is 70% of a basestocks volume.

11, in accordance with the method for claim 4, it is characterized in that pH is controlled at 4.5 in the fermenting process, with ammoniacal liquor control pH value.

12, in accordance with the method for claim 4, it is characterized in that, it is nitrogenous source that described minimal medium contains 0.6-1.4% (weight) nitrogen compound, 0.8-1.2% (weight) organic carbon compound is a carbon source, 0.4-0.7% (weight) phosphoric acid salt, 0.8-1.2% (weight) magnesium salts, copper, zinc, cobalt and the salt compounded of iodine of the iron of 1.5-4.0ppm, manganese salt, borate, vitamin substances and 0.2-1.10ppm.

13, in accordance with the method for claim 4, it is characterized in that pO in the fermenting process ₂Value is controlled at 25-50%, and rotating speed is 450-1000rpm.

14, in accordance with the method for claim 4, it is characterized in that pO in the fermenting process ₂Value is controlled at 30-40%.

15, in accordance with the method for claim 4, it is characterized in that the thalline weight in wet base is controlled at 180-300g/L.

16, in accordance with the method for claim 15, it is characterized in that the thalline weight in wet base is controlled at 230g/L.

17, in accordance with the method for claim 4, it is characterized in that the outer inulinase activity of the highest born of the same parents reaches 45-75U/ml in the fermented liquid.

18, in accordance with the method for claim 17, it is characterized in that leavening temperature is 38 ℃, the outer inulinase activity of the highest born of the same parents reaches 60U/ml in the fermented liquid.

19, in accordance with the method for claim 4, it is characterized in that described yeast is Kluyveromyces sp (Kluyveromyces marxinus).

20, in accordance with the method for claim 4, it is characterized in that described yeast is kluyveromyces marxianus bacterium (Kluyveromyces marxinus), IW9801 bacterial strain, deposit number are CGMCC NO.0360.

21, a kind of method of utilizing the inulinase glycosylated degradation inulin production high fructose syrup of producing by the described method of claim 4, comprise: thalline is with after enzyme liquid separates, inulinase liquid is mixed according to 1: 8～17 volume ratio with the inulin solution of sugar degree 8-17%, at 45-55 ℃ of following enzymatic saccharification 30-40 hour, obtain high fructose syrup.

22, in accordance with the method for claim 21, it is characterized in that the condition of inulinase degraded inulin is: synanthrin concentration 15% (weight), enzyme liquid and 8-15% (weight) inulin liquid blending ratio is 1: 15, and hydrolysis temperature is 50 ℃, and enzymolysis time is 36 hours.

23, according to the application of the described yeast strain of claim 1 in producing high fructose syrup.

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