CN101942392A - New Fusarium and application thereof in degradation of plant cell wall - Google Patents

Abstract

The invention relates to a new Fusarium and application thereof in the degradation of a plant cell wall. In the invention, 18 purified fungi having high stability and wide enzyme catalysis activity are used to produce enzymes for degrading plant straw powder. Liquid inducement is carried out on Fusarium sp.Q7-31 in a liquid nutrient medium using the plant straw powder as the unique carbon source to culture the produced enzymes; and Fusarium Q7-31 is screened out for further study by comparing the degradation effects of the produced enzymes of various bacteria. Fusarium sp.Q7-31 can produce and secrete out cell wall degrading enzymes having high activity and high stability in the liquid nutrient medium. By using 1% of plant straw powder as the substrate to react with the centrifuged supernatant, the result shows that the degradation efficiency of the natural enzyme produced by Fusarium sp.Q7-31 on paddy rice straws is 150 g/min/mL. What is more important, the enzyme can still maintain 75% of the enzyme activity at an initial state after storage at 25 DEG C or 40 DEG C for three days. Besides, the invention also provides an application method of the induced enzyme liquid of Fusarium sp.Q7-31 plant cell wall degrading enzyme.

Description

A kind of new sickle spore bacterium and the application in the degrading plant cell walls thereof

Technical field

The present invention relates to a kind of new Fusarium fungi and the application in the degrading plant cell walls thereof.

Background technology

Bioenergy is the energy inexhaustible on the earth, nexhaustible and that environment is not polluted, absorbs CO because it derives from ₂Carry out photosynthetic biomass (Schlamadinger andMarland, 1996; Cowie and Gardner, 2007).Annual 170-200 * 10 that produce on the earth ⁹70% is cell wall components among the ton biomass, and wherein human use's total amount has only 2% (Markus P.et al 2008).At present, the two mcroorganism energy-bio-ethanols and biofuel are respectively the selectable fuel of gasoline and diesel oil.They are to reducing exhaust gas emission, alleviating Global warming vital role is arranged, and therefore, bioenergy can be applied on the existing mobile facility well.

In recent years, the output of the bio-ethanol in whole world every year surpasses 10,000,000,000 gallons, and great majority obtain (Yuan et al., 2008) by starch and the carbohydrates that we produce at present.Because people constantly increase the demand of these carbohydrate, cause the grain dog-eat-dog, corresponding, the cost of bio-ethanol is also more and more higher.Therefore, the focus of research concentrates on the abundantest plant cell wall polysaccharides of occurring in nature carbon source at present.Yet it is to become fermentable monose by acting on effective degradation polymer earlier that low-cost high-efficiency is produced one of main difficult problem of bio-ethanol, and then with yeast fermentation (Szczodrak and Fiedurek, 1996; Mielenz, 2001; Anderson and Akin, 2008).Mostly be to utilize various physical and chemical effects to carry out pre-treatment in traditional starting stage, for example high temperature, high pressure, acid, alkali, organic solvent or combined action destroy cross-link bond such as xylogen and low-molecular-weight phenolic compound (Petersson et al., 2007 of polysaccharide and aromatics; Lindeet al., 2008).These pre-treatment effects are expense height, contaminate environment not only, and may restraining effect be arranged to enzymic hydrolysis and fermentation afterwards.Therefore as far as possible reduce or do not use these pre-treatment to improve bio-ethanol production efficiency, the aspect that reduces cost has important effect.

Reach this target, can realize, for example improve cell-wall component and make it to be degraded easily and find a kind of can the generation to have that extensive enzyme is lived and the microorganism of the enzyme of strong hydrolytic action by the several method acting in conjunction.In general, fungi is evolved to form and keeps its growth (Warzywoda et al., 1992 by the degrading plant cell wall polysaccharides; De Vries and Visser, 2001).It is reported now had the fungal enzyme of a large amount of degrading plant cell wallss to be applied in the industrial production.Yet, owing to have a large amount of not being found and cognitive fungi at occurring in nature.Fungi plays an important role in natural material cycle as the decomposer of maximum, yet, in about 250,000 kinds of fungies, the fungi of people's understanding only has hundreds of be used to (Peiji G..et al 2004) such as industrial fermentation, drug developments less than 40%.So, very likely be separated to more, as to have the cell wall degrading enzyme that enzymic activity is higher, more stable better, Degradation is more wide in range fungi.In order to screen strain excellent, we choose Fusarium sp.Q7-31 and further study its characteristic from 18 strain bacterium.

Summary of the invention

The initial stage of this invention, to can degrading plant the screening of cell walls bacterial strain be in China, carry out in the Xie Zhanling of Qinghai University professor's the laboratory Xining, Qinghai.Bacterial strain comprises that we are at the Aspergillus of Qinghai-Tibet separation and purification sp., Fusarium spp.F-1, F-2, F-3, Q7-21, and Q7-31; Geotrichum sp.; Morchella spp.M-1, M-2, M-3, M-4, and M-5; Mucor sp.U-1, U-2, U-3, and U-4; Penicillium sp.; Pestalotiopsissp.; With Trichoderma sp. totally 18 strain bacterium.For the generation of inducing cell wall degrading enzyme, each strain bacterium all with 0.3% rice straw powder as the liquid nutrient medium of sole carbon source on growth 3 days.By centrifugal mycelium and the remainder water rice straw powder of removing fungi from substratum, supernatant liquor is crude enzyme liquid, detects its degradation capability to plant cell wall with this crude enzyme liquid immediately then.Measuring method is that the rice straw powder of thick enzyme and 1% reacted 10 minutes for 40 ℃, adds the content that DNS measures reducing sugar then.

In 18 strain bacterium, according to the high stability and the high vigor of the thick enzyme of Fusarium sp.Q7-31, we further study its feature.By analysis (for example mycelial width and habit) and its ITS sequence (sequence number: FJ646593) identify that bacterial strain Fusarium sp.Q7-31 belongs to Fusarium and belongs to its morphological feature.Q7-31 with 0.3% rice straw powder as the agarose culture medium culturing of sole carbon source 7 days, its mycelial growth is very luxuriant, colony diameter reaches 6cm.Its this growth vigor and degradation capability be transferred on the liquid nutrient medium cultivate 3 days so same.It is maximum that cell wall degradation ability by 3 days this thick enzymes of cultivation reaches, again through maintaining higher level in 4 days.Yet the protein content cumulative growth occurs in preceding four days in the substratum.For the stability of enzyme relatively, the thick enzyme of Fusarium sp.Q7-21 and Q7-31 is kept at surveys the variation of cell wall degradation enzyme activity in 25 ℃ and 40 ℃.Under 25 ℃ and 40 ℃ of conditions, the thick enzyme of Q7-31 still keeps its enzymic activity of initial 75%, and the enzymic activity of Q7-21 just disappeared in one day.In addition, the enzyme that Fusariumsp.Q7-31 produces is for other the cereal grass stalk such as the barley of locality, wheat, and corn, oat, the Degradation of rye is stronger than paddy rice.These results show that Fusarium sp.Q7-31 is a kind of bacterial strain of the novel enzyme that can produce high stable degrading plant cell wall polysaccharides.Therefore, on cost efficiency, this bacterial strain has and utilizes the potential of cereal grass for the raw material production bio-ethanol.Biological material specimens preservation relevant information: biological material specimens is the sickle spore of survival, and Fusarium sp. deposit number is CGMCC NO.2710.Preservation date issued is on October 14th, 2008.Depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC).

Description of drawings

Fig. 1. have the liquid culture of the fungi of different rice straw powder degrees of degradation.Bacterial strain was being cultivated 3 days in as 25 ℃ of following 100rpm shaking tables of substratum of sole carbon source with 0.3% rice plants stalk powder.A，Morchella?sp.M-1；B，Fusarium?sp.Q7-31；C，Pestalotiopsis?sp.；D，Fusarium?sp.Q7-21；E，Trichoderma?sp.；F，Penicillium?sp.；G，Mucor?sp.U-1；H，Fusarium?sp.F-2；I，Geotrichum?sp.；J，Morchella?sp.M-2；K，Morchella?sp.M-3.

Fig. 2. total reducing sugars output in different fungal bacterial strain substratum.Every strain bacterial strain all is grown in the liquid nutrient medium that 5ml contains 0.3% rice stem stalk powder.Surveying enzyme with the DNS method when growing into the 3rd and the 6th day lives.

Habit and the mycelium of Fig. 3 .Fusarium sp.Q7-31. (A) mycelium is seeded in and contains the central authorities of 0.3% rice stem stalk powder as the solid medium of sole carbon source.This pictures is clapped after 5 days 25 ℃ of cultivations.(B) this width of cloth caption mycelium has barrier film.Scale is respectively 1cm and 5um at (A) and (B).

The growth curve of Fig. 4 .Fusarium sp.Q7-31 on the solid medium of making carbon source with 0.3% rice straw powder.Mean number comes from 3 parallel measuring results with standard error.

The contrast of Fig. 5 .ITS sequence.Show the difference between species, Fusarium sp.Q7-31 (sequence number: FJ646593) have 159 bases and his contiguous species consistent.All species of having reported all are included in the bracket.Arrow indicates the line of delimitation of ITS2 and 28S ribosomal RNA gene.

Fig. 6 .Fusarium sp.Q7-31 produces the vigor of thick enzyme (cell wall degrading enzyme).The content of beginning reducing sugar utilizes the DNS method, uses spectrophotometer measurement under the 575nm wavelength.Absorptivity is transformed into ug/mL according to the DNS typical curve of known glucose concentrations, thereby learns glucogenic amount.The final calculating of enzyme activity will be divided by the enzymic catalytic reaction time of 10min.Measuring result is by three parallel mean number and standard errors of drawing.

The semi-invariant of soluble protein in Fig. 7 .Fusarium sp.Q7-31 liquid nutrient medium.According to the Bradford method, under wavelength 595nm, use the spectrophotometer measurement absorbance value.The typical curve that absorbance value is done according to the bovin serum albumin of concentration known draws protein content.

The stability of the thick enzyme that Fig. 8 .Fusarium sp.Q7-31 produces.Liquid culture is passed through centrifuging and taking supernatant liquor (thick enzyme) after three days.Enzyme liquid places 25 ℃ and 40 ℃ of environment, lives at each point in time measurement enzyme.

The enzyme that Fig. 9 .Fusarium sp.Q7-31 produces is to the degradation effect of all kinds of cereal grass stalks.By the thick enzyme generation of inducible strain over 3 days of growth in containing 0.5% rice straw powder culture medium.Every kind of plant stalk powder adds under isopyknic thick enzyme 40 conditions as substrate and reacts 10min, surveys enzyme with the DNS method then and lives.

The detailed description of contrivance:

Plant cell wall polysaccharides is the abundantest carbohydrate of occurring in nature.Its main moiety comprises Mierocrystalline cellulose, hemicellulose, pectin and xylogen.Mierocrystalline cellulose is the main moiety of plant cell wall, and by β-1, the 4-D-glucosyl residue is in conjunction with being crystal fibre shape linear structure.Hemicellulose comprises multiple isomer polysaccharide, comprises xylan, dextran and xyloglucans.The main chain of these polymkeric substance has the different side chain group of multiple-limb usually, L-arabinose for example, D-semi-lactosi, ethanoyl, feruloyl, p-coumaroyl and glucuronic acid residue.The main chain of pectin is β-1,4-D-galacturonic acid residue and α-1,2-L-rhamnosyl residue also is connected with the L-arabinose and D-galactose residue side-chain radical (the De Vries and Visser of relevant length, 2001). in addition, hemicellulose and pectin, aromatics is xylogen and the lower molecular weight phenolic compound all mutually combines by covalent linkage and hydrogen bond or combine with main polysaccharide on the plant cell wall for example, this combination makes cell walls harder (Anderson and Akin, 2008).

Because the complicacy of vegetable cell wall construction and the diversity of composition, different enzymes such as endoglucanase, cytohydrolist, Glycosylase, exoglucanase, endoxylanase, the β xylosidase, the β endomannanase, the β mannosidase, pectin hydrolase and pectin lyase all are relevant for the hydrolytic action of main chain.Also have a large amount of attached enzyme such as α-xylosidase; arabinofuranosidase, araboxylan enzyme, pectinose lytic enzyme; inscribe and circumscribed arabinase; α-and beta-galactosidase enzymes, inscribe and exoglucanase, α-Pu Taotang aldehyde neuraminidase; feruloyl; ρ-coumaroyl, ethanoyl and methyl esterase also act on the substituting group or the side-chain radical (Walton, 1994) of plant cell wall polysaccharides.These cell wall degrading enzymes from many microorganisms purifying come out, particularly in keeping earth carbon cycle, play the filamentous fungus of important effect.The more important thing is that the enzyme that produces from special fungal bacterial strain has the scope of degrading widely, high enzyme is lived and high stability.Reported the substrate specificity of cell wall degrading enzyme in addition, passed through the enzyme activity (de Vriesa et al., 2000) of synergy between these enzymes to strengthen..These factors of analysis-by-synthesis, for the degradation of cell wall polysaccharide of energy maximum, we adopt the enzyme of the supernatant liquor of bacterium culture as thick enzyme rather than separation and purification.

Process of the test:

The purifying of bacterial strain:

Sample comes from Morchella sp. sporophore (mushroom) separate tissue thing during isolated strains Q7-31.Be by a series of foster purifying culture that filters out of being commissioned to train on screening culture medium.Screening culture medium comprises 0.6% peptone, 0.1% K ₂HPO ₄, 0.05% MgSO ₄7H ₂O and 1% glucose, pH 7.0. were cultivated 3-5 days under 28 ℃ of conditions.Well-grown then bacterium colony is by inferior foster being transferred on the fresh culture of being commissioned to train.From the bacterium colony of even growth, with transfering loop picking one ring transition (BLM) 28 ℃ in the basic liquid nutrient medium identical with selecting medium component, the 100rmp shaking table is cultivated.For preservation, bacterial strain is inoculated on the PDA substratum and preserves under 4 ℃ of conditions.

The bacterial strain of screening energy degrading plant cell walls

The mycelium of every kind of inoculation one ring grows into state of saturation to the basic liquid nutrient medium (BML) that 50ml contains 1% glucose conduct.Approximately the saturated culture of 50uL is transferred among the BLM that contains 0.5% rice stem stalk powder of 5ml and cultivates under 28 ℃ of conditions.At the 3rd and the 6th day, the content by reducing sugar in every kind of substratum of DNS method measurement 1ml compared 18 strain bacterium.

The DNS method is surveyed total reducing sugars content

According to the described DNS method of Miller (1959) process, change a little, measure the content and the enzyme of reducing sugar in the solution and live.DNS reagent comprises 0.5% DNS, 0.025% S-WAT, 0.5%NaOH, the KOH of 0.1% phenol and 2.5N.0.5mL fungal cultures or thick enzyme reaction solution and 100 ℃ of the DNS reagent reaction 10min down of 0.1mL.The distilled water that adds 0.4mL then is that cumulative volume reaches 1mL, reads spectrophotometric absorbance value under the 575nm wavelength, determines the content of reducing sugar in the solution.

Evaluation on Q7-31 morphology and the molecules

Mycelium continues to cultivate 5 days on PDA, is used for measuring morphological feature, as the mycelia width, and the formation of spore and the shape of spore.With CTAB method (van Burik et al., 1998) from be grown in the PDA substratum mycelium of last 5 day, extract genomic dna, utilization has forward ITS5 primer, the anti-phase primer 5 ' of 5 '-GGAAGTAAAAGTCGTAACAAGG-3 ' and ITS4-TCCTCCGCTTATTGATATGC-3 ' PCR Yahg et al. (2007) method, ITS DNA is amplified.The sequential analysis that the order-checking back produces PCR by NCBI BLAST database.

Fungal growth is grown in agarose and liquid nutrient medium

In order to determine growth rate, be inoculated in the agarose minimum medium that contains 0.3% rice stem stalk powder with transfering loop picking mycelium.The performance of fungal growth rate was measured hypha,hyphae colony diameter in three culture dish by every day, and its diameter constantly enlarges.The transfering loop mycelium of every strain bacterium is inoculated into 0.6% peptone that contains of 5mL, 0.1% K ₂HPO ₄, 0.05% MgSO ₄7H ₂O and 1% glucose or rice stem stalk powder be as sole carbon source, and pH is on 7 the substratum.Liquid nutrient medium is 100rpm in shaking table, 28 ℃ of constant temperature culture.

The preparation of plant powder:

Derive from the Dong-Jin paddy rice, the Ho-Pyum barley, the Geum-Kang wheat, the Chum-Ye rye, local corn variety and the oat of Ok-Chun, their seed 30min that sterilizes in the household bleach of a half strength is planted in the water saturated soil then.After the seed culture 20 days, the bud that results grow, grind into powder in liquid nitrogen.Wherein chlorophyll and soluble substance are containing 95% ethanol, and 65 ℃ of incubated overnight are tentatively removed.Centrifugal 5min abandons liquid phase then under the 3000rmp.Complete drying in 65 ℃ of incubators then, these bulk powder of doing utilize mortar and pestle to be changed into the soft powder of homogeneous by further powder.

Enzyme is lived and stability is measured:

In order to measure the vigor of cell wall degradation, reaction soln is by the Q7-31 upper strata nutrient solution (being defined as thick enzyme) of 0.2mL, 0.4mL contain 1.0% (w/v) rice plants powder dissolution in pH5.5, the phosphoric acid buffer of 50mM, 10min. measures reducing sugar in the enzyme reaction solution by edlefsen's reagent method (DNS method) output (Miller, 1959 take place down at 40 ℃ in enzyme reaction; Sengupta etal., 2000).Under the 575nm wavelength, utilize the spectrophotometric determination absorbance value, convert the content of reducing sugar then to according to typical curve.

The mensuration of protein content:

According to Bradford method (Bradford, 1976), 0.4mL mixes by Maas light blue solution and 1.6mL culture supernatant, measures the absorbance value of 595nm wavelength in 5 minutes, utilizes bovine serum albumin drawing standard curve to measure protein content.

The result:

Q7-31 produces the plant cell-wall degrading enzymes screening active ingredients:

The 18 strain bacterium that grow on the substratum that contains the rice plants powder present different opacities, have showed it to the different degradabilities (Fig. 1) of plant powder.Comprise Q7-31, the active disintegration cell walls powder of the bacterial strain of Fig. 1 (A) to (E) becomes colloidal state; Yet, (J) still keep the cell walls small-particle that is not decomposed in the strain cultures in (K) at Fig. 1 (I); The part strain culture (G) and (H) forms the mycelium polymkeric substance of bigger white as Fig. 1 (F), degrading plant powder that can part; In addition, Morchella sp.M-1 disintegration cell walls powder becomes colloidal state also can produce outside the higher reducing sugar; Equally, Geotrichum sp., Morchella spp.M-2 and M-3 can not change cell walls and produce a small amount of reducing sugar (Fig. 1 and 2; Table 1).Except their mode of appearance of culture degradation of cell wall, among these bacterial strains, the reducing sugar that Q7-31 produces the from the 3rd to the 6th day is all the highest, and muddiness (Fig. 1 and 2 of just becoming of the 3rd day substratum; Table 1).With the content of other bacterial strain contrast reducing sugars, by 6 days cultivation, especially the content of reducing sugar was the highest in the Q7-31 substratum.Therefore, we select Q7-31 aspect cell wall degradation to its further research.

Q7-31 morphology and molecules are identified:

As seen, the mycelium of Q7-31 presents the down-like of doing of typical white, spreads all over bacterium colony (Fig.3A) everywhere from Figure 1A.Mycelium is formed ((Fig.3B) .) by many intervals. in order to identify the growth rate of Q7-31, its mycelium is inoculated on the minimum medium solid that contains 0.3% paddy rice powder, cultivated 7 days at 28 ℃, the diameter of bacterium colony reaches about 6cm, in 1 to 3 day growth curve chart, present the abrupt slope shape, show that the growth in this period is logarithmic growth (Fig.4).

Identify at the genome relevant with 18S ribosomal gene partial sequence, genomic dna among the extraction bacterial strain Q7-31 is as the template of pcr amplification, and by round pcr, internal transcription transcribed spacer 1 increases, 5.8S ribosomal RNA gene, internal transcription transcribed spacer 2 and 28S ribosomal RNA gene partial sequence.The sequence information that pcr amplification goes out is used to do BLAST research in ncbi database.BLAST result shows that Q7-31 analyzes section, wherein 99% and Fusarium, and Gibberella, some bacterial strain respective section of Cordyceps ((Fig.5)) are consistent.The analysis of combining form and molecules, we are included into Q7-31 among the Fusarium, and be deposited in A, DaTun Road, Chaoyang District, BeiJing City, Chinese microorganism strain preservation center (Deposition No:CGMCC2710) No. 3 on October 14th, 2008, Institute of Microorganism, Academia Sinica's postcode: 100101 according to Budapest pact.

Originate in the cell wall degrading enzyme of Fusarium sp.Q7-31:

The Q7-31 bacterial strain is grown under 28 ℃ of conditions in the BLM substratum that contains 0.3% paddy rice powder.The amount of the total reducing sugars that produces at the every mL bacterial strain of identical time measurement per minute every day is measured with μ g and to be represented the cell wall degrading enzyme enzymic activity.Increase significantly cultivating enzyme work in the 3rd day, yet very big fluctuation is arranged since enzyme work in the 4th day.Enzyme work lasted till that living to the 8th day enzyme descend (Fig. 6, table 2) occurred the 7th day.When the mensuration enzyme is lived soluble protein content in the bacterial strain Q7-31 substratum is measured.At the 1st day that cultivates, middle protein content accumulation was the fastest.Total protein concentration is stable the raising at 2-5 days that cultivate, and begins to reduce in the 6th day (Fig. 7).In the enzyme variation diagram alive, the stable rising work to the of total protein content reached the peak in 5 days in the Q7-31 substratum.

The stability of Fusarium sp.Q7-31 enzyme:

The enzyme stability of living is unusual important parameters under room temperature environment, and the application facet on industrial production especially is with being like this.The thick enzyme that Q7-21 and Q7-31 bacterial strain produce in substratum is extracted and remains on 25 ℃ or 40 ℃.The vigor of the cell wall degradation of the thick enzyme of storage is at the different time point measurement of cultivating 4 days.Under 25 ℃ of conditions, can also keep the enzyme of original state 75% to live through 3 days thick enzymes of Q7-31.Yet Q7-21 lives at the time glucose-6-phosphate dehydrogenase of uniform temp next day and has just disappeared.And Q7-31 shows and is stored in 25 ℃ of down similar stability (Fig. 8, table 3) remaining under 40 ℃ of conditions.From at least three revision tests, each test is three contrast Q7-21 and is shown identical pattern with the Q7-31 enzyme stability.

The thick enzyme that Q7-31 produces is to the degraded vigor of different plant cell wall powder:

For the inducing cell wall degrading enzyme produces, Q7-31 cultivates on the BLM substratum that contains 0.3% rice stem stalk powder and cultivated 3 days.Measuring the enzyme of thick enzyme in the substratum that contains various substrates (barley of local different varieties, wheat, corn, oat and rye) lives.With the rice stem stalk is compared, the Q7-31 bacterial strain shows more high enzymatic activity (Fig. 9) except paddy rice to the degraded vigor of other cereal grass cell wallss, the cereal grass that other have been surveyed is showing different acutenesses at enzyme aspect living, main with high variability is relevant.

Discuss:

In order to improve the production efficiency of biological alcohol.Effectively the degrading plant cell wall polysaccharides has caused people's extensive interest.Therefore develop big quantity research to reduce the anti-degradation property of raw material, method comprises more effective to raw materials pretreatment with to having the research of more extensive more effective product lytic enzyme microorganism.In order to assess the degradability of selected bacterial strain, we select substratum upper strata suspension (thick enzyme) and it not to be further purified reason as follows: the lytic enzyme vigor that (1) has different substrate specificities is essential to the degrading plant cell walls.Berlin et al. (2007) spells out by the Degradation of the wooden plasticized cellulose sample of 1.5L cellulase effect by replenishing Novozym188 (Novozymes), poly zytase (Genencor) and poly polygalacturonase (Genencor) and by a large amount of reinforcements.(2) other multiple different enzymes, the synergy in crude enzyme liquid between the single enzyme also may exist.For example, cultivate in that the feruloyl esterase of purifying is collaborative with the zytase that comes from Sporotrichum thermophile with from Fusarium oxysporum, its activity is reinforced 5 times (Topakas et al., 2003).

In initial shaker test, fungi is cultured in and contains on the BIM substratum of 0.3% rice plants cell walls powder as sole carbon source.From soluble plant powder to colloidal state to degrade fully attitude they show widely cell wall degradation vigor (typical case's representative shows) among Fig. 1.The difference to these cell wall degradation effect degree of performance may be different with the content of reducing sugar in the substratum relevant.Though yet for Morchella sp. bacterial strain M-1 the rice plants powder there is higher Degradation, cultivate relatively low (Figure 1A and 2 of content of reducing sugar in connecing; Table 1).Reason may be to have consumed the reducing sugar that produces in the fungal growth process, and the amount of reducing sugar reduces in the corresponding substratum.Containing the high growth vigor on the agar plate substratum of paddy rice powder and containing high degraded vigor in the paddy rice powder liquid nutrient medium according to mycelium, we select Fusarium sp.Q7-31 to its separation and purification from 18 fungal strains.In the Fusarium of this selection spp. bacterial strain, the cultivation through 3 days and 6 days demonstrates the highest reducing sugar amount (Fig. 2) in the Q7-31 substratum.Especially passed through six days cultivation, high reducing sugar content shows that the enzyme that bacterial strain can continue to produce enzyme or generation has high stability in the substratum.As can be seen from Figure 6 the enzyme work of Q7-31 began to occur bigger fluctuation at the 3rd day.May be that this influences the vigor that whole enzyme produces reducing sugar because produce dissimilar cell wall degrading enzymes at different time points.For example, the cellulase of three kinds of main types is arranged, comprise β-endoglucanase, β-exoglucanase and beta-glycosidase.Since the specificity of these enzyme substratess, the different degradation efficiencies (Palmarola-Adrados et al., 2005) that may influence Microcrystalline Cellulose in the cell walls of these cellulases content in the substratum on particular point in time with ratio.The enzyme work of the thick enzyme of Q7-31 approximately was 66 μ g/min/mL at the 3rd day in the substratum, approximately 0.37U/mL.Khan et al reported once that suitable with it enzyme was alive, and he induces Trichoderma spp. and Phanerochaetechrysosporium to produce cellulose degrading enzyme with rice straw.The concentration that obtains cellulase from the Trichoderma sp T-3 substratum of growing 6 days is 0.65U/mL filter paper vigor, yet, the activity of the cellulase that Phanerochaetechrysosporium produces is the highest, about 1.43U/mL filter paper vigor and 2.40U/mL carboxymethyl cellulose vigor.Yet the no comparability of the linearity of the thick enzyme activity of Q7-31 and these enzymes contrast, because the difference of special parameter, for example total class and the quantity of substrate, the pre-treatment of substrate and the pH and the temperature of reacting.

Proteic accumulation has two kinds of different sources in the substratum: the protein that fungi is discharged into extracellular albumen and balances each other with the rice plants cell walls of degrading.The cell wall protein that discharges in the long more substratum of fungi incubation time in the paddy rice powder culture medium is many more.Yet the 5th day protein content that Fig. 5 is presented at cultivation begins to descend.May be since fungus secretion be not only protein that cell wall degrading enzyme also has other kinds for example proteolytic enzyme and transglycosylase, these enzymes produce negatively influencing (Hrmov á et al., 1991) to the protein accumulation in the substratum after surpassing 5 days cultivating.

Except very high enzymic activity, the thick enzyme that the Q7-31 bacterial strain produces has the Q7-21 bacterial strain of similar cell wall degradation vigor to compare with the thick enzyme that is the generation of Fusarium sp genus equally and has higher stability.25 ℃ of cultivation Q7-31 through 4 days keep at least 50% initial enzyme to live.Yet under the same terms, Q7-21 advanced one day cultivation enzyme work with regard to completely dissolve (Fig. 8 and table 3).Gerber et al. (1997) is purified into two kinds of zytases from Trichoderma sp., a kind of is molecular weight 20kD, and the iso-electric point value is low, and another kind is molecular weight 30kD, the medium value iso-electric point.Live though the zytase of macromolecule has lower enzyme, it shows the thermostability higher than having of small molecular weight.The substrate that the stability that studies show that enzyme of Raquel et al. (1997) is cultivated zytase influences.Stephens et al. (2007) points out the varient at thermostable xylanases, and its gene comes from thermophilic fungus Thermomyces lanuginosus and increases by error-prone PCRs.Find to compare with those wild-type zytases in their research, the varient zytase of these three kinds of specializations 1 or 3 amino acid on aminoacid sequence is replaced the result and is demonstrated the raising of 2 to 3 times of thermostabilitys.Similarly, Damaso et al. (2003) points out that 50 ℃ of processes of recombinant chou zytase 4h cultivation of selecting can keep 100% enzyme work.Infer that according to these aspects the high stability of the thick enzyme of Q7-31 may be because cell wall degrading enzyme molecular genetics properties changes and causes.

The detection of the zymologic property of Q7-31 is not only carried out on the rice plants powder, detects its degradation capability to other cereal grass cell walls powder simultaneously.These cereal grass powder comprise local barley, wheat, and corn, the mutation of oat and rye comes bearing results of inducible enzyme to find that these plant powders are easier of the thick enzyme liberating of Q7-31 with these powder, and the enzyme of the thick enzyme of paddy rice powder is lived higher relatively simultaneously.These cereal grasss are made up of different cell walls polymers.For example, rice straw is by 41.6% Mierocrystalline cellulose, and 31.5% hemicellulose and 12.5% xylogen are formed (Pauly andKeegstra, 2008).

Though the content of xylogen is more a lot of than height in the rice straw in wheat stalk, yet the hemicellulose of low levels may be thick enzyme is higher than the paddy rice powder to the degradability of wheat plant powder a major cause in wheat stalk.For the composition of different cell wallss, cellulosic fibre may exist with the crystal form of different levels in the cereal grass cell walls in addition, and this can influence the concentration of degradable end and the affinity of lytic enzyme (Jeoh et al., 2007).These data show that the thick enzyme of inducing Q7-31 to produce with the rice plants powder has substrate specificity relatively widely.

In a word, Fusarium sp.Q7-31 is induced by vegetation water rice plant powder easily and produces the highly active thick enzyme of high stable.The advantage of Q7-31 also comprises, vigorous through in a few days incubation growth on the liquid medium within, the cell wall degradation production of enzyme is high relatively.The thick enzyme that produces from Q7-31 in addition shows all has Degradation to most of kind plant cell walls.Show that by further optimization this bacterial classification is particularly having higher advantage on the industrial application on biological Alcohol Production cost efficiency to zymetology production and cell wall degradation condition.

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Claims (8)

1.Fusarium?sp.Q7-31(Deposition?No：CGMCC2710)。

2. the Fusarium of claim 1 can secrete the degrading plant cell wall degrading enzyme in the liquid medium within.

3. the bacterium Fusarium in the claim 1, the enzyme that its produces can become fermentable sugars by the Synergistic degradation plant cell wall.

4. the bacterium Fusariun in the claim 1, it is the carbon source that produces enzyme with plant straw powder as inductor.

5. the bacterium Fusarium in the claim 1, the enzyme that its produces is better than rice straw to the degradation effect of other grain straws such as barley, wheat, corn, oat, rye.

6. exist the application of degrading plant cell walls with the bacterium Fusarium in the claim 1.

7. the application in the claim 1 or 6, utilizing the supernatant liquor behind the Fusarium fluid suspension culture is that crude enzyme liquid comes degrading plant stalks.

8. at least 75% the enzymic activity that still can keep original state in 3 days is placed in the application in the claim 1 or 3, the thick enzyme of Fusarium sp.Q7-31 down 40 ℃ and 25 ℃.

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