CN104263667B - Streptomyces althioticus and applications thereof - Google Patents

Streptomyces althioticus and applications thereof Download PDF

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CN104263667B
CN104263667B CN201410260651.4A CN201410260651A CN104263667B CN 104263667 B CN104263667 B CN 104263667B CN 201410260651 A CN201410260651 A CN 201410260651A CN 104263667 B CN104263667 B CN 104263667B
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xylanase
fermentation
streptomycete
lmzy
different sulfur
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CN104263667A (en
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蔡俊
罗玲
王常高
杜馨
周安盛
向梦雄
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Hubei University of Technology
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
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    • C12R2001/465—Streptomyces

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Abstract

Streptomyces althioticus and applications thereof are disclosed. The strain is streptomyces althioticus LMZY, is deposited in the China Center for Type Culture Collection on March 28, 2014, and has an accession number of CCTCCM2014110. The strain can produce xylanase in a high yield after utilizing agricultural wastes, thus effectively solving recovery and reutilization of the agricultural wastes, reducing the production cost, reducing environment pollution and improving added value of products.

Description

A kind of different sulfur streptomycete and its application
Technical field
The invention belongs to microbial technology field, maize cob meal is utilized to produce xylanase ability in particular to one plant Stronger different sulfur streptomycete (streptomyces althioticus) and its application.
Background technology
Hemicellulose is the second largest regenerated resources beyond cellulose that continue, universally present in timber, grass, farming Among thing garbage.Xylan, as the important component of plant hemicellulose, accounts for 1/3rd of plant total sugar, it mainly becomes Be divided into d-2 xylose (be a kind of can by microbial cell transform into SCP and energy source pentose).
Xylanase refers to xylan degrading can become the general name of one group of enzyme of oligosaccharide and xylose, and main inclusion inscribe β- Isosorbide-5-Nitrae-xylanase, circumscribed β-Isosorbide-5-Nitrae-xylanase and xylobiase etc., wherein inscribe β-Isosorbide-5-Nitrae-xylanase are key Enzyme.As new association with pulp bleaching auxiliary agent, xylanase can reduce the consumption of bleaching chlorine, reduces the environment in paper industry Pollution problem；In food industry, xylanase is in the improvement of Semen Tritici aestivi flour and improves outside fruit and vegetable juice effective ingredient leaching rate, its The low xylan of feature that degraded is formed has significant bacillus bifiduss multiplication capacity, not digested characteristic, no dental caries, promotes people To multiple good physiology characteristics such as the absorptions of calcium as fodder enzyme preparation, xylanase can effectively release xylan to body Anti- trophism, promotes poultry that roughage is digested and assimilated, and adds xylanase simultaneously and can also reduce livestock and poultry intestinal canal diseases, increases Enter livestock birds health, make poultry body weight uniformly, reduce viscous excrement, reduce in the air ammonia and sulfide concentration；Xylanase can hydrolyze Hemicellulose in plant, the pentose being formed can be further used for producing xylitol, ethanol, the product such as organic acid, can Regenerated resources bioconversion using in there is important function.Therefore, xylanase papermaking, food, feedstuff, weaving, medicine and The fields such as the energy have a wide range of applications.
The species of xylanase is various and is widely present Yu Haiyang and land, but because obtains the former of complexity and purity Cause, currently commercial xylanase still rely primarily on fermentable and be obtained.Report that the microorganism producing xylanase mainly has carefully Bacterium, actinomycetes, funguses, aspergillosis, Trichoderma spp. etc..And studying both at home and abroad and applying most is that the wood that antibacterial, aspergillosis, Trichoderma spp. are produced gathers Carbohydrase, such as: the hemicellulose induction streptomycete strs-2 that the report such as Zhu Qizhong is extracted with wheat straw, producing enzyme vigor is less than 50 u/ ml；Also there is researcher directly to use agricultural wastes inducible strain to produce xylanase, but producing enzyme level is generally relatively low.As Report strain streptomycete ab106 such as charin techapun utilizes agricultural wastes producing enzyme vigor only 15 u/ml；Li Jiaqun Deng with rice straw powder fermentation culture streptomycete lh-3 at optimum conditions, Xylanase activity is up to 120.3 u/ml) slightly below Aspergillosis and Trichoderma spp., but usual not eccrine fiber element enzyme, therefore it has higher industrial application value.
Content of the invention
Because the report of actinomycetes product xylanase is less, therefore the present inventor utilizes agricultural wastes pair by laboratory The actinomycetes producing xylanase carry out fermentation condition optimization screening, and the actinomycetes sieved are cooked further selection-breeding, and obtaining one plant can Using maize cob meal high yield xylanase bacterial strain different sulfur streptomycete (streptomyces althioticus).The present invention is obtained The bacterial strain obtaining not only is only capable of high yield xylanase it is often more important that efficiently solving the regeneration of agricultural wastes, reduces life Produce cost, reduce environmental pollution, improve product surcharge.
In order to realize the purpose of the present invention, inventor is repeated Screening And Fermenting Cultivation research by lot of experiments, and Obtain eventually one plant utilize maize cob meal produce the stronger different sulfur streptomycete of xylanase ability (streptomyces althioticus) lmzy, it is preserved in China on March 28th, 2014. Wuhan. Wuhan University. China typical culture collection Center, deposit number is: cctcc m 2014110.
The high yield xylanase being related to of the present invention different sulfur streptomycete (streptomyces althioticus) lmzy There is following biological property:
Morphological feature: fibrillae of spores spiral type, aerial hyphae powdery, white, the back side is light brown.
Physiological property: using glucose, Fructose, rhamnose, Mannitol and galactose；In arabinose, xylose, inositol And maltose etc. is as carbon source；Produce the althiomycin (althiomycin) of suppression Gram-positive and negative bacteria.
Metabolic characteristic: metabolic process can produce xylanase.
Compared with existing different sulfur streptomycete, the different sulfur streptomycete that the present invention is obtained by mutagenesis screening (streptomyces althioticus) lmzy has the advantage that and marked improvement:
(1) bacterial strain of institute of the present invention selection-breeding can be using high yield xylanase after agricultural wastes, gained fermentation liquid after fermentation Middle xylanase activity is up to 38.86u/ml.
(2) regeneration of effectively solving agricultural wastes of the present invention, reduces production cost, reduces environmental pollution, carries High product surcharge.
Brief description
Fig. 1 is the influence curve figure to enzyme activity for the fermentation period.
Fig. 2 is the impact bar diagram to enzyme activity for the fermentation temperature.
Fig. 3 is the impact bar diagram to enzyme activity for the inoculum concentration.
Fig. 4 is the impact bar diagram to enzyme activity for the initial ph.
Fig. 5 is the impact bar diagram to enzyme activity for the liquid amount.
Specific embodiment
Form is described in further detail to the above of the present invention again by the following examples, but should not manage this Solve the scope for the above-mentioned theme of the present invention and be only limitted to below example, all technology realized based on the above of the present invention are equal Belong to the scope of the present invention.
Embodiment one: the selection-breeding of strain
(1) bacterial strain
Different sulfur streptomycete (streptomyces althioticus)
(2) culture medium
Enrichment medium described in every 1 l contains: self-control Corncob Xylan 10g, peptone 10 g, nacl 5 g, k2hpo41.5 g、 mgso4· 7h2O 1 g, described fluid medium ph is natural；
Pseudonocardia described in every 1 l contains: self-control Corncob Xylan 8g, kno31g、nacl 0.5g、 mgso4· 7h2o 0.5g、 k2hpo41g, agar 20g, described Solid media for plates ph is natural；
In shaking flask described in every 1 l, seed culture medium contains: soluble starch 20g, kno31g、k2hpo40.5g、mgso4 · 7h2o 0.5g、nacl 0.5 g 、feso4· 7h2O 0.01g, described fluid medium ph is natural；
Solid slant culture base described in every 1 l contains: soluble starch 20g/l, kno31g/l、k2hpo40.5g/l、 mgso4· 7h2o 0.5g/l、nacl0.5 g/l 、feso4· 7h2O 0.01 g/l, agar 20 g/l 0.1mpa Sterilizing 20min, described inclined-plane solid medium ph are natural；
In shaking flask described in every 1 l, secondary screening fermentation medium contains: maize cob meal 20g, yeast powder 10 g, k2hpo4 0.5g、na2hpo40.5g、mgso4· 7h2O 1.0g, described fermentation medium ph5.5.
(3) solution used in testing
Physiological saline solution: take 8.5g sodium chloride to be dissolved in dissolving in the distilled water of 1l, 0.1mpa sterilizing 20min.
(4) experimental procedure
Gather 10 parts of soil sample, 6 points of water sample from paper mill.Take certain sample first from every part of sample, use sterile physiological Sample is broken up by saline；Use enrichment medium (self-control Corncob Xylan 10g/l, peptone 10 g/l, nacl 5 g/ again l、k2hpo41.5 g/l、 mgso4· 7h2O 1 g/l 0.1mpa sterilizes 20min) 30 DEG C of culture 1d；Then use physiology salt Water gradient dilution becomes different soil supensions, takes 0.1ml to coat under the conditions of Selective Separation flat board puts 37 DEG C, cultivates 2-4 D, picking transparent circle is big and the bacterial strain of obvious actinomycetes form is as primary dcreening operation bacterial strain；Choose the single bacterium colony on flat board, access tiltedly Face solid medium preservation is standby.
By shake flask fermentation verification experimental verification, obtain one plant and utilize maize cob meal to produce the stronger different sulfur chain of xylanase ability Mycete (streptomyces althioticus) lmzy, it is preserved in China typical culture collection on March 28th, 2014 The heart, deposit number is: cctcc m 2014110.
Example two: the training systern of different sulfur streptomycete lmzy
(1) bacterial strain: the strain different sulfur streptomycete lmzy that example one filters out
(2) method and step:
Fermentation medium is initial medium (maize cob meal 20g/l, yeast powder 10 g/l, k2hpo40.5g/l、 na2hpo40.5g/l、mgso4· 7h2o 1.0g/l).Initial fermentation condition is: natural ph, inoculum concentration 10%, liquid amount 50 Ml/250 ml, 37 DEG C, 180r/min shake flask fermentation 72h.
Using the fermentation temperature of the single factor experiment different sulfur streptomycete lmzy of investigation, initial ph, fermentation period, inoculum concentration, dress The impact to xylanase yield for the liquid measure.
A. the optimization test of fermentation period
Other fermentation conditions are initial fermentation condition, every 12h sampling and measuring xylanase activity, determine different sulfur streptomycete Lmzy produces the optimum fermentation period of xylanase.
As shown in Figure 1, the enzyme activity highest when fermentation is to 120h, is 3.546u/ml.Therefore select fermentation period to be 120h.
B. the optimization test of fermentation temperature
Fermentation time takes the optimal result that above-mentioned test determines, other fermentation conditions are initial fermentation condition, respectively 25 DEG C, 28 DEG C, 31 DEG C, 34 DEG C, 37 DEG C, carry out fermentation culture in 40 DEG C and 43 DEG C of shaking tables after measure xylanase activity, Investigate the impact that temperature produces xylanase to different sulfur streptomycete lmzy.
As shown in Figure 2, when fermentation temperature is 40 DEG C, enzyme activity highest, is 4.129 u/ml.Therefore select fermentation temperature to be 40 ℃.
C. the optimization test of inoculum concentration
Fermentation time and fermentation temperature take the optimal result that above-mentioned test determines, other fermentation conditions are Preliminary fermentation bar Part, when inoculum concentration is respectively 2%, 4%, 6%, 8%, 10% and 12%, investigates inoculum concentration and produces xylanase to different sulfur streptomycete lmzy Impact.
Known by Fig. 3, when inoculum concentration is 6%, enzyme activity highest, is 4.589u/ml.Therefore select inoculum concentration to be 6%.
D. the optimization test of initial ph
Fermentation time, temperature and inoculation measure the optimal result that above-mentioned test determines, other fermentation conditions are Preliminary fermentation Condition, adjusts initial ph and is respectively 4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0 and 8.5, after shake flask fermentation culture Measure xylanase activity, investigate the impact that ph produces xylanase to different sulfur streptomycete lmzy.
As shown in Figure 4, when the initial ph that ferments is 5.0, enzyme activity highest 4.876u/ml.Therefore select initial ph to be 5.0.
E. the optimization test of fermentation shake flask liquid amount
Fermentation time, temperature and inoculum concentration and initial ph take the optimal result that above-mentioned test determines, other fermentation conditions are Initial fermentation condition, fills liquid using 250 ml shaking flasks, when shaking flask liquid amount is respectively 30ml, 50ml, 70ml, 90ml, investigates dress Liquid measure produces the impact of xylanase to different sulfur streptomycete lmzy.
As shown in Figure 5, when shaking flask liquid amount is 50ml (Flask volume 250ml), enzyme activity is 4.887u/ml.Therefore select Liquid amount is 50ml (Flask volume 250ml).
Example three: fermentation checking test
With different sulfur streptomycete lmzy as fermentation strain；Condition of culture is fermentation time 120h, 40 DEG C of temperature, inoculum concentration 6%, Initial ph5.0, liquid amount 50ml (shaking flask is 250ml)；Medium component is: maize cob meal 31.45g/l, Semen sojae atricolor powder 16 g/ L, potassium nitrate 8g/l, k2hpo40.4g/l、na2hpo40.528g、mgso4· 7h2O 0.4g/l, carries out fermentation checking examination Test.In gained fermentation liquid after fermentation, xylanase activity is 38.86u/ml.

Claims (2)

1. different sulfur streptomycete (streptomyces althioticus) lmzy, deposit number is cctcc m 2014110.
2. the different sulfur streptomycete lmzy described in claim 1 is producing answering of xylanase using agricultural wastes maize cob meal With.
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