CN109536431A - Composite bio-chemical Resuscitation promoting factor composition and its application - Google Patents
Composite bio-chemical Resuscitation promoting factor composition and its application Download PDFInfo
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Abstract
The present invention provides composite bio-chemical Resuscitation promoting factor composition and its application, the composition components include two kinds of Rpf albumen and Sodium Pyruvate；Described two Rpf albumen are the recombinant protein MLUT_RS18675 from micrococcus luteus, the recombinant protein c HU_0099 from Cytophaga hutchinsonii；The recombinant protein is obtained by Escherichia coli heterogenous expression and purifying.Stalk is added in screening and culturing medium as substrate and inoculum, while adding the composite bio-chemical Resuscitation promoting factor composition, the bacterium solution for cultivating acquisition is the straw degradative microbial flora screened, and can be used for straw degradative process.Composition provided by the present invention and its application can improve the ratio of uncultured microorganisms by the activation of the organism mulching factor and the protection mechanism of Sodium Pyruvate, and then filter out the microbial flora of high activity for fields such as the ecological agriculture, environmental improvement and derived energy chemicals.
Description
Technical field
The present invention relates to microorganism fields, and in particular to composite bio-chemical Resuscitation promoting factor composition and its application.
Background technique
Microorganism meeting when condition of culture (such as temperature, pH, osmotic pressure, oxygen concentration, nutrient concentrations) changes
Into " suspend mode " state with the variation of response environment condition, this state is referred to as living but can not cultivation conditions (Viable
But not culturable, VBNC), alternatively referred to as difficult culture microorganism.Microorganism into VBNC state can not be artificial
Culture.Show that in most of environment 90% ~ 99% microorganism can not manually be cultivated based on 16S rRNA Quantitative Study
Or difficult culture.Microorganism in VBNC state can be recovered under given conditions, and Resuscitation promoting factor reported at present is main
There are two types of, it is organism mulching factor protein and chemical Resuscitation promoting factor respectively.Kaprelyants et al. is had found for the first time from rattan
Yellow micrococcus luteusMicrococcus luteusResuscitation promoting factor(Rpf), Rpf albumen is not only
The microorganism that suspend mode can be activated may additionally facilitate division and proliferation (the Applied and of the microorganism of living body
Environmental Microbiology, 1993,59:3187-3196).The knockout of Rpf protein coding gene will lead to rattan
Yellow micrococcus luteus can not survive, it means that Rpf albumen is necessary (FEMS Microbiology for certain micro-organisms
Reviews 104:271-286).Research based on Rpf albumen is concentrated mainly on medicine and area of infectious disease, but has existed at present
Industrial wastewater pollution object degradation direction is applied.Zhejiang Normal University professor Ding Linxian is by micrococcus luteus Rpf albumen application
It is identified in the separation of VBNC special resource microorganism.Ding Linxian et al. reports a kind of more chlorine of utilization cell factor containing Rpf screening
The method of phenylbenzene degradation flora, this method can be such that the degradation rate of polychlorinated biphenyl (3500 mg/L) is increased to by 6.2%
60.8%, research finds there is a large amount of micro- life (Applied of difficulty culture for deriving from Rhod and pseudomonas in the flora
Microbiology Biotechnology, 2015,99:1989-2000).Further bacterial strain sieve is carried out using Rpf albumen
Choosing, obtain 6 plants with polychlorinated biphenyl degrading activity new strains (Bioresource Technology, 2013,146:
27-34).In addition, by means of micrococcus luteus Rpf cell factor, flocculation is had successfully been obtained in researcher from sewage disposal system
Rate is up to 98.23% bacterial strain.Micrococcus luteus Rpf albumen be reported can widely act on mycobacterium category, Rhod,
Bacillus (Microbiology, 2011,80,143-149).The research of microorganism is screened still currently with Resuscitation promoting factor
The Rpf albumen from micrococcus luteus being put forward for the first time dependent on 1993.However, research shows that difference Rpf albumen is answered
There are difference in genera by Su Zuoyong.Rpf albumen is a kind of bifunctional enzyme with proteinase activity and glycohydrolase activity,
Recovery microorganism is by cell-wall components under hydrolysis VBNC state, and then the division growth function of recovery microorganism.With point
The development of sub- bioinformatics has found Rpf albuminoid gene (BMC in Firmicutes and actinomyces Men Zhongjun in recent years
Genomics, 2005 , 6 (1) :39.；Microbiology, 2013,159 (7): 1298).
In recent years a large number of studies show that microorganism can adapt to environment-stress by way of synthesizing pyruvic acid.Pyruvic acid
As the important intermediate of glycolytic cycle, a variety of biochemical reaction processes are taken part in, are the existence of microorganism and mentioning for metabolism
For necessary precursor substance.In the research of early stage, Morishige et al. compares the Effect of Resuscitation of different acetone acid-like substances,
It was found that pyruvic acid may be the energy metabolic pathways for taking part in microorganism, and demonstrates pyruvic acid and closed in activation DNA and protein
Play a significant role (Frontiers in public health, 2014,2:103) in.
There are efficient degradation straw in the environment such as ruminant tumor gastric, straw compost area, anaerobic fermentation pond and termite gut
The microbial flora of stalk.However, being less able to drop based on the straw degradative flora that traditional cultivation is screened from above-mentioned environment
Untreated stalk is solved, this is because there is a large amount of difficult culture microorganism to participate during straw degradative.Li Fuli et al.
Two kinds of fibers that can not manually cultivate are screened from corn stover deposit by adding Bacillus licheniformis cell extracting solution
Element degradationClostridium The activity of beta-glucosidase of CS-3-2 and CS-4-4, CS-3-2 are 18 times of CS-4-4, and CS-
The beta-xylanase activity of 4-4 is 27 times of CS-3-2, and two kinds of bacterial strains of co-incubation significantly improve the degradation energy of corn stover
Power (Applied and environmental microbiology, 2014,00295-14).Therefore, pass through addition biology
The culture that chemical Resuscitation promoting factor improves difficult culture microorganism in environment is a kind of straw degradative microorganism with very high potential
Screening technique, however, at present both at home and abroad temporarily without using composite bio-chemical Resuscitation promoting factor screen or improve natural environment in straw degradative
The report of microorganism.
Summary of the invention
The purpose of the present invention is to provide a kind of composite bio-chemical Resuscitation promoting factor composition and its applications.
To realize the above-mentioned technical purpose, the present invention adopts the following technical scheme:
A kind of composite bio-chemical Resuscitation promoting factor composition includes two kinds of Rpf albumen and Sodium Pyruvate；The Rpf albumen is next
Derived from micrococcus luteus ((Micrococcus luteusNCTC 2665) recombinant protein MLUT_RS18675(Gene ID:
7985360) and from Cytophaga hutchinsonii (Cytophaga hutchinsoniiATCC 33406) recombinant protein c HU_
0099(GenBank:ABG57393.1)；The recombinant protein passes through Escherichia coliE.coli BL21 (DE3) heterogenous expression and
Purifying obtains.
Method of the invention, in the composition, recombinant protein MLUT_RS18675 concentration is 0.01 ~ 1.0 mg/L, excellent
Select 0.1 ~ 1.0 mg/L；Recombinant protein c HU_0099 concentration is 0.01 ~ 1.0 mg/L, preferably 0.1 ~ 1.0 mg/L；；Sodium Pyruvate
Concentration is 0.05 ~ 5.0 mM, preferably 0.5 ~ 5.0 mM.
The Rpf albumen is the purifying protein obtained through nickel affinity chromatography and dialysis, and purity is greater than 90%.
Another object of the present invention is to provide the applications of above-mentioned composite bio-chemical Resuscitation promoting factor composition.
The application method are as follows: the composition is added to the screening and culturing medium comprising stalk, is cultivated.
The present invention provides a kind of specific screening techniques, include the following steps:
A) screening and culturing medium is prepared, the stalk of crushing is added as substrate；
B) inoculum is added under anaerobic environment into culture medium；
C) composite bio-chemical Resuscitation promoting factor composition is added into the culture medium that inoculum is added；
D) anaerobic environment is kept to carry out stationary culture and secondary culture, each secondary culture be added the composite bio-chemical recovery because
Sub-portfolio object；Collecting thallus is gained straw degradative microbial flora.
The screening and culturing medium is PCS culture medium, ingredient are as follows:
1.0 g/L of yeast extract, 5.0 g/L of peptone, 5.0 g/L of sodium chloride, 3.0 g/L of calcium carbonate.
The present invention may be based on different substrate/inoculums and select different screening and culturing mediums.
Further, the application further include: carry out straw degradative using the straw degradative microorganism filtered out.
The mode of straw degradative specifically: the thallus of stalk and collection, closed culture are added in the medium.
There are a large amount of microbial resources, the albumen of genome encoding is in agricultural, chemical industry and energy neck in natural environment
Domain plays an important role.However, the screening technique based on culture-based method, which can not filter out in environment, can not cultivate micro- life
Object, which has limited the excavations of the depth of microbial resources.The present invention is respectively acting on gram sun using two kinds of organism mulching factors
Property and gramnegative bacterium, while the angle for using chemical Resuscitation promoting factor to synthesize from enhancing energy supply, DNA and protein with
The Culturability of environmental microorganism is improved, the microbial resources of acquisition can not be screened by excavating culture-based method.
Detailed description of the invention
Fig. 1 is that flora is obtained under different composite Resuscitation promoting factor additive amount to the degradation rate difference of wheat straw；
Fig. 2 is the wheat straw degradation rate that compost sample screening obtains different floras；
Fig. 3 is the wheat straw degradation rate that cow rumen screening obtains different floras；
Fig. 4 is the wheat straw degradation rate that forest soil screening obtains different floras.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
It applies content described in example and is merely to illustrate the present invention, without hair described in detail in claims should will not be limited
Bright content.
Embodiment 1
The present embodiment illustrates the preparation method of composite bio-chemical Resuscitation promoting factor composition.
Two kinds of specific obtaining steps of recombinant protein are as follows:
1) building can the expression cell organism mulching factor recombinant bacterium
The method that the encoding gene of Rpf albumen MLUT_RS18675 and CHU_0099 pass through one-step cloning respectively is connected to
The multiple cloning sites of pET28a vector plasmid obtain pET28a-MLUT_RS18675 and pET28a-CHU_0099 recombination respectively and carry
Constitution grain；Transfer vector plasmid is converted respectively toE.coliBL21(DE3 corresponding recombinant bacterial strain) is constructedE.coli
BL21(DE3) pET28a-MLUT_RS18675 andE.coliBL21(DE3) pET28a-CHU_0099.Picking individual colonies extract
Plasmid is sequenced, and confirmation expression vector establishment is correct.
2) inducing expression of cell biological Resuscitation promoting factor
Two kinds of recombinant bacteriums are crossed in LB solid medium containing kanamycin respectively and are cultivated, picking individual colonies, which are inoculated in, to be contained
There is activation culture in kanamycins LB liquid medium, condition of culture is 30 DEG C, 12h, obtains seed liquor；Seed liquor is pressed respectively
Ratio 1.0%(v/v) is seeded to 30 DEG C of 2 ~ 3h of culture of LB culture medium, works as OD600The IPTG induction of 0.5 mM is added when being 0.6
The expression of Rpf albumen, inductive condition are 20 DEG C, 12h.
3) cell biological Resuscitation promoting factor isolates and purifies
It after induction, is centrifuged under the conditions of 4 DEG C, 8000 rpm, collects recombination bacillus coli thallus respectively.In the ratio of 20:1
After thallus is resuspended with equilibrium liquid respectively, clasmatosis is carried out, broken condition is 350w power, and super 3s stops 6s, and ultrasonication 30 divides
Clock.With 12000rpm centrifugation 10 minutes under the conditions of 4 DEG C, supernatant is collected, and with 0.22 μm of 2 membrane filtration supernatant of water system.
Supernatant with 1mL/min flow velocity be added to nickel affinity chromatography column until A280 stablize after, use the removal of impurities liquid of 10 times of column volumes with
The flow velocity of 0.5mL/min removes foreigh protein removing.The elution that 3 times of column volumes are respectively adopted collects recombinant protein MLUT_
RS18675 and recombinant protein c HU_0099, above-mentioned albumen is placed in the bag filter of 3000Da molecular weight, is placed in dialyzate thoroughly
Analysis is stayed overnight, to obtain the recombinant protein MLUT_RS18675 and recombinant protein c HU_0099 of purifying respectively.
Equilibrium liquid: 20 mM Tris-HCl, 0.5M NaCl, pH8.0；
Clean liquid: 20 mM Tris-HCl, 0.5 M NaCl, 50 mM imidazoles, pH8.0；
Eluent: 20 mM Tris-HCl, 0.5 M NaCl, 500 mM imidazoles, pH8.0；
Dialyzate: 20 mM Tris-HCl, pH8.0.
The recombinant protein MLUT_RS18675 and recombinant protein c HU_0099 purity of acquisition are all larger than 90%.
Embodiment 2
The present embodiment illustrates various concentration composite bio-chemical Resuscitation promoting factor composition and screens wheat straw degrading microorganism in biogas slurry
In application effect
Specific steps are as follows:
1) wheat straw degrading microorganism bacterium screening
50 mL PCS medium components are weighed, are settled to 45mL with pure water.Perseverance is dried to 60 DEG C of wheat straw that crushed 20 meshes
Weight is stand-by.
The wheat straw (0.5 g) of 1.0 wt% is added as substrate, 121 DEG C sterilize 15 minutes, are cooled to room temperature spare.To
Lead to high-purity nitrogen (purity 99.999%) in PCS culture medium 2 minutes, immediately accesses the fresh biogas slurry of 5 mL as inoculation
Object, the composite bio-chemical Resuscitation promoting factor composition of addition various concentration combination, continues to be passed through high pure nitrogen 2 minutes, with aluminium lid
Rubber plug is closed.37 DEG C carry out continuous enrichment culture, and passage in every 7 days is primary, and the biochemistry that each passage is both needed to be added above-mentioned concentration is multiple
Su Yinzi.Not add the experimental group of biochemical Resuscitation promoting factor as control, adding biochemical Resuscitation promoting factor is experimental group, above-mentioned sieve
After the straw degradative microorganism of choosing is stored in anaerobism bottle under normal temperature conditions or is mixed with 30% glycerol and bacterium solution in 1:1 ratio
Packing saves.
2) different microflora degradation wheat straws are utilized
50 mL PCS medium components are weighed, are settled to 45 mL with pure water, the wheat straw (0.5 g) that 1.0 wt% are added is used as bottom
Object, 121 DEG C sterilize 15 minutes, are cooled to room temperature spare.
Lead into PCS culture medium high-purity nitrogen (99.999%) 2 minute, 5 mL difference addition sides are immediately added
The bacteria group culture liquid that acquisition is screened under formula continues to be passed through high pure nitrogen 2 minutes, closed with the rubber plug with aluminium lid.
After degradation in 15 days, remaining stalk is collected by filtration with 100 mesh gauzes, stalk cleans 3 postpositions with 50mL pure water
In 105 DEG C of baking ovens, drying to constant weight, records weight and calculates straw degradative rate.
This experiment devises 3 kinds of addition manners according to the concentration combination of Resuscitation promoting factor composition components:
Addition manner 1:0.01mg/L Rpf albumen (MLUT_RS18675), 0.01mg/L Rpf(CHU_0099), 0.05 mM
Pyruvic acid.
Addition manner 2:0.1mg/L Rpf albumen (MLUT_RS18675), 0.1mg/L Rpf(CHU_0099), 0.5 mM
Pyruvic acid.
Addition manner 3:1.0 mg/L Rpf albumen (MLUT_RS18675), 1.0 mg/L Rpf(CHU_0099), 5.0
MM pyruvic acid.
Different flora wheat straw degradation capability comparisons are as shown in Figure 1, the results showed that: addition composite bio-chemical Resuscitation promoting factor composition
The flora that screening obtains is significantly better than the degradation capability of wheat straw and does not add what the screening of composite bio-chemical Resuscitation promoting factor composition obtained
Flora, through degradation in 15 days, the experimental group wheat straw degradation rate for adding composite bio-chemical Resuscitation promoting factor composition respectively reaches 35.6%,
40.7% and 41.2%, it is only 29.1% without adding experimental group wheat straw degradation rate, wheat straw degradation rate can be improved 22.3% to 41.7%.
Meanwhile the microflora degradation rate difference that addition manner 2 and addition manner 3 obtain is smaller, therefore additive amount 2 is preferred addition concentration
Combination.
Embodiment 3
The present embodiment illustrates different composite biochemistry Resuscitation promoting factor composition additive amount in varying environment sample screening varieties wheat straw
Application effect in degradation flora
Specific steps are as follows:
1) wheat straw degrading microorganism bacterium screening
50 mL PCS medium components are weighed, are settled to 45mL with pure water.Perseverance is dried to 60 DEG C of wheat straw that crushed 20 meshes
Weight is stand-by.
The wheat straw (0.5 g) of 1.0 wt% is added as substrate, 121 DEG C sterilize 15 minutes, are cooled to room temperature spare.To
Lead to high-purity nitrogen (purity 99.999%) in PCS culture medium 2 minutes, immediately accesses 5 g During High-Temperature Composting soil, milk cow
Rumen fluid and forest soil add the composite bio-chemical Resuscitation promoting factor composition of various concentration combination, continue to be passed through as inoculum
It is high pure nitrogen 2 minutes, closed with the rubber plug with aluminium lid.37 DEG C carry out continuous enrichment culture, and passage in every 7 days is primary, passes on every time
It is both needed to that the biochemical Resuscitation promoting factor of above-mentioned concentration is added.Experimental group not add biochemical Resuscitation promoting factor is added biochemical as control
Resuscitation promoting factor is experimental group, and the straw degradative microorganism of above-mentioned screening is stored under normal temperature conditions in anaerobism bottle or with 30%
Glycerol and bacterium solution are dispensed after mixing in 1:1 ratio and are saved.
2) different microflora degradation wheat straws are utilized
50 mL PCS medium components are weighed, are settled to 45 mL with pure water, the wheat straw (0.5 g) that 1.0 wt% are added is used as bottom
Object, 121 DEG C sterilize 15 minutes, are cooled to room temperature spare.Lead into PCS culture medium high-purity nitrogen (99.999%) 2 minute,
It is immediately added under 5 mL difference addition manners and screens the bacteria group culture liquid of acquisition, continue to be passed through high pure nitrogen 2 minutes, use
Rubber plug with aluminium lid is closed.
After degradation in 15 days, remaining stalk is collected by filtration with 100 mesh gauzes, stalk cleans 3 postpositions with 50mL pure water
In 105 DEG C of baking ovens, drying to constant weight, records weight and calculates straw degradative rate.
This experiment devises 3 kinds of addition manners according to the concentration combination of Resuscitation promoting factor composition components:
Addition manner 1:0.01mg/L Rpf albumen (MLUT_RS18675), 0.01mg/L Rpf(CHU_0099), 0.05 mM
Pyruvic acid.
Addition manner 2:0.1mg/L Rpf albumen (MLUT_RS18675), 0.1mg/L Rpf(CHU_0099), 0.5 mM
Pyruvic acid.
Addition manner 3:1.0 mg/L Rpf albumen (MLUT_RS18675), 1.0 mg/L Rpf(CHU_0099), 5.0
MM pyruvic acid.
From the result of Fig. 2,3,4: the composite bio-chemical Resuscitation promoting factor composition of addition various concentration combination can be mentioned effectively
Degradation effect of the high gained flora to wheat straw.Wherein compost sample source flora highest can be improved 107.4%, cow rumen sample
Highest can be improved 54.4%, and forest soil sample highest can be improved 187.9%.Wherein, the flora in cow rumen source is to wheat straw
Degradation rate highest, reaches 53.3%.In all experimental groups, the drop of addition manner 2 and the obtained flora of addition manner 3 to stalk
Solution rate difference is smaller.
Claims (10)
1. a kind of composite bio-chemical Resuscitation promoting factor composition, which is characterized in that the composition components include two kinds of Rpf albumen and third
Ketone acid sodium；
Described two Rpf albumen are to bite fiber from the recombinant protein MLUT_RS18675 of micrococcus luteus, from Kazakhstan
The recombinant protein c HU_0099 of bacterium；The recombinant protein is obtained by Escherichia coli heterogenous expression and purifying.
2. composition according to claim 1, is characterized in that, the recombinant protein MLUT_RS18675 concentration is 0.01 ~
1.0 mg/L；Recombinant protein c HU_0099 concentration is 0.01 ~ 1.0 mg/L；Sodium Pyruvate concentration is 0.05 ~ 5.0 mM.
3. composition according to claim 1, is characterized in that, the recombinant protein MLUT_RS18675 concentration is 0.1 ~
1.0 mg/L；Recombinant protein c HU_0099 concentration is 0.1 ~ 1.0 mg/L；Sodium Pyruvate concentration is 0.5 ~ 5.0 mM.
4. composition according to claim 1, is characterized in that, the Rpf albumen is obtained through nickel affinity chromatography and dialysis
Purifying protein, purity be greater than 90%.
5. application of any one of claim 1 ~ 4 composition in straw degradative microbe to screen.
6. application according to claim 5, which is characterized in that the application method are as follows: the composition is added to packet
Screening and culturing medium containing stalk, is cultivated.
7. application according to claim 5, which comprises the steps of:
A) screening and culturing medium is prepared, the stalk of crushing is added as substrate；
B) inoculum is added under anaerobic environment into culture medium；
C) composite bio-chemical Resuscitation promoting factor composition is added into the culture medium that inoculum is added；
D) anaerobic environment is kept to carry out stationary culture and secondary culture, each secondary culture be added the composite bio-chemical recovery because
Sub-portfolio object；The thallus for collecting culture is gained straw degradative microbial flora.
8. application according to claim 7, which is characterized in that screening and culturing medium used in the step a) is PCS
Culture medium, ingredient are as follows:
1.0 g/L of yeast extract, 5.0 g/L of peptone, 5.0 g/L of sodium chloride, 3.0 g/L of calcium carbonate.
9. application according to claim 5, which is characterized in that further include: using the straw degradative microorganism filtered out into
Row straw degradative.
10. application according to claim 9, which is characterized in that the mode of straw degradative are as follows: stalk is added in the medium
With the thallus of collection, closed culture.
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