CN106635889A

CN106635889A - Geobacillus sp and application thereof

Info

Publication number: CN106635889A
Application number: CN201611049743.3A
Authority: CN
Inventors: 王琳; 黄红英; 钱玉婷; 常志州
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2016-11-21
Filing date: 2016-11-21
Publication date: 2017-05-10

Abstract

The invention discloses geobacillus sp JAAS-YTC01 and a method of generating keratinase. A strain of the geobacillus sp is preserved in China General Microbiological Culture Collection Center on July 6, 2016, and has a preservation number of CGMCC No.12740; the strain of the geobacillus sp disclosed by the invention can generate the keratinase, feathers can be completely degraded in 48 hours, the problem of recycling waste feathers can be effectively solved, available amino acid and polypeptide are obtained, environment pollution is reduced, and the generated keratinase has thermal stability and is suitable for industrial production utilization.

Description

One plant of ground bacillus and its application

Technical field

The present invention relates to microbial technology field, particularly a kind of ground bacillus (Geobacillus sp.) JAAS- YTC01 and its application in degradation of feather.

Background technology

Keratin derives from ectoderm cell, is that a water insoluble class, diluted acid or dilute alkaline soln, mechanical strength are very high, changes Learn hard protein stable in properties, be widely present in send out, hair, squama, plumage, first, hoof, angle, pawl, beak, in silk and other epidermal structures. More than 1,000,000 tons, and with the fast development of intensive culture, yield increases year by year the annual feather total amount of China.Feather In up to 90% composition be keratin.Feather Analysis of Nutritive Composition shows that amino acid content is potential excellent more than 70% Dystrophin matter resource.It is not most protease such as pawpaw egg but the disulfide bond in keratin structure makes it have anti-degradability The degradeds such as white enzyme, pepsin and trypsase.On the other hand millions of tons of waste feathers can cause in annual world wide The harm of environmental pollution, protein resource there is also waste problem, particularly China's protein resource deficiency, to its processing and utilization more It is of practical significance.

Due to keratin polypeptide interchain disulfide bond, sat linkage, being widely present for hydrogen bond, make keratic structure highly stable, Water insoluble, conventional protease, such as trypsase, papain, pepsin is all difficult degraded keratin.Traditional Biodegrading process is high steam and soda acid cooking process, and the palliating degradation degree of HIGH PRESSURE TREATMENT keratin is limited, can also destroy some tools Nutritious amino acid, such as lysine, methionine and tryptophan, and produce the unwanted amino acid of some animals, such as sulphur Change double alanine；Soda acid cooking process can equally destroy some amino acid with nutriture value, and produce in hydrolytic process Waste water can also be to environment.Using keratinase degradation of feather keratin energy effectively solving the problems referred to above, but at present Also sell without a large amount of business keratinases on the market.

It is separated at present to have more than 30 kinds to the keratic microorganism that degrade, it is belonging respectively to bacterium, actinomyces and fungi. But it is known produce keratinase microorganism belong to normal temperature bacterium mostly, enzyme reaction temperature typically between 40-60 DEG C, the angle of generation The shortcomings of there is heat endurance difference in protease, and most of microbe can not directly utilize undressed feather.It is how all Phoenix (" identification of efficient feather degradation bacteria strains and the optimization of fermentation condition ", what Zhou Feng etc., genomics and applied biology, 2015) etc. to produce keratinase producing bacillus subtilis enzyme condition optimizing after, enzyme activity is 23.6U/ml, and most suitable enzyme is anti- The temperature answered is 37 DEG C；Bacillus licheniformis (Bacillus licheniformis BBE11-1) (" originate by bacillus licheniformis Impact of the keratinase N-terminal to vigor and its heat endurance ", Liu Baihong etc., microbiology circular, protease 2014) is transformed Front 60 DEG C of half-life are 9min, and Liu Baihong et al. carries out stepwise deletion by 5 amino acid before keratinase N-terminal, and by sequence Arrange the N compared front 5 amino acid substitutions of N-terminal are the thermophilic protease from Thermoactinomyces vulgaris End, wild type and mutant keratinase gene are expressed in bacillus subtilis WB600, and 60 DEG C of half-life bring up to 20min, but enzyme activity reduces 70%.Thermophilic bacteria shows under the high temperature conditions the existence adaptability and special of uniqueness Metabolite, the enzyme that it is produced has under the high temperature conditions very high activity and heat endurance, and is difficult pollution, while thermally-stabilised Property preferable enzyme can be reacted at a relatively high temperature, be conducive to improve enzyme reaction rate, reduce other micro- lifes The pollution of thing and promotion enzyme molecule are fully penetrated in substrate and reacted.

The content of the invention

For the problems referred to above, the present invention is by high temperature bacterium, one plant of ground gemma bar that can produce high temperature keratinase is screened Bacterium, can at 60 DEG C 48 as a child degradable feathers, the keratinase that the bacterium produces has good heat endurance simultaneously, What the present invention was realized in：

One plant of ground bacillus (Geobacillus sp) JAAS-YTC01, its deposit number is CGMCCNO.12740, is protected The Tibetan date is on July 6th, 2016；The ground bacillus thalline is in shaft-like, and long 2-3 μm, Gram's staining is positive, bacterium colony size It is medium, surface wettability, neat in edge；The bacterium 16S rRNA are as shown in SEQ ID NO.1.

A kind of deposit number is application of the ground bacillus of CGMCCNO.12740 in keratin enzyme liquid is prepared, specifically Step is as follows：The ground bacillus are inoculated in fermentation medium by mass volume ratio 2%, in 60 DEG C, 180rpm is stirred After culture 24-72h, filter or be centrifuged, gained filtrate or supernatant are keratin enzyme liquid；

The fermentative medium formula is：In per 1000mL fermentation mediums, 10g containing sucrose, ammonium sulfate 4g, feather 10g, NaCl0.5g、K₂HPO₄ 0.4g、KH₂PO₄ 0.3g、MgCl₂·6H₂O 0.1g、pH7.2-7.4。

A kind of deposit number produces keratinase answering in degraded keratin for the ground bacillus of CGMCCNO.12740 With concrete grammar is as follows：

The ground bacillus are pressed during mass volume ratio 2% (W/V, g/l) is inoculated in fermentation medium, pH=is adjusted 8.5, cultivate 24-72h in 70 DEG C, you can degraded keratin；

The fermentative medium formula is：In per 1000mL fermentation mediums, 10g containing sucrose, ammonium sulfate 4g, feather 10g, NaCl0.5g、K₂HPO4 0.4g、KH₂PO₄ 0.3g、MgCl₂·6H₂O 0.1g、pH7.2-7.4。

Preferably, the deposit number for CGMCCNO.12740 ground bacillus degraded keratin in application, institute The feather that keratin refers to chicken is stated, the feather in the fermentation medium is the feather of chicken.

In the present invention, the enzyme required for 1 μ g tyrosine will be generated per 1min catalytic decompositions keratin with keratin as substrate Amount is defined as an enzyme-activity unit.

Enzyme activity assay method is the crude enzyme liquid 1ml for taking 5-10 times of dilution, adds the keratin of 1ml 1%, is entered at 60 DEG C The TCA that 2ml 0.4M are added after row enzyme reaction 15min terminates enzyme activity, 4000rpm centrifugation 10min.To be initially charged 2ml 0.4M TCA adds 1% substrate as blank.1ml supernatants are taken, the Na of 5ml 0.4M is added₂CO₃Solution, adds 1ml good fortune Woods phenol reagent, is incubated 20min by 40 DEG C, determines the absorbance of 660nm.

In the present invention, deposit number is inoculated in into fermentation training for the ground bacillus JAAS-YTC01 of CGMCCNO.12740 In foster base, inoculum concentration 2%, (W/V, g/l), 60 DEG C, 150-200rpm stir cultures are after 48 hours, whole feather quilt in fermentation flask Degraded, terminates fermentation, and filter centrifugation obtains keratin enzyme liquid, and the keratin highest enzyme activity of survey is 10.4U/ml.

Preserving number provided by the present invention is the ground bacillus JAAS-YTC01 of CGMCCNO.12740, what the bacterium produced The most suitable enzyme reaction temperature of keratinase is 70 DEG C, and optimal pH is 8.5, and enzyme liquid is incubated 1h in 60 DEG C, and enzyme activity remains to reach initially The 86.7% of enzyme activity, compared with existing bacterial classification, with higher heat endurance；The bacterial strain can directly degrade without any process Feather waste, degradation speed is fast, the characteristics of produced keratinase has high temperature resistant and good heat endurance, is suitable to extensive Popularization and application.

Description of the drawings

Fig. 1 is ground bacillus JAAs-YTC01 colonial morphology schematic diagrames.

Fig. 2 is ground bacillus JAAs-YTC01 Gram's staining result schematic diagrams.

Fig. 3 is ground bacillus JAAs-YTC01 thalline electron microscopes.

Fig. 4 is ground bacillus JAAs-YTC01 keratinase optimal reactive temperature cylindricality schematic diagrames.

Fig. 5 is ground bacillus JAAs-YTC01 heat endurance curve maps.

Fig. 6 is ground bacillus JAAs-YTC01 keratinase optimal reaction pH curve maps.

Fig. 7 is degradation effect schematic diagram of ground bacillus JAAs-YTC01 fermented and cultureds 48h to feather.

Specific embodiment

Technical scheme is further illustrated below by way of specific embodiment.

Involved culture medium in embodiment：

Enriched medium (1000ml) is beef-protein medium：Beef extract 5g, peptone 10g, NaCl 5g, plus Water is settled to 1000mL, pH natures；

Isolation medium (1000ml)：4g casein is dissolved in 20mL 0.1mol/L NaOH solutions, 20g is added Agar, adds water and is settled to 1000mL；

Screening and culturing medium (1000ml)：Feather 10g, NaCl 0.5g, K₂HPO₄ 0.4g、KH₂PO₄ 0.3g、MgCl₂· 6H₂O 0.1g, dusty yeast 0.1g, addition is settled to 1000mL, pH7.2-7.4；

Seed culture medium (1000ml)：NaCl 5g, beef extract 5g, peptone 10g, dusty yeast 2g, balance of water, pH7.2-7.4；Slant medium (1000ml)：NaCl 5g, beef extract 5g, peptone 10g, dusty yeast 2g, balance of water, pH7.2-7.4；Fermentation basal medium (1000ml)：Feather 10g, NaCl 0.5g, K₂HPO₄ 0.4g、KH₂PO₄ 0.3g、 MgCl₂·6H₂O 0.1g、pH7.2-7.4；Liquid amount 50ml/250ml, 60 DEG C, 180rpm shake flask fermentation 48h；

Fermentation medium (1000ml)：Sucrose 10g, (NH₄)₂SO₄4g, feather 10g, MgCl₂·6H₂O 0.1g, NaCl 0.5g,K₂HPO₄, 0.3g KH₂PO₄0.4g, pH7.2-7.4, liquid amount 50ml/250ml.

Above-mentioned culture medium mesoptile is chicken feather (Nanjing chicken butchers market collection).

Involved nucleotide sequence in embodiment：

SEQ ID NO.1：

SEQ ID NO.2(27F)：agagtttgat cctggctcag；

SEQ ID NO.3(492R)：ggttaccttg ttacgactt；

The strain breeding thereof of embodiment 1

1st, bacterial classification is screened

Sample from the During High-Temperature Composting (the peaceful grain Organic Fertilizer Plants in Nanjing) of feces of livestock and poultry, take a random sample from every part of sample first Product, are broken up in sample with SPSS；After gradient dilution, 60-80 DEG C of enrichment squamous subculture in enriched medium is inoculated into 2-3 days；Plate streaking is separated, and obtains high temperature bacterium, and by high temperature bacterium dibbling to isolation medium flat board, screening has hydrolysis Bacterial strain, and then will in these inoculations to screening and culturing medium, observe feather degraded situation, finally filtering out one plant has degraded The bacterial strain of feather ability, will be stand-by in the inoculation to inclined-plane solid medium.

2nd, strain idenfication

A) bacterial strain for screening step 1 is rule on beef-protein medium, 60 DEG C of culture 24h, obtains single bacterium Fall (as shown in Figure 1), and bacterium colony is of medium size, surface wettability, neat in edge；

Gram's staining is carried out to the bacterium, discovery is positive (as shown in Figure 2)；Collects thalline after Liquid Culture, glutaraldehyde After method is fixed, electron microscopic observation thalli morphology, and photo is shot as shown in figure 3, observing the thalline in shaft-like, thalline length is 2-3 μ M,；The strain gene group DNA is extracted as template, by the use of bacterial genomes DNA kit (Omega companies) with 27F and 1492R Respectively as upstream and downstream primer, 16S rRNA gene magnifications are carried out：

PCR reaction systems：The μ l of Premix Taq (Takara) 25, template 0.1-2 μ g, upstream primer 10-100pmol, under Trip primer 10-100pmol, distilled water complements to 50 μ l；

PCR response procedures：94 DEG C of denaturations 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 90s, totally 30 Circulation；72 DEG C of extension 5min.

16S rRNA amplified productions transfer to Shanghai Sheng Gong bioengineering Co., Ltd to be sequenced, sequencing result such as SEQ ID Shown in NO.1, sequencing result is compared in ncbi database, and molecular evolution phylogenetic tree analysis is carried out to it, identified It is ground bacillus category (Geobacillus sp), and it was named as JAAS-YTC01 by applicant certainly, on July 6th, 2016 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address：The Chaoyang District, Beijing City North Star The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica；Postcode 100101, deposit number is：CGMCC NO.12740.

The bacterial strain JAAS-YTC01 fermentating enzyme-producing conditions of embodiment 2 optimize

The different carbon source (1%) (W/V, g/l) of addition and different nitrogen sources and variable concentrations are investigated using single factor experiment The bacterial strain JAAS-YTC01 keratinase yield effects that the addition of feather is obtained to embodiment 1.

The screening of the most suitable carbon sources of a

In fermentation basal medium, glucose, sucrose, maltose, starch, the bran of 1% (W/V, g/l) are separately added into Skin, corn flour, in 60 DEG C, after 180rpm shake flask fermentation 48h enzyme activity are determined.As a result show to add sucrose to increase keratinase Yield.

The screening of the most suitable nitrogen sources of b

In fermentation basal medium, dusty yeast, the dregs of beans of 1% (W/V), the urea element of 0.4% (W/V), sulphur are separately added into Sour ammonium, in 60 DEG C, after 180rpm shake flask fermentation 48h enzyme activity is determined.As a result show to add ammonium sulfate to increase keratinase product Amount.

The determination of the most suitable feather concentration of c

In fermentation basal medium, carbon source and nitrogen source that addition is filtered out above, the concentration 1% (W/V) of setting feather, 2.5%th, 5%, 10%, 20%., 60 DEG C, determine enzyme activity after 180rpm shake flask fermentation 48h.As a result show that optimal feather is dense Spend for 10%.

Finally determine that the fermentation medium after optimization is：Sucrose 10g, ammonium sulfate 4g, feather 10g, NaCl 0.5g, K₂HPO4 0.4g、KH₂PO₄ 0.3g、MgCl 6H₂O 0.1g、pH7.2-7.4；Liquid amount 50ml/250ml, produces under the conditions of this Keratinase activity be 12.3U/ml.

The optimal reactive temperature of the bacterial strain JAAS-YTC01 keratinases of embodiment 3, optimal pH and heat endurance

The bacterial strain JAAS-YTC01 that embodiment 1 is screened is inoculated into fermentation medium with the inoculum concentration of 2% (W/V, g/l) In, after 180rpm stir cultures 48h, 8000rpm centrifugation 10min, gained supernatant is keratin enzyme liquid.

Keratin enzyme liquid 1ml of 5-10 times of dilution is taken, the keratin of 1ml 1% is added, enzyme reaction is carried out at 60 DEG C The TCA that 2ml 0.4M are added after 15min terminates enzyme activity, 4000rpm centrifugation 10min.Added with being initially charged 2ml 0.4M TCA 1% substrate is used as blank.1ml supernatants are taken, the Na of 5ml 0.4M is added₂CO₃Solution, adds 1ml forint phenol reagents, 40 DEG C, 20min is incubated, determines the absorbance of 660nm.

With keratin as substrate, the enzyme amount generated required for 1 μ g tyrosine per 1min catalytic decompositions keratin is defined as one Individual enzyme-activity unit.

1st, most suitable enzyme reaction temperature and heat endurance

Under conditions of identical p H values, the enzyme activity in 50-80 DEG C of temperature range is determined, with enzyme activity peak as 100% Calculate relative enzyme activity.Testing result is as shown in figure 4, most suitable enzyme reaction temperature is 70 DEG C.

Enzyme liquid is kept into 20min, 40min, 60min, 80min under condition of different temperatures, the activity of residual enzyme is determined, with Highest enzyme activity is 100% evaluating the heat endurance of enzyme.As a result as shown in Figure 5, most suitable 70 DEG C of enzyme reaction temperature, the enzyme 50,60 It is DEG C relatively stable, keep 60min, enzyme activity to retain 86.8% at 60 DEG C, illustrate that the heat endurance of the enzyme is good.

2nd, most suitable enzyme reaction pH

Under conditions of mutually synthermal, it is 50mmoll to determine enzyme liquid in concentration^-1Different pH buffer systems:Phosphoric acid delays Rush liquid (pH7.0-7.5), Tris-Cl buffer solutions (pH7.5-9.0), under glycine sodium hydrate buffer solution (pH9.0-10.5) Enzyme activity, with enzyme activity peak as 100% relative activity is calculated, and determines the optimum pH of keratinase.As a result as shown in Figure 6, Most suitable enzyme reaction pH is 8.5, belongs to basic keratins enzyme.

The feather degradation experiment of embodiment 4

Feather in the present embodiment is chicken feather.

Embodiment 1 is obtained into bacterial strain JAAS-YTC01 (liquid amount 50ml/ in fermentation medium is accessed with 2% inoculum concentration 250ml), 70 DEG C, 24-72h, fermentation observation feather signs of degradation are cultivated under conditions of pH=8.5.

As a result as shown in fig. 7, before fermentation when such as Fig. 7 A, fermentation 12h or so, the tiny lint on the main stalk of feather comes off, training Foster base is gradually muddy；When fermentation proceeds to 40h, culture medium is muddy, and light tight, feather major part is degraded, only the main stalk of remaining part point； After 48h, the main stalk of culture medium mesoptile is also degraded (Fig. 7 B).

17 kinds of free amino acids and total amino acid content increase in fermentation 48h wild Oryza species filtered fluids, and concrete outcome is shown in Table 1：

Free aminoacid content in culture medium before and after the fermentation of table 1

Concrete application approach of the present invention is a lot, and the above is only the preferred embodiment of the present invention, it is noted that for For those skilled in the art, under the premise without departing from the principles of the invention, some improvement can also be made, this A little improvement also should be regarded as protection scope of the present invention.

SEQUENCE LISTING

<110>Jiangsu Province Agriculture Science Institute

<120>One plant of ground bacillus and its application

<130> 3

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1519

<212> DNA

<213>Artificial sequence

<400> 1

agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc 60

ggactggatt ggagcttgct ctgattcggt cagcggcgga cgggtgagta acacgtgggc 120

aacctgcccg caagaccggg ataactccgg gaaaccgggg ctaataccgg ataacaccga 180

agaccgcatg gtctttggtt gaaaggcggc ctttggctgt cacttgcgga tgggcccgcg 240

gcgcattagc tagttggtga ggtaacggct caccaaggcg acgatgcgta gccggcctga 300

gagggtgacc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 360

agggaatctt ccgcaatggg cgaaagcctg acggagcgac gccgcgtgag cgaagaaggc 420

cttcgggtcg taaagctctg ttgtgaggga cgaaggagcg ccgttcgaag agggcggcgc 480

ggtgacggta cctcacgagg aagccccggc taactacgtg ccagcagccg cggtaatacg 540

tagggggcga gcgttgtccg gaattattgg gcgtaaagcg cgcgcaggcg gttccttaag 600

tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg ggacttgagt 660

gcaggagagg agagcggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 720

accagtggcg aaggcggctc tctggcctgc aactgacgct gaggcgcgaa agcgtgggga 780

gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttaga 840

ggggtcacac cctttagtgc tgcagctaac gcgataagca ctccgcctgg ggagtacggc 900

cgcaaggctg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 960

taattcgaag caacgcgaag aaccttacca ggtcttgaca tcccctgaca acccaagaga 1020

ttgggcgttc ccccttcggg gggacagggt gacaggtggt gcatggttgt cgtcagctcg 1080

tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac cctcgcctct agttgccagc 1140

attcggttgg gcactctaga gggactgccg gcgacaagtc ggaggaaggt ggggatgacg 1200

tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatggg cggtacaaag 1260

ggctgcgaac ccgcgagggg gagcgaatcc caaaaagccg ctctcagttc ggattgcagg 1320

ctgcaactcg cctgcatgaa gccggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380

aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagcttg caacacccga 1440

agtcggtgag gtaacccgca agggagccag ccgccgaagg tggggcaagt gattggggtg 1500

aagtcgtaac aaggtaacc 1519

<210> 2

<211> 20

<212> DNA

<213>It is artificial synthesized

<400> 2

agagtttgat cctggctcag 20

<210> 3

<211> 19

<212> DNA

<213>It is artificial synthesized

<400> 3

ggttaccttg ttacgactt 19

Claims

1. one plant of ground bacillus（Geobacillus sp.）, its deposit number is CGMCCNO.12740；The ground bacillus Thalline is in shaft-like, and long 2-3 μm, Gram's staining is positive, bacterium colony surface wettability, neat in edge.

2. application of the ground bacillus as claimed in claim 1 in keratin enzyme liquid is prepared, it is characterised in that concrete steps are such as Under：The ground bacillus are inoculated in fermentation medium by mass volume ratio 2%, in 60 DEG C, 180rpm stir cultures 24- After 72h, filter or be centrifuged, gained filtrate or supernatant are keratin enzyme liquid；

The fermentative medium formula is：In every 1000mL fermentation mediums, 10g containing sucrose, ammonium sulfate 4g, feather 10g, NaCl 0.5g、K₂HPO₄ 0.4g、KH₂PO₄ 0 .3g、MgCl₂·6H₂O 0.1g、pH7.2-7.4。

3. application of the ground bacillus as claimed in claim 1 in degraded keratin.

4. application as claimed in claim 3, it is characterised in that concrete grammar is as follows：

The ground bacillus are inoculated in fermentation medium by mass volume ratio 2%, pH=8.5 is adjusted, in 70 DEG C 24- is cultivated 72h, you can degraded keratin；

The fermentative medium formula is：In every 1000mL fermentation mediums, 10g containing sucrose, ammonium sulfate 4g, feather 10g, NaCl 0.5g、K₂HPO4 0.4g、KH₂PO₄ 0 .3g、MgCl₂·6H₂O 0.1g、pH7.2-7.4。

5. the application as described in claim 2-4, it is characterised in that the keratin refers to the feather of chicken.