CN106701615B - Protease-producing bacteria and application thereof - Google Patents

Protease-producing bacteria and application thereof Download PDF

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CN106701615B
CN106701615B CN201611042427.3A CN201611042427A CN106701615B CN 106701615 B CN106701615 B CN 106701615B CN 201611042427 A CN201611042427 A CN 201611042427A CN 106701615 B CN106701615 B CN 106701615B
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protease
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casein
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CN106701615A (en
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解志红
伍朝亚
李岩
刘卫
任承钢
刘晓琳
孙雨
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Yantai Institute of Coastal Zone Research of CAS
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Abstract

The invention discloses a high-efficiency protease producing bacterium and enzyme producing conditions thereof, wherein the high-efficiency protease producing bacterium is obtained by separating from sea bottom surface sediments of Bohai sea, is classified and named as Bacillus BH70049(Bacillus altitudinis), is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Siro-1 of the sunward area in Beijing city) within 2016 (5-23 days), and has the preservation number of CGMCC No. 12490. The strain has the advantages of high growth speed, strong protease production capacity and high enzyme activity, can degrade casein, gelatin and elastin, is applied to sewage treatment, decomposes suspended particles in the ocean, promotes the degradation of ocean organic nitrogen, and maintains the nitrogen circulation balance of the ocean ecosystem. The invention provides an important source of a wide range of industrial protease preparations, and has good application prospect in the industries of food, medicine, tanning, detergent, cosmetics, textile, animal and plant protein, waste treatment and the like.

Description

Protease-producing bacteria and application thereof
Technical Field
The invention relates to protease, protease-producing bacteria and applications thereof.
Background
Protease is an enzyme catalyzing protein hydrolysis, is the most widely used enzyme at present, is also the enzyme with the largest demand at present, is widely present in animals, plants and microorganisms, accounts for about 2 percent of all proteins, performs a plurality of different functions, is widely applied to washing, leather, silver washing, medicines, foods, feeds, chemical engineering and the like, and accounts for about 60 percent of the enzyme preparation market. Traditional animal and plant derived protease has the defects of long growth period, high production cost, difficulty in control and management, easiness in land and season limitation and the like, and microorganisms increasingly become a main source of industrial enzyme preparations. However, the sources of protease-producing microorganisms in China are limited, and most of the microorganisms entering protease production at home are from land or fresh water environment, so that important scientific research, application and commercial values are achieved by searching and developing marine protease-producing microorganism resources and further mastering the relevant characteristics of novel protease.
Above 2/3 on the earth's surface is covered by marine sediments, where a large number of metabolically active microorganisms, including bacteria and archaea, are present extensively. The microorganisms degrade suspended granular organic nitrogen in the ocean into dissolved organic nitrogen with high molecular weight, the dissolved organic nitrogen is decomposed, aminated (mineralized), nitrified and denitrified, and finally participates in nitrogen circulation in the sediment at the bottom of the sea, and the protein is an important component of the granular organic nitrogen and the dissolved organic nitrogen in the sediment, so that the protease-producing bacteria play an important role in the degradation process of the microorganisms.
Disclosure of Invention
The invention aims to provide a bacterium capable of efficiently producing protease and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a high-efficiency protease-producing bacterium is Bacillus BH70049(Bacillus sp) which is preserved in China general microbiological culture Collection center (CGMCC for short, No. 3 of Xilu 1 Beichen of Beijing market and Chaoyang district) in 2016, 5 and 23 days, and the preservation number is CGMCC No. 12490.
The protease-producing bacteria are obtained by separating and screening Bohai sea bottom sediment, and the 16SrRNA gene sequence of the bacteria is carried outColumn analysis and finding it by alignment in GenBank to Bacillus altitudinis strain 41KF2bT(accession No.: NR042337) the highest similarity was 99%. Thus, the isolated bacterium was named Bacillus spBH 70049.
Has been preserved in China general microbiological culture Collection center (CGMCC, address No. 3 Xilu 1 Beijing, Chaoyang district, Beijing) in 2016, 5 months and 23 days, and the preservation number is CGMCC No. 12490.
Use of a bacterium which is highly efficient in producing protease, the bacterium being for use in the production of protease.
The bacterium is Bacillus BH70049(Bacillus sp), and the application thereof in producing protease.
Further, the Bacillus BH70049(Bacillus sp) is cultured in a casein gelatin enzyme production medium to obtain the protease.
Further, the Bacillus BH70049(Bacillus sp) is cultured in a casein gelatin enzyme production medium with the pH value of 8, the casein concentration of 3g/L, the gelatin concentration of 5g/L and the yeast powder concentration of 2g/L to obtain the protease. The enzyme activity in the enzyme production culture medium is strongest, and the enzyme activity reaches 53.09U/mL by the enzyme activity of the casein enzyme activity measured by protease.
The invention has the advantages that:
1. the bacillus BH70049 has the characteristic of high activity of producing the protease, can degrade casein, gelatin and elastin, has the hydrolysis loop diameters/colony diameters of 4.8, 5.42 and 1.32 on a casein plate, a gelatin plate and an elastin plate in sequence, has high enzyme activity in a fermentation medium, and produces the protease with the enzyme activity of 53.09U/mL for the activity determination of the casein enzyme.
2. After the optimization of the invention, the enzyme production capability of the strain is obviously improved, and the enzyme activity reaches 85.98U/mL. .
3. The strain is separated from the bottom sediment of the sea of the Bohai sea, belongs to Bacillus strain, is non-toxic and harmless, is not genetically modified, and can be applied to industries such as food, medicine, tanning, detergent, cosmetics, textile, animal and plant protein, waste treatment and the like.
Drawings
FIG. 1 is a photograph showing the production of hydrolysis circles on a casein plate by the protease-producing strain Bacillus BH70049 provided in the examples of the present invention.
FIG. 2 is a photograph of the protease producing strain Bacillus BH70049 provided in the examples of the present invention producing hydrolysis rings on a gelatin plate.
FIG. 3 is a photograph showing the generation of hydrolysis circles on an elastin plate by the protease producing strain Bacillus BH70049 provided in the examples of the present invention.
FIG. 4 shows protease enzyme activity before and after optimization of culture conditions of protease-producing strain Bacillus BH70049 provided by the embodiment of the present invention.
Detailed Description
The present invention will be further described with reference to the following examples. The examples are intended to illustrate the invention, but not to limit it in any way.
1. Separating, purifying and storing the strain bacillus BH 70049.
Collecting samples from non-polluted sediments on the bottom surface layer of Bohai sea, weighing 1g of sediments, and diluting to 10 times of the sediment with 10 times of sterile seawater in a gradient manner-6Mixing, and taking 0.1mL (10)-2-10-6) The diluted sample is coated on a screening culture medium plate, inverted and cultured for 3-5 days at 28 ℃, single bacterial colonies with different bacterial colony forms and producing transparent hydrolysis rings are respectively picked, connected to a 2216E culture medium plate, the bacterial strain is purified by adopting a continuous streak plate method, and then transferred to a 2216E culture medium inclined plane. Respectively transferring the purified strains to a casein gelatin solid culture medium (3g/L casein, 5g/L gelatin, 2g/L yeast powder, 15g/L agar powder and artificial seawater), culturing for 4 days, measuring diameters of bacterial colonies and hydrolysis rings, calculating a ratio of the hydrolysis rings to the diameters of the bacterial colonies, wherein the ratio can be used as a qualitative test and directly reflects the capability of the strains for degrading proteins. Therefore, the strain BH70049 with the largest ratio of the hydrolysis ring to the colony diameter is selected as a research object in the research.
Fermentation medium (for metabolic enzyme production of strain) 3g/L casein, 5g/L gelatin, 2g/L yeast powder, artificial seawater, and pH 8.0. The preparation method comprises the following steps: weighing 0.3g of casein, soaking with a little 1M NaOH, adding a little deionized water, and heating to dissolve; weighing 0.5g of gelatin, adding a little deionized water, and heating until the gelatin is dissolved; mixing the dissolved casein and gelatin, using deionized water to fix the volume to 20mL, and adjusting the pH value to 8.0; adding 0.2g of yeast powder into 80mL of artificial seawater, and adjusting the pH value to 8.0; subpackaging to 40mL per bottle, sterilizing respectively at 121 deg.C for 20 min. At the time of inoculation, 10mL of casein gelatin mixed solution was added to each flask of the medium.
Screening medium (casein gelatin solid medium): adding 0.2g of yeast powder and 1.5g of agar powder into 80mL of artificial seawater, and adjusting the pH value to 8.0; respectively sterilizing at 121 deg.C for 30min, cooling to 45-50 deg.C, mixing with casein gelatin mixture, and pouring into flat plate; wherein the casein gelatin mixture is prepared by weighing 0.3g casein, soaking with a little 1M NaOH, adding a little deionized water, and heating to dissolve; weighing 0.5 gelatin, adding a little deionized water, and heating to dissolve; mixing the casein and the gelatin obtained by the dissolution, and fixing the volume to 20mL by using deionized water to adjust the pH value to 8.0;
2216E Medium: 2.5g/L yeast powder, 5g/L peptone, artificial seawater and 1.5g/L agar powder. (natural pH, sterilizing at 121 deg.C and 0.11Mpa for 30 times).
Artificial seawater (Smibert and Krieg,1994) NaCl 2.75%, MgCl2 0.5％、MgSO4 0.2％、CaCl2 0.05％、KC1 0.1％、FeS040.0001 percent and deionized water.
The short-term preservation mode of the strain is as follows: inoculating the purified strain to a 2216E solid inclined plane, and storing in a refrigerator at 4 ℃ after bacterial colonies grow well; the long-term preservation mode is as follows: culturing the purified strain to late logarithmic phase or stationary phase, adding glycerol with final concentration of 20%, making into glycerol tube, and freezing at-80 deg.C for long-term storage.
Identification of strains
The strain on the 2216E inclined plane is subjected to streak activation on a 2216E plate, a single colony generated on the plate is observed after the strain is cultured for 24 hours at 28 ℃, the colony is circular, yellow and opaque, the edge is relatively neat, the surface is smooth, the strain is easy to pick, and the strain has the phenotypic characteristics of typical bacteria and is preliminarily identified as the bacteria.
MoleculeAnd (3) identification: in order to further determine the phylogenetic position of the bacillus bacterium BH70049, 16SrRNA gene sequence determination and phylogenetic analysis are carried out. The PCR reaction adopts universal primers (a forward primer 27F and a reverse primer 1492R) and has the following reaction system: the volume of the reaction system is 50 mu L, and the composition of the reaction solution is as follows: mu.L of DNA template, 25. mu.L of 2 XMMastarMix, 1. mu.L of 27F, 1. mu.L of 1492R, and ultrapure water to make up to 50. mu.L. The PCR reaction conditions are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 94 deg.C for 45s, annealing at 56 deg.C for 1min, extension at 72 deg.C for 1.5min, 30 cycles, and final extension at 72 deg.C for 10 min. The amplification product is detected to be qualified by 1.0% agarose gel electrophoresis and then sent to Beijing Okoku Dingsheng Biotech Co. Comparing and analyzing the 16SrDNA sequence obtained by sequencing by using Nucleotide BLAST of NCBI, and comparing the strain to a reference strain Bacillus altitudinis strain 41KF2bTThe degree of similarity was 99%, and therefore, this strain was judged to be of the genus Bacillus.
Bacillus altitudinis BH 7004916S rRNA gene splicing sequence:
ACCGTCACTTCGGCGGCTGGCTCCAATAAAGGTTACCTCACCGACTTCGGGTGTTGCAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTAAACCTTGCGGTCTCGCAGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGTCCCCGAAGGGAAAGCCCTATCTCTAGGGTTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCAAGCAGTTACTCTTGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACTGCATGTATAGGCCGCCGC
synthesizing the identification results, and naming the strain as Bacillus sp BH 70049; has been preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 of Xilu 1 of Beijing Kogyo-south China) in 2016, 5 months and 23 days, and has a preservation number of CGMCC No.12490, strain BH70049 for short.
2. Analysis of the ability of Bacillus BH70049 to hydrolyze proteins
(1) Bacillus BH70049 hydrolysis loop assay on different protein substrate plates
The bacillus laterosporus BH70049 is streaked and activated on a 2216E solid plate, the plate is inverted and cultured for 24 hours at the temperature of 28 ℃, then the 2216E plate bacterial colony is picked and respectively inoculated on a casein plate, a gelatin plate and an elastin plate, the plate is cultured for 5 days at room temperature, and the bacterial colony and the diameter of a hydrolysis ring are measured. Since the culture medium of casein plate and gelatin plate is transparent, the generation of hydrolysis loop of strain is not easy to observe, so acidic mercuric reagent (HgCl) is added215g, concentrated hydrochloric acid 20mL, constant volume to 100mL distilled water), a significant hydrolysis ring was observed. The diameters of the hydrolysis circles and colonies on the three plates were measured and the ratio thereof was calculated. Finally, the hydrolysis loop diameter/colony diameter of the bacillus BH70049 on a casein plate, a gelatin plate and an elastin plate are sequentially measured to be 4.8, 5.42 and 1.32 (see pictures 1, 2 and 3), so that the strain is preliminarily identified to have the capability of degrading proteins and different degradation capabilities for different proteins.
(2) The determination method for determining the enzyme activity of protease produced by bacillus BH70049 is carried out by referring to light industrial standard QBT 1803-1993, which comprises the following steps:
1) preparation of standard tyrosine curve
Accurately weighing, and oven drying at 105 deg.COven drying to constant weight tyrosine O.lOOOg, preparing 1mg/mL tyrosine standard solution with 50mM Tris-HCl (pH 8.0), gradually diluting with 50mM Tris-HCl (pH 8.0) to obtain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100| μ g/mL tyrosine solution, respectively taking 100 μ L, adding 500 μ L0.4M Na2CO3After adding 100. mu.L of Folin-phenol to the solution, the solution was developed at 40 ℃ for 10min, and the absorbance was measured at 660nm to prepare a standard curve.
2) Determination of activity of protease produced by bacillus BH70049 on casein enzyme
Inoculating the strain into 50mL (100mL triangular flask) fermentation medium, performing shake cultivation at 28 ℃ and 180r/min, taking a proper amount of fermentation liquor after 3d, centrifuging at 13000r/min for 1min to obtain a supernatant, namely a crude enzyme solution, and taking 100 mu L of the supernatant in a 1.5 centrifuge tube.
② adding a2 percent casein solution (prepared by 50mM Tris-HCl (pH 8.0)) preheated at 40 ℃ into a centrifuge tube, and preserving the temperature for 10min in a water bath at 40 ℃.
③ adding 200 mu L0.4MTCA to terminate the reaction, and keeping the temperature at 40 ℃ for 10 min.
13000r/min for 3min, taking 100 mu L of supernatant, adding 500 mu L0.4M Na2CO3, then adding 100 mu L of Folin-phenol, developing at 40 ℃ for 10min, and measuring the absorbance at 660 nm. The control group reverses the steps II and III and takes denatured and inactivated enzyme as a control.
The unit of enzyme activity U is defined as: under the above conditions, the amount of enzyme required to release 1. mu.g of tyrosine per minute per ml of liquid enzymatic hydrolysate casein.
According to the casein standard curve and the measured light absorption value, the bacillus BH70049 with higher enzyme activity is obtained and is 53.09U/mL (see picture 4).
3. Optimization of enzyme production conditions of bacillus BH70049 fermentation
The production and secretion capacity of the protease has great relation with the fermentation conditions (such as temperature, pH, fermentation time, liquid loading amount and the like) of the strain, and the invention optimizes the temperature, pH, fermentation time and liquid loading amount in the process of producing the enzyme by fermentation, thereby finally obviously improving the enzyme production capacity.
The fermentation conditions before optimization were: the temperature is 28 ℃, the pH is 8.0, the liquid loading amount is 50mL (100mL triangular flask), the fermentation is carried out for 72h, the enzyme production capacity of the bacillus BH70049 is stronger under the basic fermentation condition (the enzyme activity is 53.09U/mL), but the enzyme production capacity is probably not the optimal fermentation condition of the bacillus BH70049 for producing protease, therefore, the fermentation condition is optimized, and the temperature is 25 ℃, 30 ℃, 34 ℃ and 37 ℃ respectively; setting pH 5, 6, 7, 9, 10; setting liquid loading amounts to be 20mL, 30mL, 40mL and 60 mL; a series of culture conditions such as 48h,60h,84h and 96h are set, and orthogonal tests are carried out on the set fermentation conditions, and the results show that the protease activity is highest and the enzyme production capacity is strongest when the temperature is 30 ℃, the pH is 9, the liquid loading is 30mL (100mL triangular flask) and the fermentation time is 60 h. After the fermentation conditions are optimized, the protease activity produced by the bacillus BH70049 is measured to reach 85.98U/mL (see picture 4).
SEQUENCE LISTING
<110> institute of tobacco pipe coastal zone of Chinese academy of sciences
<120> protease-producing bacteria and use thereof
<130>
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 1445
<212> DNA
<213> protease producing bacteria (Bacillus altitudinis)
<220>
<221> gene
<222> (1)..(1445)
<223>
<400> 1
accgtcactt cggcggctgg ctccaataaa ggttacctca ccgacttcgg gtgttgcaaa 60
ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct 120
gatccgcgat tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac 180
tgagaacaga tttgtgggat tggctaaacc ttgcggtctc gcagcccttt gttctgtcca 240
ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc atcccccacc 300
ttcctccggt ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag 360
atcaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca 420
accatgcacc acctgtcact ctgtccccga agggaaagcc ctatctctag ggttgtcaga 480
ggatgtcaag acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg 540
cttgtgcggg cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg 600
gagtgcttaa tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc 660
atcgtttacg gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc 720
ctcagcgtca gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta 780
cgcatttcac cgctacacgt ggaattccac tctcctcttc tgcactcaag tttcccagtt 840
tccaatgacc ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc 900
gagcccttta cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc 960
tggcacgtag ttagccgtgg ctttctggtt aggtaccgtc aaggtgcaag cagttactct 1020
tgcacttgtt cttccctaac aacagagctt tacgatccga aaaccttcat cactcacgcg 1080
gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140
agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200
gtcgccttgg tgagccgtta cctcaccaac tagctaatgc gccgcgggtc catctgtaag 1260
tgacagccga aaccgtcttt catccttgaa ccatgcggtt caaggaacta tccggtatta 1320
gctccggttt cccggagtta tcccagtctt acaggcaggt tacccacgtg ttactcaccc 1380
gtccgccgct aacatccggg agcaagctcc cttctgtccg ctcgactgca tgtataggcc 1440
gccgc 1445

Claims (4)

1. A bacterium which efficiently produces protease, characterized in that: bacillus (A), (B)Bacillus altitudinis) BH70049 has been deposited in the general microbiological culture collection center of the china committee for culture collection (CGMCC, address: west road No.1 hospital No. 3, north kyo, chaoyang district, beijing), the preservation number of which is CGMCC number 12490;
the bacillus BH70049 is obtained by separating and screening from Bohai sea bottom sediments.
2. The use of the high efficiency protease-producing bacterium of claim 1, wherein: use of the bacterium for the production of a protease.
3. The use of a high efficiency protease producing bacterium according to claim 2 wherein: the bacteria are cultured in a casein gelatin enzyme production culture medium to obtain the protease.
4. The use of a high efficiency protease producing bacterium according to claim 3 wherein: the bacteria are cultured in a culture medium with pH =8, 3g/L casein, 5g/L gelatin and 2g/L yeast powder enzyme production to obtain the protease.
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CN102533598A (en) * 2011-12-19 2012-07-04 安徽农业大学 Neutral protease enzyme-producing bacterial strain in bombyx mori intestinal tract, neutral protease gene and prokaryotic expression vector and preparation method thereof
CN103232963A (en) * 2013-05-27 2013-08-07 天津益丽康生物科技有限公司 Collagenase producing strain

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