CN105543147A - Pseudomonas aeruginosa strain and application thereof in producing proteinase - Google Patents

Abstract

The invention discloses a Pseudomonas aeruginosa strain SWJSS3 and application thereof in producing proteinase. The strain is collected by General Microorganism Center of China Committee for Culture Collection of Microorganisms on June 10th, 2015; the collection address is Institute of Microbiology in Chinese Academy of Sciences, Yard 1, Beichenxi Road, Chaoyang District, Beijing City; and the collection number is CGMCC No.10973. The application method of the strain for producing proteinase comprises the following steps: adding a fermentation culture medium into a fermentation tank, wherein the liquid filling amount is 8-12%, the initial pH value is 7.0-7.5, the strain inoculum size is 0.8-1.2%, and the initial temperature in the tank is 20-40 DEG C; and fermenting under oscillating conditions for 30-80 hours to generate abundant proteinase in the tank, and separating and extracting to obtain the proteinase. The strain SWJSS3 has high proteinase production capacity; and the obtained proteinase has the characteristics of high enzyme activity and high salt tolerance. The fermentation method disclosed by the invention has the advantages of simple conditions and stable hereditary property, and is suitable for industrial production.

Description

One Pseudomonas aeruginosa strain and the application in production proteolytic enzyme thereof

Technical field

The invention belongs to industrial microorganism field, be specifically related to the Pseudomonas aeruginosa (Pseudomonasaeruginosa) that a strain derives from deep-sea ooze, and produce the application in proteolytic enzyme.

Background technology

Ocean is the source region of life, takes up an area 70% of ball surface-area, has the Biological resources of the earth 80%.Ocean generally has the features such as high salt, high pressure, oligotrophic, low temperature, and marine microorganism is in order to adapt to these extreme environments, and some meta-bolitess formed in long-term evolutionary process and active substance and Lu Sheng microorganism also exist different.

Microbial protease is the lytic enzyme that a class is feature with protein hydrolysate peptide bond, in the production, life of the mankind, play considerable role.Along with developing rapidly and widespread use of genetically engineered and fermentation technique, utilizing microbial fermentation technology to produce enzyme has become the main method of producing commercial enzyme preparation.And the proteolytic enzyme that the marine microorganism of surviving in extreme circumstances produces generally has the features such as salt tolerant, pH value and wider range.Salt-durable microbe or salt tolerant protein enzyme still can grow under hypersaline environment because of it, and remain with greater activity, therefore are subject to the favor of investigator in the field such as sewage disposal, sauce fermentation.

For a long time, Pseudomonas aeruginosa is a kind of important industrial microorganism, is often applied to producing rhamnolipid, dissolving the research fields such as microcystic aeruginosa, degraded oil, phenol pollutent.But at present institute finds that the Pseudomonas aeruginosa of utilization is mainly derived from land, and derives from deep-sea ooze, and the work of high enzyme can be produced, the bacterial strain of high salt stability proteolytic enzyme but rarely has report.

Summary of the invention

Primary and foremost purpose of the present invention is to provide bacterial strain---Pseudomonas aeruginosa (Pseudomonasaeruginosa) SWJSS3 that a strain derives from deep-sea ooze, that can produce high enzyme high salt-tolerant trait proteolytic enzyme alive.

Another object of the present invention is to provide above-mentioned bacterial strains producing the application in proteolytic enzyme.

Object of the present invention is achieved through the following technical solutions:

Pseudomonas aeruginosa strains SWJSS3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 10th, 2015, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, and deposit number is CGMCCNo.10973.

Morphology and Physiology and biochemistry qualification are carried out to above-mentioned bacterial strains, have following characteristics:

Table 1 strain morphology feature

Table 2 physiological and biochemical property

Test event	Result	Test event	Result	Test event	Result
						Growth temperature	4～40℃	Glucose	+	Rhamnosyl	-
Aerobic	+	Indoles	-	Pectinose	-
						Oxydase	+	V-P tests	-	Sucrose	+
Lactose	-	Sorbyl alcohol	+	Melibiose	-

Note: in table 1 and 2, "+" represents that more than 90% bacterial strain is positive, and "-" represents that more than 90% bacterial strain is negative.

Through extracting and order-checking, the length of the 16SrDNA of bacterial strain SWJSS3 is 1387bp, and its sequence is SEQIDNo.1.

By the 16SrDNA sequence inputting of acquisition to Genbank, the gene order in itself and database is compared by application blast program.The homology that result shows itself and Pseudomonas aeruginosa 16SrDNA sequence is 100%.

By the sequence alignment result of 16SrDNA in conjunction with strain morphology and Physiology and biochemistry identification mark, determine that this bacterial strain is a Pseudomonas aeruginosa strain (Pseudomonasaeruginosa) bacterial strain, and called after SWJSS3.

Bacterial strain SWJSS3 can be used for liquid fermenting and produces proteolytic enzyme, and its fermentation process comprises the following steps:

In fermentor tank, add fermention medium, liquid amount is 8-12%, and initial pH value is 7.0-7.5, inoculation amount is 0.8-1.2%, initial temperature 20 ~ 40 DEG C in tank, oscillating condition bottom fermentation 30-80h, can produce a large amount of proteolytic enzyme in tank, separation and Extraction obtains proteolytic enzyme.

The present invention has following advantage and effect relative to prior art:

1, bacterial strain SWJSS3 of the present invention produces proteolytic enzyme ability by force, and gained proteolytic enzyme has the work of high enzyme, high salt-tolerant trait.

2, bacterial strain SWJSS3 fermentation process condition provided by the invention is simple, and stable hereditary property, is applicable to suitability for industrialized production.

Accompanying drawing explanation

Fig. 1 is that different fermentations temperature produces the impact of proteolytic enzyme to bacterial strain SWJSS3.

Fig. 2 produces the impact of proteolytic enzyme to bacterial strain SWJSS3 the different fermentations time.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Its formula of substratum used by embodiment is as follows:

1, enrichment medium (g/L artificial seawater): 1. peptone 5.0; 2. yeast extract powder 1.0; 3. tertiary iron phosphate 0.1; 4. sodium-chlor 100; 5. pH value 7.2 ~ 7.6.

2, seed culture medium (g/L artificial seawater): 1. peptone 5.0; 2. yeast extract powder 1.0; 3. tertiary iron phosphate 0.1; 4. pH value 7.2 ~ 7.6.

3, screening culture medium (casein plate) (g/L artificial seawater): 1. casein 10; 2. beef extract powder 3.0; 3. potassium primary phosphate 2.0; 4. agar powder 15; 5. pH value 7.2 ~ 7.6.

4, fermention medium: 1. glucose 4; 2. glycerine 8; 3. yeast extract powder 15; 4. K ₂hPO ₄0.2; 5. tween-80 0.5; 6. sea salt 10; 7. deionized water 1L; 8. pH value about 7.5.

5, test tube slant substratum: 1. extractum carnis 4; 2. peptone 6; 3. yeast extract powder 2; 4. sea salt 5; 5. agar powder 15; 6. deionized water 1L; 7. pH value 7.2 ~ 7.6.

Embodiment 1

The separation of bacterial strain SWJSS3, screening and identification, comprise the following steps:

Enrichment: take 1g ooze and be added with in the sterile centrifugation tube of granulated glass sphere in 50mL, the stroke-physiological saline solution adding 10mL0.9% is placed on shaking culture 30min in shaking table makes it fully disperse, get 1mL supernatant liquor in enrichment medium, 37 DEG C, 180r/min constant temperature culture 24h.

Primary dcreening operation: get the nutrient solution 1mL after 2 enrichments with 0.9% stroke-physiological saline solution be 10 by its gradient dilution ^-1, 10 ^-2, 10 ^-3three concentration, therefrom respectively get 100 μ L and be filled on the casein plate that solidified, coating evenly, is upside down in 37 DEG C of constant incubators and cultivates 24h.Picking has single bacterium colony of obviously hydrolysis circle, repeatedly rules and is inoculated into test tube slant substratum after three times and is stored in 4 DEG C of refrigerators.

Multiple sieve: be inoculated in seed culture medium after the above-mentioned pure growth be deposited on test tube slant is activated three times, in 37 DEG C, cultivate 12h in 180r/min constant-temperature shaking incubator, be inoculated in fermention medium with 1% inoculum size, 48h is cultivated under the same terms, take out centrifugal, get supernatant liquor and namely obtain crude enzyme liquid.Crude enzyme liquid is surveyed its enzyme after doing suitably dilution and is lived.

Crude enzyme liquid salt stability is tested: get and a certain amount ofly have the crude enzyme liquid that enzyme lives and dilute 5 times and mix with NaCl, make salt concn be 15%, in same treatment mode but the crude enzyme liquid not adding NaCl is contrast, in 4 DEG C of refrigerators, place different time, survey its remnant enzyme activity respectively, calculate relative value:

The relative enzyme thick enzyme enzyme that %=adds the remnant enzyme activity of NaCl/do not add NaCl of living is lived

Result is as shown in table 3:

The screening situation of table 3 proteolytic enzyme salt-enduring strain

Screened and fermented liquid enzyme activity determination by casein hydrolysis circle, enzyme work is diluted 5 times higher than the fermented liquid of the 10 strain bacterium of 20U/mL and is made into the NaCl solution that final concentration is 15%, 4 DEG C of refrigerators are deposited in after mixing, its remnant enzyme activity is surveyed after 3h, not add the bacterial strain fermentation liquor enzyme work of NaCl for contrast, calculate relative enzyme value (%) alive, result is as shown in table 3, the relative activity of strains A 3 is the highest as can be seen from the table, be 40.70%, so determine that A3 is the research object of subsequent experimental, be numbered SWJSS3.

The qualification of bacterial strain SWJSS3:

By the inoculation of activation in LB substratum, 37 DEG C, 180rpm cultivates 12h, gets the fresh bacterium liquid of 1.5ml 4 DEG C, the centrifugal 10min of 10000rpm, gets thalline in 2ml centrifuge tube, adopts DNA extraction kit extraction DNA.Universal primer is adopted to carry out its 16SrDNA sequence of pcr amplification after electrophoresis detection.

Primer sequence is:

Forward primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 ' (SEQIDNo.2)

Reverse primer: 5 '-GGTTACCTTGTTACGACTT-3 ' (SEQIDNo.3)

Amplification total reaction system (50 μ L), comprising: templet gene DNA1.25 μ L; Upstream and downstream primer (20 μm of ol/L) each 2.5 μ L; Taq DNA polymerase 0.5 μ L; 10 × buffer5.0 μ L; 4 kinds of deoxynucleotide mixture dNTP (each 2.5mmol/L) 4.0 μ L, 25mmol/LMgCl ₂4.0 μ L, distilled water (ddH ₂o) 30.25 μ L.

PCR response procedures adopts two-step approach: the first step denaturation 94 DEG C, 2min, sex change 98 DEG C, 10s, and anneal 55 DEG C, 1min, extends 68 DEG C, 30s (25 circulations), and the last time lengthening that extends is to 10min.

The 16SrDNA gene fragment length completing mensuration after PCR primer purifying is 1387bp, and its sequence is SEQIDNo.1, has the sequence similarity of 100% with the 16SrDNA sequence of the different strains of Pseudomonasaeruginosa in database.

In conjunction with strain morphology (table 1), physiological and biochemical property (table 2), determine that bacterial strain SWJSS3 is the new Pseudomonas aeruginosa (Pseudomonasaeruginosa) of a strain.

Embodiment 2

Bacterial strain SWJSS3 is producing the application in proteolytic enzyme, comprises the following steps:

(1) leavening temperature optimization, adopt fermention medium above, respectively at incubator temperature is 20 DEG C, 25 DEG C, 30 DEG C, 37 DEG C, 40 DEG C, 45 DEG C, initial pH value is 7.5, and liquid amount is 10%, and inoculum size is under the condition of 1%, after cultivating 48h, centrifugal survey enzyme is lived, result is as Fig. 1, and as can be seen from the figure this bacterial strain has certain yield of enzyme when temperature is 20 DEG C ~ 40 DEG C, but yield of enzyme is the highest 25 DEG C time.

(2) fermentation time optimization, at 30 DEG C, pH value is 7.5, and liquid amount is 10%, 180rpm condition bottom fermentation, from 0-80h, every 6h serial sampling, surveys biomass wherein and the protease activity variation tendency with fermentation time.

Result as Fig. 2, as can be seen from the figure: strain growth enters stationary phase after 20h.After cultivating 12h, bacterial strain starts large volume production enzyme, and production of enzyme enters stationary phase after 30h, wherein reaches maximum when 36h, tends towards stability afterwards.

Embodiment 3

The inheritance stability Journal of Sex Research of bacterial strain SWJSS3

Bacterial strain SWJSS3 continuous passage is cultivated 5 times, and surveys its proteinase activity, as the index evaluating genetic stability.

Result is as table 4, and Secondary Culture 5 times, proteinase activity all remains on about 750U/ml, shows that its genetic stability is good.

Table 4 strain passage enzyme is lived

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (3)

1. pseudomonas aeruginosa strains SWJSS3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 10th, 2015, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, and deposit number is CGMCCNo.10973.

2. bacterial strain SWJSS3 according to claim 1 is producing the application in proteolytic enzyme.

3. bacterial strain SWJSS3 according to claim 2 is producing the application in proteolytic enzyme, it is characterized in that comprising the following steps:

In fermentor tank, add fermention medium, liquid amount is 8-12%, and initial pH value is 7.0-7.5, inoculation amount is 0.8-1.2%, initial temperature 20 ~ 40 DEG C in tank, oscillating condition bottom fermentation 30-80h, can produce a large amount of proteolytic enzyme in tank, separation and Extraction obtains proteolytic enzyme.