CN117070394A - Alkalophilic strain for producing alkaline protease, alkaline protease and application thereof - Google Patents
Alkalophilic strain for producing alkaline protease, alkaline protease and application thereof Download PDFInfo
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- CN117070394A CN117070394A CN202310427590.5A CN202310427590A CN117070394A CN 117070394 A CN117070394 A CN 117070394A CN 202310427590 A CN202310427590 A CN 202310427590A CN 117070394 A CN117070394 A CN 117070394A
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/06—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
- A23L13/48—Addition of, or treatment with, enzymes
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38609—Protease or amylase in solid compositions only
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38618—Protease or amylase in liquid compositions only
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C14—SKINS; HIDES; PELTS; LEATHER
- C14C—CHEMICAL TREATMENT OF HIDES, SKINS OR LEATHER, e.g. TANNING, IMPREGNATING, FINISHING; APPARATUS THEREFOR; COMPOSITIONS FOR TANNING
- C14C3/00—Tanning; Compositions for tanning
- C14C3/02—Chemical tanning
- C14C3/08—Chemical tanning by organic agents
- C14C3/18—Chemical tanning by organic agents using polycondensation products or precursors thereof
- C14C3/20—Chemical tanning by organic agents using polycondensation products or precursors thereof sulfonated
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The application disclosesAn alkalophilic strain for producing alkaline protease, alkaline protease and application thereof relate to the technical field of microorganisms, wherein the alkalophilic strain for producing alkaline protease is alkaline halobacillus bacteriaAlkalihalobacillus sp.) PAIA262 with deposit number GDMCC NO. 63124 and with deposit time of 2023, 3 and 23 days. The alkalophilic strain for producing the alkaline protease can secrete the alkaline protease, and the obtained alkaline protease has the advantages of high reaction speed, high stability, easiness in treatment and the like, and the protease secreted by the PAIA262 strain obtained by screening is typical alkaline protease. Therefore, the alkaline halobacillus PAIA262 strain and the alkaline protease produced by the strain have biotechnological application value and provide a new source for the production of the alkaline protease.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to an alkalophilic strain for producing alkaline protease, the alkaline protease and application thereof.
Background
Alkaline proteases are a class of hydrolases that hydrolyze proteins to small peptides or amino acids under alkaline conditions, and alkaline proteases are one of the important industrial hydrolases. However, existing alkaline proteases are single in source.
Disclosure of Invention
The application mainly aims to provide an alkalophilic strain for producing alkaline protease, alkaline protease and application thereof, and solves the problem that the prior alkaline protease has single source.
To achieve the above object, the present application provides an alkaline protease-producing basophilic strain which is a basophilic strainThe alkaline strain is bacillus alcaligenesAlkalihalobacillus sp.) PAIA262 with deposit number GDMCC NO. 63124 and with deposit time of 2023, 3 and 23 days.
Alternatively, the 16S rDNA gene sequence of the alkaline protease-producing alkalophilic strain is shown in SEQ ID NO. 1.
Optionally, the alkalophilic strain for producing the alkaline protease can contain 0.03-0.09 mol/L Na 2 CO 3 Is grown in the medium of (a).
The present application further proposes an alkaline protease secreted by an alkaline protease-producing basophilic strain as described above.
Optionally, the alkaline protease is tolerant at a temperature of 30-100 ℃.
Optionally, the alkaline protease is tolerant at a pH of 7-13.
The application further provides an alkaline protease for use in any one of the following:
detergent production, leather tanning processing, food processing, pharmaceutical industry and protein waste treatment;
wherein the alkaline protease is secreted by an alkaline protease-producing basophil strain as described above.
In the technical scheme provided by the application, a strain of alkaline halobacillus bacteria which can well grow and secrete protease under alkaline conditions is separated from the intestinal tract of periplaneta americanaAlkalihalobacillus sp.) PAIA262. The alkalophilic strain for producing the alkaline protease can secrete the alkaline protease, and the obtained alkaline protease has the advantages of high reaction speed, high stability, easiness in treatment and the like, and the protease secreted by the PAIA262 strain obtained by screening is typical alkaline protease. Therefore, the alkaline halobacillus PAIA262 strain and the alkaline protease produced by the strain have biotechnological application value and provide a new source for the production of the alkaline protease.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the circles of proteins formed by strain PAIA262 of example 1 of the present application on milk agar plates;
FIG. 2 is a graph showing colony characteristics of strain PAIA262 in example 2 of the present application;
FIG. 3 is a graph showing the gram stain profile of strain PAIA262 in example 2 of the present application;
FIG. 4 is a graph showing the spore stain characteristics of strain PAIA262 in example 2 of the present application;
FIG. 5 is a 16S rDNA sequence phylogenetic tree analysis of strain PAIA262 in example 2 of the present application;
FIG. 6 is a graph showing the effect of temperature on the growth of strain PAIA262 in example 3 of the present application;
FIG. 7 is a graph showing the effect of pH on the growth of strain PAIA262 in example 3 of the present application;
FIG. 8 is a view of Na in example 3 of the present application 2 CO 3 Graph of effect of concentration on growth of strain PAIA 262;
FIG. 9 is a graph showing the effect of temperature on enzyme activity in example 4 of the present application;
FIG. 10 is a graph showing the effect of pH on enzyme activity in example 4 of the present application;
FIG. 11 is a graph showing the effect of pH on enzyme stability in example 4 of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Alkaline proteases are a class of hydrolases which hydrolyze proteins to small peptides or amino acids under alkaline conditions, and are important toolsOne of the industrial hydrolases. However, existing alkaline proteases are single in source. In view of this, the present application proposes an alkaline protease-producing basophilic strain which has been sent to the cantonese province microorganism strain collection at the address: china, guangzhou City first China No. 100 college No. 59 building 5, classified name alkali halobacillus bacteria @ floorAlkalihalobacillus sp.) PAIA262 with deposit number GDMCC NO. 63124 and with deposit time of 2023, 3 and 23 days.
In the technical scheme provided by the application, a strain of alkaline halobacillus bacteria which can well grow and secrete protease under alkaline conditions is separated from the intestinal tract of periplaneta americanaAlkalihalobacillus sp.) PAIA262 (hereinafter abbreviated as strain PAIA 262). The alkalophilic strain for producing the alkaline protease can secrete the alkaline protease, and the obtained alkaline protease has the advantages of high reaction speed, high stability, easiness in treatment and the like, and the protease secreted by the PAIA262 strain obtained by screening is typical alkaline protease. Therefore, the alkaline halobacillus PAIA262 strain and the alkaline protease produced by the strain have biotechnological application value and provide a new source for the production of the alkaline protease.
Referring to FIGS. 1 to 4, the composition contains Na 2 CO 3 On beef extract-peptone culture medium, bacillus alcaligenes bacteria @Alkalihalobacillus sp.) The colonies of PAIA262 were round, milky to pale yellow, smooth and moist on the surface, with a certain thickness (FIG. 2). Gram staining and violet baculous cells were observed under an oil microscope, gram positive (fig. 3). Spores were stained and red baculophyte and green free spores were observed under an oil microscope (fig. 4).
The 16S rDNA gene sequence of the alkalophilic strain for producing the alkaline protease is shown as SEQ ID NO. 1.
SEQ ID NO. 1：
GGACGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCAATTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTATGGGATTGGCTCCACCTCGCGGTTTCGCTGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGCCCCCGAAGGGGAAGCTCTGTCTCCAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACTGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGTGTTTACTTCGGCACTACGGGCATCGAAACCCCTAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATATCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGCTTCCCAGTTTCCAATGGCCGCTCGGGGTTGAGCCCCGAGATTTCACATCAGACTTAAGAAGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTGAGGTACCGTCAAGGTACCGGTAGTTACGCCGGTACTTGTTCTTCCCTCACAACAGAGCTTTACGACCCGAAGGCCTTCCTCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCGCCGCAGGCCCATCCCTTAGTGACAGCCGAAACCGTCTTTCAAAAGAGAATCAGGTGATTCTCTTTATTATCCCGTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTAAGGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCGCTGACTTCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTCGTAGAGGAAAAAGGA
The 16S rDNA sequence of the alkalophilic strain PAIA262 producing alkaline protease is amplified by PCR with primers 27F and 1492R as primers, and the agarose gel electrophoresis detection result of the PCR product shows that the size of the PCR product is about 1500 bp. Sequencing the PCR products, performing NCBI-Blast comparison, and constructing a phylogenetic tree, wherein the result shows that the 16S rDNA of the strain PAIA262 has higher similarity with the 16S rDNA of each strain of bacillus, and forms a big branch of phylogenetic evolution of the species (refer to figure 5). Alkaline protease-producing alkalophilic strain PAIA262 can be identified as alkaline halobacillus bacteria by combining bacterial colony morphology, gram and spore staining characteristics and 16S rDNA gene sequence analysisAlkalihalobacillus sp.）。
In the screening method of alkaline protease-producing alkalophilic strains provided by the application, the screening method of alkaline protease-producing alkalophilic strains comprises the following steps:
capturing American cockroach from the trade market of certain aquatic products in Guangzhou, cleaning the surface of the cockroach with 75% ethanol solution, dissecting, taking out the intestinal tract, and placing in sterilized 0.15 mol/L Na 2 CO 3 Grinding the solution in a homogenizer, standing for 10 min, and collecting supernatant.
Coating the above homogenate on skimmed milk powder containing bottom layer and Na containing upper layer 2 CO 3 The beef extract peptone screening plate is subjected to inverted culture at the constant temperature of 37 ℃ for 5-7d. Colony growth and protein degradation loop production were observed every 24 th h. The colony which has good growth condition and generates obvious protein degradation ring in the plate is selected for further purifying the strain by a plate streaking separation method.
Re-inoculating the purified strains to skimmed milk powder containing bottom layer and Na containing upper layer 2 CO 3 Beef extract peptone screening plate of (2) containing skimmed milk powder in bottom layer and Na-free in upper layer 2 CO 3 Placing the ordinary beef extract peptone screening plate at 37 ℃ for inverted culture for 4-5d, taking the ordinary beef extract peptone screening plate which does not obviously grow but contains Na 2 CO 3 And (5) screening strains which can form a milk proteolytic loop on the culture plate for seed preservation, and carrying out the next test.
The alkaline protease from microorganism is usually extracellular enzyme, and the alkaline protease produced by utilizing microorganism fermentation has the advantages of short period, high enzyme yield, easy extraction and separation, suitability for large-scale production and the like. At present, most industrial enzyme preparations are produced by utilizing microorganisms, and separation and breeding of alkaline protease-producing strains are always paid attention to. Some microorganisms growing in insects, especially various intestinal microorganisms, play an important role in the physiological processes of digestion, nutrition absorption, immunity and the like of hosts, and constitute a special habitat microorganism resource library for finding bioactive enzymes. Periplaneta americana (Periplaneta americana) is one of the oldest known insects, has strong physiological and environmental adaptation, is omnivorous, and usually living in a warm, moist, microorganism-rich environment, and can carry a variety of microorganisms in or on its body surface. These microorganismsThe substances, in part, have been extensively studied as human and animal pathogens. On the other hand, symbiotic bacteria of the insects have abundant diversity in composition and can be used as a beneficial microbial resource. Some studies have shown that certain alkalophilic microorganisms of biotechnological importance can also be isolated in the usual, non-extreme environments. In order to more fully excavate and utilize the intestinal fungus resource of the cockroach, the application adopts the material containing Na 2 CO 3 Screening out alcalophilic bacteria from the intestinal tract of periplaneta americana, and researching the enzymatic properties of alkaline protease produced by the bacteria.
Research shows that the alkalophilic strain for producing the alkaline protease provided by the application can contain Na of 0.03-0.09 mol/L 2 CO 3 Is grown in the medium of (a). Inoculating 5 mL of Na with a suspension containing the alkaline protease-producing alkalophilic strain in an inoculum size of 0.5% 2 CO 3 Shaking culture of 24h in beef extract peptone culture medium with concentration of 0, 0.03, 0.06, 0.09, 0.12, and 0.15 mol/L respectively under the above-determined optimal growth temperature condition, observing whether the culture solution is turbid, and determining OD of the culture solution 600 Value, examine Na 2 CO 3 The effect on the strain growth shows that the alkalophilic strain for producing the alkaline protease contains 0.03-0.09 mol/L Na 2 CO 3 Can grow well in beef extract peptone liquid culture medium, and is most suitable for Na 2 CO 3 The concentration was 0.06 mol/L.
The present application further proposes an alkaline protease secreted by an alkaline protease-producing basophilic strain as described above.
Preferably, the alkaline protease is tolerant at a temperature of 30-100 ℃, i.e. the alkaline protease can function at a temperature of 30-100 ℃. More preferably, the alkaline protease is tolerant at a temperature of 50 ℃, i.e. the protease has an optimal reaction temperature of 50 ℃, at which the protease activity is highest.
Preferably, the alkaline protease is tolerant at a pH of 7 to 13. The research shows that the alkaline protease has a certain casein degradation activity in the pH value of 7-13 and the highest activity in the pH=9-11, which indicates that the alkaline protease is an alkaline protease with an optimal reaction pH value of 9-11.
The application further provides an alkaline protease for use in any one of the following:
detergent production, leather tanning processing, food processing, pharmaceutical industry and protein waste treatment;
wherein the alkaline protease is secreted by an alkaline protease-producing basophil strain as described above.
The alkaline protease is mainly used for detergent production, leather tanning processing, food processing (protein hydrolysate production, meat processing, etc.), medical industry (catalyzing certain synthetic reactions in pharmaceutical industry), protein waste treatment, etc. For example, alkaline proteases can be added to detergents to catalyze the hydrolysis of proteins in stains to water-soluble small peptides or amino acids under alkaline conditions for stain removal; in the leather tanning process, the alkaline protease can be used as a soaking enzyme to promote the uniform dispersion of fiber yarns, so that the leather is softened, flexible, open, and the like.
The use of the alkaline protease secreted by the alkaline protease-producing strain provided by the application has all the beneficial effects of the alkaline protease-producing strain, and will not be described in detail herein.
The following technical solutions of the present application will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present application and are not intended to limit the present application.
Experimental materials
The periplaneta americana is captured in the cave of a bridge near the trade market of a certain aquatic product in Guangzhou.
Containing Na 2 CO 3 Or beef extract peptone culture medium (beef extract 3 g/L, peptone 10 g/L, naCl 5 g/L, anhydrous Na) 2 CO 3 0.15 mol/L, agar powder 20g/L, skimmed milk powder 20 g/L) as alkalophilic strain for producing alkaline protease and alkaline protease screening medium. Na (Na) 2 CO 3 And skimmed milk powder are dissolved in proper amount of water for independent sterilization.
The fermentation medium adopts Na-containing 2 CO 3 And CaCO (CaCO) 3 Corn flour-soybean cake flour culture medium (corn flour 40 g/L, soybean cake flour 30 g/L, na) 2 CO 3 0.04 mol/L，CaCO 3 10 g/L). Under the condition of ensuring that the concentration of each component is unchanged, the composition does not contain Na 2 CO 3 Adding different pH buffers or Na concentrations into beef extract peptone basal medium (beef extract 3 g/L, peptone 10 g/L, naCl 5 g/L) 2 CO 3 Test medium as corresponding indicator.
EXAMPLE 1 isolation and purification of alkaline protease-producing basophilic Strain
Capturing American cockroach from the trade market of certain aquatic products in Guangzhou, cleaning the surface of the cockroach with 75% ethanol solution, dissecting, taking out the intestinal tract, and placing in sterilized 0.15 mol/L Na 2 CO 3 Grinding the solution in a homogenizer, standing for 10 min, and collecting supernatant.
Coating the above homogenate on skimmed milk powder containing bottom layer and Na containing upper layer 2 CO 3 The beef extract peptone screening plate is subjected to inversion culture at the constant temperature of 37 ℃ for 5-7d. Colony growth and protein degradation loop production were observed every 24 hours. The colony which has good growth condition and generates obvious protein degradation ring in the plate is selected for further purifying the strain by a plate streaking separation method.
Re-inoculating the purified strains to skimmed milk powder containing bottom layer and Na containing upper layer 2 CO 3 Beef extract peptone screening plate of (2) containing skimmed milk powder in bottom layer and Na-free in upper layer 2 CO 3 Placing the ordinary beef extract peptone screening plate at 37 ℃ for inverted culture for 4-5d, taking the ordinary beef extract peptone screening plate which does not obviously grow but contains Na 2 CO 3 The strain on the culture plate, which can form a milk proteolytic loop, was selected for seed preservation, and the next test was performed, as shown in FIG. 1.
Example 2 identification of strains
Morphological identification
Pure culture of strainStreaking the material to be inoculated on Na-containing 2 CO 3 Is cultured at 37℃and the colony characteristics are observed to give FIG. 2. The culture at the proper stage is smeared and fixed, and is observed by an oil mirror after being respectively subjected to gram staining and spore staining, so that the images shown in fig. 3 and 4 are respectively obtained.
Identification of ribosomal 16S rDNA sequences
Fresh bacterial cultures were taken and PCR amplified using bacterial ribosomal 16S rDNA sequence primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'), and the PCR products were identified by agarose electrophoresis and sequenced by Beijing qingke biosciences, inc. BLAST comparison is carried out on the 16S rDNA sequence of the strain and the existing nucleic acid sequence of the NCBI nucleic acid database, and an NCBI-BLAST Tree View program is adopted to construct a evolutionary Tree (www.ncbi.nlm.nih.gov/BLAST/treeview/treeView. Cgi), so that FIG. 5 is obtained, and the classification status of the strain is determined through comparison with highly similar sequences.
EXAMPLE 3 temperature, pH and Na 2 CO 3 Effect of concentration on Strain growth
The strain contains a proper amount of Na 2 Shake culturing 72 h in CO3 beef extract peptone liquid medium, collecting fresh culture, and making OD 600 Uniform bacterial suspension=1.0 for the following assay:
1. effect of temperature on Strain growth
The bacterial suspension was inoculated in an amount of 0.5% to 5 mL Na-containing cells 2 CO 3 (0.04 mol/L) of beef extract peptone culture medium, shaking culture in a shaking table at constant temperature of 25deg.C, 28deg.C, 31deg.C, 34 deg.C, 37deg.C, and 40deg.C for 24h, observing whether the culture solution is turbid, and measuring OD of the culture solution 600 The effect of temperature on strain growth was examined (bacterial liquid concentration was OD 600 The values represent), resulting in fig. 6.
2. Influence of pH on the growth of the Strain
Inoculating the bacterial suspension into 5 mL beef extract peptone culture medium with pH value of 7, 8, 9, 10, 11, 12 according to 0.5%, shake culturing under the above-determined optimal growth temperature condition for 24h, observing whether the culture solution is turbid, and determining OD of the culture solution 600 The effect of pH on strain growth was examined and FIG. 7 was obtained.
3、Na 2 CO 3 Effect of concentration on Strain growth
The bacterial suspension was inoculated in an inoculum size of 0.5% to 5 mL Na 2 CO 3 Shaking culture of 24h in beef extract peptone culture medium with concentration of 0, 0.03, 0.06, 0.09, 0.12, and 0.15 mol/L respectively under the above-determined optimal growth temperature condition, observing whether the culture solution is turbid, and determining OD of the culture solution 600 Value, examine Na 2 CO 3 The effect on strain growth gave fig. 8.
Example 4 Strain enzyme-producing fermentation and measurement of enzyme Activity
Inoculating the strain to Na-containing strain 2 CO 3 And CaCO (CaCO) 3 In the corn flour-soybean cake flour fermentation medium, 144 h is cultured in a shaking way at 37 ℃, and the supernatant (crude enzyme liquid) is taken as the crude enzyme liquid, so that the enzyme activity under various subsequent conditions is measured.
The quantitative determination of the activity of alkaline protease is carried out by using a furin-phenol reagent method, and the operation is mainly carried out by referring to the method in the national standard of the current protease preparation (GB/T23527-2009), and is briefly described as follows: mixing crude enzyme solution and casein, performing water bath reaction for 10 min, adding trichloroacetic acid to stop reaction, centrifuging, collecting supernatant, and adding Na 2 CO 3 And a forskolin-phenol reagent, the OD of which is determined 680 Values. The blank group is that trichloroacetic acid is firstly added into crude enzyme liquid to inactivate enzyme, then casein is added, and other conditions are kept consistent. Definition of enzyme activity unit: 1 mL crude enzyme solution hydrolyzes casein for 1 min under certain temperature and pH conditions to generate 1 mug tyrosine, namely 1 activity unit expressed as U/mL. The calculation formula is as follows: enzyme activity (U/mL) =kxaxnxx (B/T). Wherein: k is a colorimetric constant, A is the difference between the OD value of the sample and the OD value of the blank control, N is the dilution of the enzyme sample, B is the dilution of the stop reaction solution when the OD value is measured, and T is the enzyme reaction time (min).
Enzymatic Property testing
The following measurement was performed using the supernatant of the fermentation broth of the aforementioned strain as a crude enzyme solution:
1. influence of the reaction temperature on the enzymatic Activity
The enzyme optimum reaction temperature was judged by using 1% casein as a substrate and testing it at 30-100deg.C (interval of 10deg.C) according to the above-mentioned alkaline protease activity quantification method to obtain FIG. 9.
2. Influence of the reaction pH on the enzymatic Activity
The activity of alkaline protease was measured under the conditions of pH 7, 8, 9, 10, 11, 12 and 13 using 1% casein prepared from buffer solution of the corresponding pH as a substrate, and the optimum reaction pH of alkaline protease was determined, and the reaction was performed under the condition of the optimum reaction temperature, to obtain FIG. 10.
3. Stability test of enzymes under different pH values
The crude enzyme solutions were mixed with buffer solutions having pH values of 7, 8, 9, 10, 11, and 12 in equal volumes, and left to stand at 4℃for 24. 24h, and then the enzyme activities were measured, and the enzyme activities (relative enzyme activities) were calculated to determine the enzyme stability under different pH conditions, to obtain FIG. 11.
Results
1. Isolation and selection of alkaline protease-producing basophilic strains
The method comprises the steps of adopting a milk flat plate transparent ring method to obtain 5 strains capable of producing milk degradation rings from American cockroach intestinal homogenate grinding liquid through preliminary screening, and finally obtaining a strain which grows on an alkaline flat plate and produces milk degradation rings (figure 1) and does not grow (or grows slowly) on a neutral flat plate through repeated screening, wherein the strain is named as bacillus alcalogenes bacteriaAlkalihalobacillus sp.) PAIA262, which is alkalophilic and produces proteases.
2. Identification of strains
(1) Morphological identification
In the presence of Na 2 CO 3 On beef extract-peptone culture medium, bacterial strain PAIA262 colony is round, milky white to yellowish, smooth and moist on the surface and has a certain thickness (figure 2). Gram staining and violet baculous cells were observed under an oil microscope, gram positive (fig. 3). Spores were stained and red baculophyte and green free spores were observed under an oil microscope (fig. 4).
(2) Molecular characterization of the Strain (ribosome 16S rDNA sequence characterization)
PCR amplification of the 16S rDNA sequence of strain PAIA262 using primers 27F and 1492R as primers,
the 16S rDNA gene sequence of the alkalophilic strain for producing the alkaline protease is shown as SEQ ID NO. 1.
SEQ ID NO. 1：
GGACGGCTGGCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCAATTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTATGGGATTGGCTCCACCTCGCGGTTTCGCTGCCCTTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGCCCCCGAAGGGGAAGCTCTGTCTCCAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACTGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGTGTTTACTTCGGCACTACGGGCATCGAAACCCCTAACACCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATATCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGCTTCCCAGTTTCCAATGGCCGCTCGGGGTTGAGCCCCGAGATTTCACATCAGACTTAAGAAGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTGAGGTACCGTCAAGGTACCGGTAGTTACGCCGGTACTTGTTCTTCCCTCACAACAGAGCTTTACGACCCGAAGGCCTTCCTCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATGCGCCGCAGGCCCATCCCTTAGTGACAGCCGAAACCGTCTTTCAAAAGAGAATCAGGTGATTCTCTTTATTATCCCGTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTAAGGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCGCTGACTTCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTCGTAGAGGAAAAAGGA
Agarose gel electrophoresis detection of the PCR product showed that the PCR product was approximately 1500 bp. Sequencing the PCR product, performing NCBI-Blast comparison, and constructing a phylogenetic tree, wherein the result shows that the 16S rDNA of the strain PAIA262 is compared with the 16S rDNA of each strain of bacillusThe similarity is high, and a big branch of the phylogenetic evolution of the species is formed (figure 5). By combining bacterial colony morphology, gram and spore staining characteristics and 16S rDNA gene sequence analysis, the strain PAIA262 can be preliminarily determined as alkaline halobacillus bacteriaAlkalihalobacillus sp.）。
3. Temperature, pH and Na 2 CO 3 Effect on Strain growth
Based on beef extract peptone culture medium, temperature, pH value and Na were examined respectively 2 CO 3 The effect on the strain growth shows that the optimum growth temperature is 37 ℃ (figure 6), the optimum growth pH value range is 9-11 (figure 7), and the strain contains 0.03-0.09 mol/L Na 2 CO 3 Can grow well in beef extract peptone liquid culture medium, and is most suitable for Na 2 CO 3 The concentration was 0.06 mol/L (FIG. 8).
4. Enzymatic Property determination of alkaline proteases
(1) Influence of the reaction temperature on the enzymatic Activity
Referring to FIG. 9, the influence of the reaction temperature on the protease activity produced by the strain was examined at a temperature ranging from 30℃to 100℃and the result showed that the optimum reaction temperature was 50 ℃.
(2) Influence of the reaction pH on the enzymatic Activity
Referring to fig. 10, the effect of pH on enzyme activity was investigated in the pH range of 7-13, and the results showed that casein degradation activity was exhibited to some extent in the pH range of 7-13, and the highest activity was found in the ph=9-11, indicating that the enzyme is an alkaline protease, and the optimal reaction pH range was 9-11.
(3) Stability of enzymes at different pH values
Referring to FIG. 11, the enzyme remained largely active after 24. 24h in buffers with pH values of 7, 8, 9, 10, 11, 12 at 4deg.C, respectively, and the results of the optimal reaction with alkaline pH values indicated that the enzyme had a certain stability under alkaline conditions.
In summary, insects are a group with wide distribution and extremely strong ecological impact, and co-epiphyte microorganisms with abundant in-vivo species are one of the focus of research.The application separates a bacillus alcaligenes bacterium which can well grow and secrete protease from the intestinal tract of periplaneta americanaAlkalihalobacillus sp.) PAIA262. The reaction condition is critical to the application of alkaline protease, especially the temperature and the pH value, and the thermostable and thermophilic enzyme has the advantages of high reaction speed, strong stability, easy treatment and the like from the aspect of temperature, the optimal reaction temperature of the alkaline protease secreted by the PAIA262 strain obtained by screening is 50 ℃, the optimal reaction pH range is 9-11, and the thermostable and thermophilic enzyme has stability under alkaline conditions, thus being typical alkaline protease. Therefore, the alkaline halobacillus PAIA262 strain and the alkaline protease produced by the strain have biotechnological application value.
The foregoing is merely a preferred embodiment of the present application and is not intended to limit the scope of the present application, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the present application.
Claims (7)
1. An alkalophilic strain for producing alkaline protease, which is characterized in that the alkalophilic strain for producing alkaline protease is bacillus alcaligenes bacteriaAlkalihalobacillus sp.) PAIA262 with deposit number GDMCC NO. 63124 and with deposit time of 2023, 3 and 23 days.
2. The alkaline protease-producing basophilic strain of claim 1, wherein the 16S rDNA gene sequence of the alkaline protease-producing basophilic strain is shown in SEQ ID No. 1.
3. The alkaline protease-producing basophilic strain of claim 1, wherein the alkaline protease-producing basophilic strain can contain 0.03-0.09 mol/L of Na 2 CO 3 Is grown in the medium of (a).
4. An alkaline protease secreted by the alkaline protease-producing basophilic strain of any one of claims 1 to 3.
5. The alkaline protease according to claim 4, wherein the alkaline protease is resistant at a temperature of 30 to 100 ℃.
6. The alkaline protease of claim 4, wherein the alkaline protease is tolerant at a pH of 7 to 13.
7. Use of an alkaline protease in any one of the following:
detergent production, leather tanning processing, food processing, pharmaceutical industry and protein waste treatment;
wherein the alkaline protease is secreted by the alkaline protease-producing basophil strain of any one of claims 1 to 3.
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