CN116042459A - Acetylmicrobacterium YD37 and low-temperature protease and biological enzyme produced by same - Google Patents

Abstract

The invention provides a strain of aceto-microbacterium YD37 for producing low-temperature protease and application thereof. The classification of the Acetobacter YD37 is named as Acetobacter Exiguobacterium acetylicum, the preservation number is CGMCC No.25293, and the colony is yellow, opaque, round, regular in edge and smooth in surface. Gram positive bacteria have both short rod-shaped and spherical forms, and the size of the bacterial cells is about 0.5-1.5 mu m, and the bacterial cells have flagella. The optimum acting temperature of the protease is 35-37 ℃, the optimum pH is 6.0-7.0, and the enzyme activity can be regulated by metal ions.

Description

Acetylmicrobacterium YD37 and low-temperature protease and biological enzyme produced by same

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a low-temperature protease produced by Acetylmicrobacterium YD37 and application thereof.

Background

The market demand for low temperature proteases is large. The optimal catalysis temperature of the low-temperature protease is close to the temperature of a human body, and compared with the use value of the medium-temperature protease, the low-temperature protease has better use value, for example, the protease preparation can promote the efficacy of medicines to be exerted by adding some medicines, and the daily chemical production of cosmetics, skin care products, soaps, hand washing solutions, detergents and the like has urgent demands for the low-temperature protease. However, the low-temperature protease on the market is more than some washing enzymes of a national company such as Norwestine, and the mature high-quality low-temperature protease products in China are relatively deficient.

The research on the low-temperature protease in China is relatively late, most of low-temperature protease preparations on the market at present are from import, the low-temperature protease industry in China is in a blank stage, and a mature technology and an industrial system for developing the low-temperature protease are not yet seen. The research on low-temperature protease in China is less, the related strains are mostly from Pseudomonas and Pseudomonas, and some strains have certain biological hazard and are not suitable for large-scale deep fermentation or meet the biological safety requirements of environmental release.

The invention starts from a safe wild strain, screens the strain producing the low-temperature protease, and explores the upstream fermentation, downstream separation, extraction and purification and enzymatic properties of the low-temperature protease so as to obtain the low-temperature protease with application potential, thereby providing resources and technology for industrial development.

Disclosure of Invention

The invention aims to provide a low-temperature protease produced by acetominibacillus YD37 and YD37 capable of producing the low-temperature protease and application thereof.

In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:

the invention provides a strain of low temperature resistant proteinase-producing Acetylmicrobacterium YD37, which is classified and named as Acetylmicrobacterium Exiguobacterium acetylicum and has the preservation number of CGMCC No.25293.

Further, the colony of the Acetylmicrobacterium YD37 is yellow, opaque, round, regular in edge and smooth in surface. Gram staining results are positive, and the gram staining results have two forms of short rod shape and sphere shape, and the cell size is about 0.5-1.5 mu m, and the flagellum is a periphyton.

Further, the 16S rRNA gene sequence of the acetoMicrobacterium YD37 is shown as SEQ ID No. 1.

Further, the Acetobacter xylinum YD37 has low-temperature protease activity, low-temperature amylase activity and low-temperature cellulase activity.

In another aspect, the present invention provides a low temperature protease produced by the Acetylmicrobacterium YD37 of claim 1.

Furthermore, the gene sequence of the low-temperature protease is shown as SEQ ID No. 2.

Further, the protein sequence of the low-temperature protease is shown as SEQ ID No.3.

The production method of the low-temperature protease comprises the following steps: the fermentation conditions for producing the low-temperature protease by the Acetylmicrobacterium YD37 are as follows: the initial pH of fermentation is 6.0-7.0, the fermentation temperature is 15-25 ℃, the liquid loading amount is 30-60%, the rotating speed is 200-300rpm, the inoculation amount is 1-2%, and the fermentation time is 20-30h.

Further, the low temperature protease has a pH of 5.0 to 9.0, preferably, a pH of 6.0 to 8.0; more preferably the pH is 7.0.

Further, the low temperature protease has an action temperature of 10℃to 50℃and preferably 35 ℃.

Further, the low-temperature protease can regulate the enzyme activity through metal ions, and specifically:

1) By Co ²⁺ And Mn of ²⁺ Activating a low temperature protease; and/or;

2) By Zn ²⁺ 、Fe ²⁺ And Cu ²⁺ Inhibiting low temperature protease.

In another aspect of the present invention, there is provided a biological enzyme having:

(I) The amino acid sequence of the low temperature protease of claim 6, SEQ ID No.3; or (b)

(II) an amino acid sequence which is obtained by substituting, deleting, adding, converting one or more amino acids in the amino acid sequence described in (I) and has the same function as the amino acid sequence described in (I).

Further, the biological enzyme is a low temperature protease.

Compared with the prior art, the invention has the following advantages and technical effects:

1) The invention provides a strain of aceto-microbacterium YD37 capable of producing low-temperature protease, and the strain can produce the low-temperature protease.

2) The invention provides a low-temperature protease which can play a role in pH at 5-9 at the temperature of 10-50 ℃, has protease, amylase and cellulase activities, and can promote or inhibit enzyme activity reaction by combining with metal ions.

3) The invention provides an amino acid sequence of biological enzyme, which comprises an amino acid sequence SEQ ID No.3 and amino acid sequence transformation thereof, and the polypeptide can prepare biological enzyme.

Drawings

FIG. 1 shows colony morphology of Acetobacter xylinum YD37. Wherein a: transparent ring on protein plate, b: colony morphology on LB plates.

FIG. 2 is a graph showing the result of transmission electron microscopy of Acetobacter xylinum YD37.

FIG. 3 is a phylogenetic tree of Acetobacter xylinum YD37.

FIG. 4 is a diagram showing purification of low-temperature protease produced by fermentation of Acetylmicrobacterium YD37.

FIG. 5 shows SDS-PAGE of crude low temperature protease and protease enzyme profiling.

FIG. 6 shows the identification of low temperature protease sequences.

FIG. 7 shows the prediction of low temperature protease structure: a: predicting a tertiary structure; b: is a tertiary structure surface prediction.

FIG. 8 shows the optimum temperature of the crude enzyme of low-temperature protease.

FIG. 9 shows the temperature tolerance of the crude enzyme of low temperature protease.

FIG. 10 shows the optimum pH of the crude enzyme of low temperature protease.

FIG. 11 shows the pH tolerance of the crude enzyme of low temperature protease.

FIG. 12 is a graph showing the effect of metal ions on crude enzyme activity of low temperature proteases.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to specific embodiments.

The experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions recommended by the manufacturer; materials, reagents, etc. not noted are commercially available products. Percentages and parts are by weight unless otherwise indicated.

Example 1: screening and identification of low temperature protease producing strains

25g of collected soil sample is taken and added into 225mL of sterile water, and shaking culture is carried out for 30min at 180rpm, so as to prepare sample suspension. 1mL of the sample suspension was taken and added to 9mL of sterile water for 10-fold gradient dilution. Selecting proper dilution gradient, coating 100 mu L on screening culture medium (skimmed milk powder 20g/L, agar 20 g/L) plate, observing its change every 24h, and separating and purifying strain capable of producing transparent ring on LB plate continuously for 3 times. And (3) selecting the single colony after purification on a screening culture medium, culturing for 24 hours at 15 ℃, and selecting the strain with a large transparent circle/colony ratio as a screening candidate strain. The re-screened strain is inoculated in LB culture medium (peptone 10g/L, yeast extract 5g/L, naCl 10 g/L) for liquid fermentation, after shaking culture for 24 hours at 180rpm, the enzyme activity of the fermentation supernatant is measured at 20 ℃, and the strain with the highest enzyme activity is screened out, and the number is YD37.

Example 2: YD37 enzyme production Activity Studies

Simultaneously preparing a starch culture medium plate, a cellulose culture medium plate, a lipase plate, a saccharifying enzyme plate and a glucanase plate respectively, inoculating YD37 single colony on the plates, culturing at 15 ℃ in an inverted mode, and observing the enzyme production type and activity of the single colony. The YD37 has good low-temperature protease activity, low-temperature amylase and cellulase activities, and no lipase, saccharifying enzyme and glucanase activities, and the ratio (R/R) of transparent circle diameter to colony diameter is shown in Table 1.

TABLE 1 Low temperature enzyme production type and Activity of YD37 strain

Example 3: identification of species

Bacterial strain YD37 was streaked on LB, and the colony morphology was observed, and the colony was yellow, opaque, round, regular-edged and smooth-surfaced (FIG. 1 a). The strain YD37 was observed by transmission electron microscopy, and the strain YD37 had both a short rod shape and a spherical shape, and the cell size was about 0.5 to 1.5. Mu.m, and the flagellum (FIG. 1 b).

The results of the physiological and biochemical characteristics of YD37, such as gram staining, anaerobism, operability, thixotropic enzyme, V-P experiment and the like, are shown in Table 2 by referring to the manual for identifying common bacterial systems:

TABLE 2 physiological and biochemical experimental results of YD37 strain

YD37 strain is cultured overnight at a temperature of 20 ℃ and 180rpm, and the bacterial liquid is used as a template for 16S rRNA amplification, and the general primers are as follows: upstream primer 27F:5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer 1492R:5'-GGTTACCTTGTTACGACTT-3'; the PCR reaction system is as follows: template 1. Mu.L; 1 mu L of each of the upstream and downstream primers; 2 XPCR Master Mix 12.5. Mu.L; ddH ₂ O 9.5. Mu.L; the components are added into a 200 mu L small centrifuge tube (carried out on ice), and after being mixed evenly, the components are centrifuged briefly to mix evenly, and the mixture is put into a PCR amplification instrument to carry out PCR amplification of target fragments. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 55℃for 90s, extension at 72℃for 1min, and repeating 34 cycles; extending at 72℃for 10min. DNA sequencing is carried out on the obtained PCR product, and the obtained strain 16S rRNA nucleotide sequence SEQ ID No.1 is input into a GenBamk database for BLAST comparison. The results showed that the similarity of the strain YD37 with other 2 strains Exiguobacterium acetylicum reached 99%, and that a phylogenetic tree of the strain YD37 was constructed by using the phylogenetic tree software MEGA7.0 as shown in FIG. 2. The strain YD37 is identified as Acetylmicrobacterium Exiguobacterium acetylicum by combining the morphological, physiological and biochemical characteristics of the strain YD37 and the identification result of 16S rRNA.

The above results are substantially consistent with the physiological and biochemical characterization of Acetobacter xylinum in the Berger's bacterial handbook and literature, and YD37 was determined to be Acetobacter xylinum. Performing strain preservation on the screened strain YD37, wherein the preservation unit of the acetobacter xylinum YD 37: china general microbiological culture Collection center (CGMCC); address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing; preservation date: 2022, 07, 13; the preservation number of the Acetylmicrobacterium Exiguobacterium acetylicum is CGMCC No.25293.

Example 4: fermentation conditions and purification of low-temperature protease produced by Acetobacter xylinum YD37

Acetylmicrobacterium YD37 was inoculated into LB medium and cultured in shake flask at 20℃for 12 hours. Biosynthesis is then carried out by a liquid deep aeration process. For example, 20L of the enzyme-producing fermentation medium (corn meal 20.2g/L, yeast extract 2g/L, tryptone 6g/L, beef extract 2g/L, naCl 7g/L, KCl 1 mmol/L) of the prepared Acetylmicrobacterium YD37 was added to a 50L fermenter and sterilized for 40 minutes, and the first seed solution was inoculated into the fermenter at an inoculum size of 1%. The fermentation temperature is set to 20 ℃, the initial pH of fermentation is 6.3, the rotating speed is 200rpm, the inoculum size is 1%, the tank pressure is 0.2-1.5MPa, and the fermentation time is 22h.

Meanwhile, in the fermentation process, various conditions of initial pH of fermentation of 6.0-7.0, fermentation temperature of 15-25 ℃, liquid loading of 30-60%, rotating speed of 200-300rpm, inoculum size of 0.2-15%, fermentation time of 20-30h and the like are respectively tested for strain fermentation, so that bacterial liquid is obtained.

The obtained fermentation broth was refrigerated and centrifuged at 8000rpm at 4℃for 15min to remove the cells, and the supernatant was retained. At 4 ℃, respectively detecting the residual enzyme activities in the supernatant fluid at 20%, 40%, 60% and 80% of ammonium sulfate, wherein the result shows that the residual enzyme activities in the supernatant fluid after 60% saturation ammonium sulfate precipitation are only 3.2% (figure 4), which shows that the effective precipitation of the low-temperature protease can be realized at the concentration of the ammonium sulfate. Dissolving the obtained precipitate with a proper amount of PBS buffer solution, removing excessive salt in a dialysis bag for intercepting the molecular weight of 1kD, and freeze-drying to prepare crude enzyme to obtain crude protease.

Example 5: identification of Low temperature protease produced by Acetylmicrobacterium YD37

The crude enzyme thus prepared was subjected to SDS-PAGE. According to the protein electrophoresis experimental technique, 5% (w/v) concentrated gel and 12% (w/v) separation gel are used, the same crude enzyme sample is spotted on 2 lanes, the gel after electrophoresis is divided into two parts, half of the gel containing markers is normally dyed, and the other half of the gel containing non-denatured samples is prepared into zymograms by a substrate soaking method. The zymogram is prepared by a substrate soaking method, and the specific method comprises the following steps: after electrophoresis, taking the separation gel, washing with 2.5% Triton X-100 at room temperature under constant speed of 60rpm for 3 times, and 15min each time to remove SDS; then washed 2 times with 50mM Tris-HCl buffer for 20min each to remove Triton X-100; then placing the separation gel in 50mM Tris-HCl buffer solution with pH 7 and 0.1% gelatin, and placing the separation gel in a constant temperature shaking table at 35 ℃ for reaction for 2 hours at 60 rpm; the rapid decolorization solution is taken out for decolorization until a blank strip appears. The protease position was determined by alignment of markers, and the result was shown in FIG. 5, in which the low-temperature protein band was around 45kDa, thereby determining that the protein was a protease produced by strain YD37.

The 45kDa protein band was excised, submitted to Shanghai enzymatic hydrolysis and mass spectrometry identified, and by database search, it was found that 5 fragment sequences could be matched to an aminopeptidase derived from Exiguobacterium enclense with a sequence coverage of 58% indicating a certain homology between the two (FIG. 6). Thus, it was initially judged that the protease may be an aminopeptidase derived from E.acetylicum. At present, no report of E.acetylicum aminopeptidase production has been found.

Aminopeptidase genes that may be present in the E.acetylicum genome-wide sequences published in the database were selected based on them. The primer is designed based on the upstream and downstream sequences of the gene, the extracted E.acetylicum YD37 genome is used as a template for PCR, and the product is sent to Shanghai industrial sequencing, so that the nucleic acid sequence SEQ ID No.2 and the translated protein sequence SEQ ID No.3 of the gene are obtained.

The three-level structure of E.acetylicum YD37 low temperature protease was predicted using bioinformatics software, and the results are shown in FIG. 7, which contains 14 alpha-helical structures and 19 beta-sheet structures (FIG. 7 a), the three-level structure of which is mostly covered by negatively charged surface (FIG. 7 b).

Example 6: enzymatic Properties of Acetobacter yD37 Low temperature protease

1. Determination of optimum catalytic temperature

And (3) respectively reacting the crude enzyme solution with casein solution at 10, 20, 30, 35, 40 and 50 ℃, adding Fu Lin Fen solution for color development, measuring absorbance at 680nm, and determining the optimal enzyme catalysis temperature. As shown in FIG. 8, the enzyme activity at 35 ℃ is 167.46U/mL at the highest, and the enzyme activity at low temperature of 10 ℃ and 20 ℃ is 36.0U/mL and 73U/mL respectively, which shows that the enzyme can still function at low temperature, and the enzyme activity difference at different temperatures reaches a significant level (P < 0.05).

2. Temperature tolerance measurement

The crude enzyme solution was incubated in a water bath at 10, 20, 30, 40 and 50℃for 90min, and the enzyme activity was measured at the optimum enzyme catalysis temperature, and the enzyme activity at the optimum enzyme catalysis temperature was set to 100%, and the other treatment groups were expressed as relative enzyme activities. The results are shown in FIG. 9, in which the enzyme activity was gradually decreased with increasing temperature, and in which the enzyme activity of the 50℃treatment group was completely lost, indicating the heat intolerance of the enzyme. Generally, low temperature proteases are difficult to withstand high temperature environments above 50 ℃. Intolerance to high temperature environments is a general feature of low temperature proteases, by which the termination of the enzymatic reaction can be achieved by means of heating and an efficient control of the enzymatic reaction can be achieved.

3. Optimum catalytic pH determination

After the crude enzyme solution and casein react under the conditions of pH 6.0, 7.0, 8.0 and 9.0 respectively, a Fu Lin Fen solution is added for color development, and the absorbance is measured at 680nm to determine the optimal catalytic pH. As a result, as shown in FIG. 10, the enzyme activity at pH 7.0 was 147.33U/mL at the highest, the enzyme activity at pH 9.0 was 72.36U/mL, and the difference in enzyme activities at different pH values reached a significant level (P < 0.05).

4. Determination of pH tolerance

The crude enzyme solution was incubated in a water bath at pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 for 90min, the enzyme activity was measured at the optimum catalytic pH, the enzyme activity at the optimum catalytic pH was set to 100%, and the other treatment groups were expressed as relative enzyme activities. As shown in FIG. 11, the enzyme activity of the low-temperature protease was maintained at 75% or more after incubation for 90min at pH 6.0-8.0. However, when the pH is <6.0 or >9.0, the enzyme activity drops sharply, and even the enzyme activity is not detected.

5. Influence of Metal ions on Low temperature proteases

Adding Cu with the final concentration of 1-2 mmol/L into the crude enzyme solution ²⁺ 、Fe ²⁺ 、Mn ²⁺ 、Ca ²⁺ 、Mg ²⁺ 、Co ²⁺ And Zn ²⁺ After incubation for 90min, residual enzyme activity was determined with Fu Lin Fenfa at the optimal enzyme reaction temperature and pH. The crude enzyme solution without metal ions was used as a control. As a result, as shown in FIG. 12, the enzyme activity was significantly affected by Mn ²⁺ And Co ²⁺ Activated, subject to Zn ²⁺ 、Fe ²⁺ And Cu ²⁺ Inhibition of (P)<0.05 While Ca) ²⁺ And Mg (magnesium) ²⁺ The effect on the enzyme activity was not significant (P>0.05)。

Example 7: site-directed mutagenesis of proteins

The amino acid site near the low-temperature protease activity center of Acetylmicrobacterium YD37 was mutated by site-directed mutagenesis of the protein sequence, and the mutated sequence was detected for the optimal enzyme activity temperature by the method of example 6, and the results are shown in Table 3.

TABLE 3 list of mutation information

As shown in Table 3, after the mutation of the amino acid near the active center of the Acetylmicrobacterium YD37 low-temperature protease, the optimum temperature of the mutant sequence 1 was 35℃as the optimum temperature of the Acetylmicrobacterium YD37 low-temperature protease; the optimal temperature of the enzyme activity of the mutant sequence 2 is increased; the optimal enzyme activities of the mutant sequence 3 and the mutant sequence 4 are reduced to 30 ℃, which is more beneficial for the protease to act at low temperature.

The biological enzyme is produced by fungus fermentation, has high safety and wide application prospect.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Claims (12)

1. The strain of low-temperature protease-producing Acetylmicrobacterium YD37 is characterized in that the strain is classified and named Acetylmicrobacterium Exiguobacterium acetylicum, and the preservation number is CGMCC No.25293.

2. The micro-acetenuibacter YD37 according to claim 1, wherein the 16S rRNA gene sequence of the micro-acetenuibacter YD37 is shown in SEQ ID No. 1.

3. The micro-acetenuibacter YD37 according to claim 1, wherein the micro-acetenuibacter YD37 has low temperature protease activity, low temperature amylase activity and low temperature cellulase activity.

4. A low temperature protease produced by the mini-acetobacter YD37 of claim 1.

5. The low temperature protease according to claim 4, wherein the gene sequence of the low temperature protease is shown in SEQ ID No. 2.

6. The low temperature protease according to claim 4, wherein the amino acid sequence of the low temperature protease is shown in SEQ ID No.3.

7. The low-temperature protease according to claim 4, which is completed by deep liquid aeration fermentation, wherein the fermentation conditions of the low-temperature protease are as follows: the initial pH of fermentation is 6.0-9.0, the fermentation temperature is 15-25 ℃, the liquid loading amount is 30-80%, the tank pressure is 0.2-1.5MPa, the rotating speed is 50-300rpm, the inoculum size is 1-2%, and the fermentation time is 20-30h.

8. The low temperature protease according to claim 4, wherein the low temperature protease has a pH of 5.0-10.0, preferably a pH of 6.0-8.0; more preferably, the pH is 7.0.

9. The low temperature protease according to claim 4, wherein the low temperature protease has an action temperature of 10 ℃ to 50 ℃, preferably 35 ℃.

10. The low-temperature protease according to claim 4, wherein the low-temperature protease can regulate the enzyme activity by metal ions, specifically:

1) By Co ²⁺ And Mn of ²⁺ Activating a low temperature protease; and/or;

2) By Zn ²⁺ 、Fe ²⁺ And Cu ²⁺ Inhibiting low temperature protease.

11. A biological enzyme, characterized in that the biological enzyme has:

(I) The amino acid sequence of the low temperature protease of claim 6, SEQ ID No.3; or (b)

(II) an amino acid sequence which is obtained by substituting, deleting, adding, converting one or more amino acids in the amino acid sequence described in (I) and has the same function as the amino acid sequence described in (I).

12. The use according to claim 11, wherein the biological enzyme is a low temperature protease.

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