CN108753637A - A kind of bacterial strain and its fermentation process producing low temperature sterol esterase - Google Patents

Abstract

The invention relates to the fields of microbiology, biochemistry, fermentation engineering and the like, and is a bacterial strain for producing low-temperature sterol esterase, which is named Q-06. The bacterial strain is derived from the low-temperature domestication of the original bacterial strain Pseudomonas fragi . The optimal temperature for the production of low-temperature sterol esterase produced by the strain Q-06 of the present invention is 18-24°C, and low-temperature sterol esterase can be produced without energy consumption such as heating/cooling, which reduces production costs and meets industrial production requirements. Requirements: The bacterial strain Q-06 producing low-temperature sterol esterase of the present invention has the ability to produce sterol esterase, and the bacteria can efficiently produce stable low-temperature sterol esterase.

Description

A strain producing low-temperature sterol esterase and its fermentation method

technical field

The invention relates to the fields of microbiology, biochemistry, fermentation engineering and the like, in particular to a method for acclimating a Pseudomonas berry strain at low temperature and obtaining sterol esterase by fermenting the new low-temperature bacterial strain.

Background technique

Sterol esterase (Sterol esterase, EC 3.1.1.13) is an ester hydrolase that can catalyze the hydrolysis of sterol esters to generate sterols and fatty acids. Under suitable conditions, it can also catalyze the synthesis of sterol esters through esterification or transesterification reactions. Sterol esterase can help the body digest and absorb oil and fat nutrients, because it is related to human lipid metabolism and cholesterol absorption, sterol esterase is one of the important enzyme preparations for detecting the total cholesterol content in blood. In the paper industry, the combination of sterol esterase and lipase can be used to effectively hydrolyze wood sterol esters and improve the problem of "resin barrier" in the paper production process. At present, the research on the production of sterol esterase by microbial fermentation mainly focuses on the isolation and identification of high-yield sterol esterase strains, mutation breeding, separation and purification of sterol esterase, and the stage of cloning and expression in engineering bacteria, all of which are medium and high temperature sterol esterase . The low-temperature sterol esterase has high enzyme activity and high catalytic efficiency at low temperature, so it has the advantage of saving energy in industrial production; the low-temperature enzyme can lose its activity after mild heat treatment, and low temperature or suitable temperature treatment will not affect The quality of the product. The application of low-temperature sterol esterase will have a profound impact on traditional food, medicine and other fields. At present, there are no research reports on the production of low-temperature sterol esterase by marine microbial fermentation at home and abroad. If the industrial production of marine microbial fermentation sterol esterase is realized, good economic and social benefits can be created.

Contents of the invention

The object of the present invention is to provide a low-temperature Pseudomonas berry, a bacterial strain producing low-temperature sterol esterase, and a method for producing sterol esterase by fermentation thereof.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

A strain producing low-temperature sterol esterase, named Q-06, which comes from the low-temperature domestication of the original strain Pseudomonas fragi (CGMCC NO.1.3349). The applicant has developed a set of cultivation techniques for the strain Q-06, which can be obtained from The bacterium produces low-temperature sterol esterase through stable fermentation.

The biochemical characteristics of the bacterial strain producing low-temperature sterol esterase are:

The morphological characteristics of the colony are:

It grows well on the sterol ester medium. The single colony of the strain Q-06 is approximately round, with neat edges, raised surface, light yellow, opaque, easy to pick with an inoculation loop, and the center is shiny;

The morphological characteristics of the bacteria are:

The strains are rod-shaped, mostly in a single arrangement, and the Gram staining result is negative;

Main physiological and biochemical characteristics:

Oxidase test and contact enzyme test are positive, can oxidize and decompose glucose, can generate indole acetic acid, cannot generate hydrogen sulfide, cannot hydrolyze starch, nitrate reduction test, VP test, methyl red (MR) test, The gelatin hydrolysis test was negative, and the arginine dihydrolase test was positive;

Genetic Traits:

The sequence identification of the sterol esterase-producing strain Q-06 was carried out, and the sequence length of the PCR amplification product was 1489bp.

A kind of method of marine microbial fermentation production low-temperature sterol esterase of the present invention specifically comprises the following steps:

1) The strain Pseudomonas fragi was acclimatized at low temperature to obtain the strain Q-06. The acclimation temperature from high to low is: 30°C → 28°C → 26°C → 24°C → 22°C → 20°C → 18°C, making it in a low temperature environment grow well;

2) Inoculate the strain obtained from 1) low-temperature acclimatization on a solid seed medium, and cultivate it at 18-24°C for 48-96 hours;

3) The low-temperature acclimated strain is used to produce sterol esterase according to conventional methods. Expand the cultivation step by step at 18~24°C to prepare liquid first-class seeds and second-class seeds;

4) Put the liquid first-class seeds or second-class seeds into the liquid fermentation medium according to the inoculum amount of 5-10% of the volume of the fermentation broth, and culture them at 18-24°C for 48-120 hours, that is, marine microorganisms ferment and produce low-temperature sterols end of esterase;

5) Centrifuge the fermentation broth from 4) at 8,000-12,000 rpm to collect the supernatant, and the collected liquid is the crude enzyme liquid;

6) According to different needs and different users, the crude enzyme solution obtained in 5) is further concentrated, separated and purified, and prepared into enzyme preparations with different activities, purity and dosage forms.

Specifically, the low-temperature acclimation medium used in step 1) is: phytosterol ester 10.0~30.0g, yeast powder 5.0~10.0g, KH ₂ PO ₄ 2.0~5.0g, MgSO ₄ ·7H ₂ O 0.5~1.5g , (NH ₄ ) ₂ SO ₄ 1.0~3.0g, glucose 5.0~15.0g, agar powder 18.0~20.0g, tap water 1.0L, pH value 6.0~8.0, sterilized at 0.1 MPa, 121℃ for 30min by high-pressure steam;

Step 2) The solid seed medium used is: yeast extract 5.0~10.0g, peptone 3.0g~5.0g, NaCl 5.0~7.0g, K ₂ HPO ₄ 1.0~3.0g, FeSO ₄ 7H ₂ O 0.05~0.1 g, MgSO ₄ 7H ₂ O 0.1~0.3 g, agar powder 18.0~20.0g, tap water 1.0L, pH 6.0~8.0, sterilize at 0.1 MPa, 121°C for 30 min by high-pressure steam;

Step 3) The liquid seed medium used for cultivation is: yeast extract 5.0~10.0g, peptone 3.0g~5.0g, NaCl 5.0~7.0g, K ₂ HPO ₄ 1.0~3.0g, FeSO ₄ 7H ₂ O 0.05~0.1 g, MgSO ₄ ·7H ₂ O 0.1~0.3 g, tap water 1.0L, pH 6.0~8.0, sterilize at 0.1 MPa, 121°C for 30 min by high-pressure steam.

Step 4) The fermented enzyme production medium used is: phytosterol ester 5.0~15.0g, yeast powder 3.0~5.0g, KH ₂ PO ₄ 1.0~2.0g, MgSO ₄ 7H ₂ O 0.3~0.5g, (NH ₄ ) ₂ SO ₄ 0.5~1.0 g, glucose 5.0~10.0 g, tap water 1.0L, pH 6.0~8.0, sterilize at 0.1MPa, 121℃ for 30 minutes by high-pressure steam.

Low-temperature sterol esterase is fermented and produced according to the enzyme-producing conditions of the present invention. The low-temperature domesticated strain Q-06 can be stored at 4°C for 2 months, and the bacterial suspension made of 10-25vt% glycerin can be stored at -80°C. It can be preserved for a long time.

Compared with prior art, the beneficial effect of the present invention is:

The optimal temperature for the production of low-temperature sterol esterase produced by the strain Q-06 of the present invention is 18-24°C, and low-temperature sterol esterase can be produced without energy consumption such as heating/cooling, which reduces production costs and meets industrial production requirements. need;

The bacterial strain Q-06 producing low-temperature sterol esterase of the present invention has the ability to produce sterol esterase, and the bacteria can efficiently produce stable low-temperature sterol esterase;

The low-temperature sterol esterase fermented and produced by the strain producing low-temperature sterol esterase of the present invention is used in the research on the preparation of functional cholesterol-lowering food, the biodegradation of polyurethane, the preparation of clinical cholesterol determination kits, etc. It is used in food, medicine, chemical industry And other fields have a very wide range of application value.

Description of drawings:

The colony morphology of Fig. 1 sterol esterase producing bacteria Q-06;

The thalline morphology of Fig. 2 sterol esterase producing bacterium Q-06;

The electrophoresis pattern of the 16S rDNA PCR amplification product of Fig. 3 sterol esterase producing bacteria Q-06;

The 16S rDNA sequence phylogenetic tree of Q-06 of Fig. 4 sterol esterase producing bacteria;

The optimum action temperature of Fig. 5 sterol esterase crude enzyme liquid;

Fig. 6 The optimal pH curve of sterol esterase crude enzyme solution.

Detailed ways:

Example 1

The acquisition of low-temperature sterol esterase-producing bacteria Q-06:

1) Activation of the original strain Pseudomonas fragi (CGMCC NO.1.3349): The activation medium is: tryptone 8.0-10.0 g, yeast powder 4.0 g-9.0 g, NaCl 8.0-10.0 g, agar 18.0-20.0 g, tap water 1.0 L, with a pH value of 6.0-8.0, was sterilized by high-pressure steam at 0.1 MPa and 121 °C for 30 min.

2) Low-temperature acclimation: low-temperature acclimatization of the strain in 1) to obtain low-temperature sterol esterase-producing strain Q-06, the acclimatization temperature from high to low is: 30°C → 28°C → 26°C → 24°C → 22°C → 20°C → 18℃, so that it can grow well in a low temperature environment; the low temperature acclimation medium used is: phytosterol ester 10.0~30.0g, yeast powder 5.0~10.0g, KH ₂ PO ₄ 2.0~5.0g, MgSO ₄ 7H ₂ O 0.5 ~1.5 g, (NH ₄ ) ₂ SO ₄ 1.0~3.0 g, glucose 5.0~15.0, agar powder 18~20.0 g, tap water 1.0 L, pH value 7.0, sterilized at 0.1 MPa, 121 °C for 30 min by high-pressure steam.

The strain Pseudomonas fragi used was purchased from China General Microorganism Culture Collection and Management Center, CGMCC NO.1.3349.

Example 2

Isolation and identification of low temperature sterol esterase producing bacteria Q-06:

The morphological characteristics of the colony are:

It grows well on the sterol ester medium. The single colony of the strain Q-06 is approximately round, with neat edges, raised surface, light yellow, opaque, easy to pick with an inoculation loop, and the center is shiny (see Figure 1);

The morphological characteristics of the bacteria are:

The strains are rod-shaped, mostly in a single arrangement, and the Gram staining result is negative (see Figure 2);

Main physiological and biochemical characteristics:

Oxidase test and contact enzyme test are positive, can oxidize and decompose glucose, can generate indole acetic acid, cannot generate hydrogen sulfide, cannot hydrolyze starch, nitrate reduction test, VP test, methyl red (MR) test, The gelatin hydrolysis test was negative, and the arginine dihydrolase test was positive (see Table 1);

Table 1 Physiological and biochemical characteristics of strain Q-06

Genetic Traits:

The sequence of the sterol esterase-producing strain Q-06 was identified, and the sequence length of the PCR amplification product was 1489bp (see FIG. 3 ).

The strain of the present invention has similarity with Pseudomonas, and its 16S rDNA sequence was analyzed systematically with Mega5.0 software, and the phylogenetic tree was constructed by Neighbor-Joining method, which showed that the strain and Pseudomonas berry belonged to The same group, the most closely related. 16SrDNA sequence analysis showed that the homology with the similar strain Pseudomonas fragi (NR 112077.1) recorded in the Genbank International Gene Sequence Database was the highest (see Figure 4).

Example 3

The fermentation process of the low-temperature sterol esterase producing bacteria Q-06 strain:

1) Culture medium preparation

① Strain activation medium: tryptone 8.0g, yeast powder 5.0g, NaCl 8.0g, agar powder 20.0g, tap water 1.0L, pH value 6.5, sterilize at 0.1MPa, 121℃ for 30 minutes by high-pressure steam;

② Liquid seed medium: yeast extract 8.0 g, peptone 3.5 g, NaCl 7.0 g, K ₂ HPO ₄ 2.0 g, FeSO ₄ 7H ₂ O 0.1 g, MgSO ₄ 7H ₂ O 0.2 g, tap water 1.0 L, pH 8.0, sterilized by high-pressure steam at 0.1 MPa and 121°C for 30 min;

③ Fermentation enzyme production medium: phytosterol ester 5.0g, yeast powder 3.0g, KH ₂ PO ₄ 1.0g, MgSO ₄ 7H ₂ O 0.3g, (NH ₄ ) ₂ SO ₄ 0.5g, glucose 5.0g, tap water 1.0 L, with a pH value of 6.0, sterilized by high-pressure steam at 0.1 MPa and 121°C for 30 minutes.

2) The strain Q-06 producing low-temperature sterol esterase was initially activated according to the strain instructions provided by China General Microorganism Culture Collection Center (CGMCC);

3) Inoculate the strain Q-06 on the solid seed medium and culture it at 18-24°C for 48-96 hours;

4) Cultivate the low-temperature acclimated sterol esterase-producing bacteria at 18-20°C for 48-72 hours according to the conventional method, and then carry out step-by-step expansion cultivation according to the inoculation amount of 5vt%, and prepare liquid first-level seeds and second-level seeds;

5) Put the secondary seeds prepared in 4) into the fermented enzyme-producing medium according to the volume of the fermentation broth at an inoculation amount of 5-7%, and culture them at 18-20°C for 48-72 hours, that is, marine microorganisms ferment and produce low-temperature End of sterol esterase;

6) Centrifuge the fermentation broth from 5) at 8,000~12,000 rpm to collect the supernatant, and the collected liquid is the crude enzyme liquid;

7) According to different needs and different users, the crude enzyme solution obtained in 6) is further concentrated, separated and purified, and prepared into low-temperature sterol esterase preparations with different activities, purity and dosage forms.

Example 3

Optimum action temperature of low temperature sterol esterase crude enzyme solution

The sterol esterase crude enzyme solution produced by the fermentation of strain Q-06 was placed under different temperature conditions (15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C °C) to measure its enzyme activity, the relative enzyme activity was calculated with the highest enzyme activity in the same group as 100%, and the temperature-relative enzyme activity line graph was drawn, and the results showed that the optimum temperature for the enzyme was 30°C. (See Figure 5)

Example 4

Optimum Action pH of Low Temperature Sterol Esterase Crude Enzyme Solution

The sterol esterase crude enzyme solution produced by the fermentation of strain Q-06 was placed in the phosphate buffer reaction system with different pH values (4.0, 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 10.0), The enzyme activity was measured, and the relative enzyme activity was calculated with the highest enzyme activity in the same group as 100%. The pH value-relative enzyme activity line graph was drawn, and the results showed that the optimum pH for the enzyme was 7.0. (See Figure 6)

Example 5

Effects of Different Chemical Substances on the Enzyme Activity of Sterol Esterase

Taking the original sterol esterase fermentation broth as a control, different chemical substances were added to the fermentation broth to make ZnSO ₄ ·7H ₂ O, AgNO ₃ , MgSO ₄ ·7H ₂ O, CuSO ₄ ·5H ₂ O, CoCl ₂ ·6H ₂ The final concentrations of O, MnCl ₂ 4H ₂ O, FeCl ₃ , CaCl ₂ and KCl were all 1 mmol/L, and the chelating agents ethylenediamine tetraacetic acid (EDTA) and sodium dodecyl sulfate (sodium dodecyl sulfate) , SDS) at a final concentration of 0.1%, the sterol esterase activity was determined, and the results are shown in Table 2.

Table 2 Effects of different chemical substances on the activity of sterol esterase

sequence listing

<110> Dalian University

<120> A strain producing low-temperature sterol esterase and its fermentation method

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1489

<212>DNA

<213> Q-06 16S rDNA

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gtttgatcct ggctcagatt gaacgctggc ggcaggccta acacatgcaa gtcgagcgga 60

tgagaagagc ttgctcttcg agttagcggc ggacgggtga gtaatgccta ggaatctgcc 120

tggtagtggg ggacaacgtt tcgaaaggaa cgctaatacc gcatacgtcc tacgggagaa 180

agcaggggac cttcgggcct tgcgctatca gatgagccta ggtcggatta gctagttggt 240

gaggtaatgg ctcaccaagg cgacgatccg taactggtct gagaggatga tcagtcacac 300

tggaactgag acacggtcca gactcctacg ggaggcagca gtggggaata ttggacaatg 360

ggcgaaagcc tgatccagcc atgccgcgtg tgtgaagaag gtcttcggat tgtaaagcac 420

tttaagttgg gaggaagggc agtaagcgaa taccttgctg ttttgacgtt accgacagaa 480

taagcaccgg ctaactctgt gccagcagcc gcggtaatac agagggtgca agcgttaatc 540

ggaattactg ggcgtaaagc gcgcgtaggt ggttcgttaa gttgaatgtg aaagccccgg 600

gctcaacctg ggaactgcat ccaaaactgg cgagctagag tagggcagag ggtggtggaa 660

tttcctgtgt agcggtgaaa tgcgtagata taggaaggaa caccagtggc gaaggcgacc 720

acctgggctc atactgacac tgaggtgcga aagcgtgggg agcaaacagg attagatacc 780

ctggtagtcc acgccgtaaa cgatgtcaac tagccgttgg aatccttgag attttagtgg 840

cgcagctaac gcattaagtt gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat 900

gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag 960

aaccttacca ggccttgaca tgcagagaac tttccagaga tggattggtg ccttcgggaa 1020

ctctgacaca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1080

ccgtaacgag cgcaaccctt gtccttagtt accagcacgt aatggtgggc actctaagga 1140

gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcatcat ggcccttacg 1200

gcctgggcta cacacgtgct acaatggtcg gtacagaggg ttgccaagcc gcgaggtgga 1260

gctaatctca caaaaccgat cgtagtccgg atcgcagtct gcaactcgac tgcgtgaagt 1320

cggaatcgct agtaatcgcg aatcagaatg tcgcggtgaa tacgttcccg ggccttgtac 1380

acaccgcccg tcacaccatg ggagtgggtt gcaccagaag tagctagtct aaccttcggg 1440

aggacggtta ccacggtgtg attcatgact ggggtgaagt cgtaacaag 1489

Claims (5)

1. a kind of bacterial strain producing low temperature sterol esterase, which is characterized in that be named as Q-06, which comes to original strainPseudomonas fragiDomestication by low temperature.

2. the bacterial strain of production low temperature sterol esterase as described in claim 1, which is characterized in that

Colony morphology characteristic is：Bacterial strain well-grown on sterol ester culture medium, single bacterium colony is approximate circle, neat in edge, Surface elevation, it is opaque in faint yellow, it is easy to picking with oese, center is glossy；Morphological features are：Bacterial strain is in bar Shape, mostly single arrangement, Gram's staining result are negative；

Major physiological biochemical character：Oxidase test, catalase test are positive, can oxygenolysis glucose, can generate Heteroauxin cannot generate hydrogen sulfide, cannot hydrolyze starch, nitrate reduction test, Fu Pu（VP）Experiment, methyl red（MR）Examination Test, gelatin hydrolysis experiment be negative, arginine dihydrolase experiment be positive；

Genetics characteristics：

The low temperature sterol ester enzyme-producing bacteria Q-06, i.e.,Pseudomonas fragiThe 16S rDNA complete genome sequences of Q-06 are total 1489bp。

3. the fermentation process of the bacterial strain Q-06 production low temperature sterol esterases of production low temperature sterol esterase as described in claim 1, It is characterized in that, the described method comprises the following steps：

1）To bacterial strainPseudomonas fragi（CGMCC NO.1.3349）It carries out domestication by low temperature and obtains bacterial strain Q-06, domestication temperature It spends and is from high to low：30℃→28℃→26℃→24℃→22℃→20℃→18℃；

2）By 1）The bacterial strain Q-06 that domestication by low temperature obtains, is inoculated in solid seed culture medium for producing sterol esterase, 18~ 24 DEG C are cultivated 48-96 hours；

3）Low temperature bacterial strain Q-06 is expanded into culture at 18 ~ 24 DEG C step by step, is prepared into liquid first order seed and secondary seed；

4）By liquid first order seed or secondary seed, liquid fermentation producing enzyme culture is accessed by 5~10% inoculum concentrations of fermentating liquid volume In base, when cultivating 48~120 h at 18 ~ 24 DEG C, i.e. marine microorganism fermenting and producing low temperature sterol esterase terminates；

5）By 4）Zymotic fluid supernatant is collected by centrifugation in 8,000~12,000 rpm, the liquid being collected into is crude enzyme liquid.

4. the fermentation process of the bacterial strain Q-06 production low temperature sterol esterases of production low temperature sterol esterase as claimed in claim 4, It is characterized in that, it is different with using object according to different needs, by step 5）Obtained crude enzyme liquid further concentrates, it is pure to detach Change, is prepared into the enzyme preparation of different activities, purity and dosage form.

5. according to the method for claim 4, wherein domestication by low temperature culture medium, solid seed culture medium, liquid seeds culture Base, fermentation medium are respectively：

Step 1）Domestication by low temperature culture medium used in middle domestication is：10.0 ~ 30.0g of phytosterin ester, 5.0 ~ 10.0g of yeast powder, KH₂PO₄2.0 ~ 5.0g, MgSO₄·7H₂O 0.5 ~ 1.5g, (NH₄)₂SO₄1.0 ~ 3.0g, 5.0 ~ 15.0g of glucose, agar powder 18.0 ~ 20.0g, tap water 1.0L, pH value 6.0 ~ 8.0, in 0.1 MPa, 121 DEG C of 30 min of high pressure steam sterilization；

Step 2）Solid seed culture medium used is：5.0 ~ 10.0g of yeast extract, peptone 3.0g ~ 5.0g, NaCl5.0 ~ 7.0g, K₂HPO₄1.0 ~ 3.0g, FeSO₄·7H₂O0.05 ~ 0.1g, MgSO₄·7H₂O 0.1 ~ 0.3 g, 18.0 ~ 20.0g of agar powder, originally Water 1.0L, pH value 6.0 ~ 8.0, in 0.1 MPa, 121 DEG C of 30 min of high pressure steam sterilization；

Step 3）Liquid seed culture medium is used in culture：5.0 ~ 10.0g of yeast extract, peptone 3.0g ~ 5.0g, NaCl5.0 ~ 7.0g, K₂HPO₄1.0 ~ 3.0g, FeSO₄·7H₂O 0.05 ~ 0.1g, MgSO₄·7H₂O 0.1 ~ 0.3 g, tap water 1.0L, pH value 6.0 ~ 8.0, in 0.1 MPa, 121 DEG C of 30 min of high pressure steam sterilization；

Step 4）Enzymatic production culture medium used is：5.0 ~ 15.0g of phytosterin ester, yeast powder 3.0 ~ 5.0g, KH₂PO₄ 1.0~ 2.0g, MgSO₄·7H₂O 0.3 ~ 0.5g, (NH₄) ₂SO₄ 0.5 ~ 1.0 g, glucose 5.0 ~ 10.0 g, tap water 1.0L, PH6.0 ~ 8.0, in 0.1MPa, 121 DEG C of 30 min of high pressure steam sterilization.