CN103937692A - Aphanocladium strain and method for producing beta-glucosidase by same - Google Patents

Abstract

The invention discloses an aphanocladium strain and a method for producing beta-glucosidase by the same, belonging to the field of food biotechnology. The invention provides a novel aphanocladium strain capable of producing beta-glucosidase. The aphanocladium strain is named as aphanocladium SK34.001 with a collection number of CCTCCNO: M2014090. According to the method disclosed by the invention, three-stage culture of slant culture, seed culture and fermentation culture is carried out on the aphanocladium strain for producing beta-glucosidase; and beta-glucosidase powder is obtained by separating and purifying from fermentation broth. The method for producing beta-glucosidase by aphanocladium SK34.001 is simple in fermentation process, easy to extract an enzyme, high in enzymatic property and stable in enzymatic property; waste resources such as bran can be directly used as a culture medium carbon source for the strain, thus saving energy consumption and reducing production cost; and the strain is wide in market application prospect.

Description

One strain silk branch trichoderma strain and the method for producing beta-glucosidase thereof

Technical field

The method that the present invention relates to a strain silk branch trichoderma strain and produce beta-glucosidase, belongs to technical field of food biotechnology.

Background technology

Beta-glucosidase (EC 3.2.1.21), also claims β-D-Glucose glycosides enzyme, and it can be hydrolyzed the β-D-Glucose glycosidic bond that is incorporated into substrate end irreducibility, discharges glucose and corresponding aglucon simultaneously.Beta-glucosidase is present in the many plants of nature, insect, yeast, mould and bacterial body, and it participates in the carbohydrate metabolism of organism, plays an important role to maintaining organism normal physiological function.

Beta-glucosidase extensively distributes at nature, almost in all organisms, has existence, but source is different, and its character is different.The specificity that this enzyme divides glucosides aglucone is not strong, can be hydrolyzed the glycosidic link of many β types.

Beta-glucosidase, except being mainly used in degraded cellulose, also can be applicable to dairy industry and reduces lactose; Catalysis glucose generation shift reaction and condensation reaction complex functionality oligose, as oligomeric dragon gallbladder sugar; Be applied to and in (really) wine, tealeaves, fruit juice, play flavouring or debitterize effect as food flavor enzyme; In addition, beta-glucosidase also can be used to hydrolyzed soy bean isoflavone, prepares highly active isoflavone genin product.

At present, both at home and abroad for the microorganism of studying and prepare beta-glucosidase mainly have Trichoderma ( trichoderma) and Aspergillus ( aspergillus).Research shows, many Aspergillus are to produce the strain excellent of high activity beta-glucosidase, and be considered to not can toxigenic cellulase producing bacteria, thereby research is more deep.Up to now, also do not have silk branch mould ( aphanocladium) report as the correlative study of beta-glucosidase generation bacterium.

Summary of the invention

The method that the object of this invention is to provide a kind of branch trichoderma strain and produce beta-glucosidase.

Technical scheme of the present invention, a strain silk branch trichoderma strain, is the silk branch trichoderma strain that the strain that previously separation screening had obtained from vegetable mould sample such as the inventor can high-yield beta-glucosidase, Classification And Nomenclature be silk mould ( aphanocladiumsp.) SK34.001, its preserving number is CCTCC NO:M 2014090, depositary institution is Chinese Typical Representative culture collection center.

The rDNA ITS sequence of described bacterial strain is as shown in SEQ ID NO:1.

The method that described silk branch trichoderma strain CCTCC NO:M 2014090 produces beta-glucosidase, step is as follows:

(1) slant culture: get a silk branch trichoderma strain CCTCC NO:M 2014090 and carry out slant culture, adopt PDA substratum, each component is in g/L: potato 200, glucose 20, agar 15 ~ 20; Nature pH, sterilizing 30min at 115 DEG C; Culture temperature is 25 ~ 35 DEG C, incubation time 3 ~ 8 days;

(2) seed culture: adopt beef-protein medium, each component is in g/L: extractum carnis 2 ~ 5, Tryptones 5 ~ 8, sodium-chlor 2 ~ 5, natural pH, 121 DEG C of sterilizing 20min; At the bottled liquid 30 ~ 50mL of 250mL triangle, the slant culture liquid of step (1) gained is carried out to seed culture, shaking flask rotating speed 150 ~ 200rpm, 25 ~ 35 DEG C of culture temperature, incubation time 18 ~ 24h;

(3) fermentation culture: fermention medium component is in g/L: wheat bran 20 ~ 40, ammonium sulfate 4 ~ 6, potassium primary phosphate 2 ~ 3, magnesium sulfate 0.3 ~ 0.5, calcium chloride 0.3 ~ 0.5, natural pH, 121 DEG C of sterilizing 20min; The seed culture fluid of step (2) gained by volume percentage ratio 2% ~ 5% inoculum size is inoculated in fermention medium, shaking flask rotating speed 150 ~ 200rpm, 25 ~ 35 DEG C of culture temperature, incubation time 3 ~ 8 days;

(4) beta-glucosidase enzyme powder preparation: the fermented liquid that step (3) is made obtains fermented supernatant fluid at the centrifugal 10 ~ 30min of 5000 ~ 10000rpm or filtration removal thalline, remove and desalt and foreign protein through ultrafiltration, through ammonium sulfate precipitation, dialysis, finally dialyzate is carried out to lyophilize and obtain beta-glucosidase enzyme powder again.

Activity of beta-glucosidase in the present invention, is by making enzyme act on p-nitrophenyl-β-D-Glucose glycosides (pNPG), decomposes the amount of the p-NP that generates and measures.The unit definition of this enzyme is the enzyme amount that per minute generates 1 μ mol p-NP.

Beneficial effect of the present invention: the present invention for adopt first silk branch mould ( aphanocladium) as the generation bacterium of beta-glucosidase, this strain culturing is convenient, zymotechnique is simple.Its enzyme that produces is extracellular enzyme, is easy to extract, and enzyme is lived high, and enzymatic property is stable, can directly utilize the waste resources such as wheat bran as silk branch mould ( aphanocladium) culture medium carbon source, save energy consumption, reduce production costs, there is wide market application foreground.

Biological material specimens preservation: a strain silk branch trichoderma strain, Classification And Nomenclature be silk branch mould ( aphanocladiumsp.) SK34.001, this bacterial strain has been preserved in Chinese Typical Representative culture collection center on March 14th, 2014, be called for short CCTCC, address: Wuhan, China, Wuhan University, deposit number is CCTCC NO:M 2014090.

Embodiment

Embodiment 1

By silk branch trichoderma strain ( aphanocladiumsp.) SK34.001 ferments under the following conditions:

Slant culture: adopt PDA substratum, each component is in g/L: potato 200, glucose 20, agar 20, natural pH, 115 DEG C of sterilizing 30min.Culture condition is: 30 DEG C of culture temperature, incubation time 5 days;

Seed culture: adopt beef-protein medium, each component is in g/L: extractum carnis 2.5, Tryptones 5.0, sodium-chlor 2.5, natural pH, 121 DEG C of sterilizing 20min.Culture condition is: the bottled liquid 30mL of 250mL triangle, shaking flask rotating speed 200rpm, 30 DEG C of culture temperature, incubation time 24h;

Fermentation culture: fermention medium component is in g/L: wheat bran 35, ammonium sulfate 4.0, potassium primary phosphate 2.0, magnesium sulfate 0.3, calcium chloride 0.3, natural pH, 121 DEG C of sterilizing 20min.Culture condition is: seed culture fluid by volume percentage ratio 2% inoculum size is inoculated in fermention medium, shaking flask rotating speed 150rpm, and 30 DEG C of culture temperature, incubation time 7 days, the enzyme that obtains fermented liquid is lived as 5U/mL.

Embodiment 2

The preparation of beta-glucosidase enzyme powder:

The fermented liquid that embodiment 1 is made centrifugal 30min under the rotating speed of 8000rpm removes thalline and obtains fermented supernatant fluid, removes and desalts and foreign protein, then through ammonium sulfate precipitation, dialysis, finally dialyzate is carried out to lyophilize and obtain beta-glucosidase enzyme powder through ultrafiltration.

<210> SEQ ID NO：1

<211> 596

<212> DNA

<213> silk branch mould ( aphanocladiumsp.) SK34.001

<400> 1

tccgtaggtg aacctgcgga gggatcatta cagagtttac aactcccaaa ccctcatgtg 60

aacataccac gatgttgctt cggcggactc gccccggcgt ccggacggcc tagcgccgcc 120

cgcggcccgg atccaggcgg ccgccggaga ccaccaaaac tattttgtat cagcagtttt 180

ttctgaatcc gccgcaaggc aaaacaaatg aatcaaaact ttcaacaacg gatctcttgg 240

ttctggcatc gatgaagaac gcagcgaaat gcgataagta atgtgaattg cagaattcag 300

tgaatcatcg aatctttgaa cgcacattgc gcccgccagc attctggcgg gcatgcctgt 360

tcgagcgtca tttcaaccct cgacttccct ttggggaaat cggcgttggg gactggcagc 420

ataccgccgg ccccgaaatg gagtggcggc ccgtccgcgg cgacctctgc gtagtaatcc 480

aacctcgcac cggaaccccg acgtggccac gccgtaaaac accccacttt ctgaacgttg 540

acctcggatc aggtaggaat acccgctgaa cttaagcata tcaataagcg gaggaa 596

Claims (3)

1. a strain silk branch trichoderma strain, its Classification And Nomenclature be silk branch mould ( aphanocladiumsp.) SK34.001, its preserving number is CCTCC NO:M 2014090, depositary institution is Chinese Typical Representative culture collection center.

2. silk branch trichoderma strain according to claim 1, is characterized in that: the rDNA ITS sequence of described bacterial strain is as shown in SEQ ID NO:1.

3. the method that described in claim 1, silk branch trichoderma strain CCTCC NO:M 2014090 produces beta-glucosidase, is characterized in that step is as follows:

(1) slant culture: get a silk branch trichoderma strain CCTCC NO:M 2014090 and carry out slant culture, adopt PDA substratum, each component is in g/L: potato 200, glucose 20, agar 15 ~ 20; Nature pH, sterilizing 30min at 115 DEG C; Culture temperature is 25 ~ 35 DEG C, incubation time 3 ~ 8 days;

(2) seed culture: adopt beef-protein medium, each component is in g/L: extractum carnis 2 ~ 5, Tryptones 5 ~ 8, sodium-chlor 2 ~ 5, natural pH, 121 DEG C of sterilizing 20min; The bottled liquid 30 ~ 50mL of 250mL triangle, carries out seed culture by the slant culture liquid of step (1) gained, shaking flask rotating speed 150 ~ 200rpm, 25 ~ 35 DEG C of culture temperature, incubation time 18 ~ 24h;

(3) fermentation culture: fermention medium component is in g/L: wheat bran 20 ~ 40, ammonium sulfate 4 ~ 6, potassium primary phosphate 2 ~ 3, magnesium sulfate 0.3 ~ 0.5, calcium chloride 0.3 ~ 0.5, natural pH, 121 DEG C of sterilizing 20min; The seed culture fluid of step (2) gained by volume percentage ratio 2% ~ 5% inoculum size is inoculated in fermention medium, shaking flask rotating speed 150 ~ 200rpm, 25 ~ 35 DEG C of culture temperature, incubation time 3 ~ 8 days;

(4) beta-glucosidase enzyme powder preparation: the fermented liquid that step (3) is made obtains fermented supernatant fluid at the centrifugal 10 ~ 30min of 5000 ~ 10000rpm or filtration removal thalline, remove and desalt and foreign protein through ultrafiltration, through ammonium sulfate precipitation, dialysis, finally dialyzate is carried out to lyophilize and obtain beta-glucosidase enzyme powder again.