CN104611314A - Heat-resistant beta-mannase and encoding gene thereof - Google Patents

Abstract

The invention discloses heat-resistant beta-mannase and an encoding gene thereof. The heat-resistant beta-mannase is selected from (a) or (b) as follows: (a) protein consisting of an amino acid sequence represented by SEQ ID No:2 in a sequence table; (b) protein which is formed by substitution and/or deletion and/or addition of the amino acid sequence represented by SEQ ID No:2 in the sequence table by the aid of one or more amino acid residues, has heat-resistant beta-mannase activity and is derived from (a). The beta-mannase has higher BGL (bean gum from locust) decomposition activity, the optimal reaction temperature is 55 DEG C, the optimal reaction pH is 5.5, the beta-mannase is treated at the temperature of 50 DEG C for 120 min, and about 90% of the enzyme activity is reserved. Research indicates that the beta-mannase has high catalytic activity and better heat resistance, is applicable to multiple slightly acidic industrial environments with higher temperature requirements and has great potential of popularization and application in production.

Description

A kind of heat-resisting 'beta '-mannase and encoding gene thereof

Technical field

The present invention relates to genetically engineered field, be specifically related to a kind of 'beta '-mannase man26 with comparatively high temps stability, and the encoding gene of this 'beta '-mannase and its high yield expressing strategy in intestinal bacteria.

Background technology

Hemicellulose is made up of multiple macromolecular compound, mainly comprises mannosans and xylan.Find the focus that the hemicellulase with very strong vigor is research hemicellulose degradation always.β ?mannase is that a class can the restriction endonuclease of β-1.4-seminose glycosidic bond in hydrolyzing mannan, belongs to hemicellulase.The microorganism that can produce mannase has a lot, comprises filamentous fungus, bacterium, actinomycetes etc.

'beta '-mannase has been widely used in the industrial circles such as food, papermaking, feed, medicine, oil production.Such as, 'beta '-mannase can be used for the extraction of vegetables oil, the purification of fruit juice, the production of soluble coffee; Make an addition in animal-feed and can lower animal intestinal viscosity, improve food conversion ratio; Also can be used for (Dhawan, S., et al., Microbial mannanases:an overview of production and applications.Crit Rev Biotechnol (2007) 27:197-216) such as laundry detergents.In recent years, along with the elimination of mannosans antinutritional factor in the exploitation to occurring in nature hemicellulose resource, feed and the discovery of mannosans physiological function, the demand of 'beta '-mannase is increasing, causes the research and development of 'beta '-mannase to enter a new climax.

In industrial application process, enzyme heat stability is more high more favourable, and generally, proteolytic enzyme is when running into high temperature, and often unstable, easy inactivation, which greatly limits the application in the industrial production of many zymins.So along with the widespread use of genetic engineering technique and DNA techniques, the 'beta '-mannase adopting genetic engineering means clonal expression from thermophile bacteria to have high reactivity and thermotolerance causes the interest of people.At present, the heat-resisting 'beta '-mannase great majority of bibliographical information derive from thermophilic fungus (Lu, H., et al., A novel thermophilic endo-beta-1,4-mannanase from Aspergillus nidulans XZ3:functional roles of carbohydrate-binding module and Thr/Ser-rich linker region.Appl Microbiol Biotechnol (2014) 98:2155 – 2163).Therefore, the heat resistance 'beta '-mannase deriving from thermophilic anaerobic bacillus has huge application prospect in the industrial production.

Summary of the invention

The object of this invention is to provide a kind of heat-resisting 'beta '-mannase and encoding gene thereof.

A kind of heat-resisting 'beta '-mannase, has good temperature capacity, derives from thermophilic anaerobic bacillus (Thermoanaerobacterium aotearoense SCUT27), is selected from following (a) or (b):

A protein that () is made up of the aminoacid sequence shown in SEQ ID No:2 in sequence table;

B the aminoacid sequence of SEQ ID No:2 in sequence table is passed through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and has the protein derivative by (a) of heat-resisting beta-mannase enzymic activity by ().

Wherein, the SEQ ID No:2 in sequence table is made up of 515 amino acid, and molecular weight is about 58.8kD.

In order to make the 'beta '-mannase in (a) be convenient at intestinal bacteria heterogenous expression, the protein N terminal of the aminoacid sequence composition in sequence table shown in SEQ ID No:2 eliminates 24 amino acid whose signal peptides.In order to make the 'beta '-mannase in (a) be convenient to purifying, the protein C end of the aminoacid sequence composition in sequence table shown in SEQ ID No:2 connects 6 × His label.And then obtain the protein that is made up of the aminoacid sequence shown in SEQ ID No:4 in sequence table.

'beta '-mannase in above-mentioned (a) and (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.B the encoding gene of the 'beta '-mannase in () is by the codon by lacking one or several amino-acid residue in the DNA sequence dna in sequence SEQ ID No:1, and/or the missense mutation carrying out one or several base pair obtains.

The encoding gene of above-mentioned 'beta '-mannase, described encoding gene is selected from following 1) or 2) or 3):

1) its nucleotides sequence is classified as SEQ ID No:1 or the DNA molecular shown in SEQ ID No:3 in sequence table;

2) under strict conditions with 1) DNA sequence dna that limits hybridizes and the DNA molecular of described 'beta '-mannase of encoding;

3) with 1) DNA sequence dna that limits has more than 90% homology and the DNA molecular of described 'beta '-mannase of encoding.

Above-mentioned stringent condition is, in the solution of 6 × SSC, 0.5%SDS, hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Recombinant vectors containing above-mentioned arbitrary described encoding gene, recombinant bacterium, transgenic cell line or expression cassette also belong to protection scope of the present invention.

Compared with prior art, the present invention has following beneficial effect:

Experiment proves, the 'beta '-mannase expression vector in the present invention is conducive to high expression and the purifying of 'beta '-mannase, and the 'beta '-mannase of expression accounts for bacterial protein and is about 15-20%, and purification efficiency, up to 51%, is lived as 193.3U/mg than enzyme.This expression vector has the features such as high expression level amount, high-recovery, high catalysis activity, is conducive to the suitability for industrialized production of beta-mannase zymin.

β of the present invention ?mannase optimal reactive temperature be 55 DEG C, optimal reaction pH is 5.5, under 50 DEG C of conditions, process 120min, enzyme live reservation more than 90%.'beta '-mannase of the present invention has good thermotolerance, has the great potential applied in the industrial production.

Accompanying drawing explanation

Fig. 1 is the electrophoretogram of pcr amplification product of the present invention; Wherein, 1: molecular weight standard (DL2000); 2:PCR amplified production.

Fig. 2 is the qualification electrophoretogram of expression vector pET-30a-man26 plasmid of the present invention; 1:pET-30a-man26 plasmid; The pET-30a-man26 of 2:Nde I and Xho I double digestion; The pET-30a of 3:Nde I and Xho I double digestion; 4:pET-30a-man26 plasmid PCR amplified production; 5: molecular weight standard (DL15000);

Fig. 3 is the SDS-PAGE figure that 'beta '-mannase purge process of the present invention collects sample.1: the broken supernatant (crude enzyme liquid) of resuspended bacterium liquid; 2: ni-sepharose purification is through liquid; Foreign protein sample contained by first protein peak of 3:160mM imidazole buffer wash-out; 'beta '-mannase protein sample contained by second protein peak of 4:300mM imidazole buffer wash-out; Protein sample after the 'beta '-mannase protein sample desalination of 5:300mM imidazole buffer wash-out; 6: protein molecular weight standard;

Fig. 4 is 'beta '-mannase zymologic property curve of the present invention, and a is optimal pH curve; B is pH beta stability line; C is optimal reactive temperature curve; D is temperature-stable linearity curve.

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method, as the method described in " Molecular Cloning: A Laboratory guide " (third edition).

Embodiment 1

The structure of 'beta '-mannase expression strain

1. the amplification of beta-mannase gene

This beta-mannase gene man26 aminoacid sequence (SEQ ID No:2) is by sequence alignment and signal peptide analysis, the N end of this enzyme known has one containing 24 amino acid whose signal peptides, relevant to the secreting, expressing of albumen, its zymologic property is not affected, is therefore removed to improve the expression efficiency of this enzyme in intestinal bacteria.

Nde I and Xho I restriction enzyme site is introduced respectively at the P1 (SEQ ID No:5) of primer, the 5' of P2 (SEQ ID No:6), and hold introducing 6 × His tag label at beta-mannase gene 3', the 'beta '-mannase given expression to like this is with histidine-tagged, utilize affinity chromatography method, the 'beta '-mannase given expression to can be carried out affinitive layer purification, be beneficial to obtain 'beta '-mannase sterling fast and efficiently.Finally determine that the present invention's beta-mannase enzyme amino acid sequence used is SEQ ID No:4, corresponding coding gene sequence is SEQ ID No:3.

P1：5'-TAGCCCCATATGTCTGGAAAGTATATTGAAAACAAA

AAAAG-3'(SEQ ID No:5)

P2：5'-TCCGTCTCGAGTTTATTGTCGACATATATTTTTAAATT

TG-3'(SEQ ID No:6)

With Thermoanaerobacterium aotearoense SCUT27 genomic dna for template, carry out this beta-mannase gene of pcr amplification man26 (SEQ ID No:3) with primer P1 (SEQ ID No:5), primer P2 (SEQ ID No:6).PCR program is as follows: first stage sex change 98 DEG C, 2min; Subordinate phase sex change 98 DEG C, 10sec, anneals 61 DEG C, and 10sec extends 72 DEG C, 1min 30sec, carries out 30 circulations altogether; Phase III extends 72 DEG C, 10min.The PCR primer obtained 1% agarose electrophoresis detects, and the results are shown in Figure 1.The nucleotide sequence of the 'beta '-mannase obtained is as shown in SEQID No:3, and its aminoacid sequence is as shown in SEQ ID No:4.

2. the structure of recombinant plasmid

The pcr amplification product of purifying and pET-30a use Nde I and Xho I double digestion respectively, product after purifying double digestion, man26 and pET-30a plasmid after being cut by enzyme with T4 ligase enzyme connects, set up following linked system: 40ng pET-30a carrier, 80ng pcr amplification product, 1 μ l 10 × ligationbuffer, moisturizing to 10 μ l, 16 DEG C connect 16h.Connect the heat-shock transformed escherichia coli DH5a competent cell of product, whether the transformant sequence verification obtained suddenlys change; The clone selecting sequence correct extracts plasmid, obtains the recombinant plasmid pET-30a-man26 containing beta-mannase gene.With 1% agarose electrophoresis qualification recombinant plasmid, the results are shown in Figure 2.

Embodiment 2

The expression of 'beta '-mannase and protein purification

Being inoculated in incubated overnight containing in the fresh LB liquid nutrient medium of 50 μ g/ml kantlex in 1:50 ratio containing recombinant bacterium E.coli BL21 (the DE3)/pET-30a-man26 of beta-mannase gene, is positioned over 37 DEG C, cultivates under 250rpm condition.As cell density OD ₆₀₀about during 0.6-0.8, adding final concentration is that the IPTG of 1mM induces the expression of 'beta '-mannase, is 25 DEG C, continues cultivation 9 hours under 200rpm condition.SDS-PAGE glue figure shows (Fig. 3), and recombinant beta-mannanase protein amount accounts for the about 15-20% of total protein concentration, reaches 19.23mg/L fermented liquid.

At 4 DEG C, collected by centrifugation thalline under 8000g condition, every 100mg wets, and to add 1ml buffer A (20mM phosphate buffered saline buffer, 500mM sodium-chlor, 20mM imidazoles, pH 7.4) resuspended for bacterium, ultrasonic 60min smudge cells under 450W ice-water bath.Collected by centrifugation supernatant afterwards, obtains 'beta '-mannase crude enzyme liquid.Further by conventional affinity chromatography method purifying 'beta '-mannase, and with conventional desalination post, desalination is carried out to 'beta '-mannase protein sample.The protein sample SDS-PAGE result that purge process is collected as shown in Figure 3.In final embodiment, the 'beta '-mannase purity of purifying gained is more than 95%, and the rate of recovery is about 51%.

Embodiment 3

The determination of activity of restructuring 'beta '-mannase

Enzyme activity determination with locust bean gum (Sigma, USA) for substrate measures gained beta-mannase enzymic activity in embodiment 2.

'beta '-mannase enzyme activity determination method is: in 1.5ml EP pipe A, add the locust bean gum solution 0.9ml of the 1g/L of the damping fluid configuration with pH 5.5 citric acid (100mM)-Sodium phosphate dibasic (50mM), 50 DEG C of preheating 10min, the enzyme night that 0.1ml suitably dilutes is added, 55 DEG C of reaction 10min in EP pipe A; Take out 100 μ L reaction solutions immediately in EP pipe B, add 100 μ L 3' fast, 5'-dinitrosalicylic acid (DNS) reagent, B pipe boils 5min, adds deionized water 300 μ L, shake up after cooling.Absorbance is measured, by inquiry reducing sugar and A at 540nm ₅₄₀between typical curve, obtain the reducing sugar amount that institute catalysis generates.

Beta-mannase enzyme activity unit is defined as (U) 1min catalysis and discharges enzyme amount required for 1 μm of ol reducing sugar.

In embodiment 2, the 'beta '-mannase of gained is more alive than enzyme is 193.3U/mg, corresponding Michaelis-Menton constant K _mfor 1.9mg/ml.

Embodiment 4

'beta '-mannase catalysis characteristics is studied

1. optimal reaction pH and pH stability study

Citric acid (100mM)-Sodium phosphate dibasic (50mM) damping fluid of preparation pH 4.0 ~ 8.0.The restructuring 'beta '-mannase enzyme measuring embodiment 2 gained respectively in different pH damping fluid is lived, and result as shown in fig. 4 a.This enzyme optimal reaction pH is 5.5, within the scope of pH 5.0-6.5, all have good catalysis activity, when pH>7.5, and enzymatic activity rapid loss.

In pH stability study, the 'beta '-mannase of embodiment 2 gained is placed 24h under 4 DEG C of conditions in the buffered soln of different pH, measure remnant enzyme activity 55 DEG C of conventional enzyme activity determination methods afterwards.Result of study shows (Fig. 4 b), and the 'beta '-mannase of embodiment 2 gained is at pH 4.5-7.5 scope endoenzyme retention rate >80% alive.

2. the suitableeest catalytic temperature and temperature-stable Journal of Sex Research

The 'beta '-mannase enzyme measuring embodiment 2 gained is at different temperatures lived.As illustrated in fig. 4 c, the optimal reactive temperature of this enzyme is 55 DEG C to result, the enzyme work 50 and 60 DEG C time be optimum condition under more than 90%.When enzymatic reaction temperature is increased to 70 DEG C, under the work of its enzyme is about optimal conditions, record 30% of enzyme work.

Restructuring 'beta '-mannase is incubated respectively under 50 DEG C and 55 DEG C of conditions different time and measures remnant enzyme activity, result shows this enzyme and be incubated 30min under 55 DEG C of condition, and enzyme retention rate alive reaches 62%.And under 50 DEG C of conditions, be incubated 120min, remnant enzyme activity about 90%.

Claims (8)

1. a heat-resisting 'beta '-mannase, is characterized in that, is selected from following (a) or (b):

A protein that () is made up of the aminoacid sequence shown in SEQ ID No:2 in sequence table;

B the aminoacid sequence of SEQ ID No:2 in sequence table is passed through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and has the protein derivative by (a) of heat-resisting beta-mannase enzymic activity by ().

2. 'beta '-mannase according to claim 1, is characterized in that, the protein be made up of the aminoacid sequence shown in SEQ ID No:4.

3. the encoding gene of the 'beta '-mannase described in claim 1 or 2.

4. encoding gene according to claim 3, is characterized in that, described encoding gene is selected from following 1) or 2) or 3):

1) its nucleotides sequence is classified as SEQ ID No:1 or the DNA molecular shown in SEQ ID No:3 in sequence table;

2) under strict conditions with 1) DNA sequence dna that limits hybridizes and the DNA molecular of described 'beta '-mannase of encoding;

3) with 1) DNA sequence dna that limits has more than 90% homology and the DNA molecular of described 'beta '-mannase of encoding.

5. the recombinant vectors containing encoding gene described in claim 3 or 4.

6. the recombinant bacterium containing encoding gene described in claim 3 or 4.

7. the transgenic cell line containing encoding gene described in claim 3 or 4.

8. the expression cassette containing encoding gene described in claim 3 or 4.