CN105602921A

CN105602921A - Chitosanase mutant

Info

Publication number: CN105602921A
Application number: CN201610212888.4A
Authority: CN
Inventors: 盛军; 沙珍霞; 郑媛; 纪晓峰; 王致鹏
Original assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Current assignee: Yellow Sea Fisheries Research Institute Chinese Academy of Fishery Sciences
Priority date: 2016-04-07
Filing date: 2016-04-07
Publication date: 2016-05-25
Anticipated expiration: 2036-04-07
Also published as: CN105602921B

Abstract

The invention provides a chitosanase EAG1 mutant with an amino acid sequence shown in SEQ ID NO:1. The chitosanase EAG1 mutant provided by the invention, compared with chitosanase EAG1, has more excellent thermal stability and higher catalytic efficiency and has capability of rapidly decomposing chitosanase. Through efficient heterologous expression on engineered yeast containing a mutant gene, large-scale preparation of the chitosanase EAG1 mutant can be realized.

Description

A kind of chitosan enzyme mutant

Technical field

The invention belongs to gene genetic renovation technique field, be specifically related to a kind of chitosan enzyme mutant.

Background technology

The enterprising one-step hydrolysis in basis of the chitosan oligosaccharide shitosan that to be chitin form after de-acetyl and obtain onePlanting natural biological macromolecule, is a large amount of unique a kind of alkaline polysaccharides that exist in living nature. It is only for chitosan oligosaccharideSpecial functional character make its wastewater treatment, food industry, weaving, chemical industry, household chemicals, agricultural,Bioengineering and medicine and other fields tool have been widely used. Ten thousand tons of China's annual output chitin and derivative 3-4, produceBe worth tens yuan. But China's chitin biotechnology industry is sold as main taking roughing and raw material midbody,In industry, be mainly as shitosan raw material low-price export or as low additional in the chitin resource of weak tendencyValue intermediate is developed outlet. The key factor of restriction chitin resources development and utilization is main concentrating at presentAspect following two: the one, the chemical method technology of traditional chitin raw material is extensive, to environmentSecondary or three times seriously polluted, cause chitin production technology can not form suitability for industrialized production always. The 2nd,Modern biological enzyme is prepared chitosan oligosaccharide has become international main trend, but current commercial specificity waterThe enzyme of separating shitosan is still monopolized by the Genencor Company of the Novi of Denmark letter and Japan, and expensive price becomesThe unstable exploitation that has limited chitin and shitosan of basis and enzyme self property.

Enzyme that can selectivity hydrolyzing chitosan is mainly present in bacterium and fungal cell, mainly comprises that shell is poly-Carbohydrase and lysozyme. Chitosan enzyme EAG1 (GenBank accession number AB008788) is from bacillusA kind of polysaccharide hydrolase of Bacillusehimensis. It can not only cut off the glycosidic bond in chitosan molecule,Be hydrolyzed into the chitosan oligosaccharide of different molecular weight, and in organic solvent and metal ion, still retained goodGood catalytic activity is a kind of industrial enzymes that has application prospect. But the heat endurance of EAG1 is poor,50 DEG C time, its activity sharply declines. Therefore, be necessary to improve the stability of chitosan enzyme EAG1, improve itsCatalytic efficiency under hot conditions.

Summary of the invention

The object of the invention is to overcome above-mentioned the deficiencies in the prior art, to chitosan enzyme EAG1(GenBank accession number AB008788) transforms, and obtained a kind of new chitosan enzyme mutant.

Chitosan enzyme mutant provided by the invention, its amino acid sequence is SEQIDNO:1;

The encode gene of above-mentioned chitosan enzyme mutant, its a kind of nucleotide sequence is SEQIDNO:2;

The present invention also protects the recombinant expression carrier that carries above-mentioned encoding gene in another aspect;

Another aspect protection conversion/transfection of the present invention has the Host Strains of above-mentioned recombinant expression carrier

As preferably, described Host Strains is Pichia pastoris.

This mutant of the present invention, compared with chitosan enzyme EAG1, has better heat endurance with higherCatalytic efficiency, thus application prospect widely there is.

Brief description of the drawings

Fig. 1: the amino acid sequence comparison result figure of chitosan enzyme EAG1 and EAG1 mutant;

Fig. 2: the SDS-PAGE electrophoretogram of chitosan enzyme EAG1 mutant;

Fig. 3: the heat endurance impact figure of chitosan enzyme EAG1 mutant;

Fig. 4: the hot deactivation lab diagram of chitosan enzyme EAG1 mutant.

Detailed description of the invention

Below in conjunction with embodiment, the present invention is described in detail

Embodiment 1: the acquisition of chitosan enzyme EAG1 mutant gene

Chitosan enzyme EAG1 (GenBank accession number AB008788) is suddenlyd change, and concrete steps are as follows:

1. manually 7 of synthetic one section of codings are amino acid whose and be rich in the nucleic acid fragment of glycine:

5 '-ggaggaggatccggaggagga-3 '; The amino acid of this fragment coding is-GGGSGGG-

2. adopt the method for pcr amplification that above-mentioned sequence is introduced to EAG1 gene; Wherein forward primer is

(italicized item is the artificial sheet that inserts to 5 '-ggaggaggatccggaggaggaatgcatatgtccaatgcgaaaccat-3 'Section), reverse primer is 5 '-cttcatttcccagttcgtgacttgag-3 ', template is EAG1 gene.

To at 16 DEG C of EAG1 genes that contains Insert Fragment after above-mentioned amplification, connect with T4DNA ligase12 hours. Reaction system is as follows:

3. the above-mentioned EAG1 mutator that connects into ring is carried out to enzyme and cut ring. Enzyme used is DnaseI, for protectingDemonstrate,prove whole gene ring and only produce an otch, the amount of DnaseI used is that every 30 μ g add 1UDnaseI.

Reaction system is as follows:

EAG1 mutator after open loop is separated by 1% (w/v) agarose gel electrophoresis, reclaimDNA fragmentation between 900-1000.

4. design secondary amplimer, wherein forward primer is 5 '-atccggtacagcgtcgaacaagcgc-3 ', oppositely drawsThing is 5 '-atacacgttatagaaggtttcccaca-3 '. The DNA sheet reclaiming with above-mentioned agarose gel electrophoresisSection is template, adopts the method amplification of PCR to obtain EAG1 mutator.

Reaction system (50 μ are l) as follows:

The gene order of the final chitosan enzyme EAG1 mutant obtaining, this sequence contains 927 base (SEQIDNO:2), 309 amino acid (SEQIDNO:1) of encoding, it compared with EAG1, not only albumenI and II structure distributing order difference, and increased one section of flexible sequence (Fig. 1) that is rich in glycine.

Expression and the protein purification of embodiment 2 chitosan enzyme EAG1 mutant genes

The chitosan enzyme EAG1 mutant gene that embodiment 1 is obtained is connected and passes through with expression vector pIC9KThe method that electricity transforms proceeds in Pichia pastoris GS115. Electroporation transforms electric shock condition: voltage: 1500V;Resistance: 400 Ω; Electric capacity: 25 μ F; Burst length: 10mS; 1-2 electric shock.

The transformant that contains mutator is linked in BMGY fluid nutrient medium to 250-300rpm, 28 DEG CWhile cultivating between OD600 to 12-16; 3000rpm, centrifugal 5 minutes, abandon supernatant, collecting cell shiftsIn 20mLBMMH fluid nutrient medium, 20mLBMMY need use the configuration of 100mL shaking flask, ensuresCertain throughput, 28 DEG C are continued to cultivate; In culture medium, added 100% methyl alcohol to dense eventually every 24 hoursDegree is 0.5%. Cultivate 96 hours, fermented supernatant fluid is centrifugal, concentrated with 10KDa milipore filter, through ionExchange column and molecular sieve carry out purifying, have obtained electrophoretically pure EAG1 mutant sample.

Embodiment 3: the zymologic property of chitosan enzyme EAG1 mutant

1. the molecular weight of chitosan enzyme EAG1 mutant

EAG1 mutant sample after purifying is carried out to discontinuous dodecyl sodium sulfate-polyacrylamide(SDS-PAGE) gel electrophoresis, result as shown in Figure 2. Detect by denaturing polyacrylamide gel electrophoresisBe an electrophoresis band, compare according to the standard protein SDS-PAGE electrophoresis pattern of known molecular amount,The molecular weight of EAG1 mutant is about 34000 dalton (34kDa), and this enzyme is monomeric protein.

2. the heat endurance of EAG1 mutant and 50 DEG C of half-life after purifying

EAG1 mutant after purifying is placed in respectively within the scope of 40-60 DEG C and is hatched 30 minutes, shift subsequentlyTo cooled on ice 5 minutes, under 37 DEG C of conditions, measure remaining chitosan enzyme vigor, the results are shown in Figure 3. EAG1The heat endurance of mutant is significantly improved compared with EAG1. Enzymatic activity at 50 DEG C remains on 81%Above.

EAG1 mutant after purifying is placed in to 50 DEG C and hatches 0-80 minute, be transferred to subsequently cooled on ice 5Minute, under 37 degree conditions, measure remaining chitosan enzyme vigor, the results are shown in Figure 4. EAG1 sudden change at 50 DEG CThe half-life of body obviously improves, and EAG1 is incubated 10 minutes at 50 DEG C, and enzyme activity has declined 50%, insulationAfter 50 minutes, enzyme activity has declined 95%. 50 DEG C of half-life of EAG1 mutant are brought up to 70 minutes, thanEAG1 has improved 7 times.

3. the vitality test of chitosan enzyme:

The vitality test of chitosan enzyme adopts dinitrosalicylic acid (DNS) method. Enzyme unit definition: 1U table aliveShow the needed enzyme amount of release 1 μ mol reduced sugar per minute under these conditions.

For measuring Km and the Vmax of chitosan enzyme EAG1 mutant to shitosan, by 0.5mL variable concentrationsChitosan solution (5,7.5,10,15,20mg/mL) prominent with 0.1mL chitosan enzyme EAG1 respectivelyVariant (final enzyme concentration 5.0U/mL) mixes. 37 DEG C of reaction 10min, add 4.0mL10%TCA moltenLiquid cessation reaction. According to double-reciprocal plot method (Lineweaver-Burk) taking 1/[S] as abscissa 1/v is as verticalCoordinate mapping, straight line slope be Km/Vmax, intercept is 1/Vmax, calculates kinetic constant KmWith maximum reaction velocity Vmax (table 1). Compared with EAG1, the maximum reaction velocity of EAG1 mutant(Vmax) improved respectively 142% and 203% with catalytic efficiency (Kcat/Km).

The kinetic parameter table of table 1:EAG1 and EAG1 mutant

Result shows that chitosan enzyme EAG1 mutant specific is strong, can effectively interrupt shell with internal-cutting way poly-β-Isosorbide-5-Nitrae glycosidic bond in sugar. Every gram of shitosan adds the EAG1 mutant of 1.0U unit, hydrolysis at 45 DEG C4 hours, shitosan can be resolved into completely to the chitosan oligosaccharide of degree of polymerization 2-8 sugar, percent hydrolysis reach 95% withOn.

Claims

1. a chitosan enzyme mutant, is characterized in that, the amino acid sequence of described mutant is SEQIDNO:1。

2. a gene, is characterized in that, described gene code chitosan enzyme claimed in claim 1 is prominentVariant.

3. gene as claimed in claim 2, is characterized in that, the nucleotides sequence of described gene is classified SEQ asIDNO:2。

4. a recombinant expression carrier, is characterized in that, described recombinant expression carrier carries claimGene described in 2 or 3.

5. a recombinant bacterium, is characterized in that, described recombinant bacterium is had the right for conversion/transfection described in requirement 4The Host Strains of recombinant expression carrier.

6. recombinant bacterium as claimed in claim 5, is characterized in that, described Host Strains is Pichia pastoris.