CN108102936A

CN108102936A - Kluyveromyces lactis mutant strain and its glycosidase and application

Info

Publication number: CN108102936A
Application number: CN201810160226.6A
Authority: CN
Inventors: 董颖军
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-02-26
Filing date: 2018-02-26
Publication date: 2018-06-01
Anticipated expiration: 2038-02-26
Also published as: CN108102936B

Abstract

The present invention relates to Kluyveromyces lactis mutant strain and its glycosidase and applications, it is starting strain with Kluyveromyces lactis ATCC 8585, one plant of Kluyveromyces lactis mutant strain is obtained by complex mutation, the produced glucosides enzyme heat stability of the mutant strain is good, take into account high temperature active, national regulations and safe source can be met simultaneously, thus be suitable in industrial production.Recombinant vector the present invention also provides polynucleotides, containing polynucleotides, the host cell containing recombinant vector and the composition containing glycosidase, in addition, additionally providing production glycosidase and the method for hydrolyzing the substance containing glycosidic bond.

Description

Kluyveromyces lactis mutant strain and its glycosidase and application

Technical field

The present invention relates to Kluyveromyces lactis, more particularly to a kind of Kluyveromyces lactis mutation for producing thermophilic glycosidase Strain.

Background technology

Glycosidase is also known as glycoside hydrolase (Glycoside hydrolase, EC3.2.1), is the various glucosides of effect or widow Sugar makes the general name of the enzyme of hydrolysis of glycoside bond.They can will account for carbohydrate (cellulose, the hemicellulose of the total biomass 70% in the world Element, starch, pectin, alginic acid, chitin etc.) monose is converted into, also non-polysaccharide glycoside compounds can be converted into aglycon.Therefore In living things catalysis and conversion field, glycosidase is in medicine, chemical industry, material, the energy, weaving, papermaking, food, feed, environment etc. All conglomeraties have extremely wide application.

The essence of enzyme is protein, and suitable reaction temperature is usually at 40 DEG C or so, however in practical application in industry Hot environment is inevitably present, this normally results in enzyme fast deactivation, causes reaction speed slack-off or even stops reaction, And then production cost is caused to rise.Zimadzhunt L 340 or thermostable enzyme refer to that one kind can keep enzyme activity for a long time in hot environment Biocatalyst, they especially have many advantages in living things catalysis industrialized production in food processing field：First, heat Stability improves, and can separate, purify and packed and transported under room temperature or tropical conditions, while can protect for a long time in high temperature environments Hold activity；Second, enzyme kinetic analysis reaction is accelerated, improves reaction efficiency；3rd, the cooling system of reaction is required to reduce, Energy consumption thus is reduced, that is, cost is reduced, so as to reduce pollution of the cooling procedure to environment；4th, due to catalytic reaction temperature Degree is high (being more than 60 DEG C), reduces the pollution of miscellaneous bacteria, so as to reduce pollution of the metabolite to product of bacterium, can improve production The purity of object simplifies its purification process；5th, enzymatic process carries out at high temperature, can increase indissoluble substance such as starch, fiber Element, the dissolubility and utilizability of lipid reduce the viscosity of organic compound and are beneficial to the diffusion and mixing of substance.Therefore, it is thermophilic Hot glycosidase has a wide range of applications in the industrial production.

Further, since particularity of the food in secure context, uses according to national food safety standard and food additives Standard (GB2760-2014) provides that (EC3.2.1.23 belongs to one kind of glycosidase to the lactase used in food industry, can incite somebody to action Lactose hydrolysis are glucose and galactolipin) it can only derive from：Kluyveromyces fragilis Kluyveromyces fragilis, black song Mould Aspergillus niger, aspergillus oryzae Aspergillus oryzae, Kluyveromyces lactis Kluyveromyces Lactis and pichia pastoris yeast Pichia pastoris (donor of enzyme must be aspergillus oryzae), thus it is next there is screening Come from the demand of the glycosidase of mentioned microorganism.

In view of major part glycosidase security and vigor is bad at present, from catalytic efficiency, substrate spectrum, high temperature active and steady Qualitative, yield etc. is far from meeting the needs of modern industry application, it is necessary to further find a kind of new, safe source And the glycosidase that thermal stability is high with activity, to meet the needs of industrial production, especially food industry production.

The content of the invention

To achieve these goals, the present invention is with currently used production glycosidase Kluyveromyces lactis ATCC 8585 Starting strain carries out complex mutation to it, and one plant of high mutation of enzyme activity at 95 DEG C is screened from obtained numerous clones Strain, is named as Kluyveromyces lactis XT1412, and is preserved on January 10th, 2018 in China typical culture collection The heart, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University, deposit number are CCTCC M 2018017, and Classification And Nomenclature is lactic acid gram Yeast Kluyveromyces lactis are tieed up in Shandong, so as to complete the present invention.

In the first aspect, the present invention provides one plant of Kluyveromyces lactis (Kluyveromyces lactis) XT1412, is preserved in China typical culture collection center, and deposit number is CCTCC M 2018017.

In the second aspect, the present invention provides a kind of glycosidase, amino acid sequence such as SEQ ID NO：Shown in 2.

In one preferred embodiment, the N-terminal of the glycosidase or C-terminal are added with sequence label.

At the 3rd aspect, the present invention provides a kind of polynucleotides, the polynucleotides

A) glycosidase of the coding present invention；

B) it is complementary with polynucleotides a).

In a specific embodiment, the sequence such as SEQ ID NO of polynucleotides of the invention：Shown in 1.

At the 4th aspect, the present invention provides a kind of recombinant vectors, contain polynucleotides of the invention.

At the 5th aspect, the present invention provides a kind of host cell, the restructuring that the host cell contains the present invention carries The polynucleotides of the present invention are integrated in body or its genome.

In a detailed embodiment, the host cell is Kluyveromyces lactis GG799-blg-XT1412, China typical culture collection center is preserved on January 10th, 2018, Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University is protected It is CCTCC M 2018018 to hide number, and Classification And Nomenclature is Kluyveromyces lactis Kluyveromyces lactis.

At the 6th aspect, the present invention provides a kind of method for producing glycosidase, including：

1) host cell of the invention；

2) glycosidase is separated from the culture obtained.

At the 7th aspect, the present invention provides a kind of composition, the composition includes the glycosidase of the present invention.

In one preferred embodiment, the composition further comprises acceptable carrier in food industry.

At the 8th aspect, the present invention provides a kind of methods for hydrolyzing the substance containing glycosidic bond, and the described method includes will The glycosidase of the present invention or the composition of the present invention and the material mixing containing glycosidic bond.

In a detailed embodiment, the reaction temperature of the method is about 10 DEG C~100 DEG C, preferably about 60 DEG C~ 100 DEG C, more preferably about 80 DEG C~100 DEG C are most preferably from about 95 DEG C.

In a detailed embodiment, the pH value in reaction of the method is about 4~8, and preferably about 4.5~6.5, it is more excellent Choosing about 5~6, is most preferably from about 5.5.

In a detailed embodiment, the substance containing glycosidic bond is selected from p- nitrobenzophenone-β-D- glucopyras Glucosides, o-nitrophenyl-β-D- glucopyranosides, p- nitrobenzophenone-β-D- galactopyranosides, o-nitrophenyl-β- D- galactopyranosides, o-nitrophenyl-β-D- mannopyranoses glycosides, o-nitrophenyl-β-D- xylopyranoses glucosides, p- nitre One or more in base phenyl-α-L- arabopyranoses glycosides, p- nitrobenzophenone-α-L- arabinofuranosidases and lactose.

Glycosidase from the Kluyveromyces lactis mutant strain XT1412 of the present invention at least has advantageous effect：

First, Kluyveromyces lactis mutant strain XT1412 of the invention is mutated from Kluyveromyces lactis ATCC 8585, So the glycosidase of its production meets national food safety standard and food additives using standard, there is very high security；

Secondly, glycosidase optimal reactive temperature of the invention for 95 DEG C and can keep 60% between 75 DEG C to 100 DEG C Above enzymatic activity, optimal pH is 5.5 and more than 60% enzyme activity can be kept between pH4~7.5, while has taken into account high temperature Under stability, not degradable；

Finally, the substrate of glycosidase of the invention composes wide and high catalytic efficiency.

Due to possessing above-mentioned characteristic, glycosidase of the invention is suitable for industrial production, especially food industry and produces, and by This bring be easy to preserve, reaction efficiency is promoted, living contaminants is reduced, reduce energy consumption, raw material Ke Li Yong Du Gao and cost-effective etc. Many advantages.

Description of the drawings

Fig. 1 is the protein purification electrophoretogram that Kluyveromyces lactis XT1412 produces glycosidase, wherein, swimming lane 1：Albumen Marker；Swimming lane 2：30%~85% saturation degree albumen of ammonium sulfate precipitation；Swimming lane 3：Enzyme solution after cation exchange chromatography；Swimming Road 4：Enzyme solution after gel-filtration purified.

Fig. 2 is the temperature profile of blg-XT1412 albumen.

Fig. 3 is the pH graphs of blg-XT1412 albumen.

Fig. 4 shows blg-XT1412 albumen thermal stability experimental results, wherein, 75 DEG C：Solid circles, 80 DEG C：It is hollow It is circular；85℃：Black triangle；90℃：Hollow triangle；95℃：Solid squares.

Fig. 5 shows pKLAC2-blg-XT1412 plasmid maps.

Specific embodiment

Below in conjunction with specific embodiment and embodiment, the present invention, advantages of the present invention and various effects are specifically described It thus will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating The present invention is not intended to limit the present invention.

Throughout the specification, unless otherwise specified, terms used herein is interpreted as usual in this field Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has and neck belonging to the present invention Field technique personnel's is commonly understood by identical meaning.If there are contradiction, this specification is preferential.

Kluyveromyces lactis

Kluyveromyces lactis is one kind in Kluyveromyces, and most bacterial strains are all isolated from primary carbon source as breast It is one of very important Non-Saccharomyces in biological technology field, meaning is mainly reflected in two in the milk product of sugar A aspect：The history of security application and the ability of industrial scale production enzyme in food service industry.

Starting strain Kluyveromyces lactis ATCC 8585 used in this application be at present common production glycosidase bacterial strain it One, the glycosidase optimum temperature of production is 55 DEG C, and when being substrate using lactose, enzymatic activity is 45U/mg, the report of other substrates It is relatively fewer.

As used in the present invention, term " high temperature " refers to 50 DEG C~100 DEG C, preferably refers to 60 DEG C~100 DEG C, more preferably 80 DEG C~100 DEG C, be most preferably 90 DEG C~100 DEG C.

Albumen and polynucleotides

Herein, " albumen of the invention ", " glycosidase of the invention ", " thermophilic glycosidase of the invention " and " blg- XT1412 albumen " may be used interchangeably, they refer to that amino acid sequence is SEQ ID NO：Albumen shown in 2；Similarly, " this The gene of invention ", " glycosidase genes of the invention ", " blg-XT1412 " and " blg-XT1412 genes " herein can be mutual Use is changed, they refer to the polynucleotide sequence or its complementary strand of the albumen of the coding present invention.

The N-terminal or C-terminal of the glycosidase of the present invention can also contain one or more polypeptide fragments, be used as label In purifying.Illustrative label is including Poly-Arg, Poly-His, FLAG, Strep-TagII etc., however, the present invention is not limited thereto.

The glucosides enzyme coding gene of the present invention can be DNA or rna form.Wherein, DNA form includes cDNA, genome DNA or artificial synthesized DNA, DNA can be single-stranded or double-strands, can be coding strand or noncoding strand, coding strand example Such as can be SEQ ID NO：1 or therebetween and sequence.

Recombinant vector

Those skilled in the art can use glucosides enzyme coding gene of the well known method structure containing the present invention and suitable The recombinant vector of transcription/translation control signal.The encoding gene is efficiently attached in the promoter of recombinant vector, guidance MRNA is synthesized, and recombinant vector further includes the ribosome bind site and transcription terminator of translation initiation.In addition, recombinant vector is also It can include one or more selected markers, the phenotypic character of the host cell of conversion is selected to provide.

Host cell

Host cell can be prokaryotic cell, eukaryocyte, insect cell, plant cell or zooblast.This field energy It is enough to select suitable recombinant vector, promoter and host cell as needed.

Decompose the method and purposes of the substance containing glycosidic bond

The glycosidase of the present invention may act on the substance containing glycosidic bond, such as may act on monoglycosides, oligosaccharides, polysaccharide, soap Glucoside and glycoprotein, but not limited to this.

In a detailed embodiment, the substance containing glycosidic bond is selected from p- nitrobenzophenone-β-D- glucopyras Glucosides, o-nitrophenyl-β-D- glucopyranosides, p- nitrobenzophenone-β-D- galactopyranosides, o-nitrophenyl-β- D- galactopyranosides, o-nitrophenyl-β-D- mannopyranoses glycosides, o-nitrophenyl-β-D- xylopyranoses glucosides, p- nitre One or more in base phenyl-α-L- arabopyranoses glycosides, p- nitrobenzophenone-α-L- arabinofuranosidases glucosides and lactose.

The method of the decomposition glycosidic bond of the present invention preferably carries out at high temperature.

The glycosidase of the present invention can be used in industrial production, be preferred in food industry production.

In one embodiment, glycosidase of the invention is under hot environment.

In another embodiment, glycosidase of the invention is in acid or alkaline environment, sour environment to be preferred The environment of pH3.0~7.0, the environment of more preferable pH5.0~7.0；The environment of preferred pH7.0~10.0 of alkaline environment, more preferably The environment of pH7.0~8.0.

In a specific embodiment, the method that the present invention decomposes glycosidic bond can be used for reducing miscellaneous bacteria in industrial production Generation.

In another embodiment, the present invention decomposes the method for glycosidic bond available for the dissolving for increasing substance hard to tolerate Property or utilizability.Wherein, substance hard to tolerate may be selected from as the one or more in starch, cellulose and lipid, but the present invention is not It is limited to this.

The present invention is described in more detail by the following examples, but the present invention is not limited to these Examples.

Culture medium involved in embodiment and preparation method thereof：

YPD fluid nutrient mediums：10g yeast extracts, 20g peptones are in 900ml deionized waters, 121 DEG C of sterilizings 20min.To be cooled to 60 DEG C or so, add in 40% glucose solutions of the 50ml Jing Guo filtration sterilization, mixing.

YPD solid mediums：10g yeast extracts, 20g peptones, 20g agar powders are in 900ml deionized waters, and 121 DEG C sterilizing 20min.To be cooled to 60 DEG C or so, add in 40% glucose solutions of the 50ml Jing Guo filtration sterilization, mixing.

5mM acetamide YCB fluid nutrient mediums：15ml 1M Tris-HCl buffer solutions, 5.85g YCB (yeast carbon source bases Plinth), add in 495ml deionized waters, 121 DEG C of sterilizing 20min.To be cooled to 60 DEG C or so, add in the sterile acetamides of 5ml 500mM Solution mixing.

5mM acetamide YCB solid mediums：15ml 1M Tris-HCl buffer solutions, 5.85g YCB, 10g agar powders, add Enter 495ml deionized waters, 121 DEG C of sterilizing 20min.It to be cooled to 60 DEG C or so, adds in the sterile acetamide solutions of 5ml 500mM and mixes It is even.

YPGal fluid nutrient mediums：10g yeast extracts, 20g peptones are added in 950ml deionized waters, 121 DEG C of sterilizings 20min.To be cooled to 60 DEG C or so, add in 40% galactose solutions of the 50ml Jing Guo filtration sterilization, mixing.

The complex mutation of 1 lactic acid yeast kluyveromyces strain ATCC 8585 of embodiment

1.1 prepare 8585 single bacterium colonies of Kluyveromyces lactis ATCC

The Kluyveromyces lactis ATCC 8585 of preservation is inoculated into YPD fluid nutrient mediums, 28 DEG C, 250rpm is continuous Culture 2~3 days.Zymotic fluid 10 is diluted with sterile water^-3, 10^-4, 10^-5It is respectively coated after times on YPD solid medium tablets, 28 DEG C static gas wave refrigerator 2~3 days, after single bacterium colony is grown, one single bacterium colony of picking is transferred in YPD fluid nutrient mediums, 28 DEG C, When the continuous cultures 48 of 250rpm are small.

1.2 UV treatment

After culture, bacterium solution is diluted with sterile saline so that barm cell concentration is about 10⁷A/ml.Take 25ml Bacterium solution is placed in sterile empty tablet, and carries out medium-speed magnetic stirring.At ultraviolet lamp (30W) 20cm respectively irradiate 1min, 2min, 3min, 4min and 5min.After the completion of irradiation 1 × 10 is diluted under red light⁶Then bacterium solution is being protected from light situation by a/ml Lower refrigerated overnight.

1.3 Ion Beam Treatment

Kluyveromyces lactis 0.1ml bacterium solutions after various dose of learning from else's experience respectively UV treatment, are uniformly coated on sterile It on tablet, is dried up with sterile wind, after microscopy ensures acellular overlapping, carries out ion implanting.Implantation Energy value is 10keV, is injected 50×10¹⁴cm^-2、100×10¹⁴cm^-2、150×10¹⁴cm^-2、200×10¹⁴cm^-2、250×10¹⁴cm^-2With 30 × 10¹⁴cm^-2, target Room vacuum degree is 10^-3Pa.Tablet carry disease germs on target platform, is injected with 10s pulseds, is spaced 50s.After the completion of ion implanting, with 4ml Sterile saline elutes, in 4 DEG C of preservations.

1.4 nitrosoguanidines (NTG) processing

Lactic acid yeast kluyveromyces liquid adds in the NTG of final concentration of 1mg/ml after 1ml Ion Beam Treatments, at 30 DEG C 250rpm handles 15min, 30min, 45min and 60min respectively.After being disposed, aseptically, with 0.22 μm of sterile filter Membrane filtration falls NTG, and cleans thalline with substantial amounts of sterile saline.1ml sterile saline suspension thallines are finally used, in 4 DEG C preservation.

The processing of 1.5 lithium chlorides

NTG is taken to handle lactic acid yeast kluyveromyces liquid 0.2ml respectively, is respectively coated containing various concentration lithium chloride On the YPD solid mediums of (0.4%, 0.8%, 1.2%, 1.6% and 2%), quiescent culture 3 days or so, have been treated at 30 DEG C After overall length goes out bacterium colony, tablet is in 4 DEG C of preservations.

The screening of 2 lactic acid yeast kluyveromyces mutant strain of embodiment

The culture of 2.1 mutant strains

Clone is chosen on the tablet in 1.5 in equipped with 96 deep-well plates in 1ml YPD fluid nutrient mediums.In 30 DEG C, 250rpm is continuously cultivated 5 days.After culture, deep-well plates are centrifuged into 5min in 5000rpm.100 μ l zymotic fluids will be taken shallow to 96 It is for use in 4 DEG C of preservations in orifice plate.

2.2 mutant strain glycosidase activities measure

The zymotic fluid that will be obtained in 2.1,30min is kept the temperature in 95 DEG C, is centrifuged 10min in 10000rpm, is collected supernatant.With P- nitrophenol β-D-Glucose glycosides (pNPG) is measured for substrate.Surveying system living is：Sodium phosphate buffer 0.05ml (100mM, pH7.0)+0.05ml+15 μ l pNPG (100mM is dissolved in DMSO) of enzyme solutions to be measured, to be not added with enzyme solution as blank pair According to reaction temperature is 95 DEG C, reaction time 1min.It is 1M Na to add in 0.1ml concentration₂CO₃Terminate reaction.With microplate reader, Absorbance (OD), the high mutant strain high for enzyme activity of wherein OD values are taken under 405nm.

2.3 culture presevation

The highest mutant strain of enzyme activity is inoculated into YPD fluid nutrient mediums in taking 2.2, and 28 DEG C, 250rpm continuously cultivates 2~3 My god.Zymotic fluid 10 is diluted with sterile water^-3, 10^-4, 10^-5It is respectively coated after times on YPD solid medium tablets, 28 DEG C of static trainings It supports 2~3 days, after single bacterium colony is grown, one single bacterium colony of picking is transferred in YPD fluid nutrient mediums, and 28 DEG C, 250rpm is continuously trained Support 48 it is small when.The 2.2 survey enzyme activity methods are repeated, confirm that enzyme activity is correct；Using micro- sem observation, after no living contaminants, Into zymotic fluid add in sterile glycerol make glycerol tube, make its final concentration of 25%, be named as Kluyveromyces lactis Kluyveromyces lactis XT1412, are preserved in China typical culture collection center, and deposit number is CCTCC M 2018017。

The acquisition of 3 Kluyveromyces lactis XT1412 glycosidases of embodiment

3.1 fermenting and producing

With the liquid YPD medium of 100ml, the Kluyveromyces lactis XT1412 of preservation in activation 2.3,28 DEG C, 250rpm is continuously cultivated 2 days.After culture, micro- sem observation is transferred to the 5L hairs of liquid YPD medium after no living contaminants In fermentation tank, 28 DEG C, 300rpm continuously ferments 5 days.After fermentation, 5000rpm centrifuges 10min, collects cell.Cell is suspended In the sterile 50mM pH7.0Na of 500ml₂HPO₄/NaH₂PO₄In buffer solution, using high-pressure homogenization instrument at 0 DEG C, crushed under 1000MPa Cell.Clasmatosis centrifuges 20min after 5000rpm, collects supernatant, and 20min is kept the temperature in 95 DEG C.Then at secondary 5000rpm from Heart 10min, collects supernatant, in 4 DEG C of preservations.

3.2 ammonium sulfate precipitation zymotic fluids

30~85% saturation degree of ammonium sulfate precipitation, ice bath are slowly added into what is ground in the supernatant collected in 3.1 Ammonium sulfate powder after being precipitated to 30%, centrifuges 8000rpm, 25min, supernatant is taken to filter, then carries out 85% precipitation again, centrifuges (8000rpm, 20min) collects precipitation, with phosphate buffer Na₂HPO₄/NaH₂PO₄(10mM, pH 7.0) dissolves.Membrane filtration is saturating Ammonium sulfate is fallen in analysis, and enzyme solution is for use in 4 DEG C of preservations, and purity is as shown in swimming lane 2 in Fig. 1.

3.3 cation exchange chromatography

DEAE-Toyopearl 650M anion chromatographies (bed volume 150ml), dress column equilibration about 500ml.3.2 are protected After hiding enzyme solution loading, Na is used₂HPO₄/NaH₂PO₄(10mM, pH7.0) buffer solution prewashing 400ml, then with containing 30mM and 60mM The Na of NaCl₂HPO₄/NaH₂PO₄(10mM, pH7.0) buffer solution elutes 400ml respectively, the Na of the NaCl containing 85mM₂HPO₄/ NaH₂PO₄(10mM, pH7.0) buffer solution elutes 300ml, the Na that NaCl containing 100mM is₂HPO₄/NaH₂PO₄(10mM, pH7.0) The Na of buffer solution and the NaCl containing 140mM₂HPO₄/NaH₂PO₄(10mM, pH7.0) buffer solution for gradient elution 500ml, flow velocity 1.5ml·min^-1.Using enzyme activity method is surveyed 2.2 Suo Shi, the activity in eluent is detected.Collect active eluent, low temperature NaCl is removed in dialysis, and for use in 4 DEG C of preservations, and purity is as shown in swimming lane 3 in Fig. 1.

It is 3.4 gel-filtration purified

Sephadex G150 (Φ 1.6cm × 100cm) are balanced overnight with basic buffer solution (about 500ml), will protected in 3.3 Enzyme solution sample concentration is hidden to 1ml (super filter tubesUltra-15,10kDa, Millipore), with substantially slow after loading Fliud flushing elutes, flow velocity 0.6mlmin^-1.Using enzyme activity method is surveyed 2.2 Suo Shi, the activity in eluent is detected.It collects active Eluent, and for use in 4 DEG C of preservations, purity is as shown in swimming lane 4 in Fig. 1.

3.5 analyses and sequencing

With the enzyme purified in polyacrylamide gel electrophoresis (the results are shown in Figure 1) and two dimensional electrophoresis analysis 3.4, as a result show The molecular weight for showing the albumen is about 60kDa.Utilize protein quantification kit measurement enzyme solution purity of protein, the results show enzyme solution sample Concentration>50pmol, purity>90%.N-terminal and C-terminal sequence analysis, result such as SEQ ID NO are carried out to the glycosidase of purifying：2 It is shown, it is named as blg-XT1412 albumen.

Embodiment 4 clones the encoding gene of blg-XT1412 albumen

4.1 design of primers

According to 3.5 sequencing result, the N-terminal protein sequence of blg-XT1412 albumen is methionine-serine-dried meat ammonia Acid-Ile-Leu, C-terminal sequence are histidine-histidine-histidine-histidine.Existed using GeneFisher softwares Line designs degenerate primer：

Sense primer：5'-ATGwsnCCnAThyTn-3'；

Anti-sense primer：5'-TTAsTGsTGsTGsTG-3'.

It is prepared by 4.2 templates

With the liquid YPD medium of 100ml, the bacterial strain of preservation in activation 2.3,28 DEG C, 250rpm is continuously cultivated 2 days.Training 5000rpm centrifugations 10min, collects thalline after supporting.The Dr.GenTLE that thalline is produced using Takra companies^TM(coming from yeast) High Recovery Yeast genomes extracts kit extracts genomic DNA, and product is for use in -20 DEG C of preservations.

4.3 gene magnification

Using product in 4.2 as template, with degenerate primer and the archaeal dna polymerase (high-fidelity of Takra companies designed in 4.1 Archaeal dna polymeraseHS (Premix)) PCR is carried out, specific reaction condition is as follows：

Confirm gene size into row agarose gel electrophoresis after reaction, and the segment of 1400~1600bp is cut respectively Glue recycles and carries out sequencing analysis, obtains gene undetermined.

4.4 DNA recombinant expressions undetermined

Gene PCR product to be determined is connected into Takra companies DNAA-Tailing Kit kits, is connected to after double digestion On pET24a (similary double digestion processing) plasmid, bacillus coli DH 5 alpha is converted.Distinguished using Takra companies plasmid extraction kit Recombinant plasmid is extracted, converts expressing protein in e. coli bl21 (DE3) after purification.

IPTG Fiber differentiations are used respectively containing recon e. coli bl21 (DE3) by above-mentioned.It is used after culture Ultrasonic disruption cell.By the sample after ultrasonication, 8500rpm centrifuges 10min at 4 DEG C, and it is for use to collect supernatant.

4.5blg-XT1412 protein coding genes determine

Using measuring enzyme activity method in 2.2, enzyme solution sample to be analyzed in detection 4.4；

Using the molecular size range of enzyme solution sample in enzyme solution sample and 3.4 to be analyzed in protein electrophoresis comparison 4.4；

Obtain the gene that length is 1512bp, expression product at 95 DEG C has enzyme activity and molecular weight and wild mutant enzyme one It causes, confirms the gene for purpose gene, sequence such as SEQ ID NO：Shown in 1, blg-XT1412 is named as.

The purifying of 5 blg-XT1412 albumen of embodiment and characterization

5.1 physical properties characterize

It is learnt by 3.5 sequencing results, blg-XT1412 albumen is made of 503 amino acid residues, and molecular weight is 58259.35 dalton, isoelectric point 5.80.

5.2 protein purification

Escherichia coli induction generates blg-XT1412 recombinant proteins and crushes liquid in taking 4.4, is splined on the good His of pre-balance Nickel purification column (10 times of volume sample-loading buffer balances, sample-loading buffer are Tris 20mM, NaCl250mM), uses 4 times of cylinders Product, about 200ml elution buffers elution target protein (elution buffer：Tris 20mM, NaCl 250mM, imidazoles 200mM). Elution is collected, 4 DEG C of dialysis remove imidazoles.It enzyme solution will freeze after purification, and obtain pure enzyme powder, it is for use in 4 DEG C of preservations.

5.3 reaction temperatures characterize

Preservation enzyme powder in taking 5.2, in the 50mM phosphate buffers of pH6.0, respectively with 10 DEG C, 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, 75 DEG C, 80 DEG C, 85 DEG C, 90 DEG C, 95 DEG C and 100 DEG C, according to method described in 2.2 measure Enzyme activity.Using peak as 100%, compare enzyme activity at each temperature.The results are shown in Figure 2,95 DEG C of the optimal reactive temperature of the enzyme, and More than 60% enzyme activity can be kept in the range of 75 DEG C~100 DEG C.

5.4 reaction pH characterizations

Preservation enzyme powder in taking 5.2, at 95 DEG C, respectively 4.0,4.5,5.5,6.0,6.5,7.0,7.5 and 8 pH value model (wherein 4.0~6.0 be citrate buffer solution, and 6.0~8.0 be phosphate buffer) measures according to method shown in 2.2 under enclosing Enzyme activity.Using peak as 100%, enzyme activity under more each pH systems.The results are shown in Figure 3, the optimum pH 5.5 of the enzyme, and More than 60% enzyme activity can be kept in the range of pH4.0~7.5.

5.5 thermal stability characterize

Preservation enzyme powder in taking 5.2 respectively when 75 DEG C, 80 DEG C, 85 DEG C, 90 DEG C and 95 DEG C heat preservations 320 are small, periodically measures residual Remaining enzyme activity, the results are shown in Figure 4.The half-life period being computed at above-mentioned temperature is as shown in table 1.

1 blg-XT1412 albumen thermal stability of table

5.6 substrate staves are levied

Enzyme activity defines：For the pure blg-XT1412 albumen of 1mg under the conditions of 95 DEG C, pH 5.5, the 1 μm of ol of hydrolysis in one minute specifies bottom Object is defined as 1U.Experiment is hydrolyzed to some common substrates containing glycosidic bond, the results are shown in Table 2, the results show blg- XT1412 albumen can hydrolyze all substrates in table 2.

2 blg-XT1412 protein substrates of table are composed

It understand that glycosidase of the invention at least may act on glucoside, galactoside, sweet dew by above-mentioned substrate spectrum Glucosides, xyloside, Arabinoside, arabinofuranosidase glucosides and lactose, however, the present invention is not limited thereto.

The glycosidase of the present invention is particularly suitable for food, medicine or health products work suitable for the commercial Application under high temperature In industry application, the substances such as flavonoid glycoside compounds, saponins glycoside compounds or lactose are hydrolyzed, to generate glycosides Member or progress enzyme process take off hardship etc..

The structure of the recombinant vector of 6 gene containing blg-XT1412 of embodiment

6.1 structure pKLAC2-bgl-XT1412 linearization plasmids

Using product in 4.2 as template, following primer (sense primer 5'-tta is usedGCGGCCGCATGTCCCCCATCCTC- 3'；Anti-sense primer 5'-ggcGAATTC) and the high-fidelity DNA polymerase of Takra companies TTAGTGATGGTGATG-3'HS (Premix) carries out PCR, and specific reaction condition is as follows：

Confirm gene size into row agarose gel electrophoresis after reaction.The segment of 1512bp is distinguished into gel extraction, And carry out sequencing analysis.

After sequencing result is correct, double digestion is carried out to PCR product using Not I and EcoR I.Endonuclease reaction terminates laggard Row agarose gel electrophoresis separate, and the segment of 1512bp are distinguished gel extraction, in -20 DEG C of preservations.

Double digestion is carried out to pKLAC2 carriers using Not I and EcoR I.Ago-Gel is carried out after endonuclease reaction Electrophoretic separation, by the segment gel extraction of 9081bp, in -20 DEG C of preservations.

In 10 μ l systems, 0.5 μ l linearisation pKLAC2 carriers are added in, 5 PCR products of the μ l through double digestion, 1 μ l 10 × Buffer, 1 μ l T4 ligases, the 2.5 sterile ddH of μ l₂O.In 16 DEG C of connections overnight.

After coupled reaction, bacillus coli DH 5 alpha is transformed into.After being accredited as positive colony, plasmid is extracted, is obtained PKLAC2-bgl-XT1412 cyclic plasmids, in -20pKLAC2 carrier preservations, the cyclic plasmid collection of illustrative plates is as shown in Figure 5A.

Utilize the above-mentioned cyclic plasmid of Sac II single endonuclease digestions.It is separated after endonuclease reaction into row agarose gel electrophoresis, it will The segment gel extraction of 7784bp, in -20 DEG C of preservations, gained linearization plasmid collection of illustrative plates is as shown in Figure 5 B.

Embodiment 7 is by blg-XT1412 genetic transformation to Kluyveromyces lactis GG799

7.1 electricity conversions

Kluyveromyces lactis competent cell on ice is placed on, adds in the linearization plasmid of preservation in 6.14, mixing It uniformly carries out electroporated.The all liq that electricity is converted in cup is transferred to sterile Eppendorf after conversion to manage, 30 DEG C of water Bath, 2h, 10000rpm centrifugation lmin, abandons supernatant, and thalline is trained containing 5mM acetamide YCB agar with being coated on after sterile aqueous suspension It supports on base tablet, 30 DEG C of quiescent cultures are cloned for 4~5 days.

7.2 recon identified for genes

Random picked clones from 7.1, containing 30 DEG C in 5mM acetamide YCB fluid nutrient mediums, 250rpm is cultivated for access 24 it is small when.5000rpm centrifuges 10min after culture, collects thalline.The Dr.GenTLE that thalline is produced using Takra companies^TM (come from yeast) High Recovery Yeast genomes extracts kit extracting genomic DNA as masterplate, using in 7.1 Same primers carry out PCR with archaeal dna polymerase, and specific reaction condition is as follows

Confirm gene size into row agarose gel electrophoresis after reaction.There is the clone of 1512bp segments, use is sweet Oil pipe is in -20 DEG C of preservations.

7.3 recon enzyme activity are identified

7.2 positive clone molecules are inoculated into liquid to contain in 5mM acetamide YCB fluid nutrient mediums, 30 DEG C, 250rpm cultures 24 it is small when.Then it is transferred in YPGal culture mediums, 30 DEG C, when 250rpm cultures 48 are small.Work as OD₆₀₀Value is more than 30, is collected by centrifugation Supernatant.Enzyme activity is measured using method described in 2.2, the bacterial strain for having an enzyme activity by one plant accesses sterile glycerol pipe, in -20 DEG C of preservations, And Kluyveromyces lactis GG799-blg-XT1412 is named as, it is preserved in China typical culture collection center, deposit number For CCTCC M2018018.

SEQUENCE LISTING

<110>Dong Yingjun

<120>Kluyveromyces lactis mutant strain and its glycosidase and application

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1512

<212> DNA

<213> Kluyveromyces lactis

<400> 1

atgtccccca tcctcggtta ctggaagatc aagggtctcg tccgcttccc caagcagggt 60

cccctcggtt tcggttactc ctggtccggt ttccagttcg agatgggtct gcccggttcc 120

gaggtcgagt ccgactggtg ggtctgggtc cacgacaagg agaacatcgc ctccggtctg 180

gtctccggtg acctgcccga gaacggtccc gcctactggc acctgtacaa gcaggaccac 240

gacatcgccg agaagctggg tatggactgc atccgcggtg gtatcgagtg ggcccgcatc 300

ttccccaagc ccaccttcga cgtcaaggtc gacgtcgaga aggacgagga gggtaacatc 360

atctccgtcg acgtccccga gtccaccatc aaggagctgg agaagatcgc caacatggag 420

gccctggagc actaccgcaa gatctactcc gactggaagg agcgcggtaa gaccttcatc 480

ctgaacctgt accactggcc cctgcccctg tggatccacg accccatcgc cgtccgcaag 540

ctgggtcccg accgcgcccc cgccggttgg ctggacgaga agaccgtcgt cgagttcgtc 600

aagttcgccg ccttcgtcgc ctaccacctg gacgacctgg tcgacatgtg gtccaccatg 660

aacgagccca acgtcgtcta caaccagggt tacatcaacc tgcgctccgg tttccccccc 720

ggttacctgt ccttcgaggc cgccgagaag gccaagttca acctgatcca ggcccacatc 780

ggtgcctacg acgccatcaa ggagtactcc gagaagtccg tcggtgtcat ctacgccttc 840

gcctggcacg accccctggc cgaggagtac aaggacgagg tcgaggagat ccgcaagaag 900

gactacgagt tcgtcaccat cctgcactcc aagggtaagc tggactggat cggtgtcaac 960

tactactccc gcctggtcta cggtgccaag gacggtcacc tggtccccct gcccggttac 1020

ggtttcatgt ccgagcgcgg tggtttcgcc aagtccggtc gccccgcctc cgacttcggt 1080

tgggagatgt accccgaggg tctggagaac ctgctgaagt acctgaacaa cgcctacgag 1140

ctgcccatga tcatcaccga gaacggtatg gccgacgccg ccgaccgcta ccgcccccac 1200

tacctggtct cccacctgaa ggccgtctac aacgccatga aggagggtgc cgacgtccgc 1260

ggttacctgc actggtccct gaccgacaac tacgagtggg cccagggttt ccgcatgcgc 1320

ttcggtctgg tctacgtcga cttcgagacc aagaagcgct acctgcgccc ctccgccctg 1380

gtcttccgcg agatcgccac ccagaaggag atccccgagg agctggccca cctggccgac 1440

ctgaagttcc agcccacccg cctcctcctc gagtacctcg tcaccaagcg ccatcaccat 1500

caccatcact aa 1512

<210> 2

<211> 503

<212> PRT

<213> Kluyveromyces lactis

<400> 2

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Arg Phe

1 5 10 15

Pro Lys Gln Gly Pro Leu Gly Phe Gly Tyr Ser Trp Ser Gly Phe Gln

20 25 30

Phe Glu Met Gly Leu Pro Gly Ser Glu Val Glu Ser Asp Trp Trp Val

35 40 45

Trp Val His Asp Lys Glu Asn Ile Ala Ser Gly Leu Val Ser Gly Asp

50 55 60

Leu Pro Glu Asn Gly Pro Ala Tyr Trp His Leu Tyr Lys Gln Asp His

65 70 75 80

Asp Ile Ala Glu Lys Leu Gly Met Asp Cys Ile Arg Gly Gly Ile Glu

85 90 95

Trp Ala Arg Ile Phe Pro Lys Pro Thr Phe Asp Val Lys Val Asp Val

100 105 110

Glu Lys Asp Glu Glu Gly Asn Ile Ile Ser Val Asp Val Pro Glu Ser

115 120 125

Thr Ile Lys Glu Leu Glu Lys Ile Ala Asn Met Glu Ala Leu Glu His

130 135 140

Tyr Arg Lys Ile Tyr Ser Asp Trp Lys Glu Arg Gly Lys Thr Phe Ile

145 150 155 160

Leu Asn Leu Tyr His Trp Pro Leu Pro Leu Trp Ile His Asp Pro Ile

165 170 175

Ala Val Arg Lys Leu Gly Pro Asp Arg Ala Pro Ala Gly Trp Leu Asp

180 185 190

Glu Lys Thr Val Val Glu Phe Val Lys Phe Ala Ala Phe Val Ala Tyr

195 200 205

His Leu Asp Asp Leu Val Asp Met Trp Ser Thr Met Asn Glu Pro Asn

210 215 220

Val Val Tyr Asn Gln Gly Tyr Ile Asn Leu Arg Ser Gly Phe Pro Pro

225 230 235 240

Gly Tyr Leu Ser Phe Glu Ala Ala Glu Lys Ala Lys Phe Asn Leu Ile

245 250 255

Gln Ala His Ile Gly Ala Tyr Asp Ala Ile Lys Glu Tyr Ser Glu Lys

260 265 270

Ser Val Gly Val Ile Tyr Ala Phe Ala Trp His Asp Pro Leu Ala Glu

275 280 285

Glu Tyr Lys Asp Glu Val Glu Glu Ile Arg Lys Lys Asp Tyr Glu Phe

290 295 300

Val Thr Ile Leu His Ser Lys Gly Lys Leu Asp Trp Ile Gly Val Asn

305 310 315 320

Tyr Tyr Ser Arg Leu Val Tyr Gly Ala Lys Asp Gly His Leu Val Pro

325 330 335

Leu Pro Gly Tyr Gly Phe Met Ser Glu Arg Gly Gly Phe Ala Lys Ser

340 345 350

Gly Arg Pro Ala Ser Asp Phe Gly Trp Glu Met Tyr Pro Glu Gly Leu

355 360 365

Glu Asn Leu Leu Lys Tyr Leu Asn Asn Ala Tyr Glu Leu Pro Met Ile

370 375 380

Ile Thr Glu Asn Gly Met Ala Asp Ala Ala Asp Arg Tyr Arg Pro His

385 390 395 400

Tyr Leu Val Ser His Leu Lys Ala Val Tyr Asn Ala Met Lys Glu Gly

405 410 415

Ala Asp Val Arg Gly Tyr Leu His Trp Ser Leu Thr Asp Asn Tyr Glu

420 425 430

Trp Ala Gln Gly Phe Arg Met Arg Phe Gly Leu Val Tyr Val Asp Phe

435 440 445

Glu Thr Lys Lys Arg Tyr Leu Arg Pro Ser Ala Leu Val Phe Arg Glu

450 455 460

Ile Ala Thr Gln Lys Glu Ile Pro Glu Glu Leu Ala His Leu Ala Asp

465 470 475 480

Leu Lys Phe Gln Pro Thr Arg Leu Leu Leu Glu Tyr Leu Val Thr Lys

485 490 495

Arg His His His His His His

500

Claims

Kluyveromyces lactis 1. (Kluyveromyces lactis) XT1412, is preserved in China typical culture collection The heart, deposit number are CCTCC M 2018017.
2. a kind of glycosidase, amino acid sequence such as SEQ ID NO：Shown in 2.
3. a kind of polynucleotides encode the glycosidase described in claim 2.
4. polynucleotides according to claim 3, sequence such as SEQ ID NO：Shown in 1.
5. recombinant vector contains the polynucleotides described in claim 3 or 4.
6. host cell, the host cell, which contains to integrate in recombinant vector or its genome described in claim 5, has the right It is required that the polynucleotides described in 3 or 4,

The host cell is, for example, Kluyveromyces lactis GG799-blg-XT1412, is preserved in Chinese Typical Representative culture guarantor Tibetan center, deposit number are CCTCC M 2018018.
7. a kind of method for producing glycosidase, including：

1) host cell described in claim 6 is cultivated；

2) glycosidase is separated from the culture 1) obtained.
8. a kind of composition, the composition includes the glycosidase described in claim 2；Preferably, the composition is further Including acceptable carrier in food industry.
9. a kind of method for hydrolyzing the substance containing glycosidic bond, the described method includes by the glycosidase or right described in claim 2 It is required that the composition described in 8 and the material mixing containing glycosidic bond.
10. according to the method described in claim 9, wherein, reaction temperature is 10 DEG C~100 DEG C, is preferably 50 DEG C~100 DEG C, Also preferably 60 DEG C~100 DEG C, more preferably 80 DEG C~100 DEG C are most preferably 95 DEG C；Optional, pH value in reaction is 4~8, excellent Elect 4.5~6.5 as, more preferably 5~6, it is most preferably 5.5.