CN110129303A - The pectase TsPec and gene of a kind of high temperature resistant acidic and application - Google Patents

The pectase TsPec and gene of a kind of high temperature resistant acidic and application Download PDF

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CN110129303A
CN110129303A CN201910371940.4A CN201910371940A CN110129303A CN 110129303 A CN110129303 A CN 110129303A CN 201910371940 A CN201910371940 A CN 201910371940A CN 110129303 A CN110129303 A CN 110129303A
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tspec
pectase
pectin
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gly
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杨江科
彭小波
韩正刚
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China Ocean Mineral Resources R & D Association (china's Ocean Affairs Administration)
Wuhan Polytechnic University
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Wuhan Polytechnic University
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    • C12Y302/01015—Polygalacturonase (3.2.1.15)

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Abstract

The invention belongs to gene engineering technology field, it is related to a kind of acid pectase TsPec resistant to high temperature and gene and application.The core amino acid sequence of the pectase TsPec is as shown in SEQ ID NO:1, and, 77 and 81 cysteines form disulfide bond in amino acid sequence shown in SEQ ID NO:1,194 and 210 cysteines form disulfide bond, and 272 and 317 cysteines form disulfide bond.The sequence of the pectase base sequence and SEQ ID NO:2 have 95% or more identity.Invention significantly improves expression quantity of the artificial synthesized pectin enzyme gene TsPec in Pichia pastoris, the activity, temperature stability and expression of pectase are significantly improved, in Pichia pastoris high level expression, high enzymatic activity, acid pectase resistant to high temperature and gene are obtained.

Description

The pectase TsPec and gene of a kind of high temperature resistant acidic and application
Technical field
The invention belongs to gene engineering technology fields, a kind of pectase TsPec more particularly, to high temperature resistant acidic and Gene order and recombinant expression carrier, recombinant strains, the application of pectase TsPec and a kind of side of hydrolysis of pectin Method.
Background technique
Pectin is one of organs such as the fruit for being present in plant natural polymer product.It is the group of plant cell wall At ingredient, it is the adhesive between flanking cell, is bonded together plant cell.Natural pectin substance usually with protopectin, The form of pectin and pectic acid is present in plant tissue and organ.Pectin is a kind of linear carbohydrate high polymer, main chain master It to be made of galacturonic acid glycan, galactan and araban galacturonic acid residues, molecular weight is about 50000~ 150000.As a kind of natural polymer, pectin has good gelatine and stable emulsifying effect.It is widely used in food Product, medicine, daily use chemicals and textile industry.On the other hand, pectin to the nutritional utilization of the processing of the beverages such as fruit juice, fruit wine, feed with And papermaking and crudefiber crop degumming etc. have a significant impact.
Pectase refers to the general name for decomposing the enzyme of pectic substance.Pectinesterase, polygalacturonic acid, pectin is generally included to split Solve enzyme etc..The ester hydrolysis that they can be catalyzed pectin depolymerization or be catalyzed in pectin molecule.Pectase can be according to its most adaptable work It is divided into acid pectase and alkaline pectase with pH value.
Acid pectase is relatively broad in food industry applications, relates generally to the squeezing and clarify of juice, natural products Extraction and feed processing etc..Pectase can have application in many industries with depolymerized pectin.Pectase juice clarify, It is related in the fields such as brewing fruit wine, natural product extraction, oil expression, feed, papermaking, weaving, environmental protection.Pectase is at first Applied in fruit and vegetable juice processing technique.Since fruit juice is generally acidity, so the predominantly acid pectase of application.Pectin is added Crushing juice rate, the viscosity for reducing juice, the light transmittance for increasing juice can be improved in enzyme.In addition pectase is mixed with amylase Clear juice is handled, can reduce by 50% filtration time.Wine can be improved behind brewing fruit wine field, grape slurry pectinase treatment Clarity and quality.Pectase can destroy the cell wall of grape to a certain degree, reduce the viscosity of fruit juice, promote the molten of pigment Solution, improves the quality of grape wine.In oil expression manufacture field, if processing before being carried out before oil expression with pectase, makes cell wall It is penetrating, oil yield can be improved, increase phenolic material content, improve turbidity, the color of oil.In field of fodder, pectase, which is added, is raised Anti-nutritional factors can be reduced in material, increases efficiency of feed utilization, reduces the viscosity of food gruel, reduce poultry manure amount.Pectase It is a kind of green feed additive, it is more prevalent in the production application of feed.
Although pectase is with a wide range of applications in fields such as food, feed, processings of farm products, but still existing need to The critical issue to be solved: 1) Rate activity of enzyme is low.Naturally occurring pectinase activity is generally lower, to the hydrolysis efficiency of pectin It is low.2) yield is lower in the industrial production for pectase, and production cost is higher.3) high-temperature stability of pectase is poor, especially In feed industry, high temperature granulating technique easily makes heat labile pectase lose activity.
Therefore, it is necessary to be transformed to pectase, to improve enzyme to the stability of temperature, its expression is improved.
Summary of the invention
The object of the present invention is to provide the high temperature resistant acidic pectase TsPec and gene order of a kind of high level expression, with And a kind of application and method of hydrolysis of pectin of recombinant expression carrier, recombinant strains, pectase TsPec.Of the invention The optimal temperature for the pectase TsPec that pectin enzyme gene TsPec gives expression to is 75 DEG C, optimum pH 5.0.
To achieve the goals above, the present invention provides a kind of pectase TsPec of high temperature resistant acidic, the pectase The amino acid sequence of TsPec contains amino acid sequence shown in SEQ ID NO:1, also, amino acid shown in SEQ ID NO:1 77 and 81 cysteines form disulfide bond in sequence, and 194 and 210 cysteines form disulfide bond, 272 with 317 cysteines form disulfide bond.According to the present invention, the amino acid sequence of the pectase TsPec contains SEQ ID Effect of the invention can be realized in amino acid sequence shown in NO:1.Under most preferred case, the amino acid of the pectase TsPec Sequence is amino acid sequence shown in SEQ ID NO:1, also, 77 and 81 in amino acid sequence shown in SEQ ID NO:1 The cysteine of position forms disulfide bond, and 194 and 210 cysteines form disulfide bond, 272 and 317 half Guang ammonia Acid forms disulfide bond.
According to the present invention, the pectase TsPec is by coming from basket bacterium (Talaromyces sp.) pectase Pec (GenBank accession number: XM_002484792.1) introduces three disulfide bond, the pectase by way of protein design The amino acid sequence of Pec is as shown in SEQ ID NO:2.Specific design includes: by 77 bright ammonia in parent pectin enzyme Pec sequence Acid and 81 threonines (T81) sport cysteine (L77C and T81C), introduce a pair of of disulfide bond；By the third of 194 Propylhomoserin and 210 valines sport cysteine (A194C and V210C)；By 272 threonines and 317 figured silk fabrics Propylhomoserin (T272C and V317C) introduces a pair of of disulfide bond.The various of this field routine can be used described in the amino acid of redesign Method realizes that concrete operation step is known to the skilled person, and details are not described herein.
The second aspect of the present invention provides a kind of pectin enzyme gene TsPec of high temperature resistant acidic, for encoding pectase TsPec.Specifically, the base sequence of the pectin enzyme gene TsPec has 95% with base sequence shown in SEQ ID NO:3 Above identity.Preferably, according to the present invention, the pectin enzyme gene TsPec, contains alkali shown in SEQ ID NO:3 Effect of the invention can be realized in basic sequence.In the most preferred case, the base sequence of pectase TsPec of the present invention is Base sequence shown in SEQ ID NO:3.
Pectin enzyme gene TsPec provided by the invention is based on pectin enzyme amino acid sequence engineer.Using complete The codon that red yeast medium-high frequency uses, so that the pectin enzyme gene TsPec for significantly improving engineer is thin in Pichia pastoris Expression quantity in born of the same parents.The present invention goes out a completely new pectase gene order according to the amino acid sequence of pectase, engineer, And the Fragments of Pectinase Gene is obtained by artificial synthesized method, base sequence is named as shown in SEQ ID NO:3 TsPec, the amino acid sequence for the pectase that the pectin enzyme gene TsPec is encoded out is as shown in SEQ ID NO:1.Of the invention The preference temperature for the pectase TsPec that pectin enzyme gene TsPec gives expression to is 65-75 DEG C, and optimal temperature is 75 DEG C, appropriate pH Value is 4.5-5.5, optimum pH 5.0.
The third aspect of the present invention provides a kind of recombinant expression carrier comprising above-mentioned pectin enzyme gene TsPec can also be wrapped Include other function unit.In the situation that the amino acid sequence of pectase TsPec and the base sequence of pectin enzyme gene TsPec determine Under, those skilled in the art can select suitable recombinant expression carrier and other function unit, for example, Pichia anomala expression Carrier.
The preparation method of the recombinant expression carrier is not particularly limited in the present invention, can be according to this field routine techniques hand Section determines.
A kind of specific embodiment according to the present invention, the preparation method of the recombinant expression carrier will be the following steps are included: will The pectin enzyme gene TsPec of synthesis is connected in expression vector.In some embodiments of the invention, the expression vector is complete Red Yeast expression carrier pPICZ α A.Certainly, in other embodiments of the present invention, other yeast expression vectors also can be selected.
A kind of more specifical embodiment according to the present invention, the preparation method of the recombinant expression carrier include following step Rapid: by the pectin enzyme gene TsPec of synthesis by restriction enzyme EcoR I and Xba I digestion, obtaining has cohesive end Pectin enzyme gene TsPec segment；Yeast expression vector pPICZ α A is passed through into restriction enzyme EcoR I and Xba I Digestion obtains the pPICZ α A segment with cohesive end；Pectin enzyme gene TsPec segment and pPICZ α A segment are passed through into T4 DNA ligase connection, obtains pPIC-TsPec recombinant expression plasmid.
The fourth aspect of the present invention provides a kind of recombinant strains, and expression product is above-mentioned pectase TsPec.In fruit In the case that the amino acid sequence of glue enzyme TsPec determines, those skilled in the art can obtain suitable recombinant strains.
Usually, the host cell can be Escherichia coli, bacillus, aspergillus, be also possible to the cells such as yeast, Either other kinds of cell, such as zooblast etc..Preferably, the host cell of the recombinant strains is to finish red ferment It is female.Meanwhile pectin enzyme gene TsPec codon optimization provided by the invention, turned using Pichia pastoris is suitable for Record accurate translation goes out the high frequency AC pulse Link of pectase, improves expression quantity of the pectin enzyme gene TsPec in Pichia pastoris.
The preparation method of the recombinant strains is not particularly limited in the present invention, can be according to this field routine techniques hand Section determines.
A kind of specific embodiment according to the present invention, the preparation method of the recombinant strains include the following steps: by Recombinant expression carrier is linearized by restriction enzyme；Above-mentioned linearized fragment is imported in Pichia pastoris, recombination table is obtained Up to bacterial strain.Optionally, turn to import recombinant expression carrier in Pichia pastoris host cell by electricity, obtain recombinant strains. In some embodiments of the invention, the Pichia pastoris is pichia pastoris yeast X-33, certainly, in other implementations of the present invention In example, other Pichi strains also can be selected.
A kind of more specifical embodiment according to the present invention, the preparation method of the recombinant strains include following step It is rapid: pPIC-TsPec recombinant expression plasmid is linearized by Bgl II restriction enzyme；It will be linearized by electrotransformation PPIC-TsPec import Pichia pastoris host cell in, obtain recombinant strains.
In the present invention, the method that recombinant strains prepare pectase can be adopted with the conventional methods in the field.According to this A kind of specific embodiment is invented, the method for producing pectase includes: by the above-mentioned recombinant strains of fermented and cultured, from fermentation Purification obtains pectase in supernatant.In some embodiments it is possible to without purification processes, directly by fermented supernatant fluid It is considered as pectin enzyme product.
A kind of more specifical embodiment according to the present invention is prepared the method packet of pectase by above-mentioned recombinant strains Include following steps: picking recombinant strains are cultivated about 24 hours into culture medium, and harvest thallus；Thallus is transferred to fresh In culture medium, about 1% methanol is added within every 24 hours, induces the expression of pectase TsPec.After inducing expression about 96 hours, from The heart collects supernatant, obtains the solution of the TsPec containing pectase.
The fifth aspect of the present invention provides the application of above-mentioned pectase TsPec.
Specifically, the sixth aspect of the present invention provides a kind of method of hydrolysis of pectin, this method comprises: by above-mentioned pectase TsPec is contacted with material containing pectin.The material containing pectin includes various types of pectin, also includes other objects containing pectin Material.
Pectase TsPec provided by the invention from parent pectin enzyme mutant, gene TsPec according to TsPec ammonia Base acid sequence carries out engineer, according to the Preference of Pichia pastoris codon, the password for selecting Pichia pastoris medium-high frequency to use Son, reduces the free energy of TsPec gene mRNA secondary structure, to significantly improve artificial synthesized pectin enzyme gene Expression quantity of the TsPec in Pichia pastoris significantly improves the high-temperature stability and expression of pectase, obtains High level expression, high enzymatic activity, pectase resistant to high temperature and gene in Pichia pastoris.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its Its purpose, feature and advantage will be apparent.
Figure 1A and Figure 1B is the structure of original pectase and three pairs of disulfide bond of introducing after redesign in the embodiment of the present invention 1 Figure.
Fig. 2A-Fig. 2 B is to examine the digestion that pectase is cloned into recombinant expression carrier pPICZ α A in the embodiment of the present invention 2 As a result.
Fig. 3 shows and produces the result that pectase bacterial strain is expressed under the conditions of shaking flask in the embodiment of the present invention 3.
The temperature curve for the pectase TsPec that Fig. 4 A and 4B are pectase Pec and the embodiment of the present invention 3 redesigns.
The pH curve for the pectase TsPec that Fig. 5 A and 5B are pectase Pec and the embodiment of the present invention 3 redesigns.
Fig. 6 A and 6B are the secondary structure figure of parent pectin enzyme gene Pec with the pectin enzyme gene TsPec after optimization.
Fig. 7 A and 7B are the protein and enzyme activity situation of change that pectase is expressed in the fermenter in the embodiment of the present invention 6.
Fig. 8 is the thin-layer chromatography testing result of product after pectase TsPec hydrolysis of pectin in embodiment 7.
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.It is real The person that is not specified actual conditions in example is applied, is carried out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not infused Bright production firm person is the conventional products that can be obtained by commercially available purchase.
Embodiment 1
The present embodiment is used to illustrate to introduce the gene design process of three pairs of disulfide bond into pectase Pec.It was embodied Journey is as follows:
According to the three-dimensional structure of pectase Pec, side that the pectase TsPec passes through protein design by pectase Pec Formula introduces three disulfide bond, and the amino acid sequence of the pectase Pec is as shown in SEQ IDNO:2.It specifically designs and includes: 77 leucines and 81 threonines in parent pectin enzyme Pec sequence are sported into cysteine (L77C and T81C), drawn Enter a pair of of disulfide bond；194 alanine and 210 valines are sported into cysteine (A194C and V210C), drawn Enter second pair of disulfide bond；272 threonines and 317 valines are sported into cysteine (T272C and V317C), Third is introduced to disulfide bond.Gene order corresponding to pectase TsPec amino acid sequence is as shown in SEQ ID NO:1.The base Because being synthesized using full genome synthetic method.
Shown in Figure 1A, 77 leucines (L77C) in pectase Pec for transformation, 81 threonines are listed (T81), 194 alanine (A194), 210 valines (V210), 272 threonines (T272) and 317 figured silk fabrics Propylhomoserin (V317)；Figure 1B show the specific location of the three pairs of disulfide bond newly introduced.
Embodiment 2
The present embodiment is used to illustrate the building of pectin enzyme gene TsPec recombinant strains.
(1) add restriction enzyme site EcoR I and restriction enzyme site Not I at the pectin both ends enzyme gene TsPec, through EcoR I and Not I digestion；Same carrier pPICZ α A is through EcoR I and Not I digestion.Wherein, digestion system are as follows: 3mg DNA, 10U EcoR I, 10U Not I, 20 μ L Buffer H add water polishing to 300 μ L volumes, digestion 4h under the conditions of 37 DEG C.As a result such as Fig. 2A institute Show that (in figure: M is DL5000 DNA Marker, and 1 swimming lane is the pPICZ α A recombinant expression carrier after digestion；2 swimming lanes is after digestions TsPec segment；As seen from the figure, the size of pPICZ α A and TsPec segment is respectively 3.7kb and 1.0kb or so, with practical phase Symbol.
(2) pectin enzyme gene TsPec endonuclease bamhi is connected with pPICZ α A endonuclease bamhi by T4 DNA ligase, is obtained Obtain pPIC-TsPec recombinant expression carrier；Wherein, linked system are as follows: genetic fragment, the pPICZ α A segment of 50ng, 1 μ of 150ng T4 buffer of L, the T4 DNA ligase of 5U plus water polishing are to 10 μ L；After connecting 8h under the conditions of 16 DEG C, by linked system It converts to Escherichia coli (DH5 α).Conversion process are as follows: in placing 10min on ice after mixing system and DH5 α bacterial strain, then set In 42 DEG C of processing 1min, after adding 800 μ L culture mediums, it is placed in 37 DEG C of culture 40min；It is sieved finally, being coated in screening flat board Positive colony is selected, extracts plasmid to get the recombinant expression carrier pPIC-TsPec with pectin enzyme gene TsPec is arrived.Extract weight Group expression vector pPIC-TsPec, carries out double digestion verifying (EcoR I, Not I), and as a result (in figure: M is as shown in Figure 2 B DL5000 DNA Marker, 1 swimming lane are the pPIC-TsPec recombinant expression carrier after double digestion, and 2 swimming lanes is after single endonuclease digestions pPIC-TsPec；3 swimming lanes are the pPIC-TsPec after double digestion).As seen from the figure, illustrate that pectin enzyme gene TsPec is successfully connected To pPICZ α A recombinant expression carrier.Recombinant expression carrier pPIC-TsPec is transformed into pichia pastoris yeast X-33, is obtained To the recombinant strains of the TsPec of enzyme gene containing pectin.The present invention is synchronous also construct parent pectin enzyme gene Pec finish red ferment Female recombinant strains.The sequence of parent pectin enzyme gene Pec is as shown in SEQ ID NO:4.Construction method and step are same as above.
Embodiment 3
The present embodiment is for illustrating fermenting and producing of the pectase TsPec in shaking flask.
(1) the red genetic engineering bacterium of finishing of the gene containing TsPec in embodiment 2 is inoculated into 5mL YPD culture medium, is cultivated 20h obtains seed liquor；
(2) above-mentioned seed liquor is seeded in the shaking flask of the fermentation medium of BMGY containing 30mL and carries out fermented and cultured, wherein BMGY fermentation medium includes: 1% yeast powder, 2% peptone, 100mM phosphate buffer (pH 6.0), 1.34% yeast nitrogen Alkali, 0.004% biotin, 1% glycerol.After culture for 24 hours, collects thallus and be transferred in 30mL BMMY culture medium and continue to cultivate. Wherein BMMY culture medium includes: 1% yeast powder, 2% peptone, 100mM phosphate buffer (pH 6.0), 1.34% yeast nitrogen Alkali, 0.004% biotin, 0.5% methanol.Per 0.15mL methanol is supplemented for 24 hours, until stopping fermentation when 96h.Fermentation liquid uses SDS- PAGE is detected.Being illustrated in figure 3 the testing result of fermentation liquid, (in figure: M is protein standards, and 1 swimming lane is non-transgenic pPICZαA；2 swimming lanes are pPIC-Pec expression product；3 swimming lanes are pPIC-TsPec expression product.Fig. 3 illustrates pectin enzyme gene Pec and the pectin enzyme gene TsPec of redesign are expressed successfully.
Embodiment 4
The present embodiment is for testing pectase TsPec optimum temperature and optimal pH.
(1) pectinase activity is measured using reducing sugar method.Steps are as follows:
A, it takes 0.5mL to dilute 500 times of fermented supernatant fluid, is added to the vinegar of pectin solution (0.5%) and 1mL of 0.5mL In the mixed liquor of sour sodium-acetate buffer solution (pH value 5.0), the water-bath 10min at specific temperature, then, with 2mL's DNS (3,5- dinitrosalicylic acid) terminates reaction, is placed in 5min in boiling water bath and reduced sugar is allowed sufficiently to develop the color；After being cooled to room temperature It is settled to 25mL with distilled water, tests the absorbance at 520nm after mixing；
B, according to measured absorbance, pectase enzyme activity is calculated referring to enzyme activity formula.Enzyme activity (U/mL) calculation formula Are as follows: U=r × Df × 1000/150.14/t, wherein Df is fermented supernatant fluid extension rate；R is according to OD520Absorbance value obtains The molar concentration (μm ol/mL) of the product reduced sugar arrived；T is enzyme digestion reaction time (min).Enzyme-activity unit is defined as: per minute Enzyme amount needed for pectin forms 1 μm of ol reduced sugar is 1 enzyme activity unit (U).
(2) the measurement system of parent pectin enzyme Pec and the pectase TsPec optimum temperature of redesign are as follows: pectin 0.25g is configured to the pectin solution of 0.5% concentration with the NaAC-HAC solution of 50mmoL pH 5.0, and it is dilute in right amount that 0.1mL is added Pectinase solution after releasing.40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 75 DEG C, 80 DEG C of isothermal gradients are respectively set, by above-mentioned reactant In the set different temperatures of system's merging, reacts 10 minutes, after reaction, reaction is measured using the DNS method in such as embodiment 3 The content of reducing sugar of generation simultaneously calculates remaining enzymatic activity.Pectin enzyme activity and anti-is drawn out according to the enzyme activity measured under different temperatures The curve of temperature is answered, optimal reactive temperature is obtained.As a result as shown in Figure 4 A, the optimum temperature of original pectase Pec is 60 DEG C. Shown in Fig. 4 B, the pectase TsPec of the three disulfide bond activity highest at 75 DEG C is introduced, therefore optimum temperature is 75 DEG C.And And temperature be 80 DEG C when, still retain 90% or so enzyme activity.Therefore, pectase TsPec of the invention has good high temperature resistant Characteristic.
(3) the measurement system of parent pectin enzyme Pec and the pectase TsPec optimal pH of redesign are as follows: pectin 0.25g, The NaAC-HAC solution that pH3.0, pH4.0, pH5.0, pH6.0, pH7.0 is respectively set is configured to the pectin solution of 0.5% concentration, The pectinase solution after 0.1mL dilutes in right amount is added.Above-mentioned reaction system is reacted 10 minutes in 75 DEG C, after reaction, is adopted The content of reducing sugar generated is reacted with the DNS method measurement in such as embodiment 3 and calculates remaining enzymatic activity.It is surveyed according under different pH Fixed enzyme activity draws out pectin enzyme activity and the curve of pH, obtains optimal reaction pH.As a result as shown in Figure 5 A and 5B, Fig. 5 A is pectin The curve of enzyme Pec, Fig. 5 B are the curve of pectase TsPec, it is seen then that the optimal pH of pectase Pec and TsPec are 5.Between the two Enzyme activity it is consistent with pH relation curve variation tendency.Therefore, pectase TsPec of the invention is acid pectase.
Embodiment 5
The present embodiment is used to illustrate that the optimization of pectase gene codon to obtain gene TsPec.
The present embodiment is based on the amino acid sequence for redesigning the pectase TsPec after introducing new disulfide bond, artificial weight Newly devise the nucleotide sequence of pectin enzyme gene.It specifically includes: low-frequency codon is substituted using high-frequency codon； Reduce the complexity and minimum free energy (Fig. 6) of the secondary structure of the mRNA of gene coding.Pectin is designed under area of computer aided Enzyme gene TsPec sequence obtains pectin enzyme gene TsPec segment by artificial synthesized method.
The present embodiment goes out a completely new pectase gene order according to the amino acid sequence of pectase, engineer, and The Fragments of Pectinase Gene is obtained by artificial synthesized method, base sequence is named as shown in SEQ ID NO:3 TsPec, the amino acid sequence for the pectase that the pectin enzyme gene TsPec is encoded out is as shown in SEQ ID NO:1.Fig. 6 A is this The secondary structure of 180 nucleotide before invention pectin enzyme gene Pec, minimum free energy are -65.00kcal/mol.Fig. 6 B is After optimization in gene TsPec preceding 180 nucleotide secondary structure, minimum free energy be -59.20kcal/mol.Therefore, originally The stability for inventing pectin enzyme gene TsPec obtained has apparent optimization.
Embodiment 6
The present embodiment is for illustrating the fermenting and producing of pectin enzyme gene TsPec in the fermenter after optimizing.
(1) the red genetic engineering bacterium of finishing of the gene containing TsPec in embodiment 5 is inoculated into 400mL YPD culture medium, is cultivated 20h obtains seed liquor；
(2) above-mentioned seed liquor is seeded in the fermentor containing fermentation medium and carries out fermented and cultured, wherein fermented and cultured Base includes: 350g KH2PO4、7g CaSO4、40g(NH4)2SO4、50g MgSO4、120g K2SO4With 560g glycerol.
(3) fermented and cultured can be divided into vegetative growth phase and methanol induction expression phase.In vegetative growth phase, fermentation ginseng Numerical control is made as: 28 DEG C of cultivation temperature, pH value 5.5, and revolving speed 300-500rpm, ventilatory capacity 3.0-5.5L/min.This stage holds Renewed treaty 30h.After nutrient growth, that is, enter methanol induction expression phase；In methanol induction phase, dissolved oxygen maintains 20- 30%, 25 DEG C of temperature, pH value 5.0-5.5, methanol flow rate 2.5-4.0mL/L/h, flow the added-time between be 105h.It is taken every 15h Sample, and measure the pectinase activity of fermentation liquid.The detection of protein composition is detected using SDS-PAGE method in fermentation liquid, detection knot Fruit is as shown in Figure 7 A.With the extension of fermentation time, the content of TsPec is gradually increased in fermentation liquid.By Fig. 7 B it is found that pectase TsPec production level is with the extension of methanol induction time, and the vigor of pectase gradually increases in fermentation liquid, is in incubation time When 96h, the vigor of pectase is up to 13,500U/mL fermentation liquid in fermentation liquid.
Embodiment 7
The present embodiment uses pectase TsPec hydrolysis of pectin for illustrating.
(1) pectase TsPec hydrolysis of pectin: the fermented supernatant fluid for taking 1mL embodiment 6 to prepare, with distilled water dilution 100 Times, take 0.5mL to be added to the mixing of pectin solution and 1mL Acetate-acetate buffer solution (pH 5.0) that 0.5mL concentration is 1% In solution, and 75 DEG C of water-bath 10min.
(2) pectin hydrolysate is detected using thin layer chromatography: takes the 10 μ L point of hydrolysate in above-mentioned steps (1) in silicon On offset plate, it is put into exhibition layer 60min in solvent；Silica gel plate is taken out, it is dry, and even application color developing agent, it is put into 108 DEG C of baking oven Middle drying colour developing.As a result (1 swimming lane is unhydrolysed pectin, and 2 swimming lanes are galactolipin, and 3 swimming lanes are hydrolyzed pectin as shown in Figure 8 0.5h, 4 swimming lanes are hydrolyzed pectin 1.0h, and 5 swimming lanes are hydrolyzed pectin 1.5h, and 6 swimming lanes are hydrolyzed pectin 2.0h).As shown in Figure 8, Pectin complete hydrolysis can be the oligosaccharide of the degree of polymerization between 1 and 3 by pectase provided by the invention.
In conclusion the present invention designs three pairs of disulfide bond of introducing into parent pectin enzyme by enzyme molecule, significantly improve Its characteristic resistant to high temperature, and after being optimized according to the Preference of Pichia pastoris to pectin enzyme gene, synthesize high temperature resistant Acid pectase gene TsPec.After the gene is connect with expression vector pPICZ α A, it is transferred in Pichia pastoris GS115 and expresses, To obtain high efficient expression, high yield, high temperature resistant acidic pectase production bacterial strain；Inducing expression 96 is small in the fermenter for it Shi Hou, the enzyme activity of pectase is up to 13,500U/mL in supernatant.The optimum temperature for the high temperature resistant pectase that the present invention obtains is 75 DEG C, and there is efficient hydrolysis ability to pectin.
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill Many modifications and changes are obvious for the those of ordinary skill in art field.
Sequence table
<110>Wuhan Polytechnic University
Chinese ocean mineral resources research and develop association (Chinese ocean transaction management office)
<120>a kind of pectase TsPec of high temperature resistant acidic and gene and application
<130> BJI1900305WHPU
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 375
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Met Arg Ser Phe Ile Gly Leu Ala Gly Leu Ala Cys Leu Ser Ser Val
1 5 10 15
Val Leu Ala Val Pro Ala Pro Ala Pro Thr Ala Pro Ala Lys Leu Glu
20 25 30
Asp Arg Ala Ala Ser Cys Thr Phe Ser Gly Ser Asn Gly Ala Ala Ala
35 40 45
Ala Ser Lys Ser Lys Thr Ser Cys Ser Thr Ile Val Leu Ser Ser Val
50 55 60
Ala Val Pro Ser Gly Thr Thr Leu Asp Leu Thr Asn Cys Asn Asp Gly
65 70 75 80
Cys Lys Val Ile Phe Glu Gly Thr Thr Thr Phe Gly Tyr Ala Glu Trp
85 90 95
Ser Gly Pro Leu Val Ser Val Ser Gly Asn Asp Ile Thr Val Glu Gly
100 105 110
Ala Ser Gly Ala Val Leu Asp Gly Asp Gly Ser Arg Trp Trp Asp Gly
115 120 125
Lys Gly Ser Asn Gly Gly Lys Thr Lys Pro Lys Phe Phe Tyr Ala His
130 135 140
Asn Leu Lys Ser Ser Ser Ile Ser Asn Ile Lys Ile Leu Asn Ser Pro
145 150 155 160
Val Gln Val Phe Ser Ile Asp Gly Ala Gln Thr Leu Thr Leu Asp His
165 170 175
Ile Thr Ile Asp Asn Ser Ala Gly Asp Ser Gln Gly Gly His Asn Thr
180 185 190
Asp Cys Phe Asp Val Gly Asp Ser Asn Gly Val Thr Ile Ser Asn Ala
195 200 205
Ile Cys Gln Asn Gln Asp Asp Cys Leu Ala Val Asn Ser Gly Thr Asn
210 215 220
Ile Ile Phe Thr Gly Gly Thr Cys Ser Gly Gly His Gly Leu Ser Ile
225 230 235 240
Gly Ser Val Gly Gly Arg Ser Asp Asn Thr Val Gln Thr Val His Ile
245 250 255
Glu Asn Ser Thr Ile Lys Asn Ser Gln Asn Gly Val Arg Ile Lys Cys
260 265 270
Val Tyr Gly Ala Thr Gly Ser Val Ser Gly Val Thr Tyr Lys Asp Ile
275 280 285
Thr Leu Ser Gly Ile Thr Lys Tyr Gly Ile Val Ile Glu Gln Asp Tyr
290 295 300
Glu Asn Gly Ser Pro Thr Gly Thr Pro Thr Asn Gly Cys Pro Ile Thr
305 310 315 320
Gly Leu Thr Ile Asp Gly Val Thr Gly Ser Val Thr Ser Ser Ala Thr
325 330 335
Glu Val Tyr Ile Leu Cys Gly Ser Gly Ser Cys Ser Asn Trp Thr Trp
340 345 350
Gln Gly Val Asn Ile Thr Gly Gly Lys Lys Ser Ser Ser Cys Lys Asn
355 360 365
Val Pro Ser Gly Ala Ser Cys
370 375
<210> 2
<211> 375
<212> PRT
<213> Talaromyces sp.
<400> 2
Met Arg Ser Phe Ile Gly Leu Ala Gly Leu Ala Cys Leu Ser Ser Val
1 5 10 15
Val Leu Ala Val Pro Ala Pro Ala Pro Thr Ala Pro Ala Lys Leu Glu
20 25 30
Asp Arg Ala Ala Ser Cys Thr Phe Ser Gly Ser Asn Gly Ala Ala Ala
35 40 45
Ala Ser Lys Ser Lys Thr Ser Cys Ser Thr Ile Val Leu Ser Ser Val
50 55 60
Ala Val Pro Ser Gly Thr Thr Leu Asp Leu Thr Asn Leu Asn Asp Gly
65 70 75 80
Thr Lys Val Ile Phe Glu Gly Thr Thr Thr Phe Gly Tyr Ala Glu Trp
85 90 95
Ser Gly Pro Leu Val Ser Val Ser Gly Asn Asp Ile Thr Val Glu Gly
100 105 110
Ala Ser Gly Ala Val Leu Asp Gly Asp Gly Ser Arg Trp Trp Asp Gly
115 120 125
Lys Gly Ser Asn Gly Gly Lys Thr Lys Pro Lys Phe Phe Tyr Ala His
130 135 140
Asn Leu Lys Ser Ser Ser Ile Ser Asn Ile Lys Ile Leu Asn Ser Pro
145 150 155 160
Val Gln Val Phe Ser Ile Asp Gly Ala Gln Thr Leu Thr Leu Asp His
165 170 175
Ile Thr Ile Asp Asn Ser Ala Gly Asp Ser Gln Gly Gly His Asn Thr
180 185 190
Asp Ala Phe Asp Val Gly Asp Ser Asn Gly Val Thr Ile Ser Asn Ala
195 200 205
Ile Val Gln Asn Gln Asp Asp Cys Leu Ala Val Asn Ser Gly Thr Asn
210 215 220
Ile Ile Phe Thr Gly Gly Thr Cys Ser Gly Gly His Gly Leu Ser Ile
225 230 235 240
Gly Ser Val Gly Gly Arg Ser Asp Asn Thr Val Gln Thr Val His Ile
245 250 255
Glu Asn Ser Thr Ile Lys Asn Ser Gln Asn Gly Val Arg Ile Lys Thr
260 265 270
Val Tyr Gly Ala Thr Gly Ser Val Ser Gly Val Thr Tyr Lys Asp Ile
275 280 285
Thr Leu Ser Gly Ile Thr Lys Tyr Gly Ile Val Ile Glu Gln Asp Tyr
290 295 300
Glu Asn Gly Ser Pro Thr Gly Thr Pro Thr Asn Gly Val Pro Ile Thr
305 310 315 320
Gly Leu Thr Ile Asp Gly Val Thr Gly Ser Val Thr Ser Ser Ala Thr
325 330 335
Glu Val Tyr Ile Leu Cys Gly Ser Gly Ser Cys Ser Asn Trp Thr Trp
340 345 350
Gln Gly Val Asn Ile Thr Gly Gly Lys Lys Ser Ser Ser Cys Lys Asn
355 360 365
Val Pro Ser Gly Ala Ser Cys
370 375
<210> 3
<211> 1128
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgcgttctt tcatcggtct ggcgggtctg gcgtgcctgt cttctgttgt tctggcggtt 60
ccggcgccgg cgccgaccgc gccggcgaaa ctggaagacc gtgcggcgtc ttgcaccttc 120
tctggttcta acggtgcggc ggcggcgtct aaatctaaaa cctcttgctc taccatcgtt 180
ctgtcttctg ttgcggttcc gtctggtacc accctggacc tgaccaacct gaacgacggt 240
accaaagtta tcttcgaagg taccaccacc ttcggttacg cggaatggtc tggtccgctg 300
gtttctgttt ctggtaacga catcaccgtt gaaggtgcgt ctggtgcggt tctggacggt 360
gacggttctc gttggtggga cggtaaaggt tctaacggtg gtaaaaccaa accgaaattc 420
ttctacgcgc acaacctgaa atcttcttct atctctaaca tcaaaatcct gaactctccg 480
gttcaggttt tctctatcga cggtgcgcag accctgaccc tggaccacat caccatcgac 540
aactctgcgg gtgactctca gggtggtcac aacaccgacg cgttcgacgt tggtgactct 600
aacggtgtta ccatctctaa cgcgatcgtt cagaaccagg acgactgcct ggcggttaac 660
tctggtacca acatcatctt caccggtggt acctgctctg gtggtcacgg tctgtctatc 720
ggttctgttg gtggtcgttc tgacaacacc gttcagaccg ttcacatcga aaactctacc 780
atcaaaaact ctcagaacgg tgttcgtatc aaaaccgttt acggtgcgac cggttctgtt 840
tctggtgtta cctacaaaga catcaccctg tctggtatca ccaaatacgg tatcgttatc 900
gaacaggact acgaaaacgg ttctccgacc ggtaccccga ccaacggtgt tccgatcacc 960
ggtctgacca tcgacggtgt taccggttct gttacctctt ctgcgaccga agtttacatc 1020
ctgtgcggtt ctggttcttg ctctaactgg acctggcagg gtgttaacat caccggtggt 1080
aaaaagtctt cttcttgcaa aaacgttccg tctggtgcgt cttgctaa 1128
<210> 4
<211> 1128
<212> DNA
<213> Talaromyces sp.
<400> 4
atgcgttcct tcattggcct ggccgggctg gcctgcttgt catcagtggt gctggccgtc 60
ccagcgccag ctcctacagc acctgctaag ttagaagatc gtgccgcgtc ttgcacattc 120
tctggctcca acggtgcagc agcagctagc aaatccaaga cttcgtgctc aaccattgta 180
ttgtcgagtg ttgctgtgcc gtcgggaacg acacttgacc taactaatct taacgatgga 240
acgaaggtca tcttcgaagg cacaaccact ttcggctacg cagagtggtc aggtcccctt 300
gtctccgtct caggaaacga catcaccgtc gaaggagcct ctggagctgt gctcgacggt 360
gacggatctc gttggtggga cggcaagggc agcaacggag gcaagacgaa gcccaagttc 420
ttttatgctc acaacctcaa gtcctcgtct atcagcaaca tcaagatcct gaactcgcct 480
gtgcaggtct tcagcattga tggtgctcag acactgactt tggaccatat cacaattgac 540
aattcggctg gtgactctca aggtggccat aatacagatg cgtttgatgt tggtgattcg 600
aatggggtta ctatctcgaa tgctattgtt cagaatcagg atgattgttt ggcagtcaac 660
tctggcacta acatcatttt cactggcggt acctgctccg gtggtcatgg tttgtccatt 720
ggctctgttg gcggccgttc tgataacacg gtccagaccg ttcacattga gaactctact 780
atcaagaact ctcaaaacgg cgtccgcatc aaaaccgtct atggcgccac cggctccgtc 840
tccggcgtaa cctacaagga catcacactc tccggcatca ccaaatacgg catcgtcatt 900
gagcaggact acgagaacgg cagccctaca ggaacaccta ctaacggcgt cccaattact 960
ggccttacca ttgatggtgt aaccggtagc gtcactagca gcgctacaga agtctatatc 1020
ctctgcggca gtggtagctg ctccaactgg acttggcagg gcgttaatat taccggtggt 1080
aagaagagta gcagttgtaa gaatgttcct agtggtgcta gctgctaa 1128

Claims (10)

1. a kind of high temperature resistant acidic pectase TsPec, which is characterized in that the amino acid sequence of the pectase TsPec contains SEQ Amino acid sequence shown in ID NO:1, also, 77 and 81 half Guang ammonia in amino acid sequence shown in SEQ ID NO:1 Acid forms disulfide bond, and 194 and 210 cysteines form disulfide bond, and 272 and 317 cysteines form two sulphur Key.
2. pectase TsPec according to claim 1, which is characterized in that the amino acid sequence of the pectase TsPec is Amino acid sequence shown in SEQ ID NO:1, also, in amino acid sequence shown in SEQ ID NO:1 77 and 81 half Cystine forms disulfide bond, and 194 and 210 cysteines form disulfide bond, and 272 and 317 cysteines are formed Disulfide bond.
3. pectase TsPec according to claim 2, which is characterized in that the pectase TsPec is led to by pectase Pec It crosses and redesigns and introduce disulfide bond and obtain, the amino acid sequence of the pectase Pec is as shown in SEQ ID NO:2.
4. a kind of pectin enzyme gene TsPec, special for encoding pectase TsPec described in any one of claim 1-3 Sign is that base sequence shown in the base sequence and SEQ ID NO:3 of the pectin enzyme gene TsPec is with 95% or more Identity.
5. pectin enzyme gene TsPec according to claim 4, which is characterized in that the base of the pectin enzyme gene TsPec Sequence contains base sequence shown in SEQ ID NO:3.
6. pectase TsPec according to claim 5, which is characterized in that the base sequence of the pectin enzyme gene TsPec For base sequence shown in SEQ ID NO:3.
7. a kind of recombinant expression carrier, which is characterized in that it includes pectin enzyme gene described in any one of claim 4-6 TsPec。
8. a kind of recombinant strains, which is characterized in that its expression product is pectin described in any one of claim 1-3 Enzyme TsPec；The host cell of the recombinant strains is Pichia pastoris.
9. the application of pectase TsPec described in any one of claim 1-3.
10. a kind of method of hydrolysis of pectin, which is characterized in that this method comprises: by described in any one of claim 1-3 Pectase TsPec is contacted with material containing pectin.
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