CN110846294B

CN110846294B - Recombinant pectinase, gene thereof, recombinant vector, preparation method and application

Info

Publication number: CN110846294B
Application number: CN201911197324.8A
Authority: CN
Inventors: 李洪波; 李露露; 何伶靖; 邓伟思; 董海丽
Original assignee: Huaihua University
Current assignee: Huaihua University
Priority date: 2019-11-29
Filing date: 2019-11-29
Publication date: 2022-05-17
Anticipated expiration: 2039-11-29
Also published as: CN110846294A

Abstract

The invention relates to a recombinant pectinase, a gene, a recombinant vector, a preparation method and application thereof, wherein the gene at least contains a DNA sheet of one of the following nucleotide sequences: 1) the nucleotide sequence of SEQ ID NO.1 in the sequence table; 2) a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function; or 3) a nucleotide sequence which is hybridized with the nucleotide sequence shown in SEQ ID NO.1 and encodes the protein with the same biological function. According to the gene sequence, a recombinant vector is further constructed and yeast is transformed, constitutive secretory expression of the recombinant pectinase under a high-density fermentation condition can be realized, active protein with the purity higher than 95% and higher protein concentration can be obtained through simple nickel affinity purification, the active protein has strong pectin hydrolysis activity at low temperature, and a novel and efficient enzyme product is provided for improving the juice yield of fruit juice.

Description

Recombinant pectinase, gene thereof, recombinant vector, preparation method and application

Technical Field

The invention belongs to the technical field of biomolecule cloning, and relates to a recombinant pectinase, a gene, a recombinant vector, a preparation method and application thereof.

Background

China is vast in vast, and the land area of China is about 960 ten thousand square kilometers, spans three areas of south and north cold zone, temperate zone and subtropical zone, and is suitable for the growth of various fruit trees, so China is a large world for fruit production, for example: hawthorn, apple, pear, peach, apricot, grape, wild jujube, sea buckthorn, blackcurrant, kiwi, mandarin, orange, pineapple, banana, guava, roxburgh rose, passionflower, and the like. In the last decade, the total fruit yield in China is kept above four thousand five million tons. The fruit resources are rich, and a good foundation is provided for the development of the fruit juice industry in China. At present, the market competition of the fruit juice is intense, the brands are numerous, a plurality of processing enterprises are in a predicament, and how to develop the fruit juice industry becomes a problem worthy of attention. How to improve the juice yield of the juice is the first major problem in juice production. Therefore, in industrial production, a certain amount of pectinase is often added to improve the juice yield. Pectinase can act to break down pectin, disrupt the cell wall and intercellular layer of the plant, and make it easier to extract juice. However, industrial pectinase is a mixture of multiple enzymes, and the addition of pectinase can increase the juice yield and bring some adverse effects, such as easy change of flavor and quality. Therefore, the development of a pure single-component pectinase with high activity is urgently needed.

In previous studies by the present inventors, a novel highly active pectinase derived from poria cocos was found, but the expression level of the enzyme in poria cocos is very low, and the enzyme cannot be obtained in large quantities by fermentation of poria cocos. Therefore, the high-efficiency expression of the novel recombinant pectinase protein by using a foreign gene expression system is a necessary way for development and application of the novel recombinant pectinase protein.

The production of recombinant pectinase by using an exogenous gene expression system is one of the main technical means for obtaining pectinase in large quantities. Currently, many expression systems have been developed such as: baculovirus expression systems, prokaryotic expression systems, yeast expression systems, filamentous fungal expression systems, insect cell expression systems, plant expression systems, mammalian expression systems, and the like. The genetic background of the escherichia coli is clear, and the escherichia coli becomes a preferred expression system of the exogenous gene due to the characteristics of short period, high efficiency, easy operation, safe use and the like. Coli is expressed in BL21(DE3) after transformation, but all obtained inclusion bodies are inactive, and soluble protein which can only be obtained by dozens to hundreds of micrograms per liter of culture medium can be obtained by dissolving, denaturing, renaturing and purifying in vitro under proper conditions. In Escherichia coli, toxic and side effects may be caused due to the presence of LPS, so that the toxicity of the expressed and purified product is often required to be analyzed and determined. Finally, to obtain a high purity protein, it is usually required to go through multiple purification steps, and the more purification steps, the lower the yield of the protein, and the more likely it is to cause inactivation of the target product, so the expression system is not suitable for mass production of recombinant pectinase. Exogenous gene expression systems such as insect cell expression systems, plant expression systems, mammalian expression systems and the like have high requirements on technology, equipment and technical level, so that the produced products are often very expensive and are not suitable for mass and low-cost production of pectinase. The methanol yeast expression system is the most widely applied yeast expression system, and the exogenous gene expression system taking Pichia pastoris as a host develops most rapidly in recent years and is most widely applied. The pichia pastoris system is widely used because the system has the most remarkable advantages besides the characteristics of common yeasts: by screening transformants for high-level secretory expression and optimizing the expression conditions, recombinant proteins can be produced and prepared inexpensively and on a large scale.

The invention obtains a transformant which can realize constitutive high-level secretory expression in pichia pastoris by repeatedly optimizing genes and changing expression vectors and strains, the transformant realizes the high-level expression and purification of the novel recombinant pectinase protein under the high-density culture condition of a fermentation tank, and the purified recombinant pectinase protein can improve the juice yield of fruits and has important application and development values.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a preparation method and application of novel recombinant pectinase for improving the juice yield of fruit juice.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a recombinant pectinase, the sequence of which comprises an amino acid sequence shown in SEQ ID NO. 2.

In a further improvement, the sequence of the recombinant pectinase is an amino acid sequence shown as SEQ ID NO. 2.

A recombinant pectinase gene, which comprises a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 2.

Further improvement, the similarity of the gene and the amino acid sequence shown in SEQ ID NO.1 is more than or equal to 90 percent

In a further improvement, the nucleotide sequence of the gene is SEQ ID NO. 1.

In a further improvement, the vector is pGAPZ alpha vector, and the pGAPZ alpha vector contains a nucleotide sequence SEQ ID NO. 1.

A preparation method of recombinant pectinase comprises the following steps:

step one, recombining and constructing a nucleotide sequence of an amino acid shown as SEQ ID NO.2 into a pGAPZ alpha vector, then transforming the pGAPZ alpha vector into pichia pastoris host bacteria, and screening a transformant of high-expression recombinant pectinase by using Zeocin;

and step two, performing high-density fermentation on the transformant in a bioreactor to obtain supernatant containing recombinant pectinase protein.

Further improvement, the method also comprises the third step of protein purification: purifying the supernatant obtained in the second step by using a nickel affinity chromatography column: the column was equilibrated with equilibration buffer, the supernatant was passed through the column, the column was pre-washed with 30mM imidazole in pH 7.4 buffer, and the fusion protein was eluted with 200mM imidazole in pH 7.4 buffer.

In the high-density fermentation, the transformant of the high-expression recombinant pectinase is cultured by an YPG culture medium until the OD600 is 10-15, and the transformant is used as a seed bacterium, and the seed bacterium is cultured according to the ratio of 1: 10 in proportion to the culture medium containing inorganic salt; continuously carrying out induction culture at 28 ℃ for 72-96 hours and supplementing 70% of glycerol as a carbon source; the pH was adjusted to 4.0 with concentrated ammonia.

The recombinant pectinase is used for improving the juice yield of fruit juice, constructing a recombinant vector, constructing a recombinant cell or constructing a kit.

The technical scheme provided by the invention has the following advantages:

firstly, the recombinant pectinase with bioactivity obtained by secretory expression and purification according to the expression method of the technical scheme can effectively prevent host bacteria from degrading expression products, and reduce the metabolic load of host cells and the toxic effect of the expression products on the hosts; secondly, the secretion signal alpha-factor signal peptide on the yeast vector pGAPZ alpha A-recombinant pectinase is utilized to guide the gene secretion expression of the target protein, the target protein can be secreted into the culture solution in a large amount, and an accurate space structure can be formed, so that the natural activity of the recombinant pectinase is maintained; thirdly, obtaining a stable yeast transformant capable of secreting and expressing the recombinant pectinase at high level by screening; fourthly, a method for expressing the recombinant pectinase by using eukaryotic host pichia pastoris and a method for quickly and efficiently purifying the recombinant pectinase are researched and explored, so that the cost can be reduced and mass production can be realized; sixthly, a constitutive expression vector is adopted, the carbon source added in the thallus culture process is glycerol which is a non-toxic and harmless substance to a human body, the biological safety of the product is ensured, and the fermented supernatant can be directly used without purification. The invention prepares a recombinant pectinase by optimizing a gene and utilizing a pichia foreign gene expression system for the first time, and the recombinant pectinase can be added into the production of grape, apple and orange juice to obviously improve the yield of the juice. The successful preparation of the recombinant pectinase has important application and development prospects in the production field of improving the juice yield of fruit juice.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a schematic diagram of the construction of an expression vector pGAPZ alpha A-recombinant pectinase according to an embodiment of the invention.

FIG. 2 is a SDS-PAGE graph of supernatants from yeast transformant cultures with high Zeocin resistance recombinant pectinase enzymes according to this invention.

FIG. 3 is a SDS-PAGE graph showing the expression of target proteins at different time points under high-density fermentation conditions in the examples of the present invention.

FIG. 4 is a SDS-PAGE result of recombinant pectinase protein eluted and purified by 200mM imidazole according to the present invention.

FIG. 5 is a SDS-PAGE detection result of the recombinant pectinase protein purified according to the example of the invention.

FIG. 6 is a photograph showing the results of SDS-PAGE detection of the target protein in the supernatant of the transformant of the comparative yeast.

FIG. 7 is a graph showing the results of mass spectrometric identification of purified recombinant pectinase protein.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples,% is by mass unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.

The Pichia pastoris strain and the integrative expression plasmid pGAPZ alpha A are both purchased from Invitrogen corporation, USA.

The formula of the culture medium is as follows:

1) YPD medium

Completely dissolving 10g of yeast extract and 20g of peptone, diluting to 900mL, autoclaving with steam at 121 deg.C for 15-20min, cooling to about 70 deg.C, and adding 100mL of 20% sterilized glucose solution. YPD solid medium can be prepared by adding 1.5-1.8% agar thereto.

2) YPG medium

Completely dissolving 10g of yeast extract, 20g of peptone and 20g of glycerol, diluting to 1000mL, and autoclaving at 121 deg.C for 15-20 min.

3) Inorganic salt culture medium

Each liter of culture solution contains 6 g of potassium sulfate, 5g of magnesium sulfate, 1 g of potassium hydroxide, 7 ml of concentrated phosphoric acid, 30 g of glycerol and 0.3 g of calcium sulfate, ammonia water is used for adjusting the pH to 4.5, and after moist heat sterilization and cooling to a room, 5 ml of trace element solution is added according to each liter of culture medium during inoculation.

4) Solution of trace elements

Each liter of the trace element solution comprises: 65g of FeSO₄·7H₂O,24g MoNa₂O₄·2H₂O,20g ZnCl₂,6g CuSO₄·5H₂O,3g MnSO₄·H₂O,0.5g CoCl₂0.2g of biotin, 0.09g of KI,0.02g H₃BO₃And 5.0mL of concentrated H₂SO₄After filtration through a 0.22 μm bacterial filter, the cells were stored in a refrigerator at 4 ℃ until use.

Example 1

The embodiment provides an optimized artificially synthesized recombinant pectinase gene with a 6 XHis tag at the C-terminal, the specific sequence of which is shown as SEQ ID No.1 in a sequence table, and the protein sequence corresponding to the gene is shown as SEQ ID No.2 in the sequence table. The optimized DNA sequence has no obvious similarity through NCBI comparison.

The DNA sequence shown in SEQ ID No.1 synthesized according to the sequence characteristics of pectinase gene and the preference of yeast codon, the natural DNA of pectinase before optimization and the artificial DNA sequence synthesized after optimization according to the preference of escherichia coli codon are respectively connected to a pichia pastoris secretory expression vector pGAPZ alpha A to obtain recombinant vectors, then the recombinant vectors are respectively transformed into pichia pastoris host bacteria X-33 by a lithium chloride transformation method provided by an operation manual of Invitrogen company, YPD plates containing 200 mug/mL Zeocin antibiotics are respectively used for screening after transformation, the transformants are verified by PCR, the pichia pastoris transformants after PCR verification are respectively streaked and respectively inoculated to YPD plates with the final concentration of 2000 mug/mL Zeocin antibiotics, the transformants are respectively screened to obtain high-resistance pichia pastoris, then 50 mL centrifuge tubes containing 1mL YPG culture solution are respectively used for culturing high-resistance transformants, culturing at 28 deg.C and 250rpm to OD₆₀₀And (3) centrifuging to obtain a supernatant, adding 40 mu l of the supernatant into 10 mu l of 5-fold loading buffer, denaturing, performing electrophoretic analysis by using 12% SDS-PAGE, dyeing and decoloring, and determining a transformant for expressing the recombinant pectinase at the highest level according to the expression condition of the target protein. Analysis results show that the pichia pastoris transformant constructed by the natural DNA of the recombinant pectinase before optimization and the artificial DNA sequence synthesized after optimization according to the codon preference of escherichia coli can hardly detect the expression of the recombinant protein, and the transformant screened by the DNA sequence synthesized by the sequence characteristics of the gene and the codon preference of yeast, namely the SEQ ID No.1 construction vector, has very high expression level of the recombinant protein.

Example 2

This example provides a method for preparing a protein, comprising the steps of:

s1: constructing an expression vector and transforming: the DNA sequence synthesized by the self sequence characteristics of the gene and the preference of yeast codons, namely the DNA in SEQ ID No.1, in the embodiment 1 is connected to a pichia pastoris constitutive secretory expression vector pGAPZ alpha A to obtain a recombinant vector pGAPZ alpha A-recombinant pectinase, the vector construction is shown as a figure 1, and the figure 1 is a schematic diagram of the eukaryotic expression vector pGAPZ alpha A-recombinant pectinase construction in the embodiment of the invention. The main vector construction steps are preferably as follows:

(1) the plasmid containing the synthesized recombinant pectinase gene (SEQ ID No.1) was digested with Xho I and Xba I to obtain the desired fragment in the following reaction system (all the enzymes and buffers were purchased from Takara, Dalian company):

(2) pGAPZ alpha A was double-digested with Xho I and Xba I to obtain vector fragments in the following reaction system (both the endonuclease and the buffer were purchased from Dalian TAKARA Co.):

(3) the target fragment and the vector fragment obtained in steps (1) and (2) were recovered by using a DNA gel retrieval kit purchased from Dalian TAKARA, and the detailed procedures were carried out according to the kit instructions.

(4) The target fragment and the vector recovered in the step (3) are connected by T4DNA ligase (purchased from TaKARA company), the target gene is accurately inserted into the reading frame of the secretory vector containing a secretory signal alpha-factor, and the reaction system is as follows:

s2: transformation of recombinant plasmid: the recombinant vector pGAPZ alpha A-recombinant pectinase is linearized by Sac I single enzyme digestion, and the recombinant vector is transformed into a pichia host strain according to a lithium chloride transformation method provided by an operation manual of Invitrogen company, wherein X-33 is selected in the embodiment. After transformation, YPD plates containing 200. mu.g/mL Zeocin antibiotic were used for selection, and transformants were verified by PCR.

S3: screening of high-level secretion expression yeast transformants and expression of proteins: the Pichia pastoris transformant after PCR verification is streaked and inoculated to a YPD plate containing 2000 mug/mL Zeocin, the transformant with high Zeocin resistance is obtained by screening, the transformant with high Zeocin resistance is cultured by a 50 mL centrifuge tube containing 1mL YPG culture solution, and the culture is carried out at 28 ℃ and 250rpm until OD is OD₆₀₀And (3) centrifuging to obtain a supernatant, adding 40 mu l of the supernatant into 10 mu l of 5-fold loading buffer, denaturing, performing electrophoretic analysis by using 12% SDS-PAGE, dyeing and decoloring, and determining a transformant for expressing the recombinant pectinase at the highest level according to the expression condition of the target protein. Culturing the transformant with high-expression recombinant pectinase to OD by using YPG medium₆₀₀10-15, and treating the seed bacterial liquid according to the ratio of 1: 10 by volume into a fermenter containing a mineral salts medium.

S4: continuously carrying out induction culture for 96 hours at 28 ℃, supplementing 60 percent V/V of glycerol as a carbon source, wherein the glycerol supplementation rate is in series connection with dissolved oxygen, and the dissolved oxygen of a fermentation tank is set to be 40 percent; the pH was adjusted with concentrated ammonia, setting the pH of the fermentor to 4.5.

It should be noted that, the transformants with high Zeocin resistance are expressed in a small amount by YPG medium, and 3 yeast transformants which stably and highly secrete recombinant pectinase are selected from 3 transformants with high resistance (the resistance level is 2000. mu.g/mL Zeocin) by SDS-PAGE analysis, while the ability of secreting and expressing the target protein from 50 transformants with resistance level lower than 500. mu.g/mL Zeocin YPD plate is significantly lower than that of the transformants with high resistance, and the SDS-PAGE result of the part of the transformants with high Zeocin resistance secreting the target protein is shown in FIG. 2.

It should also be noted that the expression of the target protein at different times in the culture conditions supplemented with glycerol in the fermentor is analyzed by SDS-PAGE, and the electrophoresis results are shown in FIG. 3, and FIG. 3 is a graph of the SDS-PAGE detection results of the expression of the target protein at different time points in the examples of the present invention. After culturing for 1-4 days, the total amount of the target protein obtained by culturing is higher by continuously supplementing glycerol, and the concentration result of the total protein in the specific supernatant is shown in the following table 1.

TABLE 1 Total protein content

Induction time	1 day	2 days	3 days	4 days
					Total protein concentration (mg/L)	320	810	1780	2200

Taking the supernatant of the fermentation liquid cultured for 1-4 days, performing SDS-PAGE, wherein the supernatant has obvious target protein expression at about 55kDa, and continuously adding glycerol to obtain a higher total amount of the target protein by culture, wherein the grayscale scanning result of SDS-PAG gel shows that the proportion of the target protein is inversely proportional to the culture time, and the proportion result of the target protein in the total protein of the supernatant is shown in the following table 2.

TABLE 2 results of percentage of target protein in supernatant protein obtained at different induction times

Induction time	1 day	2 days	3 days	4 days
					Percentage content (%)	16	21	38	37

The results of calculating the content of the target protein are shown in the following table 3.

TABLE 3 Total amount of target protein

Induction time	1 day	2 days	3 days	4 days
					Recombinant pectinase (mg/L)	51	170	676	810

Preferably, after step S4, the following steps of purifying the protein are also included:

s5: centrifuging the culture solution after S4 fermentation, taking supernate, adjusting the pH of the supernate to 7.5-8.5 by using Tris alkali, centrifuging the supernate for 10-20 minutes at a rotating speed of more than or equal to 15000g, adding the obtained supernate into a nickel affinity chromatography column balanced by a pH 7.4 Tris-HCl buffer solution, and rinsing the nickel affinity chromatography column by using a buffer solution which is 2-4 times of the volume of the chromatography column and contains 10mM Tris-HCl and 30mM imidazole and has a pH of 7.4;

s6: the nickel affinity chromatography column was eluted with a buffer containing 10mM Tris-HCl and 200mM imidazole, and the SDS-PAGE result of the eluted protein is shown in FIG. 4, from which it can be seen that the target protein has a very high purity, indicating that the recombinant pectinase gene of the present invention can be used to purify the target protein simply by nickel affinity chromatography. The resulting eluate was dialyzed against 10mM Tris-HCl buffer using a dialysis bag having a molecular weight of 10kDa, followed by concentration by ultrafiltration. Table 4 shows comparison of protein purification in the supernatant of fermentation broth and each purification stage.

TABLE 4 comparison of supernatant protein purification in fermentation broths

Preferably, after steps S5 and/or S6, the method further comprises the following steps of preserving the protein:

s7: and (3) quickly freezing the liquid containing the target protein at-80 ℃, and then freeze-drying to obtain the freeze-dried powder protein. And dissolving the freeze-dried powder in physiological saline, centrifuging at the rotating speed of 15000g for 20 minutes at 4 ℃, and taking the supernatant to perform SDS-PAGE analysis, wherein the result is shown in figure 5, the target protein is detected and has high purity, which indicates that the protein treatment method can not enable a large amount of protein denaturation to be still in a soluble state. The purified recombinant endonuclease protein is subjected to mass spectrum identification by using Nano-LC-MS/MS, 5 sequences are obtained in total, the sequences and one segment of the sequence 2 provided by the invention belong to, the detected secondary mass spectrum result and the matched sequence of one segment are shown in figure 6, and the detected peptide segment sequence is as follows: DIGPAIGKAFS, the sequence is just one segment of the full-length sequence. This result indicates that the purified protein is the target protein.

Comparative example

The expression profile of the pectinase gene of tuckahoe is analyzed by using the transcriptome technology to find the gene. Using the data obtained by the transcriptome, designing a primer, amplifying a target gene by RT-PCR and connecting the target gene to a cloning vector, wherein the natural sequence of the amplified target gene is shown as SEQ ID NO.3 in a sequence table; similarly, artificial DNA sequences synthesized after optimization according to the codon preference of escherichia coli are synthesized and are respectively connected to pichia pastoris secretory expression vectors pGAPZ alpha A, wherein one artificial sequence synthesized after optimization according to the codon preference of escherichia coli is shown as SEQ ID No.4 in a sequence table (represented by the sequence). The above recombinant pectinase gene sequence was double-digested with Xho I and Xba I and ligated to pGAPZ. alpha.A expression vector, which was also double-digested with Xho I and Xba I. The recombinant vector pGAPZ alpha A-recombinant pectinase is linearized by Sac I single enzyme digestion, transformed into a pichia host bacterium by a lithium chloride transformation method, and screened by Zeocin to obtain positive clone. The Pichia pastoris transformant after PCR verification is streaked and inoculated to a YPD plate containing 1000 mug/mL Zeocin, the transformant with high Zeocin resistance is obtained by screening, the transformant with high Zeocin resistance is cultured by a 50 mL centrifuge tube containing 1mL YPG culture solution, and the culture is carried out at 28 ℃ and 250rpm until OD is OD₆₀₀And (3) centrifuging to obtain a supernatant, adding 40 mu l of the supernatant into 10 mu l of 5-fold loading buffer, denaturing, performing electrophoretic analysis by using 12% SDS-PAGE, dyeing and decoloring, and determining a transformant which expresses the recombinant pectinase at the highest level according to the expression condition of the target protein. The results of SDS-PAGE are shown in FIG. 6, and no band of the target protein is detected at the target site. The results show that only the DNA sequences provided by the invention and synthesized according to the sequence characteristics of the recombinant pectinase gene and the yeast codon preferenceThe sequence shown in SEQ ID NO.1 is transformed into pichia pastoris to realize high-level secretory expression of the target protein.

Example 3

This example examined the enhanced apple, grape and orange juice yields of purified recombinant pectinase. The enzyme can improve the yield of the fruit juice, and the specific steps and results are as follows:

(1) adding 0, 1, 2, 4, 8, 16 and 32 mg of recombinant pectinase into 100 g of chopped apples respectively, fully pulping the apples added with the recombinant pectinase with different concentrations into pulp by using a juicer, and standing for 60 minutes at room temperature; the apple pulp was poured into 6 layers of gauze, the juice was squeezed out of the gauze until no juice was flowing out, and weighed separately. The weighed fruit juice weights are shown in table 5, and it can be seen that the fruit juice weights obtained by the experimental group added with pectinase are all higher than the fruit juice weight produced without adding recombinant pectinase, and when 32 mg of recombinant pectinase is added into 100 g of cut apples, the juice yield is about 10% higher than that without adding recombinant pectinase, which indicates that the recombinase can effectively improve the fruit juice production rate of the apples.

TABLE 5 Effect of recombinant pectinase on apple juice yield

Amount of enzyme added (mg)	0	1	2	4	8	16	32
								Weight of fruit juice (g)	60.5	60.7	60.7	61.3	63.2	64.9	66.7

(2) Adding 0, 1, 2, 4, 8, 16 and 32 mg of recombinant pectinase into 100 g of grapes respectively, fully pulping the grapes added with the recombinant pectinase with different concentrations into pulp by using a juicer, and standing for 60 minutes at room temperature; the grape pulp was poured into 6 layers of gauze, the juice was squeezed out of the gauze until no juice was flowing out, and weighed separately. The weighed weight of the juice is shown in table 6, and it can be seen that the weight of the juice obtained by the experimental group added with pectinase is higher than that of the juice produced without adding recombinant pectinase, and when 32 mg of recombinant pectinase is added to 100 g of grapes, the yield of the juice is about 7% higher than that of the juice produced by the grapes without adding recombinant pectinase, which indicates that the recombinase can effectively improve the juice production rate of the grapes.

TABLE 6 Effect of recombinant pectinase on grape juice yield

Amount of enzyme added (mg)	0	1	2	4	8	16	32
								Weight of fruit juice (g)	71.3	71.5	71.9	72.6	73.8	74.9	76.2

(3) Adding 0, 1, 2, 4, 8, 16 and 32 mg of recombinant pectinase into 100 g of navel orange pulp, fully pulping grapes added with the recombinant pectinase with different concentrations into pulp by using a juicer, and standing for 60 minutes at room temperature; pouring the navel orange pulp into 6 layers of gauze respectively, extruding the fruit juice from the gauze until no fruit juice flows out, and weighing respectively. The weighed fruit juice weights are shown in table 7, and it can be seen that the fruit juice weights obtained by the experimental group added with pectinase are all higher than the fruit juice weight produced without adding recombinant pectinase, and when 32 mg of recombinant pectinase is added into 100 g of navel orange, the yield of the juice is about 5% higher than that of the juice without adding recombinant pectinase, which indicates that the recombinant enzyme can effectively improve the fruit juice production rate of grapes.

TABLE 7 Effect of recombinant pectinase on grape juice yield

Amount of enzyme added (mg)	0	1	2	4	8	16	32
								Weight of fruit juice (g)	77.2	77.5	78.1	78.6	79.4	79.9	81.2

Although the embodiments of the present invention have been shown and described above, it is understood that the above preferred embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail through the above preferred embodiments, those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention, which should be covered in the scope of the claims and the specification of the present invention.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and those skilled in the art can make changes, modifications, substitutions and alterations to the above embodiments within the scope of the present invention without departing the essence of the corresponding embodiments from the scope of the embodiments of the present invention, which should be covered in the claims and the specification of the present invention.

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caatactggt cctctcaatc cactgctcca ggtaatggtg ttcagttgaa ggacatcact 900

ttctccaact gggacggttt cattactgac ggtgctagaa gggctccaat ccaggttttg 960

tgtgctgatg gtgctccatg taccgacatc aacatcaaca acgtcaactt gtgggctgct 1020

aacaaccagg ctaccaacaa gtgtagatcc gcttacggta ctggtgcctg tttgaagtct 1080

ggttctggtg gatcttactc ccaggtcact aagactattt ccaagccacc agctttcact 1140

actccagcta ctatgtctgg tgacttgtct gacggtttcc caactaactc cccaattcca 1200

attcctacta tcccaccatc tttcttccct ggtactcaac cattgaaggc tttggctggt 1260

aagcatcacc atcaccatca ctaa 1284

<210> 2

<211> 427

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Gln Leu Ser Gly Ser Val Gly Pro Thr Ser Ser Leu Ser Ser Lys Gln

1 5 10 15

Gly Thr Ile Cys Asn Val Leu Asn Tyr Gly Gly Ser Val Gly Ser Ser

20 25 30

Asp Ile Gly Pro Ala Ile Gly Lys Ala Phe Ser Asp Cys Val Thr Lys

35 40 45

Ala Thr Asn Gly Ala Thr Leu Tyr Val Pro Pro Gly Asn Tyr Asn Met

50 55 60

Gln Thr Trp Gln Thr Leu Asn His Gly Thr Lys Trp Ala Phe Gln Leu

65 70 75 80

Asp Gly Val Ile Thr Arg Thr Ser Thr Thr Gly Gly Asn Met Ile Val

85 90 95

Ile Gln Asn Ala Asn Asp Phe Glu Phe Phe Ser Ser Thr Gly Lys Gly

100 105 110

Ala Ile Gln Gly Asn Gly Tyr Gln Cys Arg Asn Ala Gly Pro Arg Leu

115 120 125

Ile Arg Val Val Thr Ser Thr Asn Trp Ser Leu His Asp Ile Ile Met

130 135 140

Val Asp Ser Pro Glu Phe His Leu Val Ile Gln Asp Gly Ser Asn Gly

145 150 155 160

Glu Val Tyr Asn Thr Val Ile Arg Gly Gly Asn Leu Gly Gly Ser Asp

165 170 175

Gly Ile Asp Val Trp Gly Thr Asn Tyr Trp Ile His Asp Ile Glu Val

180 185 190

Thr Asn Arg Asp Glu Cys Val Thr Val Lys Ser Pro Ala Asn His Ile

195 200 205

Gln Val Glu Gln Ile Trp Cys Asn Gln Ser Gly Gly Ser Ala Ile Gly

210 215 220

Ser Leu Gly Ala Asn Thr Thr Ile Gln Asn Val Leu Tyr Arg Asn Val

225 230 235 240

Tyr Thr Asn Gly Gly Asn Gln Ile Phe Met Ile Lys Ser Asn Gly Gly

245 250 255

Ser Gly Thr Val Gln Asn Val Asn Leu Glu Asn Phe Ile Ala Arg Asn

260 265 270

Thr Ala Tyr Gly Leu Asp Ile Asp Gln Tyr Trp Ser Ser Gln Ser Thr

275 280 285

Ala Pro Gly Asn Gly Val Gln Leu Lys Asp Ile Thr Phe Ser Asn Trp

290 295 300

Asp Gly Phe Ile Thr Asp Gly Ala Arg Arg Ala Pro Ile Gln Val Leu

305 310 315 320

Cys Ala Asp Gly Ala Pro Cys Thr Asp Ile Asn Ile Asn Asn Val Asn

325 330 335

Leu Trp Ala Ala Asn Asn Gln Ala Thr Asn Lys Cys Arg Ser Ala Tyr

340 345 350

Gly Thr Gly Ala Cys Leu Lys Ser Gly Ser Gly Gly Ser Tyr Ser Gln

355 360 365

Val Thr Lys Thr Ile Ser Lys Pro Pro Ala Phe Thr Thr Pro Ala Thr

370 375 380

Met Ser Gly Asp Leu Ser Asp Gly Phe Pro Thr Asn Ser Pro Ile Pro

385 390 395 400

Ile Pro Thr Ile Pro Pro Ser Phe Phe Pro Gly Thr Gln Pro Leu Lys

405 410 415

Ala Leu Ala Gly Lys His His His His His His

420 425

<210> 3

<211> 1263

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

cagctgtctg ggtctgtggg gcccacatcc tcgctgtcgt cgaagcaggg caccatctgt 60

aacgttctca actatggtgg ctctgtcggt tctagtgaca tcggcccagc cattggtaaa 120

gcgttcagcg actgtgtcac caaggccacc aacggtgcta cgctctacgt gccccctggt 180

aactacaaca tgcagacttg gcagacactg aaccacggta ccaagtgggc gttccagttg 240

gacggtgtca tcacccgtac cagcaccact ggaggtaaca tgatcgtcat ccagaacgct 300

aacgacttcg agttcttctc gagcacgggc aagggtgcca tccagggtaa cggctaccag 360

tgccgcaatg ctggacctcg tctcatccgt gtggtcacgt cgactaactg gtctttgcac 420

gacatcatca tggttgactc tcccgagttc caccttgtca tccaggacgg ctcgaacggt 480

gaagtgtaca acacggtcat tcgcggagga aacctcggcg gttctgacgg tatcgacgtc 540

tggggcacaa actactggat ccacgacatc gaagtcacca accgcgacga gtgcgtcact 600

gtcaagtccc ccgcgaacca cattcaagtc gagcaaatct ggtgcaacca atccggtggc 660

tccgccattg gctcgcttgg tgccaacact accatccaga acgtgctcta ccgcaatgtg 720

tacacgaacg ggggcaacca gatctttatg atcaagtcta atggtggaag tggaacggtg 780

cagaacgtga acttggagaa cttcattgcg aggaatacgg cgtatgggtt ggatattgat 840

cagtattgga gtagccagag tactgcgccg ggcaatggtg tgcagctcaa ggacatcacc 900

ttctctaact gggacggctt catcaccgac ggtgcccgcc gcgcacccat ccaagtcctc 960

tgcgcagacg gcgccccctg cacggacatc aacatcaaca acgtcaacct ctgggccgcc 1020

aacaaccaag ccaccaacaa gtgccgtagt gcgtacggta ctggcgcctg cctcaagtcg 1080

ggcagcggcg gaagttactc gcaggtgacg aagacgatca gcaagccccc tgcgttcact 1140

actccagcaa cgatgagtgg agatttgtcg gatggcttcc cgacgaactc gccgattccg 1200

attccaacta ttccaccgtc gttcttcccc ggcacccagc cgctcaaggc tttggctgga 1260

aag 1263

<210> 4

<211> 1263

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

cagctgagcg gtagcgttgg tccgaccagc agcctgagca gcaaacaggg caccatttgt 60

aatgttctga attatggtgg tagcgtgggt agcagcgata ttggtccggc aattggtaaa 120

gcatttagcg attgtgttac caaagcaacc aatggtgcaa ccctgtatgt tccgcctggt 180

aactataata tgcagacctg gcagaccctg aatcatggca ccaaatgggc atttcagctg 240

gatggtgtta ttacccgtac cagcaccaca ggtggtaata tgattgttat tcagaacgcc 300

aacgacttcg aatttttcag cagcaccggt aaaggtgcaa ttcaaggtaa tggttatcag 360

tgtcgtaatg caggtccgcg tctgattcgt gttgttacct caaccaattg gagcctgcat 420

gacattatta tggttgacag tccggaattt catctggtta ttcaggatgg tagcaatggc 480

gaagtgtata acaccgttat tcgtggtggc aatttaggtg gtagtgatgg tattgatgtt 540

tggggcacca attattggat tcacgatatt gaagtgacca atcgtgatga atgcgttacc 600

gttaaaagtc cggcaaatca tattcaggtt gagcagattt ggtgtaatca gagcggtggt 660

tcagccattg gtagcctggg tgcaaatacc accattcaga atgttctgta tcgcaacgtt 720

tataccaacg gtggcaatca gatcttcatg attaaaagca atggtggcag cggcaccgtg 780

cagaatgtta atctggaaaa ctttattgca cgcaataccg catatggcct ggatattgat 840

cagtattgga gcagccagag caccgcacct ggtaatggtg tgcagctgaa agatattacc 900

tttagcaatt gggatggctt tattaccgat ggtgcccgtc gtgcaccgat tcaggttctg 960

tgtgcagatg gtgcaccgtg taccgatatt aacattaata acgttaatct gtgggcagcc 1020

aataatcagg caaccaataa atgtcgtagc gcctatggta caggtgcatg tctgaaaagc 1080

ggcagcggtg gtagctatag ccaggttacc aaaaccatta gcaaaccgcc tgcatttacc 1140

acaccggcaa ccatgagcgg tgatctgagt gatggttttc cgaccaatag tccgattccg 1200

attcctacca ttccgcctag cttttttccg ggtacacagc cgctgaaagc actggcaggt 1260

aaa 1263

Claims

1. A recombinant pectinase is characterized in that the amino acid sequence of the recombinant pectinase is shown as SEQ ID No. 2.

2. A gene encoding the recombinant pectinase of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 1.

3. A recombinant vector which is pGAPZ alpha vector comprising the recombinant pectinase gene according to claim 2.

4. A preparation method of recombinant pectinase is characterized by comprising the following steps:

step one, recombining and constructing a nucleotide sequence shown in SEQ ID NO.1 into a pGAPZ alpha vector, then transforming the vector into pichia pastoris host bacteria, and screening a transformant of high-expression recombinant pectinase by using Zeocin;

and step two, performing high-density fermentation on the transformant in a bioreactor to obtain supernatant containing the recombinant pectinase protein.

5. The method for preparing recombinant pectinase according to claim 4, further comprising the steps of: purifying the supernatant obtained in the second step by using a nickel affinity chromatography column: the column was equilibrated with equilibration buffer, the supernatant was passed through the column, the column was pre-washed with 30mM imidazole in pH 7.4 buffer, and the fusion protein was eluted with 200mM imidazole in pH 7.4 buffer.

6. The method for producing recombinant pectinase according to claim 4 wherein the high-density fermentation is performed by culturing the transformant that expresses the recombinant pectinase to OD using YPG medium₆₀₀10-15 as seed bacteria, and the seed bacteria are mixed according to the proportion of 1: 10 in proportion to the culture medium containing inorganic salt; continuously carrying out induction culture at 28 ℃ for 72-96 hours and supplementing 70% of glycerol as a carbon source; the pH was adjusted to 4.0 with concentrated ammonia.

7. Use of the recombinant pectinase of claim 1 for increasing the yield of fruit juice, constructing recombinant cells, or constructing a kit.