CN105695492B

CN105695492B - Pectin lyase optimized gene and expression vector and application thereof

Info

Publication number: CN105695492B
Application number: CN201610179466.1A
Authority: CN
Inventors: 张宇宏; 张伟; 刘波; 徐欣欣; 秦星
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2016-03-25
Filing date: 2016-03-25
Publication date: 2020-01-14
Anticipated expiration: 2036-03-25
Also published as: CN105695492A

Abstract

The invention discloses a pectin lyase optimized gene, an expression vector and application thereof. On the premise of not changing the amino acid sequence of the pectin lyase, the invention obtains 3 optimized genes of the pectin lyase by a plurality of sequence optimization modes including codon usage frequency and distribution, codon adaptation index CAI, change of GC content of a specific gene region and the like. Further, the invention transfers the 3 pectin lyase genes and 1 wild type gene into pichia pastoris for expression, and finally screens out the pectin lyase optimized gene which has the nucleotide sequence shown in SEQ ID No.2 and obviously improved secretion expression quantity and strongest enzyme activity in the pichia pastoris. The pectin lyase expressed by the optimized pectin lyase gene can effectively degrade pectin substances in apples, improves the juice yield, and lays a foundation for further industrial expanded production.

Description

Pectin lyase optimized gene and expression vector and application thereof

Technical Field

The invention relates to a pectin lyase gene, in particular to an optimized pectin lyase gene, a recombinant expression vector containing the optimized gene and a recombinant host cell, and also relates to application of the optimized pectin lyase gene and the recombinant expression vector in preparation of pectin lyase, belonging to the field of pectin lyase.

Background

Pectin lyase (EC 4.2.2.10) can specifically catalyze the degradation of pectin with high esterification degree. The glycosidic bond is broken at the C-4 position of the pectin by a trans-elimination mode, and simultaneously, a hydrogen atom is eliminated from the C-5 position, so that an unsaturated bond-containing product is generated. The pectin lyase is the only enzyme in pectinase which can degrade pectin with high esterification degree and does not produce methanol, and can be divided into acid pectin lyase and alkaline pectin lyase according to the acidity and alkalinity of an enzyme substrate environment. It is widely used in the food industry, textile industry, paper industry, sewage treatment containing pectin substances, etc. (Yadav, s.et. pectin lysase: a review. proc Biochem,2009,44(1), 1-10.).

The fruit juice is rich in vitamins, acids, inorganic salts and other nutrients essential to human body. Along with the emphasis of people on healthy diet, the juice is popular with people, and the market scale of the juice is continuously enlarged. However, in the production of fruit juice, since fruits are rich in a large amount of substances such as pectin and cellulose, the squeezed fruit pulp is very viscous, so that the squeezing rate of the fruit juice is low and the obtained fruit juice is difficult to clarify, which is a great problem hindering the production of the fruit juice. The treatment method in the traditional process not only consumes manpower and financial resources, but also easily causes the problems of high energy consumption, secondary pollution and the like. In recent years, problems in juice production have been solved by adding pectinase by using an enzyme treatment method. The pectinase is added in the fruit juice production process, so that the viscosity of fruit pulp can be effectively reduced, the yield of the fruit juice can be greatly increased, and compared with the traditional mechanical method, the yield of the obtained fruit juice is increased, the sense of the fruit juice is improved, and more nutrient substances in the fruit can be reserved. Pectin lyase is also used in the production of fruit juices as one of the pectinases. The pectin lyase derived from fungus Penicillium expansum F16 is purified and used for treating apple juice, grape juice and the like, can effectively clarify the fruit juice, improve the light penetration rate of the fruit juice, and carry out methanol detection on the treated fruit juice, and does not detect the existence of harmful substances such as methanol and the like.

The pectin lyase used in the textile industry is alkaline pectinase, and is mainly used for degumming cotton and linen textiles. The wastewater generated in the traditional alkaline treatment process contains high COD, high BOD, high pH, high salinity and the like, and generates adverse factors such as high energy consumption, secondary pollution and the like, but the textile obtained by enzyme treatment can often avoid the defects, and the product has high water absorption rate, easy dyeing and better hand feeling. It has been reported that the treatment of cotton fibers with pectate lyase and the study of the physicochemical properties of the cotton fibers after the enzyme treatment resulted in fibers with strong wettability, better whiteness, high crystallinity index, easy dyeing, which are superior to those of fibers obtained by alkali treatment.

Pectin lyase is also used in feed, non-starch polysaccharides are composed of pectin, cellulose, hemicellulose, resistant starch, etc., and many non-starch polysaccharides are present in animal feed. The non-starch polysaccharide is an anti-nutritional factor, affects the activity of digestive enzymes in intestines and stomachs, and is not beneficial to the absorption and utilization of nutritional ingredients by intestines and stomachs. Monogastric animals lack the enzyme systems that degrade non-starch polysaccharides themselves and are therefore unable to utilize these substances, resulting in conditions such as poor appetite of the animal. The pectin lyase can degrade the non-starch polysaccharides, and the product obtained by enzymolysis can be used as a reaction substrate of other enzymes, so that the digestion and absorption of animal intestines and stomach on the feed are promoted. At present, pectinase and other enzymes are mainly prepared into compound enzyme for feed, so that the utilization rate of the feed by animals is improved, and the production performance of the feed is improved.

Pectin lyase is also used in the production of pectin oligosaccharides, Pectin Oligosaccharides (POS) mainly refer to a functional oligosaccharide consisting of 2-20 galacturonic acids linked by alpha-1, 4 glycosidic linkages, some of the galacturonic acids being methylated. The pectin oligosaccharide can promote the growth of probiotics such as lactobacilli and bifidobacteria in the intestinal tract and effectively inhibit the proliferation of pathogenic bacteria in the intestinal tract, thereby regulating the dynamic balance of microecological flora in the intestinal tract; can promote intestinal peristalsis, increase the absorption of trace elements, and improve the immunity of the organism. The pectin oligosaccharide is produced by an enzyme treatment method, other chemical reagents do not need to be added, and side reactions do not occur. Pectin oligosaccharides are mainly produced by the degradation of pectin by pectin lyase and polygalacturonase.

Natural pectin lyase is ubiquitous in plants and microorganisms, and pectinase from plants is low in yield and not easy to produce on a large scale, and microorganisms become a main source of pectinase due to the advantages of high growth speed, low nutritional requirement, easiness in large-scale culture and the like. Many microbial pectin lyases have been reported, and many of them are derived from fungi, such as Aspergillus, Penicillium, etc., and many are also reported from bacterial pectinases. At present, the production of the pectin lyase in China is mainly to carry out solid state fermentation by using a strain for producing the pectin lyase, but the yield is often not high, the enzyme activity is low and the pectin lyase is not easy to store. The pectinase with higher titer still depends on foreign import, but the imported enzyme is expensive, so that the production cost of enterprises is increased. With the continuous development of the Chinese food industry, the demand of the acid pectinase is increasing. The pectinase preparation with high yield, good stability, strong specificity and high enzyme activity is required by the market.

With the intensive research of molecular biology technology, the production of pectin lyase with excellent properties by using a plurality of efficient microbial strains becomes a new way. Researchers at home and abroad have screened a plurality of pectin lyase genes with excellent properties from different microorganisms, and the pectin lyase genes have different properties, such as high specific activity, heat resistance, low temperature resistance and the like. In order to improve the expression amount and purity of these enzymes, researchers have made heterologous recombinant expression of these pectin lyase genes in host bacteria such as Escherichia coli and Pichia pastoris. Qianghuinii and the like amplify from Aspergillus niger (EIM-6) by using RT-PCR technology to obtain pectin lyase gene A without signal peptide, insert the pectin lyase gene A into Pichia pastoris expression vector pPIC9k, construct recombinant expression plasmid pPIC9K-pelA, convert Pichia pastoris GS115 by electric shock, and obtain engineering strains with successful expression. This was induced with methanol to a final concentration of 1.5%, and the fermentation supernatant was concentrated and then its enzyme activity was measured to be 2.3U/mL by the hydrochloric acid method (Qiang, H.et. expression of a protein lysoA gene from Aspergillus niger in Pichia pastoris GS115.Chinese journal of biotechnology,2009,25(12), 1962). Claudio Perez-Fuentes et al cloned pectin polynucleotide lyase gene from Penicillium, inserted into expression vector pPICZA, constructed expression plasmid transformed yeast GS115, and obtained two strains with high expression level, with enzyme activities of 1.07U/mL and 1.16U/mL respectively, as measured by hydrochloric acid method (Perez-Fuentes, C.et al. heterologous expression of Aspergillus purpurogenum peptide in Pichia pastoris and Itscharacterization. fungal Biology,2014,118 (5-6), 507-. However, there are few reports on the acidic pectin lyase, and the expression level and the purification recovery rate are not high, so that the acidic pectin lyase cannot be widely used.

Generally, strategies for improving the secretion expression of exogenous genes in pichia pastoris include codon optimization, mRNA structure modification, GC content adjustment and the like. Wherein, the optimization of the codon of the exogenous gene is a main means (Nenie Dong Song, Beam Song Ping. strategy of high-efficiency expression of exogenous protein in Pichia pastoris, Gilbert university report 2001, 22, (3), 40-44). It has been found that species have codon preference, i.e. that different species have different codon usage frequencies, some codons are even never used, and those codons that are not used often or even never used are called rare codons or non-preferred codons. The formation of non-preferred codons is mainly due to the lack of tRNA recognizing these codons in the cell. Codon bias is different for different expression systems such as pichia and escherichia coli. If the foreign gene contains more non-preferred codons of the host bacterium, especially if the foreign gene continuously appears, the expression quantity of the foreign gene in the host bacterium is seriously influenced. In order to eliminate the influence of the rare codon on the protein expression quantity, the codon of the heterologous gene can be optimized, namely the rare codon in the foreign gene is replaced by the codon commonly used by a host under the condition of not changing the amino acid sequence of the foreign gene. The expression level of the foreign gene of the enzyme protein from different sources in pichia pastoris is greatly improved after codon optimization, for example: teng et al improved the expression level of beta-1,3-1,4-glucanase by 10-fold by optimizing the Codon composition of beta-1,3-1,4-glucanase (Teng D, et al, Codon optimization of Bacillus licheniformis beta-1,3-1,4-glucanase genes and expression in Pichia pastoris. apple Microbiol Biotechnol,2007,74: 1074-1083); chang optimizes the Codon of lipase derived from Candida, and the expression level in Pichia pastoris is increased 4.6 times (Chang SW, et al, Codon optimization of Candida rugosa lip1gene for expression in Pichia pastoris and biochemical characterization of recombinant Lipase J age Food Chem, 2006, 54: 815. sup. 822.). However, there are still many cases of the opposite, and the expression level of the gene may be reduced after codon optimization, but the research case is usually difficult to disclose. Researchers also have different opinions on the relationship of codons to expression levels (Henryian, et al., differentiating gene expression level from code use bias [ J ]. Mol biolEvol,2007,24(1):10-12.), and even studies have shown that not all Rare codons reduce expression levels (Chinnambi T. et al., Rare code priority and site specificity atthe 5' of the gene modules heterologous protein expression in Escherichia coli [ J ]. Biochem Biophys Commun,2008,376(4): 647). Therefore, simple codon optimization cannot always improve the expression level, and other technical means such as the distribution ratio of GC content in different gene regions, Codon Adaptation Index (CAI), etc. are also needed to be added, and mass verification is performed to obtain the mutant gene with high expression efficiency.

The pectin lyase gene has low secretory expression in yeast and low purification recovery rate, so that the pectin lyase gene cannot be widely applied and needs to be improved.

Disclosure of Invention

One of the purposes of the invention is to optimize the sequence of the pectin lyase gene to obtain the pectin lyase optimized gene with obviously improved secretory expression in yeast;

the other purpose of the invention is to provide a recombinant expression vector containing the pectin lyase optimized gene and a recombinant host cell of the recombinant expression vector;

the third purpose of the invention is to apply the optimized gene of the pectin lyase, the recombinant expression vector containing the optimized gene and the recombinant host cell to the production of the pectin lyase.

In order to achieve the above purposes, the invention adopts the following technical scheme:

the invention optimizes pectin lyase gene sequences by comprehensively considering a plurality of factors including but not limited to codon use frequency, GC content distribution in different gene regions, codon adaptation index CAI, deletion of unstable sequences and the like on the premise of not changing protein amino acid sequences of a pectin lyase gene pnlzj5b (SEQ ID NO.1) derived from Aspergillus niger to obtain 3 pectin lyase mutant genes pnlzj5b-m2, pnlzj5b-m3 and pnlzj5b-m4, wherein the nucleotide sequences are respectively shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

The invention further provides a recombinant expression vector containing the pectin lyase mutant gene and a host cell containing the recombinant expression vector; wherein, preferably, the recombinant expression vector is a recombinant eukaryotic expression vector, and more preferably is a recombinant pichia pastoris expression vector; preferably, the host cell is a yeast cell, more preferably a Pichia pastoris (Pichia pastoris) cell.

The invention transfers 3 pectin lyase mutant genes and 1 wild type gene into pichia pastoris for expression, and test results show that: the positive rate of each transformant is 43-46% in 100 strains of yeast transformants which are screened out with the original gene (pnlzj5b) and the optimized gene (pnlzj5b-m2, pnlzj5b-m3 and pnlzj5b-m4) of the transpectin lyase. Wherein, the strain with the highest enzyme activity of the pnlzj5b is selected, the pectin lyase enzyme activity secreted and expressed in a shake flask is 4.2U/mL, the strain with the pnlzj5b-m2 has the highest enzyme activity of 6.7U/mL, the strain with the pnlzj5b-m3 has the highest enzyme activity of 2.1U/mL, and the strain with the pnlzj5b-m4 has the highest enzyme activity of 4.4U/mL. The enzyme activity of the strain with the pnlzj5b-m2 is obviously higher than that of the strains with the pnlzj5b, the pnlzj5b-m3 and the pnlzj5b-m 4.

The pichia pastoris recombinant strain with the best expression level of the 4 strains is induced to produce enzyme for fermentation at the level of a 3-liter fermentor, and the result shows that: the activity of the glue lyase of the yeast transformant pnlzj5b of the transgene after being induced by methanol for 120h is 150U/mL, and the activity of the enzyme transformant pnlzj5b-m2 after being induced by methanol for 120h reaches 532U/mL, which is 3.5 times of that of the wild type. But the fermentation level of the mutant gene pnlzj5b-m3 is only 45U/ml, which is reduced by 70 percent compared with the wild type; the fermentation level of the pnlzj5b-m4 is 171U/ml, which is improved by 14 percent compared with the wild type, and has no significant difference with the wild type.

In the optimized genes obtained by optimizing the pectin lyase from Aspergillus niger through gene sequences of various strategies, pnlzj5b-m2(SEQ ID NO.2) can obviously improve the secretory expression of the enzyme protein in Pichia pastoris, and other optimized genes can not improve the expression level (pnlzj5b-m3) or even reduce the expression level (pnlzj5b-m4) after optimization.

The invention also provides a method for preparing pectin lyase, which comprises the following steps: operably connecting pectin lyase optimized genes shown in SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 with an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host cell to obtain a recombinant strain; culturing the recombinant strain, inducing the expression of the recombinant pectin lyase, and recovering and purifying the expressed pectin lyase to obtain the pectin lyase;

wherein, the recombinant expression vector is a recombinant eukaryotic expression vector, preferably a recombinant pichia pastoris expression vector; the host cell is preferably a yeast cell, preferably a Pichia pastoris (Pichia pastoris) cell.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the optimized pectin lyase gene is transferred into the pichia pastoris, the secretion expression amount of the optimized gene in the pichia pastoris is obviously improved, the expressed pectin lyase can effectively degrade pectin substances in apples, the juice yield is improved, and a foundation is laid for further industrial expanded production.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "recombinant host cell" or "host cell" means a cell that includes an exogenous polynucleotide, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell.Probes8:91-98 (1994)).

The term "expression" refers to the transcription and/or translation of a foreign gene in a host cell.

The term "transformation" refers to a method of introducing a foreign gene into a host cell.

The term "foreign gene" means that the gene sequence is of foreign origin or derived from the same source but modified or altered from its original sequence for a particular host cell.

Drawings

FIG. 1 shows the codon usage frequency of the pectolytic zymogen pnlzj5b gene.

FIG. 2 optimizes the frequency of codon usage of the Severe collagenase gene pnlzj5b-m 2.

FIG. 3 optimizes the frequency of codon usage of the Severe collagenase gene pnlzj5b-m 3.

FIG. 4 optimizes the frequency of codon usage of the Severe collagenase gene pnlzj5b-m 4.

FIG. 5 Secondary structural diagram of mRNA of pectin lyase original gene pnlzj5 b.

FIG. 6 is a secondary structure diagram of mRNA of the optimized Severe collagenase gene pnlzj5b-m 2.

FIG. 7 is a secondary structure diagram of mRNA of the optimized Severe collagenase gene pnlzj5b-m 3.

FIG. 8 is a secondary structure diagram of mRNA of the optimized Severe collagenase gene pnlzj5b-m 4.

FIG. 9 is a schematic diagram of construction of a yeast recombinant expression plasmid.

FIG. 10 shows the results of enzyme activity of pectin lyase in a 3-liter fermentor of yeast strain.

FIG. 11 purification of pectin lyase (M: markers 1: fermentation crude enzyme liquid 2: protein PNLZJ5B 3 purified by anion column CaptoQ: protein PNLZJ5B4 purified by gel molecular sieves: protein PNLZJ5B 5 digested with glycoside hydrolase Endo H: glycoside hydrolase Endo H, arrow refers to deglycosylated protein PNLZJ 5B).

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Description of the drawings:

the recombinant genetic techniques used in the specific examples below are all conventional in the art. Techniques not described in detail in the following test examples are performed according to the following experimental manual or relevant sections or portions of the literature, including: sambrook et al, Molecular Cloning, A Laboratory Manual (3 rd edition. 2001); kriegler, Gene Transfer and Expression: a Laboratory Manual (1990); current Protocols in molecular Biology (Ausubel et al, 1994).

Experimental example 1 optimized design and Synthesis of pectin lyase Gene

1. Test method

1.1 strains and plasmids

Aspergillus niger (Aspergillus niger), kept in the laboratory by the inventors;

escherichia coli (Escherichia coli) strain TOP10 and cloning vector pEASY-T1-simple were purchased from Beijing Quanyujin Biotechnology, Inc.;

optimized gene fragments were synthesized by Nanjing Kinshire Biotech.

1.2 optimized design of pectin lyase genes

The gene sequence is optimized according to the sequence of the original gene of the pectin lyase cloned from Aspergillus niger, and the process mainly comprises the following steps:

1) the amino acid sequence (SEQ ID NO.1) encoded by the pectin lyase gene pnlzj5b was not changed;

2) reducing the regions of stable structure that may be present in the secondary structure of the mRNA corresponding to the gene sequence of the pectin lyase, particularly near the 5 'and 3' ends.

3) Reference codon usage database (http://www.kazusa.or.jp/codon/) The codon usage frequency distribution table of Pichiapastoris, analyzing the codon usage of pectin lyase, defining the codon usage with the usage frequency of less than 15% in p.pastoris as a rare codon, replacing all or part of the rare codon in pectin lyase with a codon with a higher usage frequency, and performing various strategic changes of the usage frequency of certain amino acid codons at different positions, such as arginine.

4) The local AT content in the pectin lyase gene is balanced to prevent the occurrence of continuous high A-T content sequences (such as ATTTA, AATAAA) which would cause premature termination of transcription.

2. Test results

Through optimization, 3 pectin lyase mutant genes pnlzj5b-m2, pnlzj5b-m3 and pnlzj5b-m4 are obtained, and the nucleotide sequences are respectively shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

The changes in GC content before and after the optimization of the pectin lyase gene sequence are shown in Table 1. The nucleotide sequence similarity between the wild-type gene sequence and the mutant sequence is shown in Table 2. The codon usage before and after gene pectin lyase gene optimization is shown in FIGS. 1, 2, 3 and 4.

TABLE 1 changes in GC content before and after optimization of pectin lyase Gene sequences

TABLE 2 comparison of sequence similarity between individual pectin lyase genes

Codon optimization has many theories to improve the expression level of protein, and one reason for this is that the optimized gene has a change in secondary structure, and the stability and complexity of the secondary structure of mRNA has a large relationship with the ribosome's reading rate. And the simpler the secondary structure of mRNA, the faster the reading rate of ribosome, and the easier the translation of protein. The mRNA secondary structure of the pectin lyase gene before and after optimization is predicted, and the result is shown in FIGS. 5, 6, 7 and 8, wherein the mRNA secondary structure energy of pnlzj5b-m2(SEQ ID NO.2) is the highest and reaches-176.3 kcal/mol (FIG. 6).

Experimental example 2 construction and screening of pectin lyase recombinant Pichia Strain

1. Test method

1.1 strains and plasmids

Trans1-T1 Escherichia coli competent cells were purchased from Beijing Quanjin Biotechnology Ltd;

the expression vector pPIC9 and the Pichia pastoris receptor strain GS115 are products of Invitrogen company;

the plasmid pEASY-T1-simple-pnlzj5b with the original pectin lyase gene is constructed in the laboratory of the inventor;

plasmids pUC57-simple-pnlzj5b-m2, pUC57-simple-pnlzj5b-m3, pUC57-simple-pnlzj5b-m4 with optimized consequence gel lyase genes were synthesized by Nanjing King Shirui Biotech.

1.2 culture Medium and other solutions

YPD medium: peptone 20g/L, yeast extract 10g/L, glucose 20g/L (solid medium containing 1.7% agar powder), sterilizing at 108 deg.C for 15 min;

10 XYNB (yeast without amino acid nitrogen source): dissolving 134g YNB solid in 1000mL deionized water, filtering, sterilizing, and storing at 4 ℃;

500 × biotin: dissolving biotin 20mg in 100ml water, filtering, sterilizing, and storing at 4 deg.C;

MD solid medium: dissolving 2g of glucose and 2g of agarose in a beaker, diluting to 90mL with deionized water, carrying out damp-heat sterilization at 108 ℃, carrying out 30min, and adding 10mL of 10 XYNB and 200 μ L of 500 Xbiotin when the temperature is reduced to below 60 ℃.

BM mother liquor: dissolving 10g of yeast extract and 20g of peptone in 100mL of 1mol/L phosphate buffer solution (pH6.0), and diluting with deionized water to 900 mL;

BMGY: adding 1mL of 100% glycerol (v/v) into 90mL of BM mother liquor, carrying out damp-heat sterilization at 121 ℃, carrying out 20min, and adding 10mL of filtered 10 XYNB and 200 mu L of 500 Xbiotin after the temperature is reduced to below 60 ℃;

BMMY: 90mL of BM mother liquor, performing moist heat sterilization at 121 ℃, performing 20min, and adding 1mL of 100% sterile methanol, 10mL of filtered 10 XYNB and 200 mu L of 500 XYNB when the temperature is reduced to below 60 ℃;

1mol/L sorbitol: 182.1g D-sorbitol was dissolved in 1000mL of water, filtered, sterilized and stored at 4 ℃.

1.3 construction of recombinant expression vector for pectin lyase Yeast

pEASY-T1-simple-pnlzj5b, pUC57-simple-pnlzj 5-m b-m2, pUC57-simple-pnlzj5b-m3, pUC57-simple-pnlzj5b-m4 and pPIC9 plasmids are extracted, Eco RI and Not I are used to perform double enzyme digestion on the 5 plasmids, unmodified pectin lyase gene pnlzj5b and optimized modified 3 mutant genes and expression vector pPIC9 enzyme digestion products are recovered and connected, positive clones are identified through enzyme digestion and sequencing, and thus yeast recombinant expression vectors pPIC9-pnlzj5b and pPIC9-pnlzj5 82 b-m 56, pnzj 53-pnlzj 5-b and 869-pnlzj 865-m 4 are constructed respectively (FIG. 7-pPIC).

1.4 expression of fruit lyase in Pichia pastoris

The recombinant expression plasmids are digested by BglII enzyme respectively, the plasmids are linearized, and the 4 expression plasmids are transformed into pichia pastoris host strain GS115 respectively according to a pichia pastoris expression manual. The amount of the bacterial suspension was 200. mu.L per plate, and the plate was plated on MD plates and cultured at 28 ℃ until transformants grew.

1.5 screening of recombinant Pichia pastoris producing pectin lyase in shaker level

Firstly, a plate screening method is adopted to carry out primary screening on the transformant. Transferring the transformant growing on the MD plate to a numbered MD plate by using a sterile toothpick, culturing the MD transfer plate in a 28 ℃ incubator, inoculating the MD transfer plate to a 48-hole plate according to the number after a single colony on the plate grows well, and culturing for 2d at the temperature of 28 ℃ and 200r/min, wherein each hole contains 500 mu L BMGY; centrifuging at 4000r/min for 5min, removing supernatant, adding 500 μ L BMMY, culturing at 28 deg.C and 200r/min for 3 d; methanol was added every 12h (final methanol concentration of 1%). And after the induction is finished, centrifuging at 4000r/min for 5min, collecting supernatant, and respectively measuring the activity of the pectin lyase in the supernatant. Selecting strains with high preliminary screening enzyme activity for secondary screening, inoculating the activated strains into 10ml of BMGY culture medium, culturing for 48h at 200r/min, changing the culture medium into 5ml of BMMY, and supplementing 100% methanol once every 12h according to the addition of 1%. After 72h of induction, the enzyme solution was collected by centrifugation and used to determine chymosin activity. And (5) performing repeated tests again on the re-screening results to confirm the enzyme activity.

1.6 fermentation of pectin lyase recombinant Pichia pastoris

Taking original pectin lyase recombinant pichia pastoris as a reference strain, and respectively carrying out 3-liter fermentation tank level induction enzyme production fermentation on the 4 pichia pastoris recombinant strains with the optimal expression level in a laboratory.

Yeast transformants were picked with sterile toothpicks into 50mL YPD medium, shake-cultured (28 ℃,200 rpm) for 48h, transferred into 200mL YPD medium, shake-cultured (28 ℃,200 rpm) for 24h as fermentation seed bacterial liquid, and inoculated into 3L fermentor.

The fermentation tank parameters are set to be pH5.0, the temperature is 30 ℃, the stirring speed is 1000rpm, the aeration ratio is 2:1, the initial inoculation amount is 200ml of bacterial liquid, glucose (400ml of 40% glucose) starts to flow after 18h of culture, mixed feeding (100ml of 40% glucose +18ml of methanol) starts after 8h of sugar supplementation, after 4h of mixed feeding, methanol starts to flow to induce enzyme production, the dissolved oxygen level in the fermentation tank is controlled to be 25% -50% by adjusting the supplementing flow rate of the methanol during the induction period, after the induction is started, fermentation samples are taken every 12h to measure the wet weight of the thalli and the enzyme activity of the samples and keep the samples, the enzyme activity increases along with the extension of the induction time, and when the enzyme activity starts to decrease, the fermentation is stopped. The fermentation broth was centrifuged (10000rpm, 10min, 4 ℃ C.), and the supernatant enzyme solution was collected.

2. Test results

100 yeast transformants which are respectively screened for a pectin lyase original gene (pnlzj5b) and an optimized gene (pnlzj5b-m2, pnlzj5b-m3 and pnlzj5b-m4) have the positive rate of 43-46 percent. Wherein, a strain which is transformed with pnlzj5b and has the highest enzyme activity is selected, and the activity of the pectin lyase secreted and expressed in a shake flask is 4.2U/mL; correspondingly, the highest enzyme activity of the strain with the pnlzj5b-m2 is 6.7U/mL, the highest enzyme activity of the strain with the pnlzj5b-m3 is 2.1U/mL, and the highest enzyme activity of the strain with the pnlzj5b-m4 is 4.4U/mL. The enzyme activity of the strain with the pnlzj5b-m2 is obviously higher than that of the strains with the pnlzj5b, the pnlzj5b-m3 and the pnlzj5b-m 4.

From the 3L level fermentation result, the activity of the trans-gene yeast transformant pnlzj5b on methanol induction for 120h as a consequence of the glue lyase is 150U/mL, while the activity of the trans-gene yeast transformant pnlzj5b-m2 on methanol induction for 120h reaches 532U/mL, which is 3.5 times of that of the wild type. But the fermentation level of the mutant gene pnlzj5b-m3 is only 45U/ml, which is reduced by 70 percent compared with the wild type; the fermentation level of pnlzj5b-m4 is 171U/ml, which is improved by 14 percent compared with the wild type (figure 10), and has no significant difference with the wild type.

Test example 3 measurement of enzymatic Properties of pectin lyase

1.1 purification of pectin lyase

Firstly, the supernatant of the fermentation liquor is centrifuged at 10000rpm for 10min to remove thallus precipitates, the supernatant enzyme liquid is concentrated by adopting a tangential flow membrane filtration system of PALL company, firstly, a 0.1 mu m microfiltration membrane is adopted for filtration to remove residual cell debris and possibly existing impurities in the supernatant enzyme liquid, and the filtrate is collected. Then filtering the filtrate by an ultrafiltration membrane with the molecular weight cutoff of 10kDa, and discarding the filtrate to obtain concentrated enzyme solution. The concentrated enzyme solution was purified by anion exchange chromatography and gel filtration to obtain the purified recombinant protein (FIG. 11).

1.2 application of recombinase PNLZJ5B in apple juice

After fresh apple juice is treated by recombinant pectin lyase at 43 ℃ for 1 hour, the viscosity of the apple juice is reduced by 28.02%, the light transmittance is improved by 64.7%, and the juice yield is improved by 58.24%. The pectin lyase PNLZJ5B is shown to be capable of effectively degrading pectin substances in apples. The effect on apple juice yield can be seen in table 3: under the condition of consistent enzyme dosage, the pectinases can reduce the viscosity of apple juice and clarify the fruit juice, but the fruit juice obtained by the pectin lyase PNLZJ5B has the highest fruit juice quantity which is obviously higher than the fruit juice quantities obtained by pectate lyase PL, commercial pectin lyase CPNL, commercial polygalacturonase NPG and commercial pectin esterase PE, and the lyase PNLZJ5B has obvious advantages on improving the juice yield and clarification effect of the apple juice; by comparison, it can also be seen that pectin lyase PNLZJ5B treated juice produced less pomace than did polygalacturonase.

TABLE 3 use of pectin lyase PNLZJ5B with other different pectinases in apple juice^a

^aAdding 1U of each pectinase into 50ml of apple juice, and keeping the temperature at 43 ℃ for 1 h;

^bfiltering the treated apple juice with filter paper, and calculating the juice yield of the apple juice within 4 min; each group is made into three parallels

In conclusion, after the pectin lyase derived from Aspergillus niger is optimized by gene sequences of various strategies, in a plurality of optimized genes, pnlzj5b-m2(SEQ ID NO.2) can obviously improve the secretory expression amount of the enzyme protein in Pichia pastoris, and other optimized genes can not improve the expression level (pnlzj5b-m3) or even reduce the expression level (pnlzj5b-m4) after being optimized. Compared with the known research at home and abroad, the secretion expression quantity of the pnlzj5b-m2 pectin lyase optimized gene obtained by the experiment in pichia pastoris reaches a higher expression level, and a foundation is laid for further industrial expanded production.

Claims

1. The optimized gene of the pectin lyase is characterized in that the nucleotide sequence of the optimized gene is shown as SEQ ID NO. 2.

2. A recombinant expression vector comprising the pectin lyase optimized gene of claim 1.

3. The recombinant expression vector of claim 2, wherein: the recombinant expression vector is a recombinant eukaryotic expression vector.

4. The recombinant expression vector according to claim 3, wherein: the recombinant eukaryotic expression vector is a recombinant yeast expression vector.

5. The recombinant expression vector of claim 4, wherein: the recombinant eukaryotic expression vector is a recombinant Pichia pastoris (Pichia pastoris) expression vector.

6. A host cell comprising the recombinant expression vector of any one of claims 2-5.

7. The host cell of claim 6, wherein: the host cell is a yeast cell.

8. The host cell of claim 7, wherein: the host cell is a Pichia pastoris (Pichia pastoris) cell.

9. Use of the pectin lyase optimized gene of claim 1 in the preparation of a pectin lyase.

10. Use according to claim 9, characterized in that it comprises: operably linking the pectin lyase optimized gene of claim 1 with an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host cell to obtain a recombinant strain; culturing the recombinant strain, inducing the expression of the recombinant pectin lyase, and recovering and purifying the expressed pectin lyase.

11. Use according to claim 10, characterized in that: the recombinant expression vector is a recombinant eukaryotic expression vector; the host cell is a yeast cell.

12. Use according to claim 11, characterized in that: the recombinant expression vector is a pichia pastoris expression vector; the host cell is a Pichia pastoris cell (Pichia pastoris).