CN111254154A

CN111254154A - Pectate lyase coding gene and enzyme, preparation and application thereof

Info

Publication number: CN111254154A
Application number: CN201811458955.6A
Authority: CN
Inventors: 尹恒; 杨国军
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2020-06-09

Abstract

The invention discloses a pectate lyase gene derived from Aspergillus parasitism (Aspergillus parasiticus) and a preparation method and application of the pectate lyase gene, namely, the gene of the pectate lyase is cloned to a pichia pastoris expression vector by utilizing a technical method of genetic engineering to obtain a pichia pastoris recombinant strain capable of heterologously expressing the pectate lyase, and the pectate lyase prepared by heterologously expressing the strain can efficiently degrade pectic polysaccharide and polygalacturonic acid (PGA). The pectate lyase provided by the invention can be widely applied to the fields of agriculture, industry, food, feed additives, medicines, pectin oligosaccharide preparation and the like.

Description

Pectate lyase coding gene and enzyme, preparation and application thereof

Technical Field

The invention relates to a gene sequence of pectate lyase and a preparation method and application thereof. The invention provides a recombinant plasmid and a recombinant genetic engineering strain of the pectate lyase and application thereof in polysaccharide degradation. The pectate lyase provided by the invention can be widely applied to the fields of agriculture, food, feed addition, medicine, oligosaccharide preparation and the like.

Background

Pectin (Pectin) is a polysaccharide which is most complex in structure and function in plant cell walls and widely exists in nature, is distributed in the soft tissue cell walls, intercellular layers, cell and secondary wall connecting areas of plants in three forms of Pectin, pectic acid and protopectin, is a high-molecular compound with the average molecular weight of 20000-400000, is quite complex in structure and mainly comprises three parts of homogalacturonic acid (HG) accounting for about 65% of Pectin, Rhamnogalacturonan I (Rhamgalacturonan-I, RG-I) accounting for about 20-35% of Pectin, Rhamnogalacturonan II (Rhamgalacturonan-II, 10% of Pectin, is prepared by linear polymerization of galacturonic acid under the action of α -1,4 glycosidic bonds, is a coagulated part of Pectin, namely polygalacturonic acid (PGA), is prepared by linear polymerization of galacturonic acid under the action of low methoxyl sugar bond (RG) -65% of Pectin, and is prepared by low methoxyl amidation and high Pectin (HM) which is 70-70% of Pectin and is prepared by low Pectin.

The pectin oligosaccharide is also called oligogalacturonan, and is an acidic oligosaccharide prepared by degrading pectin polysaccharide. Oligogalacturonans not only have the biological effects of common oligosaccharides, such as inhibiting pathogenic bacteria, enhancing the immune function of the organism, improving lipid metabolism, improving the microflora in the intestinal tract and the stomach of animals, reducing the cholesterol content in serum, improving the production performance of animals, preventing constipation and abdominal distension, reducing blood pressure and the like, but also can activate the self-defense system of plants, and have the effects of resisting diseases, preventing diseases, improving the growth speed and the like on the plants. Oligogalactarates obtained by pectin degradation have been shown to have a range of biological activities in plants, such as inducing disease resistance defense responses in plants, regulating plant growth and development, and regulating rapid responses on cell surfaces. In the large intestine and colon, microbial degradation of pectin oligosaccharides releases short chain fatty acids (prebiotic effect) that have a positive impact on health.

The basic composition unit of pectin is galacturonic acid, pectin is widely distributed in the natural world, and abundant raw material sources are the most remarkable advantages of the pectin prepared by enzymolysis for oligogalacturonic acid. Most of the prior methods for preparing oligogalacturonans use pectinase to hydrolyze pectin or pectic acid to obtain a mixture of oligogalacturonans with different polymerization degrees. The oligogalacturonides prepared by the enzyme method have the characteristics of good reaction repeatability, mild conditions, simple product separation and purification process, excellent quality of the obtained oligogalacturonides and the like. Meanwhile, the enzyme is obtained by a method for enzymolysis of the pectic polysaccharide, the reaction efficiency of the enzyme is high, the method is simple and easy to control, the later separation is easy, and the enzyme is used as a biological agent and cannot bring adverse factors generated by chemical reagents. Therefore, the method for producing the oligogalacturonides by utilizing the enzymatic hydrolysis of the pectin polysaccharide is a relatively simple and easily-researched production method.

Pectate lyase (Pel) called polygalacturonate lyase (PGL) is used for cracking the structure of the isopolygalacturonic acid (HG) part of pectic polysaccharide through trans-elimination, α -1,4 glycosidic bond of galacturonic acid is broken, glycosidic bond is broken at C-4 position, and an H atom is eliminated from C-5 position to form an unsaturated double bond, thereby galacturonic acid oligosaccharides with different degrees of polymerization are generated.

Disclosure of Invention

In order to solve the problems, the invention uses a gene engineering method to obtain a structural gene for producing pectate lyase by a gene cloning technology, and the structural gene is transferred into an expression thallus to obtain a high-yield strain capable of efficiently expressing. Specifically, the first object of the present invention is to provide a novel pectate lyase Ap Pel derived from Aspergillus parasiticus (Aspergillus parasiticus) and a gene encoding the same.

The second purpose of the invention is to provide a method for preparing a novel pectate lyase Ap Pel.

The third purpose of the invention is to provide recombinant expression plasmid and recombinant genetic engineering strain containing the pectate lyase Ap Pel gene.

The fourth purpose of the invention is to provide the application of the novel pectate lyase Ap Pel in the degradation of the pectin polysaccharide.

The pectate lyase Ap Pel provided by the invention is derived from Aspergillus parasiticus (Aspergillus parasiticus) separated and purified from soil, and a pectate lyase Ap Pel coding gene (named as ApPel) amplified from the Aspergillus parasiticus, and has one or more than two of the following nucleotide sequence characteristics:

1) a deoxyribonucleic acid (DNA) sequence with the 1 st to 1065 th positions of SEQ ID NO.1 in a sequence table;

2) a deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in the sequence list; the gene sequence of the pectate lyase ApPel of the present invention is cloned from Aspergillus parasiticus (Aspergillus parasiticus) by PCR technique. The coding region of the gene is 1086bp, and belongs to PL1(polysaccharide lyase 1 family) of polysaccharide lyase. Wherein, the 1 st-1065 th site is a deoxyribonucleic acid (DNA) sequence which codes the activity of the pectate lyase Ap Pel, and the 1066 st-1086 th site is a DNA sequence which codes the restriction site, His-Tag and a stop codon;

3) a deoxyribonucleic acid (DNA) sequence encoding amino acid sequences 1 to 355 of SEQ ID NO.2 of the sequence list;

4) a deoxyribonucleic acid (DNA) sequence encoding the amino acid sequence of SEQ ID NO.2 of the sequence list;

5) a nucleotide sequence which is obtained by substituting, deleting or adding one or more than two nucleotides into a deoxyribonucleic acid (DNA) sequence of 1-1065 th sites of SEQ ID NO.1 in a sequence table and codes the DNA sequence with pectate lyase activity;

6) a nucleotide sequence which is obtained by substituting, deleting or adding one or more than two nucleotides into a deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in a sequence table and codes the DNA sequence with pectate lyase activity;

7) has a homology of 80% or more with deoxyribonucleic acid (DNA) sequence defined in the 1 st to 1065 th positions of SEQ ID No.1 of the sequence list, and can code the deoxyribonucleic acid (DNA) sequence of protein for degrading pectin polysaccharide and polygalacturonic acid.

8) Has 80% or more homology with deoxyribonucleic acid (DNA) sequence defined by SEQ ID NO.1 in the sequence table, and can code the deoxyribonucleic acid (DNA) sequence of protein for degrading pectin polysaccharide and polygalacturonic acid.

The invention also provides an amino acid sequence of the pectate lyase Ap Pel, which has one or more than two of the following characteristics:

1) 1-355 amino acid residue sequence of SEQ ID NO.2 in the sequence table from the amino terminal;

2) 1-361 th amino acid residue sequence of SEQ ID NO.2 from amino terminal in the sequence table, wherein: 1-355 is an amino acid sequence with pectate lyase Ap Pel activity, and 356-361 is an enzyme cutting site and an amino acid sequence of His-Tag;

3) an amino acid sequence with pectate lyase activity formed by substituting, deleting or adding one or more than two amino acids in the 1 st-355 th amino acid sequence of SEQ ID NO.2 in the sequence table.

4) An amino acid sequence with pectate lyase activity formed by substituting, deleting or adding one or more than two amino acids of the amino acid sequence shown as SEQ ID NO.2 in the sequence table.

The amino acid sequence and the nucleotide coding sequence of the pectate lyase Ap Pel can also be artificially synthesized according to the predicted amino acid sequence and the nucleotide coding sequence of the pectate lyase Ap Pel.

Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences disclosed herein are also part of the invention. In certain embodiments, the sequence identity at the amino acid level may be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. At the nucleic acid level, the sequence identity may be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

"substantially identical (or" substantially homologous ")" a first amino acid or nucleotide sequence contains a sufficient number of identical or equivalent (e.g., having similar side chains, e.g., conservative amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities.

The method for preparing the recombinase Ap Pel is to clone the pectate lyase gene in the technical scheme into a recombinant expression vector, and introduce the recombinant expression vector into a host cell to obtain the recombinant expressed pectate lyase.

The expression vector for recombinant expression of the pectate lyase Ap Pel can be an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a lactic acid bacteria expression vector, a streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, a mammalian cell expression vector and the like.

Recombinant bacteria or transgenic cell lines for recombinant expression of the pectate lyase Ap Pel may be e.coli host cells (e.g. Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α, etc.), yeast host cells (e.g. Saccharomyces cerevisiae, Pichia pastoris, kluyveromyces lactis, etc.), Bacillus subtilis host cells (e.g. Bacillus subtilis R25, Bacillus subtilis9920, etc.), lactobacillus host cells (e.g. lactobacillus acid COCC101, etc.), actinomycete host cells (e.g. Streptomyces spp., etc.), filamentous fungal host cells (e.g. Trichoderma viride, Trichoderma marsei, Aspergillus niger, etc.), insect cells (e.g. anaerobia, bocalaria, etc.), baby hamster ovary cells (e.g. hamster ovary cells, baby hamster ovary cells, BHK).

The invention provides the use of a pectin lyase for degrading pectic polysaccharides, polygalacturonic acids or substrates containing galacturonic acid polysaccharide linkages. In particular, the application of the pectin lyase provided by the invention comprises one or two of the following applications:

1) the application in breaking the glycosidic bond of pectin polysaccharide to obtain monosaccharide or galacturonic acid oligosaccharide;

2) the application of the polysaccharide bond of polygalacturonic acid is broken to obtain monosaccharide or galacturonic acid;

3) the application of the pectin polysaccharide glycoside bond in the aspect of cooperative breakage after being mixed with other enzymes.

The pectate lyase Ap Pel obtained by recombinant expression of Pichia pastoris can efficiently degrade pectic polysaccharide, and has good activity at 40 ℃ and pH 7-9 when the pectic polysaccharide is used as a substrate.

The pectate lyase Ap Pel can be widely applied to the fields of agriculture, industry, food, feed additives, medicines, preparation of pectic oligosaccharide and the like.

Drawings

FIG. 1: detecting pectate lyase gene Ap Pel agarose gel electrophoresis. M is nucleic acid standard; lanes 1-Ap Pel amplification products.

FIG. 2: SDS-PAGE pattern of pectate lyase Ap Pel expression and purification. The samples added in each lane are: m protein molecular weight Standard, lane 1-Ap Pel column three flow through, lane 2-20mM imidazole elution flow through, lane 3-60mM imidazole elution flow through, lane 4-80mM imidazole elution flow through, lane 5-100mM imidazole elution flow through, lane 6-200mM imidazole elution flow through, lane 7-250mM imidazole elution flow through, lane 8-500mM imidazole elution flow through.

FIG. 3: HPAEC-PAD analysis of pectate lyase Ap Pel on pectic polysaccharide and polygalacturonic acid degradation products.

FIG. 4: titration time profiles of pectate lyase Ap Pel on pectin viscosity reduction (control one: pectin degradation product with addition of commercial pectinase; control two: pectin without addition of pectinase; pectin degradation product with addition of pectate lyase Ap Pel).

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Information of SEQ ID No. 1: nucleotide sequence, length: 1086 bp; chain type: single-stranded.

Information of SEQ ID No. 2: amino acid sequence, length: 361; chain type: single-stranded.

Example 1 full-Length Gene cloning of pectate lyase Ap Pel

The Aspergillus parasiticus total RNA was extracted according to the procedure of the column type fungal total RNA extraction purification Kit (Shanghai Producer) and the first Strand cDNA was synthesized according to the procedure of RevertAId Frist Strand cDNA Synthesis Kit (Thermo Scientific). After multiple sequence alignment analysis of pectate lyase gene sequences in The National Center for Biotechnology Information (NCBI) database, degenerate primers were designed:

Ap Pel-F:5’-CAGTCTCGAGAAAAGACAGAAAGTCTCTGGTGCC-3’；

Ap Pel-R:5’-ATCTTCTAGAGATCTTGCCCGCACCAGCATTC-3’，

the first strand cDNA of aspergillus parasiticus is used as a template, a gene sequence (not including a signal peptide gene) for coding pectate lyase protein is amplified, PCR reaction conditions are that agarose gel electrophoresis analysis is carried out on PCR products (shown in figure 1) at 98 ℃ for 3min and 1 cycle, at 98 ℃ for 10s and 55 ℃ for 15s and at 72 ℃ for 2min and 35 cycles, the target gene is cut and recovered, the products are connected to a eukaryotic expression vector pPICZ α A through a double enzyme digestion method, and plasmid sequencing is extracted after amplification is carried out in escherichia coli Top 10.

Example 2 pectate lyase Gene sequence analysis

Sequencing results were analyzed using Basic Local Alignment Search Tool (BLAST) in GenBank database, DNAMAN software for multiple sequence alignments, Vector NTI for sequence information.

The obtained pectate lyase gene (named Ap Pel) has a coding region of 1086bp in length, and the nucleotide sequence of the gene is shown as SEQID NO 1. Ap Pel encodes 361 amino acids and a stop codon, the amino acid sequence is shown as SEQ ID NO 2, the theoretical molecular weight of the protein is 38.10kDa, and the predicted isoelectric point is 8.90. Ap Pel encodes an amino acid with a pectate lyase central domain, indicating that Ap Pel is a member of the polysaccharide lyase family.

Example 3 heterologous expression and purification of pectate lyase Ap Pel in Pichia pastoris

The pectate lyase Ap Pel is induced and expressed according to the method of a Pichia pastoris expression manual. The expression and purification conditions of the pectate lyase Ap Pel are detected by polyacrylamide gel electrophoresis, and the result is shown in FIG. 2, the purified pectate lyase Ap Pel is a single band on the electrophoresis gel, and the position (about 38kDa) is matched with the predicted molecular weight (38.10 kDa).

Example 4 Activity assay of pectate lyase Ap Pel

450 mu L of 0.5% (w/v) pectic polysaccharide is used as a substrate, 50 mu L of recombinase Ap Pel is added for reaction for 30min, and the activity of the product is determined by a 3, 5-dinitrosalicylic acid (DNS) method. The enzyme activity unit is defined as the amount of enzyme required to release 1. mu. mol of reducing sugar (in terms of galacturonic acid) per minute as one enzyme activity unit (U). Protein concentration was determined using a Byunnan BCA protein concentration assay kit.

Example 5 recombinant enzyme Ap Pel degraded pectin and polygalacturonic acid product analysis

0.5% (w/v) of pectic polysaccharide/polygalacturonic acid was mixed with recombinase Ap Pel in a ratio of 9:1 (volume ratio), reacted at 40 ℃ for 24 hours, deproteinized by the Sevage method, and then the product was analyzed by HPAEC-PAD and ESI-MS. As shown in FIG. 3, the degradation of pectic polysaccharide and polygalacturonic acid by Ap Pel can generate oligosaccharide mixture with DP 1-15, and by observing the peak areas of the oligosaccharide products with different degrees of polymerization, the degradation capability of Ap Pel on polygalacturonic acid is obviously higher than that of pectin, and the degradation capability of Ap Pel on polygalacturonic acid produced by different companies has certain difference. Therefore, the Ap Pel can be used for the preparation of the pectin oligosaccharide and the research on the aspects related to the degradation of the pectin polysaccharide, and comprises the fields of agriculture, industry, food, feed addition, medicine, pectin oligosaccharide preparation and the like.

Example 6 viscosity reduction analysis of enzymatic hydrolysate of pectate lyase Ap Pel

And comparison one: adding commercial pectinase (given by other laboratories) 10uL (1mg/mL) and 90uL ultrapure water into 1mL of 0.5% (w/v) citrus low-ester serving as a substrate, and oscillating and reacting for 8h at 40 ℃ by a shaking table 300 r; comparison two: adding 100uL of ultrapure water into 1mL of 0.5% (w/v) citrus low ester serving as a substrate, and oscillating and reacting for 8 hours by using a shaking table at 40 ℃ for 300 turns; PL1 was prepared by adding 100. mu.L of recombinase Ap Pel (0.4mg/mL) to 1mL of 0.5% (w/v) citrus low ester substrate and shaking the mixture in a shaker at 40 ℃ for 8 hours. After the reaction, boiling for 10min to inactivate the enzyme, cooling and centrifuging, and taking the supernatant to measure the viscosity. The titration time of the pectate lyase Ap Pel for reducing the viscosity of pectin is shown in FIG. 4, and the viscosity titration time of the pectate lyase Ap Pel is the shortest and is significantly lower than that of the control I and the control II, which indicates that the pectate lyase Ap Pel has significant viscosity reduction effect on pectin.

It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.

Sequence listing

<110> institute of chemistry and physics, large connection of Chinese academy of sciences

<120> pectate lyase coding gene, enzyme, preparation and application

<130>2013

<160>2

<170>PatentIn version 3.3

<210>1

<211>1086

<212>DNA

<213> nucleotide sequence

<400>1

CAGAAAGTCT CTGGTGCCGC CCAAGGTTTC GCTTCTGGCG TGACGGGTGG TGGTAACGCT 60

TCACCTCAGA CCCCGAAGGA TATCAACGAA CTCAAGAAAT TGCTCGCCGA TCCCTCCCCT 120

CGTGTGATTG TCCTTGACAA GTTGTACGAC TACACCGGAA CGGAGGGCAC CTCGAAGGGA 180

ACGGTCTGTG CCAACTGGGG TGAAGGTGCA AAATGCCAGA AGATCATCCA GGATGACTGC 240

GGAAGCGCCG CCAAATCCAC TGGAACCTGG GACACTGCTG CCAAGACCCC AATCGATGTC 300

GCCTCACACA AGACCATCAT CGGTGTTGGT AACAAGGGTA TCATCAAGGG CAAGGGCTTG 360

AGATTCCGCG GTGGTGCTAC CAACATTATT GTCCAGAACA TCCAGATTAC CGACCTGAAC 420

CCCCAGTACG TGTGGGGTGG TGATGCCATC TCTTTTGATG GCGCTGATCT GATCTGGGTT 480

GACCACGTTA CTACTGCTCG TATCGGACGT CAGCACTACG TTTTCGGCTT CAACACCAGC 540

AAGCGTGTGA CTCTTTCCAA CAACTTCATC AACGGCGACA GCCCTTTCTC TGCCGGATGC 600

AACGGATACC ACTACTGGAC CTTCGAGATG GTTGGCAAGG GAGACCAGAT CACCCTTCAG 660

AACAACCACA TTTACCACAC CTCTGGCCGT AGTCCTGCCC TGAGCGGCGG TACCCTCCTG 720

CATGCAGTGA ACAACGTCTG GGACGGCAAC ACTGGCCACG CCCTTGAGGG AGGCGAGGCC 780

ACTGCCAATG GTATCTTCGA GGGTAACGTT TTCACCGATG TCAAGGCCAT TGTCGCCGAC 840

TTCAAGGGCA AGGCTTTCTT CTCCCCCGAT GCCAATTCCA ACAAGCAGTG CAGCTCCGCC 900

CTTGGCCGAG CCTGCGAAGT CAATGTTCTT ACCAAGTCCG GTACTCTTCC CCCTCTCAAG 960

GATACCTCCT TCTTCGGTGA CTTCAAGGGC CTCAAGATTG CCCCTGCTAC TCCGGCTTCT 1020

CAGGCCGCGG TTAACGTCCC CAAGAATGCT GGTGCGGGCA AGATCCATCA TCATCATCAT 1080

CATTGA 1086

<210>2

<211>361

<212>protein

<213> amino acid sequence

<400>2

QKVSGAAQGF ASGVTGGGNA APQTPKDINE LKKLLADPSP RVIVLDKLYD YTGTEGTSKG 60

TVCANWGEGA KCQKIIQDDC GSAAKSTGTW DTAAKTPIDV ASHKTIIGVG NKGIIKGKGL 120

RFRGGATNII VQNIQITDLN PQYVWGGDAI SFDGADLIWV DHVTTARIGR QHYVFGFNTS 180

KRVTLSNNFI NGDSPFSAGC NGYHYWTFEM VGKGDQITLQ NNHIYHTSGR SPALSGGTLL 240

HAVNNVWDGN TGHALEGGEA TARGIFEGNV FTDVKAIVAD FKGKAFFSPD ANSNKQCSSA 300

LGRACEVNVL TKSGTLPPLK DTSFFGDFKG LKIAPATPAS QAAVNVPKNA GAGKIHHHHH 360

H 361

Claims

1. A pectate lyase gene, the nucleotide sequence of which has one or more of the following characteristics:

2) has a deoxyribonucleic acid (DNA) sequence of SEQ ID NO.1 in a sequence table;

2. A pectin lyase encoded by the pectin lyase gene of claim 1, wherein: the amino acid sequence of the polypeptide has one or two of the following characteristics:

2) the amino acid residue sequence of SEQ ID NO.2 in the sequence table;

3) an amino acid sequence with pectate lyase activity formed by substituting, deleting or adding one or more than two amino acids in the 1 st-355 th amino acid sequence of SEQ ID NO.2 in the sequence table;

3. A method for preparing pectate lyase according to claim 2, comprising: cloning the pectate lyase gene of claim 1 into a recombinant expression vector, and introducing into a host cell to obtain a recombinantly expressed pectate lyase.

4. The production method according to claim 3, characterized in that: the recombinant expression vector is selected from an escherichia coli expression vector, a yeast expression vector, a bacillus subtilis expression vector, a lactic acid bacteria expression vector, a streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector or a mammalian cell expression vector.

5. The method of claim 3, wherein: the host cell, namely a recombinant bacterium or a transgenic cell line for recombinant expression of pectate lyase, refers to one of an escherichia coli host cell, a yeast host cell, a bacillus subtilis host cell, a lactic acid bacteria host cell, an actinomycete host cell, a filamentous fungus host cell, an insect cell and a mammalian cell.

6. The method of claim 5, wherein the host cell is selected from the group consisting of Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis, Bacillus subtilis R25, Bacillus subtilis9920, Lactic acid bacteria COCC101, Streptomyces spp.

7. Use of a pectin lyase according to claim 2 for degrading pectic polysaccharides, polygalacturonic acids or substrates containing galacturonic acid polysaccharide linkages.

8. Use according to claim 7, characterized in that: including one or both of the following applications: