JP2017060424A - Lipases, polynucleotides, recombinant vectors, transformants, methods of producing lipase, methods of hydrolyzing glycerolipid and methods of producing glycerolipid hydrolysate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lipase which exerts activity in the wide region of pH of 4 to 11 and exerts sufficient activity even in alkalinity of pH of about 10, to provide a polynucleotide encoding the lipase, to provide a recombinant vector containing the polynucleotide, to provide a transformant in which the recombinant vector is introduced, to provide a method of producing the lipase, to provide a method of hydrolyzing glycerolipid, and to provide a method of producing the glycerolipid hydrolysate.SOLUTION: The present invention provides a lipase having the following properties (1) to (3): (1) having galactolipase activity and phospholipase-B activity; (2) having a molecular weight of 20,000 to 50,000; and (3) being derived from microorganisms belonging to Streptomyces genus.SELECTED DRAWING: None

Description

  The present invention relates to a lipase, a polynucleotide encoding the lipase, a recombinant vector containing the polynucleotide, a transformant introduced with the recombinant vector, a method for producing the lipase, a method for hydrolyzing glycerolipids, and glycero The present invention relates to a method for producing a hydrolyzate of lipid.

  “Lipase” is a general term for enzymes that hydrolyze glycerolipids.

  “Glycerolipid” refers to a lipid having a glycerol skeleton. Examples of the glycerolipid include glyceroglycolipid, glycerophospholipid, and neutral fat.

  “Glyceroglycolipid” refers to a compound in which a sugar chain is covalently bonded to a free hydroxyl group of diacylglycerol. Examples of the glyceroglycolipid include galactolipids such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG).

  “Glycerophospholipid” refers to a compound in which serine, choline, ethanolamine, inositol and the like are bonded to a free hydroxyl group of diacylglycerol via a phosphate group.

  An enzyme exhibiting lipase activity against glyceroglycolipid is particularly referred to as galactolipase, and the activity is referred to as galactolipase activity. The presence of galactolipase is known, for example, in higher plants such as spinach having chloroplasts (Non-patent Document 1). As galactolipase, galactolipase derived from Aspergillus japonicus (patent document 1), galactolipase derived from Chlamydomonas reinhardtii (non-patent document 2), Streptomyces sp (Streptomyces sp. .)-Derived galactolipase (Patent Document 2) is also known.

  An enzyme that exhibits lipase activity against glycerophospholipid is also referred to as phospholipase. Furthermore, the enzyme that hydrolyzes the fatty acid ester bond at the sn-1 position of the glycerol group in glycerophospholipid is called phospholipase A1 (this activity is called phospholipase A1 activity), and the fatty acid ester bond at the sn-2 position of the glycerol group is hydrolyzed. This enzyme is called phospholipase A2 (the activity is called phospholipase A2 activity). An enzyme having both phospholipase A1 activity and phospholipase A2 activity is referred to as phospholipase B (the activity is referred to as phospholipase B activity).

  However, the known galactolipase has a problem that, for example, its production amount is low and it is difficult to apply it to industries. In addition, known enzymes exhibit activity from a weakly acidic pH of 5 to a weakly alkaline pH of 9, but the activity is significantly reduced at pH 9 and above. For this reason, application to a noodle making process (pH 10) cannot be expected. Furthermore, since the galactolipase described in Patent Document 1 is derived from mold, it is not suitable for processing food materials because it requires removal of antibiotics and allergens in order to use it in the food field. Further, the galacto lipase described in Patent Document 1 has a drawback that there is no mass production method since an expression method by a gene recombination method has not been established. Furthermore, since it also acts on neutral lipids such as triglycerides, this may be a disadvantage.

JP 2008-206515 A International Publication No. 2006/008653

Sakaki T, et al., Plant Physiol. 1990 Oct; 94 (2): 773-80. Li X, et al., Plant Cell 11,4670 (2012)

  The present invention relates to a lipase that exhibits activity in a wide range of pH 4 to pH 11, and exhibits sufficient activity even in an alkaline region around pH 10, a polynucleotide encoding the lipase, a recombinant vector containing the polynucleotide, It is an object to provide an introduced transformant, a method for producing the lipase, a method for hydrolyzing glycerolipid, and a method for producing a hydrolyzate of glycerolipid.

  As a result of intensive studies to solve the above problems, the present inventors have found a novel lipase from microorganisms belonging to the genus Streptomyces, and have completed the present invention. The homology of the amino acid sequence between this lipase and the known lipase was extremely low, and this lipase was a novel lipase that was completely different from the known lipase.

That is, the present invention is as follows.
[1]
Lipases having the following properties (1) to (3):
(1) having galactolipase activity and phospholipase B activity;
(2) the molecular weight is 20,000 to 50,000;
(3) Derived from microorganisms belonging to the genus Streptomyces.
[2]
The lipase, wherein the Streptomyces genus is Streptomyces sanglieri.
[3]
Lipase, which is a protein described in the following (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 7;
(B) A protein having an amino acid sequence containing substitution, deletion, insertion, or addition of 1 to 50 amino acid residues in the amino acid sequence shown in SEQ ID NO: 7, and having galactolipase activity and phospholipase B activity.
[4]
A polynucleotide encoding the lipase.
[5]
The polynucleotide according to (A) or (B) below:
(A) a polynucleotide comprising the base sequence represented by SEQ ID NO: 4;
(B) A polynucleotide comprising a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 4 and encoding a protein having galactolipase activity and phospholipase B activity.
[6]
A recombinant vector comprising the polynucleotide.
[7]
A transformant introduced with the recombinant vector.
[8]
A method for producing a lipase, comprising culturing the transformant in a medium to produce the lipase, and collecting the lipase from the culture.
[9]
A method for hydrolyzing glycerolipid, comprising allowing the lipase to act on glyceroglycolipid and / or glycerophospholipid.
[10]
Selected from the group consisting of sugar glycerol, lyso-type sugar glycerol, 1-acyl lysoglycerophospholipid, 2-acyl lysoglycerophospholipid, and glycerol-3-phosphate, which comprises allowing the lipase to act on glycerolipid and / or glycerophospholipid. A method for producing a hydrolyzate of glycerolipid comprising one or more of the above.

  According to the present invention, the activity is exhibited in a wide range from pH 4 to pH 11.

It is a photograph which shows the analysis result by SDS-PAGE of Streptomyces sanglirei A14 origin lipase (GL). It is a graph which shows the relative activity of GL in various temperature on pH7.2 conditions. It is a graph which shows the relative activity of GL in various pH under 37 degreeC conditions. It is a graph which shows the substrate specificity (relative comparison of hydrolysis activity) of GL in pH 10.5 and 45 degreeC. It is a graph which shows the residual activity about GL after processing at various temperature on the conditions of pH 8.0 for 30 minutes as a relative activity. It is a graph which shows the residual activity about GL after processing by various pH on the conditions for 2 hours at the temperature of 4 degreeC as a relative activity. It is a graph which shows the relative activity about GL when a metal ion or EDTA is added.

<1> Lipase of the present invention The present invention provides a novel lipase. The lipase provided in the present invention is also referred to as “the lipase of the present invention”.

  In the present invention, “lipase” refers to a protein having an activity of catalyzing a reaction of hydrolyzing an ester bond (carboxyl ester bond) of glycerolipid.

  In the present invention, the lipase is not limited to a purified product, and includes a crude product and an immobilized product. The lipase can be purified using methods well known to those skilled in the art to obtain lipases of various degrees of purification (including lipases purified to almost a single level).

  In the case of a lipase derived from a microorganism, for example, the culture solution of the microorganism can be purified by a method such as ammonium sulfate precipitation, ion exchange chromatography, or hydrophobic chromatography.

  “Glyceroglycolipid” refers to a compound in which a sugar chain is covalently bonded to a free hydroxyl group of diacylglycerol. “Free hydroxyl group” refers to a hydroxyl group that is not ester-bonded to a fatty acid. For example, when diacylglycerol is 1,2-diacylglycerol, the hydroxyl group at the 3-position of the glycerol skeleton is a free hydroxyl group. The type and length of the sugar chain are not particularly limited. Examples of the glyceroglycolipid include galactolipid. “Galactolipid” refers to a compound in which a galactose chain (for example, one molecule or two molecules of galactose) is covalently bonded to a free hydroxyl group of diacylglycerol. Examples of the galactolipid include monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG). Other glyceroglycolipids include sulfoquinovosyl diacylglycerol (SQDG). Monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG) are each composed of one molecule of galactose, two molecules of galactose, and one molecule of sulfoquinoose. Is a glyceroglycolipid.

  “Glycerophospholipid” refers to a compound in which a phosphoryl base is covalently bonded to a free hydroxyl group of diacylglycerol. Examples of the base include choline, ethanolamine, inositol, serine, and glycerol.

  That is, fatty acid is ester-bonded to the α-position and β-position hydroxyl group of glycerol, and choline, ethanolamine, inositol, serine, glycerol, etc. are bonded to the other α-position hydroxyl group via a phosphate group. It is a compound. Examples of the glycerophospholipid include lecithin, 1-palmitoyl-2-oleoylphosphatidylserine (POPS), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), phosphatidylinositol (PI), 1 , 2-Dimyristoylphosphatidic acid (DMPA), dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoylphosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), lyso Examples thereof include phosphatidylethanolamine (LPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC).

  “Neutral lipid” is a general term for monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG). “Monoacylglycerol (MAG)” refers to a compound in which a fatty acid is ester-bonded to one of the three hydroxyl groups of glycerol. “Diacylglycerol (DAG)” refers to a compound in which a fatty acid is ester-bonded to two of the three hydroxyl groups of glycerol. “Triacylglycerol (TAG)” refers to a compound in which fatty acids are ester-bonded to all three hydroxyl groups of glycerol.

  The lipase activity for glyceroglycolipid is also called “galactolipase activity”, the lipase having the same activity is also called “galactolipase”, the lipase activity for glycerophospholipid is called “phospholipase activity”, and the lipase having the same activity is called “phospholipase”.

  More specifically, the galactolipase activity refers to an activity that catalyzes a hydrolysis reaction of an ester bond between a hydroxyl group of a glycerol skeleton constituting a glycerolipid and a fatty acid. Carboxylic acid (fatty acid) is liberated by hydrolysis of the ester bond.

  For phospholipase, an enzyme that hydrolyzes the fatty acid ester bond at the sn-1 position of the glycerol group in glycerophospholipid is called phospholipase A1 (the activity is called phospholipase A1 activity), and the fatty acid ester bond at the sn-2 position of glycerol The enzyme that hydrolyzes is called phospholipase A2 (the activity is called phospholipase A2 activity). An enzyme having both phospholipase A1 activity and phospholipase A2 activity is referred to as phospholipase B (the activity is referred to as phospholipase B activity). The glycerol phospholipid from which only one of the fatty acid acyl groups at the sn-1 position or the sn-2 position in the phospholipid is removed is called lysoglycerol phospholipid, but remains acting on the lysoglycerol phospholipid. The activity that catalyzes the hydrolysis reaction of the fatty acid ester bond (lysophospholipase activity) is also included herein in the phospholipase B activity.

  The position and number of ester bonds to be hydrolyzed are not particularly limited. Either one or both of two ester bonds of glyceroglycolipid and glycerophospholipid may be hydrolyzed.

  The lipase may have a molecular weight measured by SDS-PAGE of 20,000 to 50,000.

  Although the supply source of the lipase of the present invention is not particularly limited, it can be obtained from living cells such as microorganisms. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces. Preferably, Streptomyces sanglieri (Streptomyces sanglieri (Accession Number: NITE BP-1392)) is mentioned, but it is not limited thereto, and any microorganism that can produce the lipase of the present invention may be used.

  In addition, natural or artificial mutants of these biological species or gene fragments necessary for the expression of the lipase activity of the present invention are artificially extracted and used for the present invention even in other biological species incorporating them. Can do.

  For example, Streptomyces sanglieri (Streptomyces sanglieri) A14 strain (NITE BP-1392) secretes lipase outside the cells by culturing in an appropriate nutrient medium, so that the culture supernatant is lyophilized, salted out, What was processed with the organic solvent etc. can be manufactured as a lipase formulation.

  In the present invention, the microorganism is any of wild strains, mutant strains (for example, induced by ultraviolet irradiation), or recombinants induced by genetic engineering techniques such as cell fusion or genetic recombination. It may be a stock. Genetically engineered microorganisms such as recombinants are known to those skilled in the art, for example, as described in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989). Can be easily created using various techniques. The culture solution of microorganisms means both a culture solution containing microbial cells and a culture solution from which microbial cells have been removed by centrifugation or the like.

  The amino acid sequence of the lipase of the present invention derived from Streptomyces sanglieri strain A14 (NITE BP-1392) and the base sequence of the gene encoding it are shown in SEQ ID NOs: 5 and 4, respectively. That is, the lipase of the present invention may be a protein having the amino acid sequence shown in SEQ ID NO: 5, for example. The lipase of the present invention may be a protein encoded by a gene having the base sequence shown in SEQ ID NO: 4, for example. The expression “having an (amino acid or base) sequence” includes the case of “including (amino acid or base) sequence” and the case of “consisting of (amino acid or base) sequence”.

  The amino acid sequence shown in SEQ ID NO: 5 includes a signal sequence. The lipase of the present invention may be, for example, a protein having a portion of the amino acid sequence shown in SEQ ID NO: 5 excluding the signal sequence (that is, the mature protein amino acid sequence, SEQ ID NO: 7). In addition, the lipase of the present invention may be a protein encoded by a gene having a base sequence encoding the amino acid sequence of the mature protein in the base sequence shown in SEQ ID NO: 4, for example.

  The lipase of the present invention may be a variant of the lipase exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NO: 5 or 7) as long as the original function is maintained. Similarly, the gene encoding the lipase of the present invention (also referred to as “lipase gene”) is the lipase gene exemplified above as long as the original function is maintained, that is, as long as it encodes a protein maintaining the original function. (For example, the gene having the base sequence shown in SEQ ID NO: 4) may be a variant. Such a variant in which the original function is maintained may be referred to as a “conservative variant”. Examples of conservative variants include the lipases exemplified above, homologues of genes encoding them, and artificially modified variants.

  “The original function is maintained” means that the variant of the protein has an activity (or property) corresponding to the activity (or property) of the original protein. That is, “the original function is maintained” means that, in lipase, a protein variant has lipase activity.

  Examples of lipase homologs include proteins obtained from public databases by BLAST searches provided by UniprotKB, SwissProt, etc. using the above amino acid sequences as query sequences. The homologue of the lipase gene can be obtained, for example, by PCR using chromosomes of various microorganisms (prokaryotes) as templates and oligonucleotides prepared based on these known gene sequences as primers. On the other hand, in the case of a eukaryotic microorganism, a homolog of the above lipase gene can be obtained by PCR using a cDNA library as a template.

  In the lipase of the present invention, as long as the original function is maintained, one or several amino acids at one or several positions in the amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 7) are substituted or missing. It may be a protein having an amino acid sequence deleted, inserted or added. The above “one or several” varies depending on the position and type of the protein in the three-dimensional structure of the amino acid residue, and specifically, for example, 1 to 50, 1 to 40, 1 to 30, Preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3.

  One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences in the organism from which the protein is derived. Also included by

  In addition, as long as the original function is maintained, the lipase is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably relative to the entire amino acid sequence. It may be a protein having a homology of 99% or more. In the present specification, “homology” may refer to “identity”.

  In addition, as long as the original function is maintained, the lipase is a probe that can be prepared from the base sequence (for example, the base sequence shown in SEQ ID NO: 4), for example, a complementary sequence to the whole or a part of the base sequence, and a string. It may be a protein encoded by DNA that hybridizes under a gentle condition. Such a probe can be prepared, for example, by PCR using an oligonucleotide prepared based on the base sequence as a primer and a DNA fragment containing the base sequence as a template. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more. Is hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C, 1 × SSC, 0.1% SDS, preferably 60 ° C, 0.1 × SSC , 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, a salt concentration and temperature corresponding to 0.1% SDS, and conditions for washing once, preferably 2 to 3 times. For example, when a DNA fragment having a length of about 300 bp is used as the probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  The lipase gene may be any gene in which any codon is replaced with an equivalent codon as long as the original function is maintained. For example, the lipase gene may be modified to have an optimal codon according to the codon usage frequency of the host to be used.

  In the present invention, the term “gene” is not limited to DNA as long as it encodes a target protein, and may include any polynucleotide. That is, the “lipase gene” may mean any polynucleotide encoding lipase. The lipase gene may be DNA, RNA, or a combination thereof. The lipase gene may be single-stranded or double-stranded. The lipase gene may be single-stranded DNA or single-stranded RNA. The lipase gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand composed of a DNA strand and an RNA strand. The lipase gene may contain both DNA and RNA residues in a single polynucleotide chain. When the lipase gene contains RNA, the description regarding DNA such as the exemplified base sequence may be appropriately read according to RNA. The mode of the lipase gene can be appropriately selected according to various conditions such as the usage mode.

<2> Manufacture of lipase Lipase can be manufactured using the host which has the capability to produce lipase. That is, the present invention provides a method for producing lipase, comprising culturing a host capable of producing lipase in a medium to produce lipase, and collecting lipase from the culture. The lipase can also be produced by expressing the lipase gene in a cell-free protein synthesis system.

  The host having the ability to produce lipase may be one that inherently has the ability to produce lipase, or may have been modified to have the ability to produce lipase.

  Examples of the host having the ability to produce lipase include microorganisms belonging to the genus Streptomyces as described above, for example, Streptomyces sangulieri A14 strain (NITE BP-1392).

  Examples of a host having the ability to produce lipase also include a host into which a lipase gene has been introduced.

  The host into which the lipase gene is introduced is not particularly limited as long as it can express a functional lipase. Examples of the host include bacteria, actinomycetes, yeast, fungi, plant cells, insect cells, and animal cells. Preferred hosts include microorganisms such as bacteria and yeast. More preferred hosts include bacteria. Examples of bacteria include gram negative bacteria and gram positive bacteria. Examples of gram-negative bacteria include bacteria belonging to the Enterobacteriaceae family, such as Escherichia bacteria, Pseudomonas bacteria, Enterobacter bacteria, Pantoea bacteria, etc. It is done. Examples of Gram-positive bacteria include coryneform bacteria such as Bacillus genus bacteria, Brevibacillus genus bacteria, and Corynebacterium genus bacteria. Examples of actinomycetes include bacteria belonging to the genus Streptmyces. As the host, among them, Streptomyces lividans and Escherichiacoli can be preferably used.

  The lipase gene can be obtained by cloning from an organism having the lipase gene. For cloning, nucleic acids such as genomic DNA and cDNA containing the same gene can be used. The lipase gene can also be obtained by chemical synthesis (Gene, 60 (1), 115-127 (1987)).

  Moreover, the variant can also be obtained by appropriately modifying the obtained lipase gene. The gene can be modified by a known method. For example, a target mutation can be introduced into a target site of DNA by site-specific mutagenesis. That is, for example, the coding region of a gene can be modified by site-directed mutagenesis so that an amino acid residue at a specific site of the encoded protein includes substitution, deletion, insertion or addition. As site-directed mutagenesis, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 ( 1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 (1987 )).

  The method for introducing the lipase gene into the host is not particularly limited. In the host, the lipase gene may be retained so that it can be expressed under the control of a promoter that functions in the host. In the host, the lipase gene may be present on a vector that autonomously replicates outside the chromosome, such as a plasmid, or may be introduced on the chromosome. The host may have only one copy of the lipase gene or may have two or more copies. The host may have only one type of lipase gene, or may have two or more types of lipase genes.

  The promoter for expressing the lipase gene is not particularly limited as long as it functions in the host. The “promoter that functions in the host” refers to a promoter having promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a native promoter of the lipase gene or a promoter of another gene.

  A terminator for termination of transcription can be arranged downstream of the lipase gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a specific terminator of the lipase gene or may be a terminator of another gene.

  The lipase gene can be introduced into the host using a vector containing the gene, for example. A vector containing a lipase gene is also referred to as a lipase gene expression vector or a recombinant vector. A lipase gene expression vector can be constructed by, for example, ligating a DNA fragment containing a lipase gene with a vector that functions in a host. By transforming the host with a lipase gene expression vector, a transformant into which the vector has been introduced can be obtained, that is, the gene can be introduced into the host. As the vector, a vector capable of autonomous replication in a host cell can be used. Moreover, the vector may be provided with a suitable promoter or terminator for expressing the inserted gene. When constructing an expression vector, for example, a lipase gene containing a unique promoter region may be incorporated into the vector as it is, or the lipase coding region may be incorporated downstream of the above promoter and then incorporated into the vector. A lipase coding region may be incorporated downstream of the promoter inherent in the vector.

  For vectors, promoters, and terminators that can be used in various microorganisms, see “Basic Course of Microbiology 8 Genetic Engineering, Kyoritsu Shuppan, 1987”. Especially for those that can be used in actinomycetes, “PRACTICALSTREPTOMYCES GENETICS (Kieser et al. , JohnInnes Foundation, 2000) ”, and so on, can be used.

  Lipases can also be expressed as fusion proteins with other amino acid sequences (proteins and peptides). The type of other amino acid sequence is not particularly limited. Other amino acid sequences can be appropriately selected according to various conditions such as the type of host and the intended use of the lipase.

  The transformation method is not particularly limited, and a conventionally known method can be used. As a transformation method, for example, a method of increasing the permeability of DNA by treating a recipient cell with calcium chloride as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), as described for Bacillus subtilis, preparing competent cells from proliferating cells and introducing DNA (Duncan, CH, Wilson, GA and Young, FE., 1997. Gene 1: 153-167). In particular, for actinomycetes, transformation can be performed with reference to “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Innes Foundation, 2000)”.

  The lipase can be expressed by culturing a host having the ability to produce the lipase as described above in a medium. At that time, gene expression may be induced as necessary. Host culture conditions and gene expression induction conditions may be appropriately selected according to various conditions such as marker type, promoter type, and host type. The medium used for the culture is not particularly limited as long as the host can proliferate and can express lipase. As the medium, for example, a normal medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions, and other organic components as required can be used.

  Examples of the carbon source include sugars such as glucose, fructose, sucrose, molasses, and starch hydrolysates, alcohols such as glycerol and ethanol, and organic acids such as fumaric acid, citric acid, and succinic acid.

  Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, and aqueous ammonia.

  Examples of the sulfur source include inorganic sulfur compounds such as sulfate, sulfite, sulfide, hyposulfite, and thiosulfate.

  Examples of inorganic ions include calcium ions, magnesium ions, manganese ions, potassium ions, iron ions, and phosphate ions.

  Other organic components include organic trace nutrient sources. Examples of organic micronutrients include required substances such as vitamin B1, yeast extract containing them, and the like.

  The culture method may be liquid culture or solid culture, but liquid culture is preferred. The culture is preferably performed aerobically. Examples of the aerobic culture method include a shaking culture method and an aerobic deep culture method using a jar fermenter. The oxygen concentration at that time may be adjusted to, for example, 5 to 50%, preferably about 10% with respect to the saturation concentration. The culture temperature may be, for example, 10 to 50 ° C, preferably 20 to 45 ° C, more preferably 25 to 40 ° C. The pH of the medium may be adjusted to, for example, 3 to 9, preferably 5 to 8. For the pH adjustment, inorganic or organic acidic or alkaline substances such as calcium carbonate, ammonia gas, aqueous ammonia and the like can be used. The culture period may be, for example, 12 hours to 20 days, preferably 1 day to 7 days.

  By culturing under the conditions as described above, a culture containing lipase can be obtained. The lipase accumulates in, for example, the host cell and / or medium. “Cells” may be appropriately read as “cells” depending on the type of host.

  The lipase may be used as it is contained in the microbial cells or the like, and may be appropriately separated and purified from the microbial cells and used as a crude enzyme fraction or a purified enzyme.

  That is, for example, when lipase accumulates in the host cell, the cell can be appropriately disrupted, dissolved, or extracted, and the lipase can be recovered. The cells can be recovered from the culture by centrifugation or the like. Cell disruption, lysis, extraction or the like can be performed by a known method. Examples of such methods include ultrasonic crushing, dynomill, bead crushing, French press crushing, and lysozyme treatment. One of these methods may be used alone, or two or more thereof may be used in appropriate combination. For example, when lipase accumulates in the medium, the culture supernatant can be obtained by centrifugation or the like, and the lipase can be recovered from the culture supernatant.

  The lipase can be purified by a known method used for enzyme purification. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation. One of these methods may be used alone, or two or more thereof may be used in appropriate combination. The lipase can be purified to a desired degree.

  The purified lipase can be used as a “lipase” for hydrolysis of glycerolipids. The lipase may be used in a free state or may be used in the state of an immobilized enzyme immobilized on a solid phase such as a resin.

  Further, the lipase is not limited to purified lipase, and any fraction containing lipase may be used as “lipase” for hydrolysis of glycerolipid. The fraction containing lipase is not particularly limited as long as the lipase is contained so that lipase can act on glycerolipid. As such a fraction, for example, a culture of a host having the ability to produce lipase, a microbial cell recovered from the culture (cultured microbial cell), a crushed product of the microbial cell, a lysate of the microbial cell, Extracts of the same cells (cell-free extract), treated cells such as immobilized cells obtained by immobilizing the same cells with acrylamide, carrageenan, etc., culture supernatants recovered from the same culture, and partial purification thereof Product (crude product), and combinations thereof. Any of these fractions may be used alone or with a purified lipase.

  The collected lipase may be appropriately formulated. The dosage form is not particularly limited, and can be appropriately set according to various conditions such as the intended use of lipase. Examples of the dosage form include solutions, suspensions, powders, tablets, pills, and capsules. In formulation, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, flavoring agents, fragrances, diluents, surfactants and other pharmacologically acceptable additives. Can be used.

<3> Utilization of Lipase In the present invention, glyceroglycolipid and / or glycerophospholipid can be hydrolyzed using lipase. That is, the present invention provides a method for hydrolyzing glyceroglycolipid and / or glycerophospholipid, which comprises allowing lipase to act on glyceroglycolipid and / or glycerophospholipid. This method is also referred to as “the method of the present invention”. One embodiment of the method is a method for producing a hydrolyzate of glycerolipid, which comprises allowing lipase to act on glyceroglycolipid and / or glycerophospholipid.

  By hydrolysis of glyceroglycolipid and / or glycerophospholipid, fatty acids and glycerolipids in which fatty acids are liberated (that is, ester bonds are hydrolyzed to generate hydroxyl groups) are produced. These components may be obtained together as a hydrolyzate of glycerolipid and used. Of these components, a desired component may be separated and recovered and used. The components can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method and a membrane treatment method. These methods can be used in appropriate combination.

  For example, in the case of a glyceroglycolipid, hydrolysis of the glyceroglycolipid can produce sugar glycerol and / or lyso-type sugar glycerol (a sugar glycerol having an acyl ester at the 1-position or the 2-position). That is, one embodiment of the method of the present invention may be a method for producing sugar glycerol and lyso-type glycoglycerol, which comprises allowing lipase to act on glyceroglycolipid.

  The type of sugar glycerol produced corresponds to the type of glyceroglycolipid to be hydrolyzed. That is, for example, hydrolysis of monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol can produce the corresponding sugar glycerol and / or lyso-type sugar glycerol, respectively. The position of the sugar chain in the produced sugar glycerol and lyso-type sugar glycerol corresponds to the position of the sugar chain in the glyceroglycolipid to be hydrolyzed. That is, for example, by hydrolysis of a glyceroglycolipid having a sugar chain bonded to the 3rd position of the glycerol skeleton, a sugar glycerol having a sugar chain bonded to the 3rd position of the glycerol skeleton can be generated.

  For example, in the case of glycerophospholipid, hydrolysis of glycerophospholipid may produce 1-acyl lysoglycerophospholipid or / and 2-acyl lysoglycerophospholipid or / and glycerol-3-phosphate. That is, one aspect of the method of the present invention is a method for producing 1-acyl or / and 2-acyl lysoglycerophospholipid or / and glycerol-3-phosphate, which comprises allowing lipase to act on glycerophospholipid. May be.

  The glyceroglycolipids and / or glycerophospholipids may be themselves or a material containing them. In other words, the glyceroglycolipid and / or glycerophospholipid may be subjected to hydrolysis by lipase alone (ie, in an isolated state), or may be subjected to hydrolysis by lipase while contained in an arbitrary material. May be. For example, agricultural and aquatic products such as wheat flour containing glyceroglycolipid and / or glycerophospholipid, processed products thereof, isolates from them, artificially synthesized glyceroglycolipid and / or glycerophospholipid. The material may contain one type of glyceroglycolipid and / or glycerophospholipid, and may contain two or more types of glyceroglycolipid and / or glycerophospholipid.

  The glyceroglycolipid and / or glycerophospholipid can be subjected to hydrolysis by lipase in the form of, for example, a solution, a suspension, a slurry, or a paste. As the solvent or dispersion medium, for example, an aqueous medium such as water or an aqueous buffer can be used. Further, the glycerolipid may be appropriately subjected to a pretreatment such as emulsification and then subjected to hydrolysis with lipase. The reaction conditions of the glyceroglycolipid and / or glycerophospholipid in the reaction system (enzyme amount, reaction time, reaction temperature, reaction pH, etc.) are not particularly limited as long as hydrolysis is achieved to a desired degree. The reaction system (reaction solution and the like) may be composed of glyceroglycolipid and / or glycerophospholipid and a solvent, and may contain other components.

  The physical properties and functions of glyceroglycolipid and / or glycerophospholipid can be modified by hydrolysis. Therefore, for example, by hydrolyzing a food or drink containing glycerolipid or a raw material thereof with lipase, the physical properties or functions of the food or drink or the raw material can be modified. Therefore, the lipase may be provided as a glycerolipid modifier (physical property modifier or functional modifier). For example, as a food or drink or a raw material modifier (physical property modifier or functional modifier), for example, It may be provided as a quality improving agent for processed wheat foods and processed soybean foods.

  The lipase may also be provided as a test and research reagent such as a glycerolipid hydrolysis reagent.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to these.

Example 1: Culture of Streptomyces sanglieri strain A14
Prepare 800 mL of ISP2 medium (1% malt extract (Oriental Yeast), 0.4% yeast extract (Oriental Yeast), glucose (Wako Pure Chemical Industries, Ltd.), pH 7.2), 500 mL Erlenmeyer flask with baffle 100 ml each, and steam sterilized at 121 ° C. for 15 minutes. An appropriate amount of a colony of Streptomyces sanglieri A14 strain (NITE BP-1392) previously grown on a flat plate medium was taken, and inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of ISP2 medium. The culture was shaken until good growth was obtained at 0 ° C. 1 ml of this culture was inoculated into 100 mL of the sterilized medium, and cultured at 28 ° C. for 3 days.

Example 2: Purification of lipase derived from Streptomyces sanglieri A14 strain Streptomyces sanglieri A14 strain (NITE BP-1392) culture solution obtained in Example 1 was centrifuged. Was used to recover the supernatant from this culture solution.

(A) Ammonium sulfate fraction Ammonium sulfate was added to the culture supernatant collected in Example 1 so as to be 80% (w / v) saturation, and the resulting precipitate was centrifuged (10,000 rpm, 30 minutes, 4 ° C.). It was collected by. This precipitate was suspended in 80 ml of 20 mM Tris-HCl buffer (pH 9.0) and dialyzed with 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution.

(B) DEAE-Toyopearl column chromatography The crude enzyme solution obtained in (a) was previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0). A DEAE-Toyopearl 650M column (inner diameter 25 mm, height 38 mm, Applied to Tosoh Corporation). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of sodium chloride (from 0 M to 1 M).

(C) TOYOPEARL PPG-600M
The crude enzyme solution obtained in (b) was applied to a “TOYOPEARL PPG-600M” column (inner diameter 25 mm, height 20 mm) column (manufactured by Tosoh Biosciences) pre-equilibrated with 20 mM Tris-HCl buffer (pH 8.0). After applying and washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (from 1 M to 0 M).

(D) Active fractions obtained by HiTrap Q column chromatography (c) were collected and concentrated and desalted using Vivaspin (manufactured by Sartorius). To this, 20 mM Tris-HCl buffer (pH 8.0) was added. This was applied to a HiTrapQ (5 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM Tris-HCl buffer (pH 8.0), washed with the same buffer, and then sodium chloride ( The active fraction was eluted with a linear gradient from 0M to 1M.

(E) Mono Q column chromatography The active fraction obtained by (d) was concentrated and desalted using Viva spin. To this, 20 mM Tris-HCl buffer (pH 8.0) was added. This was applied to a “Mono Q” (1 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM Tris-HCl buffer (pH 8.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium chloride (0M to 1M).

  The eluted active fractions were collected and analyzed by SDS-PAGE (12% (w / v) polyacrylamide gel). FIG. 1 is an electrophoresis photograph showing the results of analysis by SDS-PAGE of this eluted fraction. Lane 1 (Marker) is a molecular weight marker, and Lane 2 (purified enzyme) shows a band of the eluted fraction. As a result, a single band was observed. As a result, a single band was observed at approximately 29 kDa.

  In this way, an electrophoretically purified enzyme was obtained from Streptomyces sanglieri strain A14.

  Table 1 shows the yield and yield in each purification step.

Example 3 Characteristic Evaluation of Streptomyces sanglieri A14 Strain-Derived Enzyme (GL) In this example, enzyme activity was basically measured based on the following method. This method is hereinafter referred to as “enzyme activity standard measurement method”.

Purified enzyme (GL) is added to a predetermined reaction solution (for example, 0.14M Tris-HCl buffer (pH 7.2) containing 1 mM POPS, 10 mM CaCl ₂ ) containing 10% (v / v) and a reaction solution having a total volume of 25 μL was prepared. Then, this reaction solution was stirred for 10 minutes under a temperature condition of a predetermined temperature (for example, 37 ° C.) to perform an enzyme reaction. In the enzyme reaction, the composition of the reaction solution such as the base mass, temperature, and pH were appropriately selected as shown in each experiment.

  After the enzyme reaction, the reaction was stopped by adding an equal amount of 1N hydrochloric acid to the reaction solution. The free fatty acid contained in 5 μL of the reaction solution was measured using a free fatty acid measurement kit “NEFA C-Test Wako” (Wako Pure Chemical Industries, Ltd.) as described in the instructions attached to the kit. Under these conditions, the enzyme activity that produces 1 μmol of free fatty acid per minute was defined as 1 unit (U) of galactolipase activity.

(Enzymological properties)
The enzymatic properties of the purified enzyme (GL) were examined.

(1) Working temperature In a solution containing 1 mM of substrate (POPS), 0.14 M of Tris-HCl (pH 7.2) and 10 mM of CaCl ₂ , purified enzyme (GL) is 10% (v / v). In addition, a reaction solution having a total amount of 25 μL was prepared. This reaction solution was subjected to an enzyme reaction at pH 7.2 for 10 minutes under each temperature condition. Thereafter, lipase activity was measured according to the enzyme activity standard measurement method.

  FIG. 2 is a graph showing enzyme activities at various reaction temperatures as relative activities based on the activity (100%) when the reaction temperature is 45 ° C. As shown in the graph of FIG. 2, this enzyme exerted activity at least at 20-60 ° C. and the optimum temperature for the reaction was in the range of 30-55 ° C.

(2) Working pH
A purified enzyme (a solution containing 0.14 M of a buffer (each pH) selected from 1 mM of a substrate (POPS), acetic acid-Na acetate, Bis-Tris, Tris-HCl, or Glycine-NaOH and 10 mM of CaCl ₂ is used. GL) was added to 10% (v / v) to prepare a reaction solution with a total volume of 25 μL. This reaction solution was subjected to an enzyme reaction at a temperature of 37 ° C. for 10 minutes under each pH condition. Thereafter, lipase activity was measured according to the enzyme activity standard measurement method.

Each pH condition is as follows.
Acetic acid-Na acetate buffer: pH 4, pH 4.8, pH 5.6
MES buffer: pH 5.6, pH 6, pH 6.5, pH 7
Tris-HCl buffer: pH 7.2, pH 7.5, pH 7.7, pH 8, pH 9
Glycine-NaOH buffer: pH 9, pH 10, pH 10.5

  FIG. 3 is a graph showing enzyme activities at various reaction pHs as relative activities based on the enzyme activity when the reaction pH is 10 (100%). As can be seen from the graph of FIG. 3, this enzyme exhibited activity in a wide range from pH 4 to pH 11, and the optimum pH of the reaction was around 10 (for example, 7 to 10).

(3) Substrate specificity The substrate (POPS or the like) is 1 mM, Glycine-NaOH (pH 10.5) is 0.14 M, a solution containing 10 mM CaCl ₂ aqueous solution, and the purified enzyme (GL) is 10% (v / v). In addition, a reaction solution having a total amount of 25 μL was prepared. This reaction solution was subjected to an enzyme reaction at pH 10.5 and 45 ° C. for 10 minutes. Thereafter, lipase activity was measured according to the enzyme activity standard measurement method.

  FIG. 4 is a graph showing enzyme activity when the following are used as substrates. This graph shows the relative activity when POPS is 100%.

Details of the substrate in the graph TAG: Triacylglycerol Olive oil (manufactured by Wako Pure Chemical Industries, Ltd.), Part No. 150-00276)
DAG: Diacylglycerol 1,2-DIPALMITIN (GL Sciences Inc., product number M338: 11)
MAG: Monoacylglycerol Monopalmitin (GL Sciences Inc., product number J364: 11)
PlsEtn: 1-O-1 '-(Z) -octadecenyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, product number 852758C, purity 99% or more)
MGDG: monogalactosyl diacylglycerol (LarodanFine Chemicals, # 59-1200)
SQDG: Sulfoquinoboxyldiacylglycerol (LarodanFine Chemicals, # 59-1230)
DGDG: Digalactosyldiacylglycerol (LarodanFine Chemicals, # 59-1210)
POPC: 1,2-diacyl-sn-glycero-3-phosphocholine (AvantiPolar Lipids, product number 850457P, purity 99% or more)
LPE: Lysophosphatidylethanolamine (L-α-Lysophosphatidylethanolamine, Egg) (DOOSAN Serdary Research Laboratories part number A340 purity 99% or more)
POPE: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, product number 850757P, purity 99% or more)
DPPE: 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, product number 850705P, purity 99% or more)
DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids, product number 850725C, purity 99% or more)
DMPA: 1,2-Dimyristoyl-sn-glycerol-3-phosphate (1,2-Dimyristoyl-sn-glycerol-3-phosphate) (AvantiPolar Lipids, product number 830845P, purity 99% or more)
PI: L-α-phosphatidylinositol (Enzo Life Science product number BML-PH100 purity 98%)
POPG: 1-palmitoyl-2-oleyl-sn-glycero-3-phospho- (1-rac-glycerol) (1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho- (1-rac-glycerol)) (Avanti Polar Lipids, product number 840457C, purity 99% or more)
POPS: 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine
(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) (Avanti Polar Lipids, product number 840034C, purity 99% or more)
As substrates other than DGDG, MGDG (monogalactosyl diacylglycerol), SQDG (sulfoquinovosyl diacylglycerol), POPG (1-palmitoyl-2-oleyl-sn-glycero-3-phospho-rac- (1-glycerol) )), POPC (1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine), POPS (1-palmitoyl-2) -Oleyl-sn-glycero-3-phosphoserine), DMPA (1,2-dimyristoylphosphatidic acid), Monopalmitin (monopalmitin), 1,2-dipalmitin (dipalmitin), olive oil were used. The lipase activity for each substrate other than DGDG was measured according to the standard measurement method for galactolipase activity, except that the same amount ((w / v)%) of each substrate was used instead of DGDG. Under these conditions, the enzyme activity that produces 1 μmol of free fatty acid per minute was defined as 1 unit (U) of lipase activity for each substrate. The lipase activity against olive oil was measured according to the method described in Japanese Patent No. 5179208. Specifically, olive oil (produced by Nacalai Tesque) 200 mg and gum arabic (produced by Wako Pure Chemical Industries, Ltd.) 100 mg are added with 10 ml of water, and the ultrasonic generator (Fine, Ultra sonic cleaner, 100W) Sonication for 1 minute and vortexing for 1 minute were emulsified under a total of 2 conditions, and this was used as a substrate solution.

  From the graph of FIG. 4, it was confirmed that the purified enzyme acts substrate-specifically and exhibits galactolipase activity and phospholipase activity.

  Specifically, the hydrolysis activity of GL was almost 0% with respect to PlsEtn, MAG, DAG, and TAG, assuming that the activity when POPS was used as a substrate was 100%. Moreover, POPG, PI, DMPA exceeded 70%, phosphatidylethanolamine or its phospholipase hydrolyzate DOPE, DPPE, POPE, LPE was 30% or more, and POPC showed activity exceeding 20%. .

  The glycerol glycolipids DGDG, SQDG, and MGDG showed an activity of about 20% or more.

(4) Temperature stability The purified enzyme (GL) was incubated at each temperature for 30 minutes. In a solution containing 1 mM of substrate POPS, 0.14 M of Glycine-NaOH (pH 10.5), 10 mM of CaCl ₂ and 0.1% (w / v) of Triton X-100, 10 enzyme after treatment at each temperature was added. % (V / v) was added to prepare a reaction solution with a total volume of 25 μL. This reaction solution was subjected to an enzyme reaction for 10 minutes under the conditions of pH 10.5 and temperature 45 ° C. Thereafter, lipase activity was measured according to the enzyme activity standard measurement method.

  FIG. 5 is a graph showing the activity of the enzyme after the treatment at each temperature as a relative activity based on the activity (100%) when the enzyme reaction is carried out at a temperature of 4 ° C. As shown in the graph of FIG. 5, after the treatment at a temperature from 4 ° C. to 55 ° C., the enzyme maintained an activity of 80% or more before the treatment.

(5) pH stability Purification to a buffer (each pH) of 50 mM selected from acetic acid-Na acetate (AceTate in the graph), MES, Tris-HCl (Tris in the graph), or Glycine-NaOH (Glycine in the graph) Enzyme (GL) was added to 20% (v / v) and treated at 4 ° C for 3 hours at each pH. Thereafter, the enzyme after treatment at each pH was added to a solution containing 10 mM of substrate POPS, 0.14 M of Glycine-NaOH (pH 10.5), 10 mM of CaCl ₂ and 0.1% (w / v) of Triton X-100. Was added so that it might become 10% (v / v), and the reaction liquid used as a total amount of 25 microliters was prepared. This reaction solution was subjected to an enzyme reaction for 10 minutes under the conditions of pH 10.5 and temperature 45 ° C. Thereafter, lipase activity was measured according to the enzyme activity standard measurement method.

  FIG. 6 is a graph showing the activity of the enzyme after treatment at various pHs as relative activity based on the activity (100%) when the enzyme reaction is performed by pH treatment at pH 8 with Tris-HCl. . As shown in the graph of FIG. 6, this enzyme was confirmed to have an activity of 50% or more even after pH treatment at least from pH 5.5 to 8.

(6) Effects of metal ions and EDTA 10% enzyme in a solution containing 1 mM substrate (POPS), 0.14 M Glycine-NaOH (pH 10.5) and 0.1% (w / v) Triton X-100 In addition to adding (v / v), 10 mM of various metal ions or EDTA was further added to prepare a reaction solution having a total amount of 25 μL. Here, metal chloride (MCln: M is a metal and n is 1 or 2) was added as a metal ion. This reaction solution was subjected to an enzyme reaction for 10 minutes under the conditions of pH 10.5 and temperature 45 ° C. Thereafter, lipase activity was measured according to the enzyme activity standard measurement method.

FIG. 7 shows the results of relative activities with various additives added, assuming that the condition (free) in which no additives (metal ions and EDTA) are added is 100%. The above enzyme showed higher activity in the presence of Na ⁺ , Al ³⁺ , Mg ²⁺ , Li ⁺ than when no additive was added. In the presence of Ca ²⁺ and K ⁺ , the activity was almost the same as when no additive was added. In the presence of EDTA, the activity tended to decrease slightly. In the presence of Zn ²⁺ , the activity was greatly reduced and activity inhibition was observed.

(7) Confirmation of phospholipase B activity of glycerol phospholipid 0.1% (w / v) Triton X-100 (manufactured by Nacalai Tesque) 1 mL of phosphoserine (Avanti polar lipids, 1-palmitoyl) -2-oleoyl-sn-glycero-3-phospho-L-serine) was dissolved to prepare 10% (w / v) phosphoserine. To 2.5 μL of 10% (w / v) phosphoserine, 17.5 μL of 0.2 M Glycine-NaOH (pH 10.5) and 2.5 μL of 10 mM NaCl were added. And after preheating at 45 degreeC for 5 minute (s), 2.5 microliters of samples containing purified enzyme (GL) were added, and it was made to react at 45 degreeC for 10 minutes. Thereafter, 100 μL of chloroform: methanol solution (2: 1, v / v) was added to 25 μL of this reaction solution, and after vigorous stirring, centrifugation was performed (21600 × g, 5 min, 4 ° C.). After centrifugation, only the chloroform layer (lower layer) was collected, concentrated under reduced pressure, and adjusted to 25 μL of a chloroform: methanol solution (2: 1, v / v).

  The prepared solution was subjected to capillary gas chromatography (cGC) analysis (using a gas chromatograph GC-14B manufactured by Shimadzu Corporation) to identify and quantify free fatty acids. Palmitic acid and oleic acid were produced by an enzymatic reaction, and the amounts produced were almost the same. Thus, it was confirmed that the enzyme (GL) of the present invention exhibits phospholipase activity B.

Example 4: Determination of amino acid sequence of lipase derived from Streptomyces sanglieri A14 [analysis of internal amino acid sequence of purified enzyme]
About said purified enzyme, after SDS-PAGE, the target enzyme was cut out and digested in gel using trypsin. And the internal amino acid sequence was analyzed with the mass spectrometer (nanoLC-MS / MS) about the obtained peptide sample. Thereby, it was confirmed that the internal amino acid sequences are those shown in SEQ ID NOs: 1 to 3.

  Here, SEQ ID NO: 1 is the sequence shown from position 141 of SEQ ID NO: 5. SEQ ID NO: 2 is the sequence shown from position 175 of SEQ ID NO: 5. SEQ ID NO: 3 is the sequence shown from position 209 of SEQ ID NO: 5. In addition, since de novo amino acid sequence analysis by nanoLC-MS / MS cannot discriminate between Leu and Ile, there are places where these amino acids do not partially match.

Example 5: Isolation of chromosomal DNA from Streptomyces sanglieri A14 strain Streptomyces sanglieri A14 strain was prepared by adding 100 μl of 2.5 M magnesium chloride, 1.25 ml of 20% glycine 1.25 ml YEME medium (peptone 0). 5%, yeast extract 0.3%, malt extract 0.3%, glucose 1%, sucrose 34%), cultured at 28 ° C. for 3 days, collected by centrifugation, sterile physiological saline, etc. Washed with.

  Subsequently, this microbial cell was suspended in 5 ml of a solution consisting of 75 mM NaCl, 25 mM EDTA, 20 mM Tris-HCl buffer (pH 7.5) and 1 mg / ml lysozyme, and treated at 37 ° C. for 1 hour. To this, 750 μl of 10% (w / v) SDS and 3.75 mg of proteinase K were added and treated at 55 ° C. for 2 hours. To this solution, 2 ml of 5M NaCl and 5 ml of chloroform were added and stirred, and 5 ml of the aqueous phase was collected by centrifugation.

  To this aqueous phase, 3 ml of isopropanol was added and mixed to collect the DNA fraction, which was rinsed with 1 ml of 70% ethanol and then centrifuged. This precipitate was collected, dried under reduced pressure, dissolved in 500 μl of a solution consisting of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA, and left at 55 ° C. overnight. RNaseA was added to this so that it might become 0.1 mg / ml, and after processing for 20 minutes at 37 ° C., 500 μl of 13% PEG solution containing 0.8 M NaCl was added and stirred, and left at 4 ° C. for 1 hour. The precipitate was recovered by centrifugation. This precipitate was dissolved in 500 μl of 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA. 500 μl of a phenol / chloroform mixed solution was added thereto and stirred, and 500 μl of an aqueous phase was collected by centrifugation.

  To this aqueous phase, 50 μL of 3M sodium acetate buffer (pH 5.2) and 1 ml of 100% ethanol were added and mixed to recover DNA.

  This DNA was immersed in 70% (v / v) ethanol for 10 minutes and then dissolved in 200 μl of a solution consisting of 10 mM Tris-HCl buffer (pH 8.0) and 1 mM EDTA.

Example 6: Genomic DNA sequence analysis of Streptomyces sanglieri strain A14
(a) The recovered DNA was treated according to the manufacturer recommended protocol of Ion shear plus enzyme mix (Life Technologies) and fragmented to about 200 bp. The obtained DNA fragments were subjected to size selection using E-gel safe imager (Invitrogen) and Solidlibrary size selection gels. Electrophoresis was performed for 18 min, and a DNA fragment of about 200 bp was collected in an LB tube as compared with the DNA size marker that was simultaneously run.

  The obtained DNA fragment was column purified using MonoFas DNA purification kit I (GL science). When eluting the DNA fragment from the column, it was eluted with 20 μl of autoclaved Tris-HCl buffer (pH 8.0).

  Thereafter, the size, quality and concentration of the prepared DNA fragment sample were confirmed using D1k screen tape (Agilent Technology).

  (b) Next, two kinds of DNA adapter primers necessary for sequencing are ligated to both ends of the prepared DNA fragment using Takara Dice thermalcycler (Takara), and nick translation using polymerase is performed. Thus, a library was prepared and column purified using MonoFas DNA purification kit I (GL science).

  Thereafter, the size, quality and concentration of the prepared DNA fragment sample were confirmed using D1k screen tape (Agilent Technology).

  (c) The prepared DNA library was subjected to emulsion PCR (hereinafter abbreviated as ePCR) using the IonOneTouch 2 system (Life Technologies). In ePCR, in a water-in-oil emulsion, a single molecule DNA library binds to DNA capture beads (Ion sphere particles, hereinafter abbreviated as ISP beads) via an adapter ligated to DNA fragments in 5-3-3. . Thereafter, by performing a PCR reaction in a water droplet, only the DNA library bound to the ISP beads is PCR amplified. This is purified by enrichment with Ion OneTouch ES and used as template DNA for the next-generation sequencer.

  (d) The fragmented DNA sample prepared as described above was sequenced using ION PGM (Life Technologies) to obtain a large amount of base sequence information of the fragmented genomic DNA. Numerous contig sequences were obtained by assembling the obtained base sequences on a computer. Using getorf (free software), orf (reading frame) was extracted from the acquired contig sequence and converted to an amino acid sequence. By searching for orf corresponding to the internal amino acid sequence obtained by nanoLC-MS / MS analysis, orf (total amino acid sequence) of GL and the base sequence of the gene were identified.

Example 7: Determination of base sequence of GL gene derived from Streptomyces sanglieri A14 strain Region containing GL gene derived from Streptomyces sanglieri A14 strain based on the base sequence determined above Was determined (SEQ ID NO: 4). SEQ ID NO: 5 is an amino acid sequence corresponding to the codon of this base sequence. Signal P (free software) was used to predict the secretory signal sequence. As a result, it was found that it has a signal region shown in SEQ ID NO: 6. Therefore, it was presumed that a portion obtained by removing positions 1 to 29 of SEQ ID NO: 6 from SEQ ID NO: 6 was a mature enzyme portion. When the isoelectric point (pI) of the mature enzyme portion was calculated using GenetyxMac, the pI was calculated to be 7.57. On the other hand, the pI of NA297 strain phospholipase A1 described in WO2013 / 145289 is 6.06, the pI of NA684 strain phospholipase B described in WO2012 / 104988 is 6.44, and is described as No. 4 sequence in Patent Document 2. The pI of galactolipase was 7.19.

  As a result of the sequence analysis, it was revealed that 264 amino acids were encoded. The amino acid sequence of SEQ ID NO: 6 is a secretory signal sequence.

  The N-terminal and internal amino acid sequences of the purified enzyme derived from Streptomyces sanglieri strain A14 determined above were present in the deduced amino acid sequence and were almost completely identical.

  That is, the amino acid sequence of SEQ ID NO: 7 is a mature enzyme part excluding the signal part. The amino acid sequence of SEQ ID NO: 1 is the sequence shown from position 112 of the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of SEQ ID NO: 2 is the sequence shown from the amino acid sequence 146 of the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of SEQ ID NO: 3 is the sequence shown from position 180 of the amino acid sequence of SEQ ID NO: 7. However, since de novo amino acid sequence analysis by nanoLC-MS / MS cannot distinguish Leu and Ile, there are places where these amino acids do not partially match.

  Further, the purified enzyme (ie, GL) derived from Streptomyces sanglieri strain A14 was calculated based on the amino acid composition of this deduced amino acid sequence, and was estimated to have a molecular weight of about 30,000. .

  According to the present invention, a novel GL is provided.

Name of the trustee organization: National Institute of Technology and Evaluation Patent Microorganism Depositary Center Depositary Organization: Japan 2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818
Date of deposit: July 24, 2012 Deposit number: NITE BP-1392
<Explanation of Sequence Listing>
SEQ ID NO: 1: GL internal sequence SEQ ID NO: 2: GL internal sequence SEQ ID NO: 3: GL internal sequence SEQ ID NO: 4: GL gene base sequence SEQ ID NO: 5: amino acid sequence corresponding to the codon of the GL gene base sequence No. 6: Secretion signal sequence of GL gene SEQ ID No. 7: Amino acid sequence of mature enzyme part of GL gene

Claims (10)

Lipases having the following properties (1) to (3):
(1) having galactolipase activity and phospholipase B activity;
(2) the molecular weight is 20,000 to 50,000;
(3) Derived from microorganisms belonging to the genus Streptomyces.   The lipase according to claim 1, wherein the genus Streptomyces is Streptomyces sanglieri. Lipase, which is a protein described in the following (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 7;
(B) A protein having an amino acid sequence containing substitution, deletion, insertion, or addition of 1 to 50 amino acid residues in the amino acid sequence shown in SEQ ID NO: 7, and having galactolipase activity and phospholipase B activity.   A polynucleotide encoding the lipase according to any one of claims 1 to 3. The polynucleotide according to (A) or (B) below:
(A) a polynucleotide comprising the base sequence represented by SEQ ID NO: 4;
(B) a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 4, and
A polynucleotide encoding a protein having galactolipase activity and phospholipase B activity.   A recombinant vector comprising the polynucleotide according to claim 4 or 5.   A transformant into which the recombinant vector according to claim 6 has been introduced.   A transformant according to claim 7 is cultured in a medium to produce the lipase according to any one of claims 1 to 3, and the lipase is collected from the culture. Manufacturing method.   A method for hydrolyzing glycerolipid, comprising causing the lipase according to any one of claims 1 to 3 to act on glyceroglycolipid and / or glycerophospholipid.   A glycoglycerol, a lyso-type glycoglycerol, a 1-acyl lysoglycerophospholipid, a 2-acyl lysoglyceroline, which comprises allowing the lipase according to any one of claims 1 to 3 to act on a glyceroglycolipid and / or a glycerophospholipid. A method for producing a glycerolipid hydrolyzate comprising one or more selected from the group consisting of lipids and glycerol-3-phosphate.