JPH02174680A - Gene of protein having lipase activity - Google Patents

Abstract

PURPOSE:To obtain vector for lipase production by synthesizing the gene coding the protein having lipase activity with messenger RNA stemmed from Geotrichum candidum ATCC 34614. CONSTITUTION:Complementary DNA (c DNA) is first prepared from messenger RNA (m RNA) stemmed from Geotrichum candidum ATCC 34614 stored in the American type culture collection. The vector for Escherichia coli, yeast, Bacillus subtilis, etc., is then incorporated with the above-mentioned lipase c DNA and its upstream portion is inserted with an appropriate promoter, thus obtaining the objective manifestation vector. Said promoter, for Escherichia coli, is pref. tryptophan beta-galactosidase, alkali phosphatase or beta-lactamase.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a gene encoding a protein having lipase activity. For more information, see Geotrichum preserved in the American Type Culture Collection
Candidum (Geo-trichun+ candi)
The base sequence of complementary DNA (hereinafter referred to as cDNA) prepared from metsenger RNA (hereinafter referred to as mRNA) derived from ATCC34614.

(Prior art) Lipase is an enzyme that acts on triglycerides and hydrolyzes them into fatty acids and glycerin. When the water content of the reaction system is reduced, it also catalyzes the reaction of synthesizing esters from fatty acids and alcohol [Journal of General Applied Microbiology (J, Gen, Appl,
Microbiol, ) River, 13 (1964))
. Currently, lipase is used in food processing as a flavoring agent, in the pharmaceutical field as a digestive agent and a clinical test reagent for quantifying neutral lipids in blood, and recently in the decomposition of fats and oils to produce fatty acids for detergents. However, its utilization is extremely low compared to amylase and protease, which are called the three major digestive enzymes along with lipase.

This is largely due to the fact that the cost of the enzyme is high due to the low enzyme productivity of lipase-producing bacteria, and the fact that the target of lipase's action is fats and oils. For example, fats and oils are not only insoluble in water, but also fats and oils made of long-chain fatty acids are solid at room temperature. Therefore, reactions using lipase, unlike other enzymatic reactions, often require considerably high temperatures or the addition of organic solvents, and such reaction systems require extremely high stability of the enzymes used. be done.

On the other hand, when lipases are classified from the viewpoint of specificity of action on the position of the ester bond in triglyceride, there are two types: those that hydrolyze the ester bond at the 1, 2, and 3 positions of triglyceride, and those that hydrolyze the ester bond at the 1 and 3 positions. We can separate. However, there are few reports on lipases that preferentially hydrolyze only the ester bond at the 2-position.If a lipase with such positional specificity could be obtained, it would be possible to perform advanced processing of raw oils and fats through transesterification reactions. It can be expected that the applications for this will expand.

In this way, when considering the industrial use of lipase, in addition to increasing enzyme productivity by microorganisms, lipases that are stable against heat, lipases that have a high optimum temperature of action, and lipases that work stably even in organic solvents are important. A lipase with characteristics such as substrate specificity and position specificity is desired. Furthermore, enzymes that are stable against acids and alkalis naturally have high industrial utility value. Many researchers are currently searching for such lipases in nature, but none have yet been found. Therefore, one method would be to use protein engineering techniques to improve existing lipases to suit industrial purposes.

(Problem to be solved by the invention) In order to improve existing lipases using protein engineering technology, the three-dimensional structure of the lipase must be elucidated, and the base sequence of the gene encoding the lipase must be elucidated. There must be.

In the present invention, Geotrichum, whose three-dimensional structure has been elucidated,
Geotrichum candidum
m) lipase [Journal of φBiochemistry (J, Biochem, ) 1821 (1979
)), mRNA was extracted and purified from lipase-producing microbial cells, and the lipase gene was cloned using the mRNA to complete the present invention.

(Means for Solving the Problems) The present invention is a gene encoding a protein with lipase activity, which is characterized by having the following base sequence.

CAG GCCCCA CGG CCG TCT CT
CAAT GGCAACGAG GTCATCTCT
GGT GTCCTT GAG GGCAAGGTT
GAT ACCTTCAAG GGA ATCCCA
TTT GCTGACCCT CCT TTG AAT
GACTTG CGA TTCAAGCACCCCCC
AG CCT TTCACT GGA TCCTACC
AGGGT CTT AAG GCCAAT GAT
TTT AGCCCT GCTTGT ATG CAG
CTT GAT CCT GGCAAACTCT CT
CACT TTG CTT GACAAA GCT C
TG GGA TTG GCAAAA GTCATCC
CCGAA GAA TTT AGA GGT CCC
CTT TAT GAT ATG GCCAAG GG
T ACCGTG TCGATG AAT GAG G
ACTGT CTT TACCTCAAT GTTTT
CCGCCCT GCT GGCACCAAG CCT
GAT GCTAAG CTCCCCGTCATG
GTT TGG ATT TACGGTGGT GCG
TTT GTT TACGGT TCT TCT G
CT GCCTACCCT GGT AACAGCTA
CGTT AAG GAA AGTATCAACATG
GGCCAG CCCGTT GTG TTT GT
TTCCATCAACTACCGT ACCGGT C
CA TTT GGATTCCTG GGT GGT
GAT GCCATCACCGCT GAGGGCAA
CACC CGCAAG GGT ATT GCCAAC GTCATG ATT ATG AGT GTT GGT GGT GAC CTT TTCCAC GGCCCT CTT GTT GGT CCC GCT CAG CAT AGT GCCAAC AG CAAG TCC CAG AACTCT GGT CTA CTC CCCAGA CCC GCCGCT TAT TACGCCAAG CAG GAG GAT GTT GCT CTC GTT AAG AAG AACGCT GGT CTCGAG TGG TTT GGT GGT TTCGGT GAG GCT CACCAG AACACCTAC TCT GCCATT CCT TACCAC GAT ATT TCC GCCGGA TGT GACACT CTG AGCTCT GTC TACGAT CTC CCT CAA TTC GACGGCAAC GAG CTCTTC GTT CCCTAC GAA GGT ACT AACGCT ACC TGG TTG CAG CTG CACGAC GTT AGCGAC GAT CCCGAT TCCGCT GGT CTT ATT GCC AACGGA AAG CTT CAG TCT GACTCT ACC TACAACAGA GACACT AGT GAG TGT CTC TTG CACGAT AAA GAT CTG CTT GGA TTT ATT ATT CCC CGCAGCG GT ATT AGCGGT GCT TTT GCT ACG ACT CCC TACATT TTC AAC AAC AAC CCC CAT AAC CCT CCC TTT CCC CCC CCC TTT CCT CAT AGA AAC CCT CAT AAC GAT GCT TCCGAG GCT TCCATT
GACCGT GTTTTG TCG CTG TA
CCCG CAG ACCCTCTCT GTTGGC
TCG CCCTTCCGCACT GGCATT C
TT AATGCCCTG ACCCCCCAG TT
CAAG CGT GTT GCGGCCATCTTG
TCCGAT ATG CTT TTCCAG TC
TCCCCGCCGCGTG ATG CTT AGC
GCCACCAAGGACGTTT AACCGCTG
ACT TACCTT TCG ACCCAT (:
TG CACAACCTG GTG CCA TTT
TTG GGTACT TTCCAT G to CAACG
AG CTT ATCTTCCAATTCAAT GT
G AACATT GGCCCCGCT AACTCC
TACCTT CGT TACTTT ATT
TCCTTT GCCAACGAG CAT GA
CCCT AAT GTT GGT ACT AACC
TGCTCCAG TGG GAT CAA TACA
CT GAT GAA GGCAAG GAG ATG
CTT GAG ATT CACATG ACCGA
TAAT GTCATG AGA ACT GAT G
ACTACAGA ATTGAG GGA ATCTC
A AACTTT GAG ACT GACGTTAA
T CTCTACGGT Where, A is adenine, G is guanine, T is thymine, C
represents cytosine.

The present invention will be explained in detail below.

■When the amount of lipase and mRNA in the microbial cell reaches its maximum value at 1 bar', the production rate of lipase reaches its maximum.

Therefore, Geo, candidum was extracted using a known method (Ag
ric.

Biol, Biochem, 31 145? (
1973)), and when the specific production rate of lipase reaches its maximum value (15 to 18 hours), the bacterial cells are collected from the culture solution by centrifugation. In order to minimize enzymatic degradation of RNA, the recovered bacterial cells are immediately frozen in acetone-dry ice and then stored frozen at -80°C until immediately before use. Note that the lipase activity was determined by Fukumoto
to J, ) et al.'s method (j, Gen, Appl,
Microbiol, Ji257 (1964)
) can be measured by rotary stirring, etc.

■ Extraction of total RNA from the bacterial cells obtained in step (■) was performed using the guanidine thiocyanate method (Biochen+1 try).

1J15294 (+979) ) and the like.

The separation of mRNA from total RNA was performed using a known method (Proc, Natl,
Acod, Sci, USA...! JL140B (197
2) ) etc.

Isolation and purification of mRNA In order to prevent degradation of RNA by bonuclease, use equipment that has been sterilized by dry heat or autoclave, and use reagents that have been sterilized by autoclave as much as possible. It is also recommended to wear vinyl gloves during operation.

■ Using the mRNA obtained by o, dil-■ as a template, the well-known Okayama-Barb method (M
ol, Ce11. Biol, 2-, 161 (1
9B2)) to produce a pod plasmid. This recombinant plasmid was used as a large 111m (E.
Transfect it into a host such as HI strain, select ampicillin-resistant strains, and create a Geocandidum cDNA library.

■ ・i, I per pe+
1 1 By chemically synthesizing DNA corresponding to a partial amino acid sequence of lipase and performing a colony hybridization test using this as a probe, the cDNA created in
A clone containing the target lipase cDNA can be isolated from the library.

The amino acid sequence of the peptide constituting Geo. candidum lipase can be determined by the following method.

For proteins whose N-terminal amino acids are not blocked, for example, the Edman degradation method (Acta Chem, 5
cand,, 14.283. (1950)), the amino acid sequence from the N-terminus can be determined. However, since the N-terminus of Geocandidum lipase is blocked, the amino acid sequence cannot be determined from the N-terminus. In such cases, purified Novase (Agric, Biol. ,Chem,'J1
．． 1457 (1973)] and fractionated and purified the peptide by high performance liquid chromatography or gel filtration using a reversed-phase C4 column, etc. Among these, for peptides whose N-terminus is not blocked, the amino acid sequence from the N-terminus can be determined by Edman degradation.

(1) Chemically synthesize NA complementary to the base sequence corresponding to the amino acid sequence of lipase determined by colony hybridization ■, and use T4 polynucleotide kinase to 5
A 3P-labeled probe is prepared by transferring the phosphate group of [γ-''''P)ATP to the hydroxyl group at the ' terminal. To isolate clones with lipase cDNA from the cDNA library created in step ①, after growing large mi in the cDNA library on a nylon filter, colony hybridization is performed to ensure that the probe DNA is strongly detected. Select the combined clones.

(2) Confirmation that the cloned cDNA is lipase cDNA.

2) It can be confirmed that the positive clone isolated in -(+) has the target lipase cDNA by, for example, determining the base sequence and then determining the amino acid sequence based on this. That is, the probe sequence used for colony hybridization is present in the determined base sequence, and Ge0triChul+candidum is present in the amino acid sequence determined from the base sequence.
It is only necessary to confirm the existence of a sequence that partially matches the amino acid sequence of lipase. Therefore, a restriction enzyme map was created for the cloned cDNA fragment, and Sanger (Sange)
rF,) et al. (Proc, Natl.

Acad, Sci, USA, 1! L, 5463
(1977) ), the base sequence is determined according to the dideoxysiefens method. By analyzing this base sequence and selecting a cDNA containing a base sequence complementary to the probe used for colony hybridization and a base sequence encoding the amino sequence of lipase, lipase cD
NA can be cloned.

(Action of the Invention) A vector for lipase production can be created by incorporating the cloned cDNA of the present invention into an expression vector or an expression/secretion vector.

In other words, vectors such as Escherichia coli, yeast, and Bacillus subtilis,
This lipase cDNA is integrated, and an appropriate promoter (for large plants, nitributophan β-galaftosidase, alkaline phosphatase, β-lactamase, etc.; for yeast, acid phosphatase, alcohol dehydrogenase, galactokinase, α-factor, etc.) is inserted upstream of the lipase cDNA. , in the case of Bacillus subtilis: α-amylase, protease, etc.). Furthermore, in some cases, an increase in expression efficiency can be expected by inserting an appropriate terminator downstream of the lipase cDNA. As hosts, in addition to microorganisms such as Escherichia coli, yeast, and Bacillus subtilis, cultured cells such as cos cells can be used.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to these examples.

〔Example〕

Ge0triChul candidum was grown in a 500-liter Yosaka flask and mixed with 60-liter lipase production medium (corn staple liquor 5%, NH4No, 0.5%, soybean oil 1
%, pH 5, 8) at 27° C., the culture solution was cultured with shaking while the lipase activity of the culture solution was measured over time. When the specific production rate of lipase reaches its maximum value (lipase activity is 20-30 U)
/N, immediately cool with water to stop the culture, and centrifuge (8
, 000 rpm, 5 minutes). The recovered bacterial cells were immediately frozen with acetone-dry ice to obtain lipase-producing bacterial cells. In addition, this bacterial cell should be kept at 180 °C until just before use.
Stored frozen at ℃.

(1) Preparation of total RNA Total RNA from bacterial cells whose specific production rate of lipase has reached the highest value
A was extracted using the guanidine thiocyanate method (Bioch
emistry, LIIL, 5294 (1979)
) was followed. That is, 4 g of frozen bacterial cells obtained in step 1
Add 20ml of guanidine thiocyanate solution (4M guanidine thiocyanate, 5mM sodium citrate (p
H7,0) 0.5% Sarcosyl Sodium, 0.1 M
After suspending in β-mercaptoethanol) and disrupting with a homogenizer, the homogenate supernatant is collected by centrifugation. 5-polyaromer tube for ultracentrifuge
3 volumes of 5.7M cesium chloride (0.1M EDTA,
(f) containing H7,5), and then layered the homogenate supernatant on top of this, and added Hitachi Kogyo 1! Ultracentrifuge at 35,000 rpm and 25° C. for 12 hours using a RPs50-20-tar. Total RNA recovered as a precipitate was diluted with 10mM Tr.
is-HCj! (pI7.5) -1mM
It was dissolved in EDTA (hereinafter abbreviated as TE) buffer.

(2) Isolation and purification of mRNA from total RNA Preliminary TE containing 0.5M LiCl and 0.5% SDS
onto an oligo(dT)+5-12 cellulose (Pharmacia) column (capacity 0.71R1) equilibrated with a buffer solution.
The total RNA from step 2 (1) dissolved in the same buffer was heated at 65° C. for 6 minutes and then supplied. Same buffer 5
M! After washing out non-adsorbed substances, elution was carried out with a TEIt buffer containing 0.5% SDS. Collect the peak fraction with absorption at 260 μm, add 1/10 amount of 3M sodium acetate, extract with phenol/chloroform (1; 1 ν/v), add 245 times the amount of ethanol, and leave at 20°C overnight. do. After collecting the precipitate by centrifugation, 0.3M
It was dissolved in TE buffer containing sodium acetate and 0.5% SDS, added with 2.5 volumes of ethanol, and stored at -20°C. The recovery rate of mRNA was 14 mRNA per 1μ wet heavy bacterial cell.
It was 5 μg.

Reverse transcriptase buffer (0,5M Tris-HC1 (p
) [8,3) , 80mM MgCI2,300m
M KCI)2uJ! , 2μ of 20mM deoxynucleotide triphosphate mixture! , 6mM DTT
1μl, Ca-”P)dCTP (400C/mn+
ole) 10 μCi, 5 μg in a mixture containing 1.4 μg of 3'-oligo(αT)-tilt pSV7186-Derived Plasmid Brimer ()7-Macia Co., Ltd.
mRNA (dissolved in TE buffer and heated at 65°C for 5 minutes) and about 10 amounts of reverse transcriptase were added to make a total volume of 2
0μ2 and allowed to react at 37°C for 1 hour. 1μJl! (
7) After stopping the reaction by adding 0.5M EDTA and 1μ of 10% SDS, add phenol/chloroform (1:1
v/V) Extract and add twice the amount of ethanol.

The precipitate was diluted with 15μ2 of terminal deoxytransferase buffer (140mM sodium cacodylate, 30mM
M Tris-HCl (pH 6,8), 1mM
CoCl, 0.1mM DTT 0.2μg PolyA
, 66℃M Ca-”P)dCTP (1011C1)
] and added about 18 amounts of terminal deoxytransferase. When the dG chain has been added with 10 to 15 residues (approximately 5 minutes), add 0.5 M EDTA and 10% SDS.
The reaction was stopped by adding 0.5 μβ of each, followed by phenol/chloroform extraction and ethanol precipitation.

The precipitate was diluted with trindm buffer (20mM Tr) at 10°C.
is-HCJ! (pH 7,4), 7mM MgC
1z, 60mM NaCl2, 0.1 mgBsA)
About 10 amounts of Hind m was added to the mixture, and the mixture was digested at 37°C for 1 hour.

After stopping the reaction, phenol/chloroform extraction and ethanol precipitation were performed. This precipitate was dissolved in 10 μm of buffer solution M and 3 μm of ethanol was added and stored at -20°C.

This sample lμk? ng 3'-oligo (dG) tilt psV1932-private Hind m linker (
Pharmacia) with lOμ2 of 0.1M NaC
1 in TE11 buffer at 65°C for 5 minutes, then at 42°C.
After being left closed for 30 minutes at ℃, it was cooled on ice. Tris-
HCJ2 buffer (pH 7,5) (20mM), Mg
CJ! 2 (4mM), (NH4) 2S04
(10rnM), KCJ! (0,1M), β-
NAD (0.1mM), BSA (50℃g/J)
, E. coli DNA ligase (0.6 Mg) was added to bring the volume to 100 μ, and the mixture was incubated at 12° C. overnight.

Deoxynucleotide triphosphate mixture (40℃M) and β-NAD (0.15mM) were added to the above reaction mixture.
, E. coli DNA ligase (0.4 μg'), large intestine i [
lDNA polymerase I (0.3μg), large II loop R
Add Nase (1 unit) and incubate sequentially at 12°C and 25°C.
Incubated for hours.

Using the obtained recombinant plasmid solution,
The H1 strain was transformed. That is, E. coli was grown in a 100-ψ medium (Bacto tryptone 20 g/λ, Bacto yeast extract 5 g/l, Mg5O, δg/2, KOH'''Qp
The cells were cultured at 37°C in a medium (adjusted to H7.6) until the absorbance (550 μm) reached 0.5. Centrifugation (6000 rpm
, 5mln), then add 40-ml water-cooled solution (30m
M potassium acetate, 100mM Rb C1,10m
M CaCJ! ,,50mM MnCJ! 2.15
% glycerin, adjusted to pH 5.8 with acetic acid),
After standing at ℃ for 5 minutes, centrifugation at 4℃ (8000 rpm)
a+, 5 minutes). Remove the supernatant and add solution ■ (10mM
MOPS, 75mM CaCj! z, 10rnM
RbC1, 16% glycerin, pH 6,5 in KOH
(adjusted to ) and left at 0°C for 155 minutes. This 2
The sample was divided into 00 μg portions, frozen with acetone-dry ice, and stored at −80° C.

The preserved bacterial cells were thawed under water-cooling, 10 μλ of the silk recombinant plasmid solution described above was added, and the cells were allowed to stand at 0° C. for 30 minutes. 42
A heat shock was applied at 37° C. for 90 seconds, the mixture was left cooled with water for 1 to 2 minutes, ψ medium aoul was added, and the mixture was incubated at 37° C. for 60 minutes. Add this to 50 times the volume of L-broth (Bacto Tryptone 10 g/fl, Ys! Extract 5 g/2,
NaCJ! 10g/l, pH 7.2) and 37°C.
I was shaken for several hours.

Ampicillin was added to 100 μg/-, and cultured with shaking for one night.
A DNA library was created. A portion of the culture solution before inoculation into L-broth was spread on a single gloss agar plate medium containing 100 μg/- of ampyridine and cultured overnight at 37°C, and the frequency of appearance of ampicillin-resistant bacteria was investigated. The size of the library was approximately 6 x 10' clones.

Geo created in step 3, candidum c
Target lipase cD from DNA library
In order to isolate clones having NA, it is effective to chemically synthesize DNA corresponding to the amino acid sequence of lipase and perform colony hybridization using this as a probe.

First, Edman decomposition method (Acta Cheap, 5c
aIIId,.

1, 283 (1950)), the N of this lipase was
An attempt was made to determine the amino acid sequence from the end, but the N-terminal amino acid was blocked and was not successful. Therefore, purified lipase 50a+g was added to 3-O, 1M Tris
-HCf (pH 9,2) buffer, 180 μl
The mixture was digested with gluendopeptidase at 30°C for 6 hours. 1 mg of this freeze-dried decomposition product was dissolved in 100μ of 0.05% trifluoroacetic acid and subjected to high performance liquid chromatography using a Synchrovac RP-4 (46 mm φ x 250 mm, Synchro 11 Co., Ltd.) column. did. Elution was with 0-6 isopropanol/acetonitrile (7/3, v/v) containing 0.05% trifluoroacetic acid.
A 0% linear concentration gradient was used at 40° C. and a flow rate of 0.81 d/min. From the elution pattern shown in FIG. 1, it was possible to fractionate about 20 types of peptides. Note that Beak 3 could be separated into Beaks 3-1 and 3-2 when the concentration gradient of the eluting solvent was set to 5-20% (FIG. 1, inset). Of these, the peptides in beaks 2.3-1.5 were analyzed by amino acid analysis (e.g. J, chromatogr
,.

4, 143 (1981)), the molar ratio of the constituent amino acids showed an integer ratio, so it was determined that it was a single peptide. Therefore, the amino acid sequences from the N-terminus of these three peptides were determined by the Edman degradation method. The results are shown in Table 1.

Table 1 (+) Colony hybridization No. 1 corresponding to the seven amino acid sequences of the peptide (beak 3-1) determined in step 4 to isolate a clone with lipase cDNA from the cDNA library created in step 3. The DNA shown in Figure 2 was chemically synthesized, and the 5' end was
Colony hybridization was performed using a probe labeled with 2P.

601 mole of chemically synthesized DNA was mixed with 10 units of T4 polynucleotide kinase and [γ-18P] ATP (
5μci/pmo! e, 300, uci) using 50 mM Tris-HcI buffer (pH 8,0).
C10mM MgC1,,5mM D T T,0
．． containing 1 mM spermidine and 0.1 mM EDTA] at 37°C for 60 minutes, and the 5' end was converted to 1! P
Labeled with.

Colony hybridization was performed using this 11p-labeled probe in the following manner. Several hundred colonies were grown on a nylon filter (Hybond-N, Amarjam) according to Amarjam's protocol (Menpuran Transfer and Detection Method), and after lysis, the DNA was immobilized on the filter. 0.1% SDS and 0.1mg/- calf thymus DN
6XSSC containing A (composition of IXSSC; O, 15M
N a CR, 0,015M sodium citrate)
Prehybridization was performed in a solution at 45° C. for 3 hours. Then in the above hybridization solution containing the 22p-labeled probe at 45°C 40
Time hybridization was performed. After this, 0.
Wash at 45°C with 5X SSC solution containing 1% SDS,
After further washing at room temperature with a 4XSSC solution containing 0.1% SDS, autoradiography was performed. As a result of performing a piperidization test on several thousand colonies, positive clones were isolated. The plasmid possessed by this clone will hereinafter be referred to as pGCL2.

(2) Preparation of a restriction enzyme map To confirm that the positive clone obtained in step 5 (1) has lipase cDNA, the nucleotide sequence may be determined. Therefore, we attempted to create a restriction enzyme map for the purpose of determining the base sequence of the cDNA integrated into pGCL2.

The above positive clones were cultured overnight in L-broth,
Plasmid DNA was extracted from the bacterial cells by cesium chloride-equilibrium density gradient ultracentrifugation [Molecular cloning (Mo1
ecular C! tu1in3) 8B(+980)
, Cold Spring Harbor Laboratory]
Phage Ml 31Ipl, which is recovered by
A restriction enzyme map was created using restriction enzymes that can be incorporated into Olmpl 9. The restriction enzyme used was from Takara Shuzo.

The restriction enzyme map prepared is shown in FIG. 3.

(3) Determination of base sequences of cloned cDNAs Prior to determining the base sequences of two types of cloned cDNAs, the method of Spring et al. [Gene 3] was used.
3. The cDNA fragments were cloned into M13mplB or mp19 according to the method described in 2010, 269 (1982)), and various deletion phages were produced using a kilobase deletion kit (Takara Shuzo KK [)]. Using this, the dideoxy chain termination method of Sanger et al. (Proc, Natl, Ac
ad, Sci, IJsA7LL, 5463 (19
7? ), J, Mo1. Biol,, IJ),?
29 (19B2)), Brimer annealing, D
Complementary strand synthesis ([α-”P)dCTP (400 μci/pm
The base sequence was determined by urea-denaturing 8% polyacrylamide gel electrophoresis and autoradiography using urea-denatured 8% polyacrylamide gel (20 μCi).

Figure 4 - cD integrated into pGCL2 in (1) and (2)
The translated region of NA is shown. Note that this translation region contains AT
No initiation codon corresponding to G was observed. Furthermore, the amino acid sequence determined from this is number 51! J-(1),
Shown in (2).

The beak 2 peptide mentioned in step 4 is 3 in Figure 5.
The amino acid sequence of the peptide, located in the 4th to 38th regions, determined from this base sequence matched the amino acid sequence determined by Edman degradation (Table 1). In addition, in order to ensure that the amino acid composition of the peptide calculated from the base sequence matched that actually measured using butide of Beak 2, a similar analysis was performed on the peptide of Beak 3-1 described in Table 2, Table 3, Slap 4. As a result, this peptide was located in the 54th to 66th regions of FIG. 5, and these matched the amino acid sequence of the peptide of Beak 3-1 (Table 1). Furthermore, the calculated value and the measured value of the amino acid composition of the peptide matched (Table 3).

In this region, there was a base sequence homologous to the probe used for colony hybridization, and its presence was confirmed in the 160th to 179th region of FIG. 4.

A similar analysis was performed on the peptide of beak 5 described in step 4. This peptide is 357-38 in Figure 5.
Amino acid sequence determined from base sequence and amino acid sequence determined by Edman degradation (Table 1)
The calculated values and the measured values also matched for the amino acid composition of the peptide (Table 4).

Table 4 From this, it was revealed that the pGCL2-derived cDNA was a lipase gene.

To further confirm this result, Geo, can-d
The amino acid sequence from the N-terminus and the amino acid sequence from the C-terminus of idum lipase were determined and compared with those determined from the base sequence. Geo, candi-dum lipase was decomposed with cyanogen bromide, the N-terminally blocked peptide was fractionated by high performance liquid chromatography using a reversed phase CI8 column, and this was decomposed with pyroglutamate aminopeptidase. The amino acid sequence from the N-terminus was determined. As a result, the amino acid sequence from the N-terminus was pyroglutamic acid-alanine-proline. This sequence was present in the 14th to 16th regions in FIG.

After translation, this result is 13th and 14th as shown in Figure 5.
This means that a cleavage occurs between the second amino acid, alanine and glutamine, and mature lipase is produced.

Since this lipase is an enzyme secreted extracellularly,
The peptide composed of amino acids 1 to 13 in FIG. 5 also serves as the - signal peptide.

On the other hand, for the C-terminal amino acid, when Motosuba Yutomi is degraded with carboxypeptidase A, glycine is first released,
It was then observed that leucine and tyrosine were liberated at the same rate. pact.

The C-terminus determined from analysis of the cDNA derived from 2 was monoleucine-tyrosine-glycine (Figure 5). This result also revealed that the cDNA incorporated into pGCL2 was a gene encoding a protein with lipase activity.

(Effects of the Invention) The cloned lipase cDNA of the present invention serves as a material for producing lipase-producing microorganisms or cells. Furthermore, the X-ray crystal structure of this lipase (J, Bioc.
hem, FM, 1B21 (1979)), we adopted a position-specific mutagenesis method, etc. to create a lipase with increased stability against heat, alkali, acid, and organic solvents, or a position-specific lipase. Materials for making lipase with characteristic substrate specificity

[Brief explanation of the drawing]

Figure 1 is a high-performance liquid chromatogram of lipase produced by Geotrichum candidum digested with lysyl endopeptidase, Figure 2 is the base sequence of the probe used for colony hybridization, and Figure 3 is the pGC
Restriction enzyme map of cDNA integrated into L2, Figure 4-(
1), (2) ((2) is a continuation of (1)) are pGC
Base sequence of the translated region of the cDNA integrated into L2, No. 5
Figures (1) and (2) ((2) is a continuation of (1)) show the amino acid sequences determined from the base sequence in Figure 4.

Claims (2)

[Claims] (1) A gene encoding a protein with lipase activity, characterized by having the following base sequence. [There is a gene sequence] (2) Geotrichum candidum
The gene according to claim 1, which is prepared from messenger RNA derived from M. candidum) ATCC 34614.