JPH0387175A - Lipase and gene of the same lipase - Google Patents

Abstract

NEW MATERIAL:A protein having an N-terminal amino acid sequence expressed by the formula and having lipase activity. USE:Used for the production of fatty acid, decomposition of beef tallow, production of cacao-substituting fat, synthesis of glyceride, synthesis of terpene alcohol ester, synthesis of sugar ester or optical resolution of racemic ester. PREPARATION:For instance, lipase producing fungus Rhizopus delemar AJ 6045 (FERM P-10550) is cultured in a medium and the fungus is separated from the culturing process, then concentrated hydrochloric acid is added to the supernatant and adjusted to pH4.6. Thus formed precipitation is removed and ammonium sulfate is added to the supernatant in an amount of 70% saturation, then the salted-out substance is separated and redissoluted to an aqueous solution of acetic acid. The resultant solution is de-salted and treated with an adsorbent, and the eluted fraction is purified by a high-performance liquid chromatography and gel filtration to afford the objective protein having N-terminal amino acid sequence and lipase activity.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a lipase enzyme used in ester decomposition reactions, ester synthesis reactions, and transesterification reactions, and its gene.

Genes make it possible to produce lipase in large quantities by recombinant DNA methods, and also make it possible to create more functional lipases by genetic modification. Applications of lipase include production of fatty acids, decomposition of beef tallow, production of cocoa fat substitutes, glyceride synthesis, terpene alcohol ester synthesis, sugar ester synthesis, and optical resolution of racemic esters.

[Conventional technology]

Organisms that produce lipases are widely distributed among animals, plants, and microorganisms, and these lipases have characteristic properties depending on their source and tissue. Among these lipases, there are those whose entire amino acid sequences are known, and those whose sequences are partially known, as listed below. From animal sources, rat Li
ngal 1ipase (Docherty, A.J.
P, HBodmer, M, W, ;8 nga1. s, ;V
ergerR, HR4viere, c, ;Lowe
, P, A, ;Lyons+ A, ;Emtage
, J.S.

;l1arris, T, J, R,: Nucleic A
c1ds Res,, 13(6)+1891-903
(1985)), rat hepatic lipas
e(Komaromy, Michael C,; 5ch
otz, Michael C.: Proc. Natl.
^cad, sci, U, s, 8, 84 (6), 152
30 (1987)), human gastri
clipase (Bodmer, Mark H,; eight gal, five arojani; Yarranton, G
eoffrey T, ;l1arris, Timot
hy J, R,; Lyons, 81an; King,
David J,; I'1eroni, Gerard;
R4viere, C1aude; Verger+Rob
ert; Lowe, Peter A.: Biochim
, BiophyS, octa, 909 (3),
237-44 (1987)), bovine bra
in diacylglycerol 1ipase
(Farooqui, Akhlaq A, HCeng
, Sh 1yuan; Rammohan, Ko
tti I; KoIa ttukudy, Papa
chan;1larrocks, Lloyd A, :B
iochem, Soc, Trans, 16(3), 2
93 (1988)), human hepatic 1
ipase (Datta, 5antanu; Luo,
Chi Cheng; LiJen Ilsiung;
Van TuinenPeterHLedbette
r, David H.; Broiyn, Mary A.
;Chen, San HwanHLiu, 5hyan
Woei; Chan, Lawrence: J. Biol
,chem,, 263(3), 110740(19
88)), and the entire amino acid sequences of these are known. Also, from bacteria, 5taphyloc
Occus hyicus 1ipase (Goet
z, Fr1edrich; Popp, Fri
tz ; Korn + Edda ; Sch 1e
ifer+Karl He1nz: Nucleic A
c1ds Res, 13(16), 5895-906.
(1985)) + Pseudomonas frag
i lipase (Kugimima + Ha tar
u ; Otan i, Yasuo ; llashim
oto, Yukiko; Takagi, Yasu
yuk i: Biochem, Biophys
,Res,Commun,+141(1),
185-90 (1986)), and the entire anoic acid sequences of these have also been determined. From filamentous fungi, R
hizomucor m1ehei 1ipase(B
oel, Esper HHugejensen.

Birgitte ;Chris jensf3n+
Mogens HThim, Lars ;Fi 1
1°N1els P, : Lipids, 23,701(
198B)), Rh1zopus n1veus 1i
pase (Japanese Unexamined Patent Publication No. 64-80290), Geot
r4chum candidum 1ipase (Japanese Unexamined Patent Publication No. 63-32489), and the entire amino acid sequence of the former two has been clarified, and the sequence of the DNA encoding the latter has been clarified.

[Problem to be solved by the invention]

By the way, unlike lipases derived from bacteria, lipases derived from filamentous fungi have the property of specifically acting on the 1.3 position of the substrate triglyceride. This property is essential for the production of cacao fat substitutes using transesterification. Many animal-derived lipases have similar specificity, but filamentous fungal lipases are considered easier to use in terms of ease of culturing.

The purpose of the present invention is to purify lipase, determine its N-terminal amino acid sequence, and clarify the molecular structure of this lipase. The objective is to provide a means by which it can be supplied in pure form in large quantities at low cost.

[Means to solve the problem]

Therefore, the present inventors purified lipase produced by Rhiz delemar, which is one of the filamentous fungi, in order to clarify its properties and structure in order to use it for transesterification reactions, etc. The terminal amino acid sequence was determined. This sequence was completely different from the previously known lipases mentioned above, and it was found that this lipase has a new protein structure.

In addition, the present inventors have also found Rh as a lipase-producing bacterium.
1zopus delemar AJ 6045 (this strain has been deposited as FERM P10550).

), and the lipase present in this culture supernatant was purified. Regarding the purification of lipase produced by this strain, Iwai et al.
a+ YosiO: 8 gr, Bio1. Chem, ,
38.1241 (1974)), but the present inventors greatly simplified the purification method and
Moreover, they succeeded in obtaining a sample with sufficient purity to determine the N-terminal amino acid sequence, and determined the N-terminal amino acid sequence.

Based on this data, a synthetic DNA probe was created and R
The lipase genome gene was extracted from the genomic DNA extracted from h1zopus delemar AJ 6045 bacterial cells, and the bottom c DNA was extracted from the extracted and purified mRNA.
The lipase genome gene was obtained from among the genes. The entire structure of the DNA was then determined.

The method will be explained below.

The culture method is the conventional one! The cultivation method is not particularly different from that of the genus. That is, the culture medium is a conventional medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, nitrates, phosphates, etc. As the carbon source, glucose, starch hydrolyzate containing these, molasses, etc. are used. Polypeptone, tryptone, meat extract, yeast extract, etc. are used as nitrogen sources. Cultivation is carried out under aerobic conditions with pl+ of the medium
Although it is desirable to adjust the temperature and temperature appropriately, it is not necessary. The culturing time is continued until the lipase activity in the medium reaches its maximum.

The following method is used to purify lipase from culture supernatant. Adjust the pl+ of the culture solution to 4.6 with hydrochloric acid and remove the precipitate. The range of pn to be adjusted at this time may be around 4.6. Moreover, the acid used does not have to be hydrochloric acid. The obtained supernatant was precipitated with ammonium sulfate, desalted, and then treated with Pharmacia Phenyl 5ephar.

5eCL4B open column to perform fractionation. This column can be used with other carriers as long as they perform separation based on the interaction between the hydrophobic groups attached to the base material of the packing material and the hydrophobic functional groups of the sample. It is also possible to omit this step. The fractionated sample is processed using high-performance liquid chromatography (here, high-performance liquid chromatography is an apparatus that performs chromatography at atmospheric pressure or higher, and includes Pharmacia FPLC).
, Tosoh TSK-GEL,, Phenny
Fractionation was performed using a l-5PW column. This column can also be used with other chromatographs that separate samples based on the interaction between the hydrophobic groups attached to the base material of the packing material and the hydrophobic functional groups of the sample. A sample that has undergone this step has already become a single band in SDS polyacrylamide gel electrophoresis.

Next, high-performance liquid chromatography was performed using Tosoh G300.
Gel filtration was performed using an 0SW column. This column can also be used as long as it has gel filtration characteristics. Also, this step can be omitted. However, if omitted, desalting will be required for the next step. In order to further separate the sample obtained here into two components, fractionation was performed by high performance liquid chromatography using a Pharmacia cation exchange column Mono S. Other columns can be used as long as they have cation exchange properties to separate cations.

The purified sample is run on an amino acid sequence analyzer,
The two N-terminal amino acid sequences shown below were determined.

8sp Asp Asn Leu Val
Gly Gly Met Thr Leu
AspLeu Pro Ser Asp Ala Pr
o Pro Ile Ser Leu+ and Val S
er Gly Lys Ser Gay Ser Se
r Asn Thr AlaVat Ser Al
a Ser Asp Asn Ala Al
aLeu This amino acid sequence has been revealed for the first time. Even in the same lipase, it is possible to substitute several of the above agonoic acid sequences with different agonoic acids.

The gene encoding the lipase of the present invention is Rh1zopu.
It may be extracted from S. delemar AJ6045 bacterial cells, or it may be synthesized. In the case of extraction, the bacterial cells are destroyed by means such as grinding and treated with protease, and then the DNA is extracted. The obtained DNA is cut with a restriction enzyme, ligated to a λ vector, and packaged in vitro to create a library. On the other hand, from the above amino acid sequence, the corresponding DNA is synthesized using an automatic DNA synthesizer.
Place the probe on the bottom. Therefore, this platform probe is used to isolate genomic DNA clones from the library.

Is RNA extraction similar to genomic DNA extraction?
It is necessary to take measures to prevent RNase contamination or inhibit RNase activity at all stages. The extracted RNA was purified into a polyA RNA fraction using an oligo dT cellulose column, and based on this, cDNA was extracted using Rehearsal transcription enzyme.
Bottom A. The obtained cDNA is ligated to a λ vector and packaged in vitro to create a library. After that, isolate clones using the same method as for genomic DNA.

As a result of genetic structure analysis of the genomic DNA and CDNA obtained as described above, it was found that there are two types of encoded proteins, one of which is not disclosed in JP-A-64-80290, 1ipase and 1. They were identical except for one amino acid.

[Example] Example 1 (1) Lipase producing bacterium Rh1zopus delema
r Cultivation of AJ6045 (this strain has been deposited as FERMP-10550).

For lipase-producing bacteria Han dish 1 纺口 AJ6045 wo 20
0I! , cultured at scale. Medium polypeptone 5%,
NaNO30.1%, KtlzPOa 0.1%, Mg
5Oa 0.05%, Glucose 2%, pl+approximately 6°Temperature 27°C0 (2) Lipase purification activity measurement method is exclusively a simple method (Dainippon Pharmaceutical Co., Ltd. Purase Kit)
S), but for the final purified preparation, the activity assay method using olive oil (Fkumoto, Juichiro
;rwai, former eko;Tsujisaka, Yosh
io:J, Gen, Appl, Microbiol,
10, 257 (1964)). The U (unit) shown below is River Zegi+-
The difference in absorbance between the sample and the blank when measured with S is converted into the amount per minute. Moreover, B means a buffer.

(2) The obtained culture broth 180 j2 was shaken through a 24φ shaker to separate 8 kg of bacterial cells. Add concentrated hydrochloric acid to the supernatant to adjust the pH.
When adjusted to 4 and 6, a slight precipitate was produced.

The resulting precipitate was removed using a Sharpless, and the supernatant liquid was
I got 1. Add 80 kg of ammonium sulfate (70% saturation) to this and add 4
When the solution was left at °C for 116 hours, salted-out substances floated on the surface of the solution. This precipitate was skimmed off to obtain 780 g of ammonium sulfate precipitate.

Take 50g of this precipitate and add it to the minimum liquid volume of 0.02M
After dissolving in acetic acid B (p++5.2) and removing insoluble matter by centrifugation, 5ephadex G-25Medium
Desalted with The desalted sample was lyophilized and 4.72
g of lipase powder was obtained. This lipase powder was 1600 u/g powder, had an activity recovery rate of 85%, and had a specific activity of 65 u/■ when dissolved in 0.02 M acetic acid B (pH 5, 6).

2.36 g of the lipase powder obtained in 2.3 was dissolved in 23.6 d of 30.02 M Yamashita B (pH 5.2), and 14.75 m of PEM ammonium sulfate was added thereto. was added to adjust the ammonium sulfate concentration to 1.5M. This is a Pharmacia Phenyl CL 4 with an inner diameter of 2.3 cm, a length of 50 cm, and a head volume of 210 cm.
B, 1.5M ammonium sulfate, 0.02M acetic acid B
After washing with (pl+5.2), 0.02M acetic acid B (pH
5,6), the adsorbed fraction was eluted. All activities are 0.02M
It was present in the acetic acid B (pH 5, 6) elution fraction, and after collecting the active fraction, it was concentrated with a follower fiber to a liquid volume of 130.

At this time, the specific activity was 77 u/■, the total activity was 2000 u, and the activity recovery rate was 53%. Next, 71 mft of this was
LC (Waters M600 multi-solvent system), column T S Kgel Phenyl -
Using 5P W7.5 mm I DX7.5c+n,
Conditions O minutes → 30 minutes, 1.5M ammonium sulfate, 0.02M acetic acid B
(pH 5,2) Isocratic, flow rate 1mR/m1n
Fractionation was carried out from 030 minutes to 90 minutes using 1.500 M ammonium sulfate, 0.02 M acetic acid B1 gradient end, and a flow rate of 1 mR/min.

(Charge is 20m1 + 11mfl + 11mR-1-1
7mQB was divided into 4 times and 4M ammonium sulfate was added to each time. ) The active fractions were not mixed with each other and stored at -70°C after flash freezing in liquid nitrogen. The specific activity 4 of this sample was 240 to 300 u/■, the total activity was 500 u, and the activity recovery rate was 44%. SDS the sample at this stage.
Figure 1 is the result of PAGE. In the figure, a is a molecular weight marker, 97400.66200 from the top.
42699.31000.21500.14400 (
7) Indicated by molecular weight.

b is a sample. At this point, it had already become a single band with Kumazi staining.

Without mixing each active fraction obtained in
M600 multi-solvent system), column, T
S Kgel G3000SW 7.5mm ID
Gel filtration was performed by charging 0.5 mfl to 0.1 mR each at x60 cm. (Conditions, equilibrium B 0.2M NaC
l, 0.02M acetic acid B1 flow rate 1 ml/min, )
The sample thus obtained had a specific activity of 142 u
/■, activity recovery rate was 80%. The molecular weight by gel filtration was calculated to be 98,000 based on the results of running a standard sample under the same conditions.

Of the samples obtained in
15 (cation exchange). Since the gel-filtered sample contains 0.2M NaCl, part (1) was used for FPLC, the Fast Deutsulting column was desalted using IIRIO/10, and part (1) was passed through Mono S by diluting the sample 3 times. The sample was prepared first. The activity recovery rate when passed through the Fast Deutsulting column was 75%. The conditions are a flow rate of 2 ml.
/min, equilibrium B, 0.02M acetic acid B0 (Mono S
The conditions are 0.02M acetic acid B → I MNaC], 0
．． 02M acetate B (pH 5,2) 60 minutes gradient end, flow rate 1 mR/min 0) The protein peak was divided into two by Mono S, and lipase activity was found in both. These are called A lipase and C lipase.

The specific activity of this sample is 280 u/mg of A lipase.
, C lipase is 100u/mg, activity recovery rate is 50%
Met.

A, C lipase is predicted to have a molecular weight of 33,000 based on electrophoresis. A summary of the purification so far is shown in Table 1.

Table 1 Summary of lipase purification (each step is converted to the amount required to obtain the final sample) (3) Determination of N-terminal amino acid sequence As a result, this protein was composed of two types of subunits, with a composition ratio of 1:1. The sequences are shown in Table 2. Furthermore, this sequence was the same for both A and C pases. Ta.

Table 2 N-terminal amino acid sequence of MonoS sample 1
2 3 4 5 6 7 8 9 10 1112 1
3 14 15 16 17 18 19 20 21
7. Since the sample purified with Mono S contains two types of subunits, it was separated using isofocus electrophoresis. A+C lipase fraction (A+C lipase fraction is P
sample after henyl SPW) with an inner diameter of 6 cylinders,
After isoelectric focusing in a 13 cm long glass tube column and Kumazi staining, a single hand at pH 9.30 was cut out. By plotting, this protein was transferred to a milliporeimopyrone transfer membrane (material: Po1y).
vinylidene difluoride, Cat
.

No. IPVH304FO), and the whole membrane was subjected to an agonoic acid sequencer to determine the N-terminal anoic acid sequence. The results are shown in Table 3.

Table 3 N-terminal amino acid sequence of T E F pH 9,30 band 1 2 3 4 5 6 7 8 9 10
118sp Asp Asn Leu Val
Gly Gly Met Thr Leu
Asn12 13 14 15 16 17 18
19 20 21Leu Pro Ser A
sp Ala Pro Pro Ile S
er Leu This result is consistent with one of the sequences in Table 2.

From the sequences in Table 3 and the sequences in (Table 2) to (Table 3), the α and β NN terminal amino sequences shown below were determined (Table 4).

Table 4 N-terminal amino acid sequences of α and β subunits 1 2
3 4 5 6 7 8 9 10 1112 13
14 15 16 17 18 19 20 21 Example 2 (1) Preparation of genomic DNA 16 g of Rh1zopus delemar AJ6045 bacterial cells were crushed using a mortar and pestle in the presence of liquid nitrogen.
50m 0.5MEDTA (pH 8,0), 10mM
Tris-11clz< roughy-(pl+8.0
) was added and suspended well. 150 mj after applying polytron for a few seconds! of 0.5 M EDTA (pH 8,0), 1
0mM Tris (pH 8,0) 200μg/rdPr
Oteinase K and 1% 5arcosy+ were added and left at 50°C for 3 hours. The operation of adding an equal amount of buffer-saturated phenol, stirring, centrifuging, and then removing the aqueous phase (hereinafter referred to as phenol treatment) was repeated three times.

The aqueous phase was mixed with 50mM Tris (pl+8.0), 10mM
The sample was dialyzed against EDTA and 10mM NaCl, and 100 μg/mfl of RNase was added to the resulting sample and allowed to react at 37°C for 13 hours.

After phenol treatment, 10mM Tris (pH 8,0
), dialyzed against 1mM TiDTA solution, 1/10
volume of 2.5MSodium Acetate (pH 5
, 2), add twice the amount of ethanol, and heat at -20°.
It was left overnight at C. Centrifuge and collect the precipitate (hereinafter this operation is referred to as ethanol precipitation).
Br equilibrium density gradient ultracentrifugation to collect the DNA band,
It was mixed with 10mM Tris (pH 8°0), 1mM
Genomic DNA was obtained by dialysis against DTA.

(2) Preparation of genomic library Genomic DNA (230μg/m℃) 230μi!
, 10 x A buffer (manufactured by Boehringer Mannheim 5
-Buffer system) 46μj, IIzO230μ
After adding 1.6 μe of ff, 5au3^I (restriction enzyme manufactured by Takarasha) and reacting at 37° C. for 165 seconds, phenol treatment was performed, and the phenol in the solution was removed with ether. After ethanol precipitation, 1 ml of 10mM Tris (pH
8,0), add 1 mM EDTA, and 6 μe of it
Then, λ phage EMBL3 (purchased from Promega)
2 μe (l μg), 1 p (l of 0.5 M Tris(
pl+7.4), 0.1MDTT.

10mM ATP, 0.1 MMgCIz, and T4
Add DNA ligase (purchased from Takarasha) and incubate at 11°C.
I left it overnight.

This reaction solution was pancaged according to the instructions of the in vitro packaging kit (purchased from Amajam).
A library was created.

(3) Preparation of synthetic NA probe Based on the determined N-terminal amino acid sequence, design DNA as shown in Figure 2, and perform the phosphoramidization method using a 3BOA DNA synthesizer manufactured by Applied Biosystems. A probe was made.

However, the β subunit probe was prepared based on the initial N-terminal amino acid sequence data.

From the results of subsequent analysis using an accurate anoic acid sequencer, β
The N-terminal sequence is VSGKSGSSNTAVSASDDNA
It was determined as A.

(4) Isotope label synthesis of probe 0.5MT of 34 to 50pmole of DNA probe
ris (pH 7,6), 0.1MMgCh, 0.05
MDTT, 0.001M IEDT8, 1μm! T4
Polynucleotide Kinase (purchased from Takarasha) 10μe
[γ-''P) ATP (purchased from Amajam Co., Ltd. 1) was mixed and the liquid volume was adjusted to 30 μe at 11□0.

After reacting at 37°C for 160 minutes, the mixture was treated with phenol and gel filtrated using Sephadex G-50 to obtain an isotope-labeled probe.

(5) Brate the phages according to the method shown in the Screening In Vitro Packaging Kit (manufactured by Amajam), cool the plate to 4°C, and then transfer the phages onto the filter by placing a nitrocellulose filter in close contact with the surface. did. Filter with 1.5M NaCl, 0,5M NaOH
Immerse in a solution to denature the attached DNA, and add 1.5 MNa.
Cl, neutralized with 0.5M Tris (pH 8,0) solution. Additionally 0.36M NaCl, 20mM Na1lz
PO4 (pi(7,4), 2 mM EDTA (
After rinsing with pH 7.4), it was dried. This filter was immersed in the solution shown below and hybridization was performed at 50° C. for 11 nights. [Solution: 5mR Carrier
DNA (Salmon 5 rng/mR)
, 25m1. Add 1 mM EDTA and incubate at 100°C.
Divide into cubes and cool.

50 mN of 10X SSC (10X SSC and Na
Cl37.5g, Na"citrate 44g
, add 1lzo to make 11) 0.2% SD2S, add 20mM EDTA, 1% of 20mfl
BSA, 1% pvp (pvp-360) 1% Fic
ol1400 plus isotope-labeled probe. ]2.5X after hybridization
It was washed with SSC, dried and autoradiographed. As a result of autoradiography, approximately 60 positive clones were obtained.

(6) Nucleotide sequencing from positive clones by plate lysed method (Molecular CIoning, Co1d S
pring 1larb.

r Laboratory (1982)) Phage DN
A was adjusted and the inserted DNA was transferred to p[Jc18 (purchased from Takarasha) to produce PLDAIII. (The E.coli JM109 strain containing this plasmid is F.
It has been deposited as ERM P-11212. )PL
The base sequence of the portion of DAIII that has affinity with the probe was determined. Base sequencing was carried out using DIEAZ 8 Sequence 1-L (manufactured by FF1) and following its instructions. The determined base sequence is shown in Figure 3. In FIG. 3, the portion presumed to be the coding w4 region of each subunit is translated into amino acids. Further, CCT88 from 203 to 208 is a eukaryotic promoter sequence to TATA8 from 261 to 265, and AATAAA from 1601 to 1606 is a polyazonylation signal.

Furthermore, 456 to 512 are sites corresponding to the βN-terminal probe, and 658 to 714 are sites equivalent to the αN-terminal probe. Additionally, there is a region between 1153 and 1194 that encodes an amino acid sequence homologous to other lipases.

Example 3 (1) Microbial cells that had been frozen for three cycles of mRNA were crushed using a mortar and pestle in liquid nitrogen, and 5 μm of DTT was added to the Ig bacterial cells, and 2 μm of DTT was added to the Ig bacterial cells. .6ml of 0.2M NaBorate (p
l+9.0), 1% SDS, 30mM
IEGTA, 10mM Vanadyl-ri
bonucleoside (this is called XTB, 90
~95°C) and a polytron was applied. Protect
Add inasek to 0.5■/rM,
It was left at 0°C for 2 hours.

80 pe of 2M KCl was added to 1 mfl of XTB, and the mixture was left on ice for 1 hour. 20 minutes at 1200 rpm,
It was centrifuged at 4°C and the precipitate was washed twice with 2M LiCl.

Approximately 2 mg/in 10mM Tris (pH 7,5)
ml of potassium acetate (200mM).
was added, followed by ethanol precipitation. Precipitate with 11□O
and heated at 65°C for 1 minute. 2 rtrR's 1.
5M NaCl, 2 mfl of 20ml Tri
s (pH 7,6) was added, the mixture was passed through a cellulose column, and the non-adsorbed fraction was adsorbed onto an oligo dT cellulose column. At this time, the column was equilibrated with 0.4M NaCl, 10m
M Tris (pH 7,6) (this is the former nding B
) was used. Wash with 30mfl BindinB and remove PolyA with 10mM Tris (pH 7,6).
RNA was eluted. 5 MN to be 0.5M
Add aCI, adsorb onto the 01igo dT cellulose column again, wash with 20mR Binding B,
Additionally 10mR of 0.4M NaCl.

Washed with 10mM Tris (pH 7,6). 10mM
Elute with Tris (pH 7,6), PolyA RN
I got an A.

(2) Preparation of cDNA library cDNA synthesis was performed using cDNA synthesis system plus manufactured by Amajam. The method was according to the instructions of the kit. Created c
A library was constructed using DNA as a material using the cDNA cloning system λgt10ver, 2.0 manufactured by Amajam. The method was according to the instructions of the kit.

(3) Screening and base sequencing Based on the prepared cDNA library, (
3) A large number of cDNA clones were obtained according to the methods described in (4) and (5). The base sequence was determined according to method (6). POCl2 containing cDNA insert is pl, c
DNA6-1-2 and E, c containing this plasmid.
oli JM109 has been deposited as FIl+RM P-11213. The determined base sequence is shown in Figure 4. In FIG. 4, the portions considered to be the α and β unit coding regions are translated into amino acids. 118-17
4 sites correspond to the N-terminal probe of β, 320-376
The position corresponds to the N-terminal probe of α. In addition, it was the same as the genome gene sequence except for one base (458-G).

(458-A in the genome).

As a result, in the genome there are 47Lys in the α sequence, but in the cDNA it is 47GIu. In addition, in the β sequence, amino acid sequences 1 to 28 are considered to be the β signal sequence, and in the α sequence, amino acid sequences 1 to 20 are considered to be the α signal sequence, and 21 to 51 are considered to be the pro sequence 6. .

〔Effect of the invention〕

According to the present invention, lipase can be easily mass-produced with high purity by genetic engineering means. Furthermore, by using protein engineering techniques, it is also possible to create new lipases with higher activity or stability.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing SDS polyacrylamide gel electrophoresis. FIG. 2 is a diagram representing synthetic DNA probe design. D.N.
The A sequence is an antisense sequence. Figure 3 shows the genome gene sequence. The coding regions of the two subunits were deduced and translated into agonoic acids. The underlined amino acid sequence indicates the N-terminal amino acid sequence whose structure was determined from the protein side. Note that part of the sequence between 1 and 300 has not been determined.

Claims (10)

[Claims] (1) The N-terminal amino acid sequence is [There is a gene sequence. ] A protein with lipase activity consisting of (2) The N-terminal amino acid sequence is [There is a gene sequence. ] A protein with lipase activity consisting of (3) N-terminal amino acid sequence [There is a gene sequence. ] as well as [There is a gene sequence. ] A protein with lipase activity consisting of (4) Claim (1) (2) or (3) produced by filamentous fungi
Proteins listed in (5) Gene encoding lipase (6) Claim (5) containing a signal sequence and a prostructure
Genes listed in (7) There is a gene sequence of the gene described in claim (5) that includes the promoter region shown in the CCTAAT and TATAA sequences shown below. ] (8) Amino acid sequence [There is a gene sequence. ] A gene encoding a polypeptide consisting of (9) The amino acid sequence is [There is a gene sequence. ] [There is a gene sequence. ] A gene encoding a polypeptide consisting of (10) Claims derived from filamentous fungi (5) (6) (7) (8)
or the gene described in (9)