TW295602B - DNA fragment used for manufacturing alkaline lipase - Google Patents

Abstract

DNA fragment with a base sequence encoding alkaline lipase gene and a base sequence controlling the expression of the encoded alkaline lipase gene have the base sequence as listed in an attached sequence table.

Description

Printed 295602 A7 B7 by the Employees ’Consumer Cooperative of the Central Standard Falcon Bureau of the Ministry of Economic Affairs 5. Description of the invention (1) The present invention relates to a method for manufacturing alkaline lipase, new microorganisms used in this method, and those used for manufacturing this novel microorganism Novel microbial DNA fragments. Technical background Lipase is an important metabolic enzyme of general organisms, which can hydrolyze fat to generate free fatty acids. In recent years, it has been found that lipase can not only break down fat but also carry out many diversified reactions. Fatty neck factor is more stable in organic solvents, and can even react at 100 ° C, and can carry out more diverse reactions. Many breakthrough research and developments have also caused widespread problems in pharmaceuticals, oils, spices, chemicals, pesticides , Food and other industries. Adding alkaline lipase to the cleaning agent is another application direction. Adding enzyme to the cleaning agent has been popular since the early addition of alkaline proteinase. A few years ago, the addition of alkaline fiber enzyme Commodities have also stimulated the so-called washing powder revolution, and the use of alkaline lipase to remove grease and dirt on clothes to enhance washing power is another industrial use [Satsuki, T., et al (1990) Bio. Industry, 7 : 501] 〇Summary of the invention The strain used in the present invention is pseudoalcaligenes pseudoalcaligenes Fl 11) ^ CCRC 910008 (t ^ Republic of China Food Industry Development Research Institute, Microbiological Depository Center). The present invention relates to a DNA fragment derived from the chromosome of Pseudomonas sp. F_ 丨 11, which contains a base sequence encoding a test fat digestion gene and regulates the expression of a base sequence encoding a test lipase gene. The DNA fragment has the following base sequence: This paper scale is suitable for the rate of Chinese interpreters (（, NS） 八 4 坭 格 (2 丨 0 乂 2 叼 公 部) --------- Installation --- ---. 玎 ------ Φ (Please read the precautions on the back before filling in this page) 5. Description of the invention (of) 10 A7 B7 j hA / «1 30

GGATCCCCAG CAGGTCAGGA CTCCGACAAT CAGCGCGCCC 50

AGGCCAAAGA AAGCCGCAAT 90

CGGAAATCAG CAGGGCGAAC 130

ATACCCGTTG ACATAACTGC 170

TTGCATTAAC GGTGGCAGGC 210

TTGCCCTGGC TCGGGGGCGC 250

CATTCACAAT TTCGCTGCTG 290 70 CAGCCCCGGG ACGAAGAATT 1 10 CCCAGTAGCA GCCAGAAGGC 150 TCAGCAGCAT GATGACATCC 190 AAAGCTGAGA GTCTACCAGA 230 TCAATCGGGT TTGCGGAACC 270 CTGACACACC CAAACTCTGA 3 10

ATTACCGTTC ATCGGATTCA TCCTGACAAT ATCCGGATGC 330 350

CAGAGCGCAC GACTAATTGG TCACTTTTCA GCCGCCTAGG 3 70 390

TCAACCCCGC TGAACCCTGC GCCGCGCCTT CAATTCATCA 410 430 --------- f ------ lsτ ------ line '(please read the notes on the back first #fill this page) Central Bureau of Standards, Ministry of Economic Affairs ; Printed by 5 industrial and consumer cooperatives

CCAGGCCTGA CTATGCTCCC GGACAGCCTC TGTGATGCAG ORF 1

450 470 TCAGAGACAC AACAACAATA AAACCGCACA AGGACTCGCA

The standard of this paper is the General Chinese Standard (CNS) Λ4 ^ 格 (2 丨 0'X2W public yak). The A7 B7 is printed by the Employee Consumer Cooperative of the Central Bureau of Economics of the Ministry of Economic Affairs. 5. Description of the invention ()) 490 TTATGCGCAA CAAGACTCGC 510 GTCTCGCTCC GCCTCGGGCT 530 GGCCACTACG CTGGGCATCA 550 GCACCCAGCC CAGGCCTTCC 570 TGTTCGGCTC CTCGAACTAC 590 ACCAAGACCC AGTACCCGAT 6 10 CGTCCTGACC CGCGGCATGC 630 TCGGCTTCGA CAGCCTGCTT 650 GGGGTCGACT ACTGGTACGG 670 CATTCCCTCA GCCCAGCGTA 690 AAGACGGCGC CACCGTCTAC 710 TTCACCGAAG TCAGCCAGCT 730 CGACACCTCC GAAGCCCGGG 750 GTGAGCAACT GCTGACCCAG 770 GTCGAGGAAA TCGTCGCCAT 790 CAGCGGCAAA CCCAAGGTCA 810 ATCTGTTCGG CCACAGCCAT 830 GGCGGGCCTA CCATCCGCTA 850 CGTTGCCGCC GTGGCCCCGG 870 ATCTGGTCGC CTCGGTCACC 890 AGCATTGGCG CGCCGCACAA 910 GGGTTCGGCC GCCGCCCACT 930 TCATCCGCCA GGTGCCGGAA 950 GGATCGGCCA GCGAAGCGAT 970 TCTGGCCGGG ATCGTCAATG 990 GTCTGGGTGC GCTGATCAAC 5 IIIII CNS) Λ4 specification ( 210Χ2 () 7g t) 295602 A7 B7 V. Description of the invention (bucket) 1010 1030

TTCCTCTCCG GCAGCAGTTC GGACACCCCA CAGAACTCGT 1050 10 7 0

TGGGCACGCT GGAGTCGCTG AACTCCGAAG GCGCCGCACG 1090 II 10

GTTCAACGCC CGCTTCCCCC AAGGTGTGCC GACCAGCGCC 130 I 50

TGCGGCGAGG GTGATTACGT AGTCAATGGC GTGCGCTATT 1 70 1 90

ACTCCTGGAG CGGCACCAGC CCGCTGACCA ACATACTCGA 12 10 1230

CCCTTCCGAC CTGCTGCTCG GCGCCACCTC CCTGCCATTC 1250 丨 270

GGTTTCGAGG CCAACGATGG TCTGGTCGGA CGCTGCAGCT I 290 13 10

CCCGGCTGGG TATGGTGATC CGCGACAACT ACCGGATGAA 1330 1350

CCACCTGGAT GAGGTGAATC AGACCTTCGG GCTGACCAGC 1370

ATATTCGAGA CCAGCCCGGT

1390 ATCGGTCTAT CGCCAGCAAG --------- ^ ------ iT ------- ^ (Please read the precautions on the back before filling this page) Employee Consumer Cooperative of Bureau of Loss of Standards, Ministry of Economic Affairs Inze

1410 CCAATCGCCT GAAGAACGCC

1430 GGGCTCTGAA ATAGGCTCCA 1450 1470

CAACCAGACA GGGCTGGCCT CAGGGGCCAG TCACAGGTAC

1490 CGATATCGAC ATGAAGCCGC

Eco RV site ORF 2 15 10

TGATTTATCT GCCGTTGCTA This paper scale is applicable to the Chinese National Standard (CNS) Λ4 specification (2 丨 0'X 2 < > 7 g). 5. Description of the invention (exaggerated) A7 B7

1530 1550 CTCGGCCTCG GCCTGCTCGG CTGGCACCTG AGCACACCGA 1570 1 590 CACCCAGCCC CTCCGCGCCC ACATCAACGC CGCTACAAGC 1 6 1 0 1630 CGGCAGTGAA CAACCCGCCA CAACTCCTGT GAGTCTGACC 1 650 1670 CGTCCGACCA CGCGCAGCAC CGACCAGCAC CTGCCCGCCT 1 690 1 7 10 CACTGCGCGA TACCGACATC GATGGTCAAC TGGAAGTCGA 1730 1750 CGCCCAGGGC AATCTGGTGA TTACCGACCA ACTGCGTCAC 177 0 1 790 CTGTTCGATT ATTTCTTCAG CACCGTCGGC GAACAGTCGT

1810 1830 Printed by TCGAGCAGGC CAGCAGCGCT ATCCGTGACT ATCTGGCCAG Ministry of Economy Central Standards Bureau employee consumer cooperative

1850 1870 CCAGCTGCGT GACGCGGCTC TGGCTCAGGC CCTTGATCTT 1890 1910 CTGGATCGCT ATATCGACTA CAAAACTGAG CTGGTGGAGC 1 930 1950 TGGAGCGACG CTTCCCGATG GTGACCGAAC TGGACGGCCT 1970 1990 GCGCGCCCGC GAAGATGCCG TACAACGCCT GCGCGCGCCA (Fill in this page) r This paper scale is applicable to Chinese National Standard (CNS) 8% 4% (21 () X2 () 7)) A7 B7 2 1 90 Printed by Beigong Consumer Cooperative of Central Bureau of Economics of the Ministry of Economy V. Inventions Description (6) 2050 2070

AAGAGGTCTA TAACCAGTTC ACTCTTGAGC GTCTGGCGAT 2090 21 10

ACTGCACGAT CCGTCGCTGG ATCCGCAGAC ACAGGCCGAA

J ——1 ^ —— ill · · brain I 2130 2150

CGGATTGAAC GGCTGCGCGA AGGGCTGCCC GACGAGTTGC 2 170

AACAATTGCT GGTACCGCAA TTGCACCTGA CCCTGCGCCA 2210 2230

CGACCCAGCA GTTGCTGACC AAGGTGCCGA GCCGGAACAG 2250 2270

CTACGCCAGT TGCGCCTGAA CCTGTTCGGG CCTCAGGCAA 2290 2310

CCGAGCGGCT GGAACGCTGG ACCGCCAACG CAGCGAATGG

The paper scale is applicable to the Chinese National Standard (CNS) Λ4 specification (2Ι〇Χ2 < > 7 public broadcasting) --------- installation ------ order ------. 4. ( (Please read the notes on the back before filling this page) A7 B7

Printed by Ff, Ministry of Economic Affairs, Central Bureau of Standards, Beigong Consumer Cooperative V. Description of the invention (up) The base sequence of the gene encoding alkaline lipase (named ORF 1) is as follows: 310

GGATTCA TCCTGACAAT ATCCGGATGC 330 350

CAGAGCGCAC GACT_AATT_GG— _1CACTTTTCA · i £ LG ...? IlAGG putative promoter 370 390

TCAACCCCGC TGAACCCTGC GCCGCGCCTT CAATTCATCA 410

CCAGGCCTGA CTATGCTCCCGGACAGCCTCTGTGATGCAGTC — — — — — — — — — ------- -------------------------— ·

putative MLPDSLCDAV S-D sequence Signal peptide

AiAG_AC_A_c_Aj_aALAilA_MLc_LGiMM? AAilM_ciTl ^

R D T T T I K P Η K D S Η Y

_G_c_5_cJM_A_AJ_A_nCJ_CJ_T__CJ_LG_aCJ_GJ_C_jy A Q Q D S R L A P P R A G H 1

TACGCTGGGCATCAGCACCCAGCCCAGGCCTTCCTGTTCGGC Y A G H Q Η P A Q A F L F G

Mature alkaline 10 lipase

TCCTCGAACTACACCAAGACCCAGTACCCGATCGTCCTGACC S S N Y T K T Q Y P I V L T 20 30

CGCGGCATGCTCGGCTTCGACAGCCTGCTTGGGGTCGACTAC ^ GMLGFDSLLGVDYM's Zhang scale is applicable to the Chinese National Standard (CNS) Λ4 specification (210X297 male yak) --------- ^ ------ lsτ ------. ^ (Please read the back first (Notes and fill in this page again) A7 A7 Printed B7 of the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs B. V. Invention Description (孓) 40

TGGTACGGCATTCCCTCAGCCCAGCGTAAAGACGGCGCCACC

WYGIPSAQRKDGAT 50 60

GTCTACTTCACCGAAGTCAGCCAGCTCGACACCTCCGAAGCC

VYFTEVSQLDTSEA 70

CGGGGTGAGCAACTGCTGACCCAGGTCGAGGAAATCGTCGCC E G E Q L L T Q V E E I V A 80

ATCAGCGGCAAACCCAAGGTCAATCTGTTCGGCCACAGCCAT 1 "-II I > ..... 1 ^ — I S G K P K V N L F G H S Η putative active 90 100

GGCGGGCCTACCATCCGCTACGTTGCCGCCGTGGCCCCGGAT G G P T I fl Y V site no

CTGGTCGCCTCGGTCACCAGCATTGGCGCGCCGCACAAGGGT L V A S V T S I G A P Η K G 120 130

TCGGCCGCCGCCGACTTCATCCGCCAGGTGCCGGAAGGATCG

S A A A D F I R Q V P E G S 140

GCCAGCGAAGCGATTCTGGCCGGGATCGTCAATGGTCTGGGT

ASEAILAGIVNGLG __ (¾_ scale is applicable to China National Standards (CNS) Λ4 specifications (21〇X2 < > 7 公 # ·) (please read the precautions on the back before filling out this page). Install. -Se Central Ministry of Economic Affairs A7 _B7_ printed and printed by industrial and consumer cooperatives V. Description of invention (5) 150

GCGCTGATCAACTTCCTCTCCGGCAGCAGTTCGGACACCCCA A L I N F L S G S S S D T P 160 170

CAGAACTCGTTGGGCACGCTGGAGTCGCTGAACTCCGAAGGC

Q N S L G T L E S L N S E G 180

GCCGCACGGTTCAACGCCCGCTTCCCCCAAGGTGTGCCGACC A A H F N A R F P Q G V P T 190 200

AGCGCCTGCGGCGAGGGTGATTACGTAGTCAATGGCGTGCGC

S A C G E G D Y V V N G V R 210

TATTACTCCTGGAGCGGCACCAGCCCGCTGACCAACATACTC Y Y S W S G T S P L T N I L 220

GACCCTTCCGACCTGCTGCTCGGCGCCACCTCCCTGCCATTC D P S D L L L G A T S L P F 230 240

GGTTTCGAGGCCAACGATGGTCTGGTCGGACGCTGCAGCTCC

G F E A N D G L V G R C S S 250

CGGCTGGGTATGGTGATCCGCGACAACTACCGGATGAACCAC RLGMVIRDNYRMNH _ 0_ The paper size is applicable to the Chinese National Standard (CNS) A4 specification (2 丨 OX 297 public health) ---------- ^ ------ iT ------ ^ ( Please read the precautions on the back before filling out this page) 295602 A7 B7 5. Description of the invention (/ Μ 260 270

CTGGATGAGGTGAATCAGACCTTCGGGCTGACCAGCATATTC L D Ε V N Q T F G L T S I F 280 GAGACCAGCCCGGTATCGGTCTATCGCCAGCAAGCCAATCGC E T S Ρ V S V Y R Q Q A Ν Η 290 1429 CTGAAGAACGCCGGGCTCTGA L K N A G

T Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs This paper standard applies to the Chinese National Standard (CNS) A4 (210X297mm) A7

Please read the "Important I" item first, and then fill out this page. I Binding I Ball. The Central Standards Bureau of the Ministry of Economic Affairs Employee Consumer Cooperation Du Printed A7 _B7 V. Invention Description (A)

L R H L F D Y F F S T V G E 110

CAGTCGTTCCAGCAGGCCACCAGCGCTATCCGTGACTATCTG Q S F E Q A S S A 1 R D Y L 120

GCCAGCCAGCTGCGTGACGCGGCTCTGGCTCAGGCCCTTGAT A S Q L R D A A L A Q A L D 130 140

CTTCTGGATCGCTATATCGACTACAAAACTGACCTGGTGGAG L L D R Y I D Y K T E 1 V E 150

CTGGAGCGACGCTTCCCGATGGTGACCGAACTGGACGGCCTG L E R R F P Μ V T E L D G L 160 170

CGCGCCCGCGAAGATGCCGTACAACGCCTGCGCGCCAGTCTG H A R E D A V Q R L R A S L 180

TTCAACGCGCAGGAGCACGCCGCCTTCTTCGCCAGCGAAGAG

F N A Q E il A A F F A S E E 190

GTCTATAACCAGTTCACTCTTGAGCGTCTGGCGATACTGCAC VYNQFTLERLA1LH 200 210 GATCCGTCGCTGGATCCGCAGACACAGGCCGAACGGATTGAA

DPSLDPQGQAERIE 14- This paper wave scale is suitable for China National Standard (CNS) Λ4 specification (210X297mm) --------- ^ ------, order ------ ^ (please read first Note on the back and then fill in this page) A7 B7 V. Description of the invention (l >) 220

CGGCTGCGCGAAGGGCTGCCCGACGAGTTGCAACAATTGCTG R L R E G L P D E L Q Q L L 230 240

GTACCGCAATTGCACCTGACCCTGCGCCACGACCCAGCAGTT

V P Q L H L T L R H D P A V 250

GCTGACCAAGGTGCCGAGCCGGAACAGCTACGCCAGTTGCGC A D Q G A E P E Q L R Q L R 260

CTGAACCTGTTCGGGCCTCAGGCAACCGAGCGGCTGGAACGC LNLFGPQ ί \ TERL Ε 270 280 2333 TGGACCGCCAACGCAGCGAATGGGATCAGCGCCTGA WTANAANG 1 SA * II i Order (please read the notes on the back of the page 4) China National Standards Bureau Employee Consumer Cooperative Printed this paper standard CNS) Λ4 specification (210X 297mm) A7

Description of the invention () 85. 8. t The printing of the invention by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs also relates to a method of using the above-mentioned DNA fragments to manufacture test fat and anti-brewing. This method includes: ^ (a), self-fake The chromosomal DNA was isolated from the genus F_ni: (b), the chromosomal DNA of Pseudomonas Flu was partially digested with the restriction enzyme Sau 3 A, and the gene sequence encoding the test fat fermentation gene and the control code test were obtained " The DNA fragment showing the base sequence of the lipase gene; (Ο. The DNA fragment is cloned into a plastid that can be expressed in E. coli (for example, Xiyi / "+>), and the vector is obtained; (d), Transform the expression vector into coliform cells; (e), cultivate the transformed E. coli; and (0, isolate alkaline lipase. Other features and superiority of the present invention can be shown by the following diagram Description and Examples for a better understanding. Brief description of the diagram: The first picture: shows the restriction enzyme cleavage diagram containing the gene segment encoding alkaline lipase. The second picture: shows the steps for preparing pCj] j (4 Fig. Third: Show TIT-7 selection strains in tributyl oleate sieve The performance of the agar gum medium. The fourth figure: shows the analysis of SIT-polyacrylamide gel electrophoresis analysis of the production of test fat produced by TIT-7 strains. (1) Gene selection strategy The gene selection strategy in this experiment As shown in the second picture, the cadaver Μί / ί / ο monas plant-/// chromosomal DNA was partially cut with Sau 3A, and then the household G £ M-3Z / ^ + j plastid was used for shotgun colonization (shutgun) cloning) Yu Dait This paper scale is applicable to China National Standard (CNS) M specifications (210X297mm) II 1-I. m I — ^ 1 " ―Shirt ...... n (please read the back (Notes and then fill this page)

'1TI-. 1. I n 1 II 295602 A7 ____ B7 V. Description of the invention (β) Enterobacteriaceae; selected on oleoresin tributyric acid oleoresin culture medium selected to express alkaline lipase Transgenic strains of genes. (2) Selection of genes The plastids suitable for this DNA implantation are [Promega, USA] from E. coli, pBR3 22 [Gene, 2: 95 (1 977)], pBR325 [Gene, 4: 1 2 1 (1978) and pUC13 [Gene, 19: 259, (丨 982)] can also use any other plastid that can be replicated and maintained in the host. The method of congratulating this DNA 'is described for example in Miniatis, T. eta 1., Molecular Cloning, Cold Spring Harbor Laboratory, page 2 3 9 (1 9 8 2). Examples using E. coli, for example, E. coli DH10B [Lorow, D., and Jessee, J., (1990) Focus, 12:19], HB101 [J. Moi. Biol., 4 1, 459 (1969)], KI2, DH1 [Proc. Natl. Acad. Sci., USA, 60, 160 ( 1 968)] ° The method of transforming the host with plastids, such as the gasification method described in Miniatis, T. et al., Molecular Cloning, Cold Spring Harbor Laboratory, page 239 (1982), or by Calcium chloride / gasification method, which can be obtained by the above method, etc., containing Ρλλί?? / 〇 / wi; w α λ. Pseudoalcali ge η es F gene library. Falcon wing of central wing economy Bureaucratic consumption Printed by the cooperative (please read the precautions on the back # fill in this 1) If necessary or appropriate, the selected DNA containing alkaline lipase can be further cloned to such as pBR322, pUC12, pUCl3, pUCl8, pUC19 (3) Screening containing genes encoding alkaline lipase Use enzyme activity to directly look at the gene expression of alkaline lipase, and select agar gum medium in tributyric acid oil ester (丨% tributyric acid) Oil vinegar, 1% B actotrypt ο ne, 0.5% yeast extract, 0.5% vaporized sodium and 〇〇〇〇〇〇. (Ampicillin, mixed with a juice machine emulsified evenly), pick the strain that will produce a transparent ring, Or according to Pseudomonas A7 B7 Printed by the Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economy V. Invention Instructions (/ 6) 1; · ^^ 菌 ^^ 1 1 1 (Pseudomonas pseudoalcaligenes F-111) amino acid sequence of derivatized fat 1. A colony hybridization method using chemically synthesized oligonucleotides as probes. ® Same as (4) pCKH4 plastid and its sub-selected carbohydrates on the performance of the gene for basic fatty stomach is to understand the position of the gene containing the encoded basic fat_ in pCK_ plastid, delete test and co-transform for you ( {： 0 & 3115 € 〇1: 0 ^ 1 丨 〇11), as shown in Table 1, it is known that the expression of this gene encoding basic fat requires the assistance of another regulatory DNA fragment, and this reverse regulation There are many phenomena of trans-acting

Pseudomonas belongs to% of fat produced [Jane Gilbert, E., (1993) Enzyme Micrib. Technol., 15: 634]. The base sequence that regulates the gene expression of alkaline lipase is approximately 1.5 to 2.9 Kbp. Table]: PCKH4 plastid and its sub-selection and alkaline lipase performance of the sterilized plastid name position size activity (test base pair) (transparent ring performance) PCKH4 full length 4000 PCKH-EE3.9 0.1-4.0 3900 + PCKH -PE2.7 1.3-4.0 2 70 0-PCKH-EP1.3 0. 0-1 · 3 1300 PCKH-SS2.2 0.7-2.9 220 0 PCKH-XC1. 7 0.0-1. 7 1700 — PCKH-CSa2. 3 1.7-4.0 2300 — PCKH-BB2.〇0.0-2. 0 2000 — r, j The scale of this paper is applicable to the Chinese National Standard (CNS) Λ4 specification (210X2 (m > ^) m ml H m In HI ml ^ i TV ,-° (please read the precautions on the back before filling in this page) Printed Bag A7 B7 of the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economy V. Description of Invention (〇) PCKH-BE2.〇2.0-4.0 2000 PCKH-ESO.7 0.0 -0.7 700 PCKH-HER2.5 1.5-4.0 2500 PCKH-PE2.7 + PCKH-XC1.7-— PCKH-XC1.7 + PCKli-CSa2. 3-PCKH-XC1. 7 + PCKH-HER2.5-PCK11 -SS2. 2 + PCKH-XC 1 .7 B: BamHI, C: CLaI, E: EcoRI, ER EcoRV, H: HindIV, S: SphI, Sa: SacI, X: XbaI ° (5) with coded alkaline The DNA of the lipase gene base sequence is determined by the method of dideoxy seguencing [Sanger, F, Nicklen, S., and Coulson, AR, 1 979, Proc. Natl. Acad. Sci., 74: 5463.] Discharge 2910 base pair sequence, and then this base sequence, and then with the known basic fatty acid amino acid Sequence comparison, and confirmed the existence of the gene encoding alkaline lipase. The gene encoding alkaline lipase is a complete ORF (open reading frame) sequence (413 to 1426 bps), which can translate 3 3 8 amine groups

II The paper scale is applicable to the Chinese National Standard (CNS) Λ4 specification (210X2 ^ 7mm) I n I n III «nnn I ni T nn« I n I _ (please read the precautions on the back before filling this page) Economy A7 ___ B7_ Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Foreign Affairs 5. Description of the invention (imitation) ~ The protein composed of acid is compared with the N-terminal sequence of the protein, and the secretion signal sequence is subtracted, and the complete mature alkaline lipase sequence is From the 290 amino acid sequences starting from FLFGSS, the calculated molecular weight is 30,82 6 Dalton, which is similar to the enzyme molecular weight measured by SDS-PAGE of 32,000 Dalton. The DNA encoding alkaline lipase obtained in Example 1 described below is a typical example of the DNA fragment encoding alkaline lipase according to the present invention, and its restriction enzyme cleavage diagram is shown in the first figure. (6) The determination of the base sequence that regulates the expression of the gene encoding alkaline lipase results from the enzyme activity of the transformed strain of Example 3. This expression, which contains the gene encoding the test fat, requires the assistance of another DNA fragment, According to Example 3 (Table 1), this sequence containing the gene encoding alkaline lipase is located between 1.5 and 2.9 Kbp, and there is another gene base sequence U 4 6 9-2 4 9 1 bp that regulates the performance of alkaline lipase. , Can translate protein composed of 3 40 amino acids. Example 1 _ Pseudomonas F-1 1 1 FW / U Chromosome DNA Isolation

Pseudomonas pseudoalcaligenes A 400 ml LB liquid culture medium in a 1 1 Erlenmeyer flask, shake at 37 ° C for 24 hours. Centrifuge at 4 ° C, 6000 rpm for 30 minutes to obtain cell sediment. Resuspend in 36 ml TE buffer (pH 8) After breaking up the cells, add protein lysozyme to make the final concentration 1 mg / ml, and shake gently at 37 ° C for 30 minutes. Then add SDS to make the degree 1%, at 60 ° C Rotate and gently shake for 30 minutes. Add the same volume of phenol and gaseous imitation (volume ratio 1: 1) and slowly rotate for 60 minutes. Centrifuge at 9000 rpm for 20 minutes to extract the DNA dissolved in the water layer. Repeat three times. 1 / 丨 〇 volume of 3 mM potassium acetate and double volume of ice-cold ethanol to D Ν Α solution to precipitate d NA, centrifuged to collect the precipitated 10 _> ^ wave scale is applicable to China National Standard (CNS) Λ4 specifications (210X 297 Gongxiong) --------- Refer to ----- 1T ------ {Please read the note $ item on the back and then fill in this page) Printed by the Staff Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs Poly A7 B7 V. Description of invention (i.e.) DNA, washed with 70% ethanol, dried in the air, added 2 m of distilled water to dissolve the DNA, and stored at -20. (:. Example 2. Selection of genes containing genes encoding basic fat (A) Preparation of gene library

(a) To limit the partial digestion of S au 3 AI ρ λ. t? i / ί / o / w ο «α λ · pie μ do α / / fa e« e .V F- / / / chromosome DNA to limit S au3 A Partial Digestion Example 1 Pseudomonas pseudoaligenes DNA isolated »33 mM Tris-acetate buffer (pH 7.9), 10 mM magnesium acetate, 66 mM potassium acetate, 0.5 mM dithiothreitol, 10 ug stained DNA and 1.2 units The active Sau 3A, with a total volume of 200 μl, was reacted at 37 ° C for 20 minutes and treated with powder-air imitation. The reaction mixture was freed of protein plus two volumes of ethanol to recover the digested DNA. (b) Digestion with restriction enzyme 8 & 丨 1 ^ 1 (; £ from -32 / broad) vector 〇1 ^ eight vector DNA (Promega USA) containing 10 mM Tris-HCl buffer (PH8.0) , 5 mM vaporized magnesium, 100 mM sodium chloride, 1 mM 2, hydrogen thioethanol, 1 ug of chromosomal DNA and 1 unit of active BamHI, total volume of 20 ul reaction system, under 37 X: reaction After 2 hours, the protein was removed from the reaction mixture by phenol-aeroform treatment, and double volume of ethyl fermentation was added to recover the digested DNA. (C) Ligation and transformation The chromosomal DNA partially digested by Sau3A obtained in step (a) above 500 ng and (b) step BamHI cut vector DNA 100 ng, containing 66 mM Tris-HCl buffer (pH7.5) 5 mM vaporized magnesium, 1 mM dithiothreitol, 1 mM ATP and 1 unit live paper size Applicable to the Chinese National Standard (CNS) Λ4 specification (2IOX 297 male aquarium) (please read the precautions on the back before filling this page) 'Rate' A7 B7 295602 —_ V. Description of the invention (T4 DNAligase of the sex, total volume In the reaction system of 2〇u 丨, the conjugation reaction was carried out at 4 minutes, and then treated with calcium chloride according to Manda 丨 et al. Method [Mandal, M., and Higa, A. 1970, J. Miol., 53: 159], fed into DH 10 B coliform bacteria and then smeared it on the LB agar gum medium containing x_ga 丨 and ampici 丨 in In the middle of picking a single strain of white plaque, about 2000 transformants can be obtained. (B) Screening of genes containing alkaline lipase. The transformants of more than 2000 strains obtained from the above are selected from the dental deposits. Into the screening media containing oleyl tributyrate (丨 ❶ / 〇 oleyl tributyrate 丨% B acto tryptone, 0.5% yeast extract, 05% sodium vapor and 〇〇〇〇〇〇〇〇. ampicilhn 'use juice machine emulsified mixed juice to pick up the strains that will produce a transparent ring' obtained a total of more than 10 active transformants. After purification, one of the transformants was named TIT-7. The tributyric acid oleic acid ester screening has a clear ring in the nutrient medium of the cabbage, as shown in the third figure, there may be genes containing basic fat. According to the method of Birnboim et al. [Birnboim, H.CVfLnH Doly, J. , 1 979, Nuclei AcidRes., 7: 7 15.], the extraction of the plastid DNA of TIT-7 transformants' results showed that the plastid DNA extracted The size determined on the glue is 7 2 Kbp, minus the size of the pGEM-3Zf (+) vector DNA (3 2 Kbp). The size of the dyed hybrid DNA inserted in pGEM_ 3Zf (+) is 4.0 kbp, depending on its size The plastid extracted from the transformed strain of Ding Ding 7 was named pCKH4. Example 3. Regulatory performance of transformed strains] > Table paper scale is applicable to Chinese national standard (CNS> Λ4 specifications (2) OX; 297 public miscellaneous --------- ^ ------ ir- ------ ^ (Please read the notes on the back before filling in this page) Printed by the Ministry of Economic Affairs Central Standards Bureau Employee Consumer Cooperative Printed by the Ministry of Economic Affairs Central Standards Bureau Employee Consumer Cooperative Du Yinzi A7 B7 V. Invention Description (冽In order to sequence out the nucleus and acid sequence, the second selection of each fragment was carried out according to the map of pCKH4 plastid. After the limit @treatment was obtained, a total of 10 different second selection strains were grown in comparison. Oleic acid butylate_ The results of enzyme activity in nutrients are shown in Table 〖Except PCKH-EE3.9 plastid can express activity, the other plastids can not show enzyme activity. 'Presumed reason' may be required for the expression of this gene Other proteins are too busy. If the plastids of other sub-selected strains are mixed and fed into the HB 10D host, the results of co-transformation of PCKH-PE2.7 and PCKH-XC1.7 and other plastids are shown. This regulatory gene may be located after 1.3 Kbp. The results of pCKH-XCl.7 and pCKH-CSa2.3 plastid co-transformation show that this regulatory gene can Before 丨 .7 Kbp, the results of pCKH-XCl.7 and PCKH-HER2.5 plastid co-transformation showed that this regulatory gene may be located after 1.5 Kbp, pCKH-SS2.2 and pCKH-XC1.7 plastid The result of the transformation shows that this regulatory gene may end before 2.9 Kbp. Example 4. Determination of the base sequence of the gene encoding alkaline lipase p CK Η 4 strains of the selected strains, and extract DNA separately, with dide ο X y seguencing method [Sanger, F., Nicklen, S., and Coulson,

AR, 1979, Proc. Natl. Bajie. Sci., 74: 5463.] The sequence of the inserted DNA sequence has 29,10 nucleotides sequenced in a total length of 4000 nucleic acids, including 2 ORFs, At 1429 bp (ORF1) and 1469 to 2491 bp. (ORF ^ | and the two ORFs are only 41 base pairs apart and have an EcoRV restricted cleavage position (GATATC), due to the 5 'end sequence of this base Partial amino acids; and the first 20 amino acid sequences from the N-terminal of the purified alkaline lipase: Phe-Leu-Phe-Gly-Ser-Ser-Asn-Tyr-Thr-Lys-Thr-Gln-Tyr -Pro-Ile-Val- Leu-Thr-Arg-Gly coincides with each other, complete LIP This paper scale is suitable for the National Threshold National Standard (CNS) Λ4 尭 格 (2 丨 OX 297 male front) -------- -Install— (Please read the precautions on the back before filling in this page) Order A7 B7 G. V., (1 9 8 6 ucleic Acids Res., 14: 4¾ The DNA sequence encoding basic lipid anti-g also contains One fat; I HSHG [Wj ^ kler, FK, et al, (1 990 & V. Description of invention (n) Gene of A, including (1 ^ terminal regulatory region; such as promoter, TAATTGGTCACTTTTCAGCC [Deretic V., et al (1 988) Bio / Technology 7: 1249.] and Shine-Dalgarno Sequence, AGGC.] (2) Secretion signal sequence; a peptide of 48 amino acids [V. Heijne, Γ]. (3) Structural gene, here, the common active center G-ature ♦ 343: 771] ° Based on these characteristics, the gene encoding alkaline lipase should be located at this OAF f .. The complete sequence containing the gene encoding the test fat lipase (413 to 1 426 bp_s) 'deducts the secretion signal sequence, which can translate 290 amino acids The actual molecular weight of the composed protein is 3 0,826, which is similar to the size measured by SDS-PAGE of 32,000. Example 5. The determination of the base sequence that regulates the expression of genes encoding basic fat is based on Example 3 (Table 1). The base sequence of the sex lipase gene is between 2.9 Kbp, and there is another ORF2 (1469-2491) in this area. It is only 41 base pairs away from the gene encoding alkaline lipase (ORFI), it is speculated This regulatory expression gene is linked to the gene encoding basic fat brewing and shares the same promoter and Shine-Dalgarno sequence. The base sequence of this regulatory expression gene is known

The base sequence of Pseudomonas belongs to [jane Giibert E, (1 993) Enzyme Micrib. Techno 1., 15: 634] alignment, there is no similarity, and it is another new regulatory sequence. Example 6 · Production of alkaline lipase with E. coli transforming strain (TIT-7) 14 This paper scale is suitable for China National Falcon (CNS) ΛΟ 见 格 (21〇χ2 (Π 公 录) -------- -Installed ------, -ST ------ Φ .. (Please read the notes on the back before filling in this page) A7 _____B7 printed by the Employee Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of invention ( Ο) The E. coli transformed strain (τι τ_7) obtained in Example 2 was picked with bamboo sticks, planted in 10 ml LB medium containing ampicillin (50 Ug / ml), and cultured at 37 ° C for 18 hours with shaking , 0.5 ml portion of the culture was planted into 50 ml LB medium containing ampicillin (50 ug / mi), incubated at 37 ° C for 5 hours, and added 0.5 ml 0.1 M IPTG ( After the final concentration of isopropyl B-thiogalactan-pyranose 1 mM), continue to culture for another 5 hours' to collect cells (1 ^ 00 rpmJ〇 minutes), suspended in 20ml Tris-HCI buffer (50 mM, PH8.5), ultrasonic treatment for 20 minutes, centrifugation (1 0,000 rpmgo) to remove cell ruptures, supernatant can be measured by @ 知 Method, alkaline fatness can be measured. SDS polyacrylamide gel electrophoresis shown in the fourth figure In the picture 2 TIT clones are, the I 3 _ _ is a host coliform purified alkaline fat, 5. Molecular weight markers). The molecular weight produced by the tit selection strain is 32,000 lanes. The extra protein ribbon cannot be formed by the DH10B host, and it is confirmed. ^ ------ 1T ------ ^ (Please read the notes on the back before filling out this page) Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs _____ ^

The standard of this paper is applicable to the Chinese national standard (CNS "Eight-four current format (210 X

Claims (1)

A8 B8 C8 D8 VI. Scope of patent application --------- ^ ------ 1T ------ i (Please read the precautions on the back before filling in this page) Central Ministry of Economic Affairs Bureau of standards staff consumer cooperative printed GGATCCCCAG CAOGTCAGGA 50 AGGCCAAAGA AAGCCGCAAT 90 CGGAAATCAG CAGGGCGAAC 130 ATACCCGTTG ACATAACTGC 1 70 TTGCATTAAC GGTGGCAGGC 2 10 TTGCCCTGGC TCGGGGGCGC 250 CATTCACAAT TTCGCTGCTG 290 ATTACCGTTC ATCGGATTCA 330 CAGAGCGCAC GACTAATTGG 370 TCAACCCCGC TGAACCCTGC 410 CCAGGCCTGA CTATGCTCCC ORF 450 30 CTCCGACAAT CAGCGCGCCC 70 CAGCCCCGGG ACGAAGAATT 1 10 CCCAGTAGCA GCCAGAAGGC 150 TCAGCAGCAT GATGACATCC I 90 AAACCTGAGA GTCTACCAGA 230 TCAATCGGGT TTGCGGAACC 270 CTGACACACC CAAACTCTGA 310 TCCTGACAAT ATCCGGATGC 350 TCACTTTTCA GCCGCCTAGG 390 GCCGCGCCTT CAATTCATCA 430 The DNA fragment of the enzyme gene expression sequence has the following base sequence: iL · 本 纸The standard is applicable to the Chinese National Standard (CNS) Λ4 specification (210X297 public f) 295602 VI. Patent application range 490 510 TTATGCGCAA CAAGACTCGC GTCTCGCTCC GCCTCGGGCT 530 GGCCACTACG CTGGGCATCA 550 GCACCCAGCC CAGGCCTTCC 570 TGTTCGGACCCAGTCACACTCAGCACCAC 590 CGTCCTGACC CGCGGCATGC 630 TCGGCTTCGA CAGCCTGCTT 650 GGGGTCGACT ACTGGTACGG 670 CATTCCCTCA GCCCAGCGTA 690 AAGACGGCGC CACCGTCTAC 710 TTCACCGAAG TCAGCCAGCT 730 CGACACCTCC GAAGCCCGGG 750 GTGAGCAACT GCTGACCCAG 770 GTCGAGGAAA TCGTCGCCAT 790 CAGCGGCAAA CCCAAGGTCA 810 ATCTGTTCGG CCACAGCCAT 830 GGCGGGCCTA CCATCCGCTA 850 CGTTGCCGCC GTGGCCCCGG 870 ATCTGGTCGC CTCGGTCACC 890 AGCATTGGCG CGCCGCACAA 910 GGGTTCGGCC GCCCCCGACT 930 TCATCCGCCA GGTGCCGGAA 950 GGATCGGCCA GCGAAGCGAT 970 TCTGGCCGGG ATCGTCAATG 990 GTCTGGGTGC GCTGATCAAC 21 This paper scale is applicable to China National Standard Rate (CNS) Λ4 specification (210X297mm) --------- ^ ------ ^ ----- -1 ^ (Please read the note $ item on the back before filling in this page) Apply Patent Fan Park 010 TTCCTCTCCG GCAGCAGTTC 1050 A8 B8 C8 D8 1030 GGACACCCCA CAGAACTCGT 070 TGGGCACGCT GGAGTCGCTG AACTCCGAAG GCGCCGCACG 1090 GTTCAACGCC CGCTTCCCCC AAGGTGTGCC GACCAGCGCC 130 I 50 TGCGGCGAGG GTGATTACGT AGTCAATGGC GTGCGCTATT 1 70 190 ACTCCTGGAG CGGCACCAGC CCGCTGACCA ACATACTCGA mo 230 CCCTTCCGAC CTGCTGCTCG GCGCCACCTC CCTGCCATTC 1250 270 GGTTTCGAGG CCAACGATGG TCTGGTCGGA CGCTGCAGCT 1290 3 10 CCCGGCTGGG TATGGTGATC CGCGACAACT ACCGGATGAA 1330 350 CCACCTGGAT GAGGTGAATC AGACCTTCGG GCTGACCAGC 1370 390 ATATTCGAGA CCAGCCCGGT ATCGGTCTAT CGCCAGCAAG 14 10 1430 ^ Pack I i II-. Order line (please read the notes on the back of the central office and then fill in the page) Printed by the consumer cooperative CCAATCGCCT GAAGAACGCC GGGCTCTGAA ATAGGCTCCA 1450 470 CAACCAGACA GGGCTGGCCT CAGGCCCCAG TCACAGCTAC 1490 CGJXAKGAC ATGAAGCCGC ORF 2 15 10 TGATTTATCT GCCGTTGCTA Application of the Chinese national standard (CNS) 8 for this paper size 8 A8 Printed by the Employee Consumer Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs 1530 丨 550 CTCGGCCTCG GCCTGCTCGG CTGGCACCTG AGCACACCGA 1 570 1 590 CACCCAGCCC CTCCGCGCGCCC ACATCAACGC CGCTACAAGC 1 6 I 0 1 630 CGGCAGTGAA CAACCCGCCA CAACTCCTGT GAGTCTGACC 1CC 0CC 1CC 0CC TACCGACATC GATGGTCAAC TGGAAGTCGA 1730 1750 CGCCCAGGGC AATCTGGTGA TTACCGACCA ACTGCGTCAC 1770 1790 CTGTTCGATT ATTTCTTCAG CACCGTCGGC GAACAGTCGT 1 8 1 0 1830 TCGAGCAGGC CAGCAGCGCT ATCCGTGACT ATCTGGCCAG 1850 1 870 CCAGCTGCGT GACGCGGCTC TGGCTCAGGC CCTTGATCTT 1 890 Ϊ 9 Shu 0 CTGGATCGCT ATATCGACTA CAAAACTGAG CTGGTGGAGC 1930 1950 TGGAGCGACG CTTCCCGATG GTGACCGAAC TGGACGGCCT 1970 1 990 GCGCGCCCGC GAAGATGCCG TACAACGCCT GCGCGCCAGT 20 丨 0 2030 CTGTTCAACG CGCAGGAGCA CGCCGCCTTC TTCGCCAGCG (please read the note on the back first and then fill in this page) This paper size is applicable to China National Standards (CNS) Λ4 specification (210X297 mm) Patent scope Λ8 B8 C8 D8 2050 2070 AAGAGGTCTA TAACCA GTTC ACTCTTGAGC GTCTGGCGAT 2090 ACTGCACGAT CCGTCGCTGG 2 ATCCGCAGAC 110 ACAGGCCGAA 2 130 CGGATTGAAC GGCTGCGCGA 2150 AGGGCTGCCC GACGAGTTGC 2 1 70 AACAATTGCT GGTACCGCAA 2190 TTGCACCTGA CCCTGCGCCA 22 I 0 CGACCCAGCA GTTGCTGACC 2230 AAGGTGCCGA GCCGGAACAG 2250 2270 CTACGCCAGT TGCGCCTGAA CCTGTTCGGG CCTCAGGCAA 23 Shu 0 2290 CCGAGCGGCT GGAACGCTGG ACCGCCAACG CAGCGAATGG 23 30 GATCAGCGCC TGA (please read the $ item on the back and then fill in this page) The paper standard printed by the Employee Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs is applicable to the Chinese National Standard (CNS) Λ4 specification (210X 297mm) A8 B8 C8 D8 Patent scope • the DNA fragment of item 1 of the scope of the patent application, wherein the gene encoding the lipase test sequence. 310 GGATTCA TCCTGACAAT ATCCGGATGC 330 350 CAGAGCGCAC GACTAATTGG TCACTTTTCAGCCGCCTAGG 370 390 TCAACCCCGC TGAACCCTGC GCCGCGCCTT CAATTCATCA 410 CCAGGCCTGA CTdw_cj_GJALA_GJ_c_T_c_I_G_l_mGJ_AJI ?. MLPDSLCDAV _AlAG_Aaa_AXAACH_lAaA_C_CG_C_A_C_A_A_GGja RDTTTIKP 11 KDS II Y GIL c_LA_LAiLAC_TC_G_C_G_T__C_TC_G_C_TCC_G_C_CT_C ^ AQQDSRLAPPRAGH 1 Ministry of Economic Affairs Bureau of the Central-like quasi-negative workers consumer cooperative printing and binding (Read the back of the note term M $ Complete this page) TACGCTGGGCATCAGCACCCAGCCCAGGCCTTCCTGTTCGGC YAGHQ Η PAQAFLFG TCCTCGAACTACACCAAGACCCAGTACCCGATCGTCCTGACC SSNYTKTQYPIVLT 20 30 CGCGGCATGCTCGGCTTCGACAGCCTGCTTGGGGTCGACTAC RGMLGFDSLLGVDY this paper by China National Qiu Bu-scale prospective ( CNS) Λ4 specification (2I0X29'7 mm) Ministry of economic Affairs Bureau of standards employees consumer cooperatives printed A8 B8 C8 D8 six patented range of 40 TGGTACGGCATTCCCTCAGCCCAGCGTAAAGACGGCGCCACC WYGIPSAQRKDGAT 50 60 GTCTACTTCACCGAAGTCAGCCAGCTCGACACCTCCGAAGCC VYFTEVSQLDTSEA 70 CGGGGTGAGCAACTGCTGACCCAGGTCGAGGAAATCGTCGCC RGEQLLTQVEEIVA 80 ATCAGCGGCAAACCCAAGGTCAATCTGTTCGGCCACAGCCAT 1 SGKPKVNLFGHSH 90 100 GGCGGGCCTACCATCCGCTACGTTGCCGCCGTGGCCCCGGAT GGPT 1 RYVAAVAPD 110 CTGGTCGCCTCGGTCACCAGCATTGGCGCGCCGCACAAGGGT LVASVTS 1 GAP Η KG 120 130 TCGGCCGCCGCCGACTTCATCCGCCAGGTGCCGGAAGGATCG SAAADFIRQVPEGS 140 GCCAGCGAAGCGATTCTGGCCGGGATCGTCAATGGTCTGGGT ASEAILAGIVNGLG Binding points (please read the precautions on the back before filling in this page) This paper size is applicable to China National Standards (CNS) 297 4 printing equipment ^ 602 as B8 C8 D8 __ six, patented range 150 GCGCTGATCAACTTCCTCTCCGGCAGCAGTTCGGACACCCCA ALINFLSGSSSDTP 160 170 CAGAACTCGTTGGGCACGCTGGAGTCGCTGAACTCCGAAGGC QNSLGTLESLNSEG 180 GCCGCACGGTTCAACGCCCGCTTCCCCCAAGGTGTGCCGACC AARFNARFPQGVPT 190 200 AGCGCCTGCGGCGAGGGTGATTACGTAGTCAATGGCGTGCGC SACGEGDYVVNGVR 210 TATTACTCCTGGAGCGGCACCAGCCCGCTGACCAACATACTC YYSWSGTSPLTNIL 220 GACCCTTCCGACCTGCTGCTCGGCGCCACCTCCCTGCCATTC DPSDLLLCATSLPF 230 240 GGTTTCGAGGCCAACGATGGTCTGGTCGGACGCTGCAGCTCC GFEANDGLVGRCSS 250 CGGCTGGGTATGGTGATCCGCGACAACTACCG GATGAACCAC RLGMVIRDNYRMNH This paper scale is applicable to the Chinese National Standard (CNS) Λ4 present grid (210X297mm) --------- ^ ------ IT ------ Φ (please read the back page first (Notes and then fill out this page) A8 B8 C8 D8 夂, patent scope 260 270 CTGGATGAGGTGAATCAGACCTTCGGGCTGACCAGCATATTC LDEVNQTFGLTSIF 280 GAGACCAGCCCGGTATCGGTCTATCGCCAGCAAGCCAATCGC ETSPV SVYHQQANR 290 1429 CTGAAGAGCGCGAGAGC Printed garment 3 of the Central Standards Bureau ’s employee consumer cooperatives. For example, the DNA fragment in item 1 of the patent application, in which the regulatory heel sequence of the gene encoding the basic lipid-preventing enzyme is: 1430 CTCTGAA ATAGGCTCCA CAACCAGACA GGGCTGGCCT 1491 1 CAGGGGCCAG TCACAGGTAC CGATATCGAC ATGAAGCCC -The size of the line paper is in accordance with the Chinese National Standard (CNS) Λ4 specification (210X297 male). Patent scope A8 B8 C8 D8 Printed by the Employee Consumer Cooperative of the Central Bureau of Economic Affairs of the Ministry of Economic Affairs 10 CTGATTTATCTGCCGTTGCTACTCGCCCTCGGCCTGCTCGGC LIYLPLLLGLGLLG 20 30 TGGCACCTG AGCACACCGACACCCAGCCCCTCCGCGCCCACA WHLSTPTPSPSAPT 40 TCAACGCCGCTACAAGCCGGCAGTGAACAACCCGCCACAACT STPLQAGSEQPATT 50 CCTGTGAGTCTGACCCGTCCGACCACGCGCAGCACCGACCAG PVSLTRFTTESTDQ 60 70 CACCTGCCCGCCTCACTGCGCGATACCGACATCGATGGTCAA II LPASLRDTDIDGQ 80 CTGGAAGTCGACGCCCAGGGCAATCTGGTGATTACCGACCAA LEVDAQGNLVITDQ 90 100 CTGCGTCACCTGTTCGATTATTTCTTCAGCACCGTCGGCGAA (Please read the back of the note and then fill in item f of this page) - Pack - This paper line scale applicable to Chinese National Standard (CNS) A4 size ( 210X 297 envy male) 8 8 8 8 ABCD Ministry of economic Affairs Bureau of standards consumer cooperative printing six employees, the scope of patented L Η HLFDYFFSTVGE 110 CAGTCGTTCGAGCAGGCCAGCAGCGCTATCCGTGACTATCTG QSFEQASSA 1 RDYL 120 GCCAGCCAGCTGCGTGACGCGGCTCTGGCTCAGGCCCTTGAT ASQLRD i \ ALAQ 130 140 CTTCTGGATCGCTATATCGACTACAAAACTGAGCTGGTGGAG LLDRYIDYKTELVE 150 CTGGAGCGACGCTTCCCGATGGTGACCGAACTGGACGGCCTG LERHFP Μ VTELDGL 160 170 CGCGCCC GCGAAGATGCCGTACAACGCCTGCGCGCCAGTCTG RAREDAVQRLRASL 180 TTCAACGCGCAGGAGCACGCCGCCTTCTTCGCCAGCGAAGAG FNAQEHAAFFASEE 190 GTCTATAACCAGTTCACTCTTGAGCGTCTGGCGATACTGCAC VYNQFTLERLAIL 200 210 GATCCGTCGCTGGATCCGCAGACACAGGCCGAACGGATTGAA DPSLDPQGQAERIE this paper scale applicable Chinese National Standard (CNS) A4 size (210X297 mm) IIIIIIIIIIII set - - II n each (please read the Notes on the back to fill out this page) repair ΐ] AS Fou ^ · day of Shu Shu, B8 change .8. DS to six, patented range 220 CGGCTGCGCCAAGGGCTGCCCGACGAGTTGCAACAATTGCTG RLRECLPDELQQLL 230 240 GTACCGCAATTGCACCTGACCCTGCGCCACGACCCAGCAGTT VPQLHLTLRHDPAV 250 GCTGACCAAGGTGCCGAGCCGGAACAGCTACGCCAGTTGCGC ADQGAEPEQLRQLR 260 CTGAACCTGT butoxy CGGGCCTCAGGCAACCGAGCGGCTGGAACGC tL-arg ^ -ass8 ^ = JlMII, e =! g :: == * 8ale! aa »* a = sgg = c ^^ a ^^^ aB ^« aes3i · — L. NLFGPQATERLER 270 280 2333 TGGACCGCCAACGCAGCGAATGGGATCAGCGCCTGA WTANAANGISA * 4. One The method of manufacturing alkaline lipase includes: (a). Isolation of chromosome 〇να from Pseudomonas sp. F_1u; (b). Partial digestion of chromosome DN of Pseudomonas sp. F_nl with restriction enzyme Sau 3A A, and may contain the coded basic fat in the Ministry of Economy, Central Standards Bureau of the Ministry of Economic Affairs, Cooperative Consumer Cooperative Printing Li --II ----- ^, ------ order (please first Read the precautions on the back and then fill out this page) Enzyme gene sequence and the DNA fragment that regulates the expression of the base sequence of the alkaline lipase gene; (c). Colonize the DNA fragment into the plastid pGEM that can be expressed in coliforms -JZyY + J * to obtain the carrier; (d). Transform the expression vector into coliform cells; (e). Cultivate the transformed E. coli; and (f). Separate the alkaline lipase. This paper wave scale it with towel SSi standard rate (CNS) eight 4 specifications (21GX297. Public love)