CN106497896B - The recombination carboxy-lesterase DMWf18-558 and its encoding gene in a kind of deep-sea source and application - Google Patents
The recombination carboxy-lesterase DMWf18-558 and its encoding gene in a kind of deep-sea source and application Download PDFInfo
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Abstract
The present invention relates to a kind of deep-sea source carboxy-lesterase DMWf18-558 and its encoding gene and applications.Carboxy-lesterase DMWf18-558 encoding gene is screened by macro genome and is obtained, and nucleotide sequence and amino acid sequence are respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.After this carboxylesterase gene heterogenous expression, the catalytic activity highest when substrate is p-nitrophenol capronate (C6), enzyme activity is up to 367U/mg.Carboxy-lesterase DMWf18-558 catalyzing hydrolysis temperature range is 15~40 DEG C, preferably 15~35 DEG C；Catalyzing hydrolysis pH value is 3.0~10.0, preferably 6.5~9.0；Catalyzing hydrolysis NaCl concentration is in 0.5~3M, preferably 0.5-1M.The esterase can be widely applied to the fields such as grease hydrolysis, pharmaceutical synthesis metabolism, food processing, wastewater treatment, detergent industry.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to a kind of halmeic deposit source carboxy-lesterase, its encoding gene
And its application.
Background technique
Lipid hydrolyzing enzyme includes carboxy-lesterase (EC3.1.1.1) and lipase (EC3.1.1.3), is widely present in microorganism
In, the hydrolysis and synthesis of ester type compound can be catalyzed.Carboxy-lesterase major catalytic is less than the short chain acyl glycerol of 10 carbon, rouge
Fat enzyme major catalytic is greater than the long acyl glycerol of 10 carbon.Lipid hydrolyzing enzyme has many good characteristics, such as catalysis reaction not
Confactor is needed, hand-type selection specificity is high, possesses wider substrate spectrum, and keep high stability in organic solvent
Deng.Lipid hydrolyzing enzyme is catalyst important in industrial production, can be widely applied to chiral drug catalysis, leather silk spinning raw material takes off
Rouge, wastewater treatment, detergent industry and food processing etc..
It is commercially produced at present there are many microbe-derived lipase, and in each side of production and living
Face is applied.And the industrial enzymes that carboxy-lesterase is important as one kind, the type and quantity being applied in actual production
It is also extremely limited.The excavation and accumulation of novel carboxylic acid esterase resource are to meet the industry such as food and chemical industry increasingly to increase carboxy-lesterase
The necessary condition of long demand.The present invention obtains deep-sea source with the technology of the building of macro genome and Carboxylesterase Activity screening
Novel carboxylic acid esterase, and study its zymologic property.
Summary of the invention
The object of the present invention is to provide a kind of new deep-sea source carboxy-lesterases, its encoding gene and preparation method thereof, should
Carboxy-lesterase can be used for esters degradation and the biocatalysis and conversion of other ester type compounds.
The present invention is screened from the macro genomic library of Pacific seamounts halmeic deposit, using tributyrin as substrate
Carboxylesterase Activity obtains a kind of new carboxy-lesterase DMWf18-558, the nucleotide sequence of encoding gene such as SEQ ID
Shown in No.1, amino acid sequence is as shown in SEQ ID No.2.
The carboxylesterase sequence is subjected to Homology search in GenBank, highest similar property is also macro genome
The esterase in source, similitude are 82% (its number of registration in GenBank database is AFB82690).Phylogenetic Analysis knot
Fruit shows the Section IV family that carboxy-lesterase DMWf18-558 belongs in hydrolysis of lipid enzyme family.Speculated according to amino acid sequence, carboxylic
The catalytic center of acid esters enzyme DMWf18-558 is by serine, glutamic acid and histidine (amino acid position 144,238 and 268) group
At wherein serine is located at glycine, aspartic acid, serine, alanine and glycine (amino acid position is 142 to 146)
In the conserved sequence of composition, therefore carboxy-lesterase DMWf18-558 belongs to Section IV family GDSAG subfamily.Oxonium ion hole is located at 75
With 76 two glycine.In conclusion DMWf18-558 should be a newcomer in lipid hydrolyzing enzyme Section IV family.
It, can be in separate catalysis shown in SEQ ID NO:2 under the premise of not influencing esterase DMWf18-558 protein active
The amino acid sequence of heart amino acid position (amino acid position of preferably 142-146,238 and 268) carry out it is various replace, addition and/
Or one or several amino acid of missing are obtained with the active derived protein of esterase DMWf18-558.According to art technology
Common knowledge, the biological activity of protein are closely related with its functional domain.In general, only occur in function
The site mutation of structural domain may have an impact the 2 and 3 dimensional organization of protein, to influence its biological activity.And
For the amino acid sites in separate functional domain (preferably away from the amino acids position of 142-146,238 and 268) occur, by
It is not involved in protein function conformation in this region, thus individual point mutation of amino acid will not produce the biological activity of protein
Raw substantial effect, so as to retain the biological function of crude protein substantially.Described can retain crude protein substantially
Biological function refer to derived protein with 80% or more esterase DMWf18-558 biological activity, preferably have
The biological activity of 90% or more esterase DMWf18-558 more preferably has the biology of 95% or more esterase DMWf18-558
Learn activity.
Preferred esterase DMWf18-558 mutant have at least with amino acid sequence 90% shown in SEQ ID NO:2 with
On homology, more preferably at least 95% or more homology, most preferably at least 99% or more homology.
Similarly, the present invention also provides the gene orders of coding amino acid sequence as shown in SEQ ID NO.2, with SEQ
Nucleotide sequence shown in ID NO.1 is consistent；The present invention also provides in nucleotide sequence shown in SEQ ID NO.1 remove 424-
438, other nucleotide outside 712-714 and 802-804 nucleotide are replaced, add and/or lack one or several cores
Thuja acid can retain the mutant gene of esterase DMWf18-558 biological activity of albumen to obtain coding substantially.Preferred esterase
DMWf18-558 mutant gene has the homology at least with 90% or more nucleotide sequence shown in SEQ ID NO:1, more
It is preferred that most preferably there is at least 99% or more homology at least 95% or more homology.
Using gene clone technology, the esterase DMWf18-558 gene being cloned into can be connected on suitable carrier, and
Convert or be transfected into prokaryotes or eucaryote host expresses preparation and reorganization esterase DMWf18-558.Suitable prokaryotes place
Main includes various bacteriums such as E.coli etc., and suitable eucaryote host includes yeast (such as methanol yeast) and mammalian cell
(such as Chinese hamster ovary cell), it is preferred to use prokaryotic expression system E.coli.
The protokaryon or eukaryotic expression for the various commercially viable purchases that suitable carrier is well known to those skilled in the art carry
Body, prokaryotic expression carrier such as pET serial carrier, pQE serial carrier；Yeast expression carrier pPICZ- α-A, pHIL-D2, pPIC9,
pHIL-S1(Invitrogen Corp.San Diego.California.USA)；Animal cell expression vectors pSVK3, pMSG
(Amersham Pharmacia Biotech Inc.USA) etc..One preferred example is that the present invention is screened carboxy-lesterase
The encoding gene of DMWf18-558 is connected on coli expression carrier pET28a (Novagen), and is transformed into Escherichia coli
In Rosetta (DE3), the Recombinant esterase of high activity is gone out through inducing expression.
The present invention also provides carboxy-lesterase DMWf18-558 or the host strain that can express carboxy-lesterase DMWf18-558 to exist
Industrial application, such as can be used for being catalyzed ester-type hydrolysis.By esterase activity measurement show carboxy-lesterase DMWf18-558 or
The above-mentioned host strain that can express carboxy-lesterase DMWf18-558 can be used for hydrolyzing short-chain aliphatic ester, such as C2-C8 short carbon chain rouge
Fat acid esters.Preferred short-chain aliphatic ester is the p-nitrophenyl phenolic ester with C2-C8 short carbon chain, such as p-nitrophenol acetic acid
Ester, p-nitrophenol butyrate, p-nitrophenol capronate or p-nitrophenol caprylate etc., wherein substrate is p-nitrophenol
Catalytic activity highest when capronate (C6), enzyme activity reach 367U/mg.
Carboxy-lesterase DMWf18-558 catalyzing hydrolysis temperature range is 15~40 DEG C, preferably 15~35 DEG C；The hydrolysis
PH value be 3.0~10.0, preferably 6.5~9.0.In 20 DEG C or less heat preservation 1h, 70% or more remnant enzyme activity can be kept；?
1h is kept the temperature in 30 DEG C, 50% or more remnant enzyme activity can be kept.Novel carboxylic acid esterase and its encoding gene provided by the invention exist
Medicine preparation, food processing and flavor improvement, wastewater treatment, washing industry have important application potential.
Detailed description of the invention
Fig. 1 is the policapram gel electrophoresis analysis figure of purification of carboxylic acids esterase DMWf18-558.
Fig. 2 is the substrate specificity figure of carboxy-lesterase DMWf18-558.C2: p-nitrophenol acetic acid esters；C4: p-nitrophenyl
Oxyphenisatin acid esters, C6: p-nitrophenol capronate；C8: p-nitrophenol caprylate；C10: p-nitrophenol decylate；C12: right
Nitrophenol dodecanoate；C14: p-nitrophenol myristinate；C16: p-nitrophenol Palmitate；When definition substrate is C6
Measured value is 100%.
Fig. 3 is carboxy-lesterase DMWf18-558 optimal reactive temperature figure.
Fig. 4 is carboxy-lesterase DMWf18-558 optimal reaction pH figure.
Fig. 5 is carboxy-lesterase DMWf18-558 optimal reaction NaCl concentration figure.
Fig. 6 is bivalent cation to carboxy-lesterase DMWf18-558 activity influence figure.
Fig. 7 is organic solvent and detergent to carboxy-lesterase DMWf18-558 activity influence figure.
Fig. 8 is the temperature stability figure of carboxy-lesterase DMWf18-558.
Specific embodiment
The acquisition of 1 carboxy-lesterase DMWf18-558 encoding gene of embodiment
Halmeic deposit sample is acquired by the visual multitube sampler in deep-sea from Pacific seamounts edge.Macro genomic library structure
It builds using CopyControlTM HTP fosmid library production kit(Epicentre
Biotechnologies, the U.S.), host strain is E.coli EPI300 (Epicentre Biotechnologies, beauty
State), carrier is pCC2FOS fosmid vector (Epicentre Biotechnologies, the U.S.).Through pulsed field gel electrophoresis
Detection, Insert Fragment size are 36~48kb.It takes 10 library μ l bacterium solutions to be diluted to 100 μ l, is coated on carboxy-lesterase screening flat board,
30 DEG C are cultivated 2 days.Culture medium prescription is LB culture medium (10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH
7.2)；After sterilizing, chloramphenicol is added under aseptic condition, makes its final concentration of 12.5 μ g ml-1。
Picking monoclonal is simultaneously packaged, and is packaged library to fosmid and is sequenced, and open reading frame is predicted after splicing and is annotated
Gene therefrom screens lipid hydrolyzing enzyme related gene.Compared by Blastx (http://blast.ncbi.nlm.nih.gov/)
To the homology of known esterase gene sequence in sequence and database.Analysis, which is compared, through database obtains DMWf18-558 coding base
Cause, size 909bp, base composition are as follows: 141A (15.51%), 146T (16.06%), 331C (36.41%) and 291G
(32.01%), nucleotide sequence is as shown in SEQ ID No:1.Coding albumen size is 302 amino acid residues, amino
Acid sequence is as shown in SEQ ID No:2.The amino acid sequence is subjected to Homology search, similar property highest in GenBank
Carboxy-lesterase be also macro genome, similitude 82%, the number of registration in GenBank database is AFB82690.
Phylogenetic Analysis the result shows that, carboxy-lesterase DMWf18-558 belongs to the Section IV man in hydrolysis of lipid enzyme family
Race.Speculated according to amino acid sequence, the catalytic center of carboxy-lesterase DMWf18-558 is by serine, glutamic acid and histidine (ammonia
Base acid position is 144,238 and 268) forms that wherein serine is located at glycine, aspartic acid, serine, alanine and sweet ammonia
In the conservative region of acid (amino acid position is 142 to 146) composition, therefore carboxy-lesterase DMWf18-558 belongs to Section IV family
GDSAG subfamily.And oxonium ion hole is located at 75 and 76 two glycine.
In conclusion DMWf18-558 should be a newcomer in lipid hydrolyzing enzyme Section IV family.
The building of the recombinant expression plasmid and recombinant bacterial strain of 2 carboxy-lesterase DMWf18-558 of embodiment
The carboxy-lesterase DMWf18-558 encoding gene that the present invention obtains is cloned on expression vector, building recombinant expression
Bacterial strain.The open reading frame sequence for the esterase gene that ORF analysis based on NCBIORF Finder obtains, design amplification esterase are complete
The upstream primer 558F (5 '-TCGCGGATCCATGGCGAGTCCACAGCTCC-3 ', BamHI) and downstream primer 558R of gene
(5 '-ATTTGCGGCCGCCTAGCGTGCGGCGGC-3 ', XhoI), for being packaged, fosmid Library PCR amplification confirmation gene is complete
Long sequence.Expression plasmid is constructed using the method for enzyme cutting clone, that is, uses BamHI and XhoI double digestion PCR product, piece after purification
Duan Yujing BamHI is connected with the plasmid pET28a of XhoI double digestion, using CaCl2Conversion method is converted into E.coliDH5 α, card
That chloramphenicol resistance screening positive clone.The plasmid of positive colony, warp are extracted using plasmid extraction kit (Axygen, the U.S.)
The identification of BamHI and XhoI double digestion, obtains the DNA fragmentation of 1000bp or so, is accredited as carboxy-lesterase DMWf18-558 through sequencing
Encoding gene.Recombinant expression plasmid is transformed into E.coliRosetta (DE3) expression bacterial strain, building expression recombinant bacterial strain.
Embodiment 3 utilizes recombinant strains expression recombination carboxy-lesterase DMWf18-558
The 3ml recombinant strains built are transferred to 100ml contains 20 μ g/ml kanamycins and 34 μ g/ml chlorine are mould
In the LB liquid medium of element, 37 DEG C of shaken cultivations to OD600Reach 0.6, the IPTG that final concentration of 0.5mM is added is induced
Expression, is transferred to 20 DEG C with 150r/min shaken cultivation 8h.Low-temperature centrifugation collects thallus, is resuspended in (the 500mM chlorination of NTA-10 solution
Sodium, 10mM imidazoles, 20mM Tris hydrochloric acid, pH 8.0) in, ultrasonic disruption processing is carried out on ice.On low-temperature centrifugation is collected
Clearly, using NTA-Ni2+Affinity column chromatography purifying expression albumen.Expressed recombinant protein contains the 6 × His tag of N-terminal, affable
It is inhaled on column with layer is adsorbed onto, by the imidazole solution gradient elution of various concentration, collects eluent.It detects, obtains through SDS-PAGE
To electrophoretically pure recombination carboxy-lesterase DMWf18-558, molecular weight 36kDa or so (Fig. 1).Protein compression is measured with Lowry method
Degree, obtains the expression quantity of about 28.9mg/100ml fermentation liquid.
The Activity determination of the recombination of embodiment 4 carboxy-lesterase DMWf18-558
Utilize the carboxy-lesterase DMWf18-558 activity of p-nitrophenol caproic acid ester process measurement purifying.Concrete operations: 1ml is anti-
Answering includes the own phenols acids of 1mM p-nitrophenyl in system, 100mM Tricine buffer (pH 9.0) and 48ng pure enzyme protein (for
The diluted purifying enzyme solution of 10 μ l), using ultra-violet and visible spectrophotometer (Beckman DU800 type, the U.S.) in 25 DEG C of items
METHOD FOR CONTINUOUS DETERMINATION light absorption value A under part4052min uses the enzyme solution of inactivation as control for returning to zero.One enzyme activity unit is defined as
The required enzyme amount of l μm of ol p-nitrophenol is generated from p-nitrophenol ester catalysis per minute.The esterase active measured is
367U/mg。
Embodiment 5 recombinates the analysis of carboxy-lesterase DMWf18-558 substrate specificity
The substrate specificity analysis of carboxy-lesterase DMWf18-558 uses system: 100mM Tricine buffer (pH
9.0) 48ng pure enzyme protein, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C is added in, 1mM substrate4052min.Measure the substrate used are as follows:
P-nitrophenol acetic acid esters (C2), p-nitrophenol butyrate (C4), p-nitrophenol capronate (C6), p-nitrophenol octanoic acid
Ester (C8), p-nitrophenol decylate (C10), p-nitrophenol dodecanoate (C12), p-nitrophenol myristinate (C14)
With p-nitrophenol Palmitate (C16).Show that carboxy-lesterase DMWf18-558 is shorter to acyl group carbochain or longer after measured
P-nitrophenyl phenolic ester (C2, C4, C6, C8, C10, C12 and C14) has catalytic activity, and wherein substrate is p-nitrophenol caproate
(C6) catalytic activity highest when, it is more difficult to hydrolyze p-nitrophenol Palmitate (C16) (Fig. 2).The result shows that carboxy-lesterase
DMWf18-558 is to the shorter lipid material of acyl group carbochain catalytic activity with higher, and for the hydrolysis vigor of short-chain lipid
Better than long-chain lipid.
Embodiment 6 recombinates the analysis of carboxy-lesterase DMWf18-558 optimum reaction conditions
Carboxy-lesterase DMWf18-558 optimal reactive temperature measures within the scope of 15~60 DEG C.Concrete operations are as follows: 100mM
48ng pure enzyme protein is added in Tricine buffer (pH 9.0), 1mM p-nitrophenol capronate, respectively 15,20,25,30,
35, METHOD FOR CONTINUOUS DETERMINATION light absorption value A under the conditions of 40,45,50,55 and 60 DEG C4052min.Measurement result shows carboxy-lesterase DMWf18-
558 range of reaction temperature is 15~40 DEG C, 25 DEG C of optimal reactive temperature (Fig. 3).
Carboxy-lesterase DMWf18-558 optimal reaction pH is measured in 3.0~10.0 ranges.Concrete operations are as follows: in different pH
1mM p-nitrophenol capronate and 48ng pure enzyme protein, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C are added in buffer3482min。
Measure the buffer used are as follows: 100mM citric acid-sodium citrate buffer solution (pH 5.0~6.0), 100mM potassium dihydrogen phosphate-hydrogen
Sodium oxide molybdena buffer (pH 6.0~7.5), 100mM Tricine buffer (pH 7.5~9.0) and 100mM 2- cyclohexylamino
Ethanesulfonic acid-sodium hydrate buffer solution (pH 9.0~10.0).Measurement result show carboxy-lesterase DMWf18-558 pH 3.0~
Active in 10.0 ranges, optimal reaction pH is 9.0 (Fig. 4).
The NaCl concentration of carboxy-lesterase DMWf18-558 optimal reaction measures within the scope of 0.5~5M.Concrete operations are as follows:
48ng pure enzyme protein is added in 100mM Tricine buffer (pH 9.0), 1mM p-nitrophenol capronate, respectively 0.5,1,
2, under the conditions of 3,4 and 5M NaCl, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C4052min.Measurement result shows carboxy-lesterase
The NaCl reaction density range of DMWf18-558 is 0.5~3M, and optimal reaction concentration is 0.5M (Fig. 5).
Embodiment 7 recombinates carboxy-lesterase DMWf18-558 zymetology stability analysis
Measurement concrete operations of the bivalent cation to carboxy-lesterase DMWf18-558 activity influence are as follows: divide in the reaction system
It Jia Ru not 10mM Co2+、Cu2+、Ca2+、Mg2+、Zn2+、Sr2+、Mn2+、Ni2+、Ba2+With ethylenediamine tetra-acetic acid (EDTA), enzyme is measured
Activity.Survey enzyme activity system are as follows: 100mM Tricine buffer (pH 9.0), 1mM p-nitrophenol capronate, the pure enzyme egg of 48ng
It is white, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C4052min.Measurement result shows that added metal ion can inhibit carboxy-lesterase
The activity of DMWf18-558, but its activity remains at 40% or more, in Mg2+In the presence of relative activity keep 80% or more (figure
6)。
The measurement concrete operations of organic solvent and detergent to carboxy-lesterase DMWf18-558 activity influence are as follows: in reactant
15% (v/v) organic solvent (isopropanol, acetonitrile, ethyl alcohol, methanol, acetone, dimethyl sulfoxide and dimethyl methyl is separately added into system
Amide) and 1% detergent (w/v or v/v) (SDS, polysorbas20, Tween 80 and Triton X-100), measure the activity of enzyme.It surveys and lives
System are as follows: 100mM Tricine buffer (pH 9.0), 1mM p-nitrophenol capronate, 48ng pure enzyme protein, at 25 DEG C
METHOD FOR CONTINUOUS DETERMINATION light absorption value A4052min.Measurement result shows that SDS and Tween 80 can inhibit the work of carboxy-lesterase DMWf18-558
Property (relative activity < 10%), and under 20 existence condition of acetonitrile dimethyl sulfoxide and Tween, DMWf18-558 be still able to maintain compared with
Strongly active (> 50%；Fig. 7).
The active thermal stability determination concrete operations of carboxy-lesterase DMWf18-558 are as follows: enzyme is placed in 10,20,30,40,50
1h is kept the temperature at 60 DEG C, measures the activity of enzyme.Live body system are as follows: 100mM Tricine buffer (pH 9.0), 1mM is to nitro
Phenol capronate, 48ng pure enzyme protein, the METHOD FOR CONTINUOUS DETERMINATION light absorption value A at 25 DEG C4052min.Measurement result shows carboxy-lesterase
DMWf18-558 can keep 70% or more remnant enzyme activity in 20 DEG C or less heat preservation 1h；1h is kept the temperature in 30 DEG C, 50% can be kept
Above remnant enzyme activity (Fig. 8).
Claims (15)
1. a kind of carboxy-lesterase, amino acid sequence is consistent with sequence shown in Seq ID NO.2.
2. encoding the gene of carboxy-lesterase described in claim 1, nucleotide sequence is as shown in SEQ ID NO.1.
3. carrying the carrier of gene described in claim 2.
4. carrier according to claim 3, it is characterised in that: the carrier is selected from pET serial carrier, and pQE series carries
Body, Yeast expression carrier pPICZ- α-A, pHIL-D2, pPIC9, pHIL-S1, animal cell expression vectors pSVK3 or pMSG.
5. carrier according to claim 4, it is characterised in that: the carrier is coli expression carrier pET28a.
6. a kind of host, by the described in any item carriers of claim 3-5 are inverted or transfected prokaryotic is biological or eucaryote
Host obtains, and the host is bacterium, yeast or mammalian cell.
7. host according to claim 6 is E.coli bacterium, methanol yeast or Chinese hamster ovary cell.
8. host according to claim 7 is E.coli bacterium.
9. carboxy-lesterase described in claim 1 or the host as claimed in claim 6 that can express carboxy-lesterase are in catalysis C2-C8
Application in short chain fatty acids ester hydrolysis.
10. application according to claim 9, which is characterized in that the C2-C8 short-chain aliphatic ester is with C2-C8
The p-nitrophenyl phenolic ester of carbochain.
11. application according to claim 10, which is characterized in that the p-nitrophenyl phenolic ester with C2-C8 carbochain
For p-nitrophenol acetic acid esters, p-nitrophenol butyrate, p-nitrophenol capronate and p-nitrophenol caprylate.
12. according to the described in any item applications of claim 9-11, which is characterized in that the esterase catalyzed temperature range for hydrolysis
It is 15~40 DEG C.
13. application according to claim 12, which is characterized in that the esterase catalyzed temperature range for hydrolysis is 15~35
℃。
14. according to the described in any item applications of claim 9-11, which is characterized in that the pH value of the esterase catalyzed hydrolysis is
3.0~10.0.
15. application according to claim 14, which is characterized in that the esterase catalyzed hydrolysis pH value is 6.5~9.0.
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| CN105505898A (en) * | 2015-12-28 | 2016-04-20 | 国家海洋局第二海洋研究所 | Carboxylesterase sourcing from deep sea, and coding gene and application thereof |
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| Croceicoccus marinus gen. nov.,sp. nov.,a yellow-pigmented bacterium from deep-sea sediment,and emended description of the family Erythrobacteraceae;Xue-Wei Xu 等;《International Journal of Systematic and Evolutionary Microbiology》;20091231(第59期);全文 |
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