WO2001079475A1 - Phosphorylase de laminaribiose, procede de production de ce dernier et de laminarioligosaccharide au moyen de l'enzyme - Google Patents

Phosphorylase de laminaribiose, procede de production de ce dernier et de laminarioligosaccharide au moyen de l'enzyme Download PDF

Info

Publication number
WO2001079475A1
WO2001079475A1 PCT/JP2001/003253 JP0103253W WO0179475A1 WO 2001079475 A1 WO2001079475 A1 WO 2001079475A1 JP 0103253 W JP0103253 W JP 0103253W WO 0179475 A1 WO0179475 A1 WO 0179475A1
Authority
WO
WIPO (PCT)
Prior art keywords
glucose
producing
laminaribiose
phosphorylase
laminari
Prior art date
Application number
PCT/JP2001/003253
Other languages
English (en)
Japanese (ja)
Inventor
Kouhei Yamada
Katsuyuki Okamoto
Shinsuke Miyoshi
Original Assignee
Showa Sangyo Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Showa Sangyo Co., Ltd. filed Critical Showa Sangyo Co., Ltd.
Priority to AU46925/01A priority Critical patent/AU4692501A/en
Publication of WO2001079475A1 publication Critical patent/WO2001079475A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01031—Laminaribiose phosphorylase (2.4.1.31)

Definitions

  • Laminariviose phosphorylase method for producing the same, and method for producing laminarioligosaccharide using the enzyme
  • the present invention relates to a novel laminaribiose phosphorylase. More specifically, the present invention relates to laminaribiose phosphorylase, a method for producing the same, and a method for producing laminarioligosaccharide.
  • Laminari-oligosaccharide is a generic name of oligosaccharides in which glucose is bound by several molecules to several tens of tens of ⁇ 3 bonds, and includes, for example, laminaribiose, laminaritriose, laminaritetraose and the like. Curdlan, pakiman, and the like are known as polysaccharides having 31 and 3 bonds, and among them, substances having anticancer activity are known. Therefore, various biological activities are also expected for laminari oligosaccharides, which are oligosaccharides having 31 and 3 bonds.
  • Laminari-oligosaccharides can be produced by methods such as acid decomposition of 1,3 glucan (Methods in Carbohydrate Chemistry, 1, 330-333, 1962), enzymatic decomposition (JP-A-59-162897), and high-concentration glucose.
  • a method of performing a condensation reaction using a solution and using / 3 dal cosidase is known.
  • the method based on the acid decomposition of 131,3 glucan has the disadvantages that the raw material / 31,3 glucan is expensive, has poor handling due to its viscosity, and has a low reaction yield, making it difficult to make a special synthesis. Therefore, it cannot be used as a means for inexpensively producing laminari-oligosaccharides.
  • the condensation reaction with j3 dalcosidase Laminari-oligosaccharides cannot be produced efficiently, and genyo-oligosaccharides with i3 1,6-linkage are usually produced as the main component, which requires a high level of separation technology.
  • the present invention has been made to solve the above problems.
  • INDUSTRIAL APPLICABILITY The present invention provides a heat-resistant laminaribio-phosphorylase, which can produce laminari-oligosaccharide, which is a very expensive saccharide, at a low cost, and is used for foods and drinks.
  • substrate specificity producing laminari-oligosaccharides from ⁇ -glucose-1-phosphate and glucose
  • Laminaribiose phosphorylase having the formula: In a preferred embodiment, laminaribiose phosphorylase has the following additional properties:
  • Isoelectric point about 4.7 (isoelectric focusing);
  • the present invention further relates to a method for producing laminarioligosaccharide, comprising a step of reacting a fungus having the ability to produce laminaribiose or a treated product thereof with ct-glucose-monophosphate and glucose.
  • the processed material has the following characteristics:
  • Substrate specificity produces laminari-oligosaccharides from ⁇ -glucose-1-phosphate and glucose;
  • the laminaribiose phosphorylase further has the following characteristics:
  • Isoelectric point about 4.7 (isoelectric focusing);
  • the fungus is a microorganism belonging to the genus Bacillus.
  • the microorganism belonging to the genus Bacillus is Bacillus subtilis K2145, Bacillus' circulans IAM12462 (Bacillus circulans IA 12462), or Bacillus circulans IA12462.
  • This is Bacillus coagulans I AMI 194.
  • the present invention further relates to a method for producing laminaribiose phosphorylase, which comprises a step of culturing a fungus having the ability to produce laminaribiose, and a step of collecting laminaribiose phosphorylase from the culture.
  • FIG. 1 is a diagram showing the synthesis of laminari-oligosaccharide.
  • FIG. 2 is a diagram showing the optimum temperature of laminaribiose phosphorylase of the present invention.
  • FIG. 3 is a graph showing the optimum pH of laminaribiose phosphorylase of the present invention.
  • FIG. 4 is a diagram showing the temperature stability of laminaribiose phosphorylase of the present invention.
  • FIG. 5 is a diagram showing the pH stability of laminaribiose phosphorylase of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
  • the laminaribiose phosphorylase of the present invention has been collected for the first time from a fungus. It has been discovered for the first time that laminaribiose phosphorylase, which was previously known only in Euglena, is also produced in microorganisms.
  • a microorganism refers to a microorganism belonging to a bacterium, a deformed bacterium, a trichome, or a fungus in taxonomic terms.
  • the present inventors searched the soil for microorganisms that produce laminarioligosaccharides from glucose and ⁇ - glucose-1-phosphate, and obtained for the first time a novel microorganism that produces laminaribiose phosphorylase.
  • This microorganism was identified as a microorganism belonging to the genus Bacillus on the basis of its bacteriological properties. Named K2145 (Bacillus subtilis K2145), deposited at National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, 1-1-1, Tsukuba, Higashi, Ibaraki, Japan. Date: February 2, 2000).
  • Bacteriological properties of Bacillus subtilis strain K2145 are as follows.
  • Colony shape irregular, curly hair, convex, matte, cream
  • the laminaribiose phosphorylase of the present invention has an optimum temperature around 55 ° C., and is characterized in that laminarioligosaccharide is produced from ⁇ - glucose monoi-phosphate and glucose. It is characterized by higher heat resistance than conventional laminaribiose phosphorylase from Euglena.
  • Isoelectric point about 4.7 (isoelectric focusing).
  • the laminaribiose phosphorylase of the present invention can be obtained by the following method, for example, using Bacillus subtilis K2145 as an example.
  • Bacillus subtilis K2145 was replaced with fructose, glucose, laminaribiose, galactose, isomerized sugar, sucrose, maltose, xylose, sorbitol, mannitol, Various sugars such as starch, sugar alcohols, starch or dextrin, etc. as a carbon source, and organic nitrogen sources such as yeast extract, fish meat extract, peptone, amino acid, etc.
  • ammonium sulfate, ammonium chloride Aerobic conditions at 20-40 ° C, preferably around 30 ° C, in a medium supplemented with inorganic nitrogen such as ammonium, urea, ammonium nitrate, etc., and minerals as required
  • the culture is performed for 1 to 5 days, preferably 1 to 3 days.
  • the cells obtained by culturing can be used as they are or after washing to produce laminari-oligosaccharides.
  • the cells obtained by culturing can be further processed, and the resulting processed product can also be used for the production of lamina oligosaccharides.
  • the processed material is: 1) microbial cells immobilized by a method commonly used by those skilled in the art; 2) mechanical or enzymatic treatment of the microbial cells, or a surfactant Alternatively, those treated with an organic solvent or the like, or those immobilized on them; 3) Crushed microbial cells to remove crushed residues, or those immobilized on them; 4) 2) or 3 ), Or an enzyme obtained by further purifying the enzyme.
  • an enzyme obtained by further purifying the enzyme means commonly used by those skilled in the art are used alone or in an appropriate combination.
  • polyacrylamide for immobilization of the treated material, polyacrylamide, calcium alginate, carrageenan, an ion exchange carrier, chitosan beads, etc. are used.
  • the obtained microbial cells and processed products thereof are mixed with a reaction solution containing ct gnorecose-i-phosphate and glucose, and used for the production of laminarioligosaccharide.
  • a Glucose-1-phosphate and glucose are about 100 ::! ⁇ 1: It is preferable that they are contained at a ratio of 100.
  • the total amount of glucose and ⁇ - glucose-monophosphate is 1 to 75% by weight in the reaction solution. /. Preferably, it is included. Preferably, it is 5 to 50% by weight, more preferably 20 to 40% by weight.
  • ⁇ of the reaction solution is not particularly limited, it is preferably about 5.0 to 8.0, and more preferably ⁇ about 6.0 to 7.0.
  • Reaction temperatures range from about 40-65 ° C, preferably about 50-60 ° C, more preferably about 50-55 ° C. Considering industrial practicality, about 50-55 ° C, pH about 6.0-7.
  • the ⁇ -glucose-1-phosphate as a substrate used in the present invention can also be obtained from an oligosaccharide or polysaccharide having a ct — 1,4 bond.
  • Oligosaccharides or polysaccharides having a 1,4-linkage include, but are not limited to, amylose and starch. By reacting amylose, starch and the like with glucan phosphorylase and the like, glucose-1-monophosphate is produced. When starch is used, a glucose-11-phosphate can be produced by simultaneously treating a branching enzyme or an amylase with these enzymes and simultaneously reacting these enzymes with glucan phosphorylase.
  • a glucose _ 1 to be able to obtain a monophosphate
  • a glucose one 1 monophosphate derived from such a top Shikamono root vegetables and milk, such as Jiyagaimo Acids can also be used.
  • aGlucose-11-monophosphate and glucose may be added at the start of the laminari-oligosaccharide production reaction or may be added sequentially. By changing the concentration of both substrates or the timing of addition, a product containing a relatively large amount of a specific polymerization degree component can be obtained.
  • laminarioligosaccharides can be produced using oligosaccharides or polysaccharides having a-1,4 bonds and glucose as substrates.
  • the presence of glucan phosphorylase together with laminaribiose phosphorylase produces laminarioligosaccharide.
  • the reaction conditions at this time are not particularly limited, and the above reaction conditions are applied.
  • the obtained crude enzyme was appropriately diluted, and 200 ⁇ l thereof was added to 200% 2% laminaribiose solution 2001 dissolved in 20 mM phosphate buffer (pH 7.0), and the enzyme reaction was carried out at 55 ° C. Was. Next, the reaction solution was boiled for 10 minutes to stop the enzyme reaction, and the sugar composition was analyzed by HP LC. Under these conditions, the amount of the enzyme that degrades 1 // mol of laminaribiose per minute was defined as 1 unit. Laminaribiose phosphorylase activity of the crude enzyme solution was 0.17 units / ml.
  • the molecular weight of the purified enzyme by gel filtration chromatography was about 170 kDa, and the isoelectric point by isoelectric focusing was about 4.7.
  • Example 1 crude enzyme solutions of B. circulans (IAM12462) and B. coagulans (IAM1194) were prepared.
  • the crude enzyme solution (10 ⁇ l) was mixed with 2 OmM phosphate buffer ( ⁇ 6.0) 100 // 1 containing 2% laminaribiose and reacted at 55 ° C for 24 hours.
  • the reaction solution was analyzed by HP LC, and glucose-1 monophosphate was measured.
  • ⁇ -glucose-1-phosphate was detected in both the reaction solutions of B. circulans (IAM12462) and B. coagulans (IAM1194), and laminaribiose phosphorylase activity was observed.
  • the laminaribiose phosphorylase of the present invention has higher thermostability and wider action ⁇ ⁇ region than those known so far, and efficiently produces laminarioligosaccharides from glucose and hydarco-su-1-phosphate. .
  • the enzyme laminaribiose phosphorylase of the present invention can be produced from microorganisms derived from the genus Bacillus, which has high food safety. By using this enzyme, laminari-oligosaccharides can be produced from starchy material at low cost and in large quantities under industrial production conditions.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un laminarioligosaccharide obtenu par réaction d'un phosphorylase de laminaribiose thermotolérant obtenu en cultivant un micro-organisme appartenant à l'espèce Bacillus avec α-glucose-1-phosphate et glucose. Un laminarioligosaccharide peut également être obtenu en ajoutant du phosphorylase d'amidon et de glycane en tant que substitut pour α-glucose-1-phosphate. Il en résulte un procédé sûr et peu coûteux de production d'un laminarioligosaccharide.
PCT/JP2001/003253 2000-04-19 2001-04-16 Phosphorylase de laminaribiose, procede de production de ce dernier et de laminarioligosaccharide au moyen de l'enzyme WO2001079475A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46925/01A AU4692501A (en) 2000-04-19 2001-04-16 Laminaribiose phosphorylase, process for producing the same and process for producing laminarioligosaccharide by using the enzyme

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2000117421A JP2001299338A (ja) 2000-04-19 2000-04-19 ラミナリビオースホスホリラーゼ、その製造方法、並びに該酵素を用いるラミナリオリゴ糖の製造方法。
JP2000-117421 2000-04-19

Publications (1)

Publication Number Publication Date
WO2001079475A1 true WO2001079475A1 (fr) 2001-10-25

Family

ID=18628740

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/003253 WO2001079475A1 (fr) 2000-04-19 2001-04-16 Phosphorylase de laminaribiose, procede de production de ce dernier et de laminarioligosaccharide au moyen de l'enzyme

Country Status (3)

Country Link
JP (1) JP2001299338A (fr)
AU (1) AU4692501A (fr)
WO (1) WO2001079475A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2397557A4 (fr) 2009-02-11 2013-09-04 Univ Mie Procédé de fabrication d'un bêta-1,3-glucane
CN110819667A (zh) * 2018-08-08 2020-02-21 中国科学院天津工业生物技术研究所 一种淀粉转化制备昆布二糖的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0466092A (ja) * 1990-07-04 1992-03-02 Natl Food Res Inst ラミナリオリゴ糖の製造方法
JPH04370099A (ja) * 1991-06-18 1992-12-22 Natl Food Res Inst ラミナリビオースの製造方法
JPH06343484A (ja) * 1993-06-10 1994-12-20 Nippon Petrochem Co Ltd ラミナリオリゴ糖の製造方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0466092A (ja) * 1990-07-04 1992-03-02 Natl Food Res Inst ラミナリオリゴ糖の製造方法
JPH04370099A (ja) * 1991-06-18 1992-12-22 Natl Food Res Inst ラミナリビオースの製造方法
JPH06343484A (ja) * 1993-06-10 1994-12-20 Nippon Petrochem Co Ltd ラミナリオリゴ糖の製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M. KITAOKA ET AL.: "Purification and properties of laminaribiose phosphorylase(EC 2.4.1.31) from euglena gracilis Z", ARCH. BIOCHEM. BIOPHYS., vol. 304, no. 2, 1993, pages 508 - 514, XP002941798 *

Also Published As

Publication number Publication date
AU4692501A (en) 2001-10-30
JP2001299338A (ja) 2001-10-30

Similar Documents

Publication Publication Date Title
CA1199292A (fr) Enzyme pour l'hydrolyse des amidons, preparation et utilisation
WO1991018974A1 (fr) HEMICELLULASE ACTIVE A DES VALEURS EXTREMES DE pH ET DE TEMPERATURE AINSI QUE SES MOYENS DE PRODUCTION
CN106068327A (zh) 使用α‑葡糖苷酶来酶水解二糖和低聚糖
WO2001090335A1 (fr) Nouvelle cyclodextrine glucanotransferase, procede de production de celle-ci et procede de production de cyclodextrine au moyen de cette enzyme
JP2711095B2 (ja) ビフイドバクテリウム菌の増殖促進剤の製造法
KR100913587B1 (ko) 이눌린의 제조방법
Wang et al. Purification and characterization of dextranase from Penicillium cyclopium CICC-4022 and its degradation of dextran
Yoshida et al. Production and application of maltose phosphorylase and trehalose phosphorylase by a strain of Plesiomonas
JP4473402B2 (ja) デキストランの製造法
JP3559609B2 (ja) 組換え型酵素とその製造方法並びに用途
WO2001079475A1 (fr) Phosphorylase de laminaribiose, procede de production de ce dernier et de laminarioligosaccharide au moyen de l'enzyme
JP5314955B2 (ja) 新規微生物、イヌリナーゼ、イヌリン分解剤、イヌロオリゴ糖の製造方法及びイヌリナーゼの製造方法
US5861289A (en) Heat-resistant β-galactosyltransferase, its production process and its use
US5153128A (en) Heat-resistant β-galactosyltransferase, its production process and its use
JP3635133B2 (ja) トレハロースホスホリラーゼおよびその調製法
Chi et al. Characterization of two thermostable β-agarases from a newly isolated marine agarolytic bacterium, Vibrio sp. S1
JP2006223268A (ja) ガラクトシル２糖類の製造法
JP2004261132A (ja) プルラン分解酵素とその製造方法並びに用途
JP3920073B2 (ja) 新規α−１，２−マンノシダーゼ及び該酵素を用いたα−マンノシル糖化合物の製造法
WO1995034570A1 (fr) Processus de production de disaccharides et de nouveaux saccharides
Abdel-Aziz et al. Partial purification and some properties of the chitosanases produced by Bacillus alvei NRC-14
Ismail et al. Nutrition requirement for the production of Penicillium chrysogenum α-galactosidase and its potential for hydrolysis of raffinose family oligosaccharides
JP3870264B2 (ja) 果糖二糖類及び果糖二糖類合成酵素の製造法
JP4826824B2 (ja) オリゴ糖
US6355467B1 (en) Saccharification enzymes from hyperthermophilic bacteria and processes for their production

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA KR US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase