CN1206344C - Method for producing fucan sulfatase by means of bacteria - Google Patents
Method for producing fucan sulfatase by means of bacteria Download PDFInfo
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- CN1206344C CN1206344C CN 03138866 CN03138866A CN1206344C CN 1206344 C CN1206344 C CN 1206344C CN 03138866 CN03138866 CN 03138866 CN 03138866 A CN03138866 A CN 03138866A CN 1206344 C CN1206344 C CN 1206344C
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- bacterial strain
- enzyme
- sulfatase
- producing
- bacteria
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- 241000894006 Bacteria Species 0.000 title claims abstract description 9
- 102000005262 Sulfatase Human genes 0.000 title claims abstract description 9
- 108060007951 sulfatase Proteins 0.000 title claims abstract description 9
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 7
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 229920000855 Fucoidan Polymers 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 5
- 241000607291 Vibrio fluvialis Species 0.000 claims abstract description 4
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 3
- 241001474374 Blennius Species 0.000 claims description 3
- 239000013049 sediment Substances 0.000 claims description 2
- 238000004108 freeze drying Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 8
- 230000001580 bacterial effect Effects 0.000 abstract description 7
- 238000000034 method Methods 0.000 abstract description 7
- 239000002244 precipitate Substances 0.000 abstract description 4
- 150000002016 disaccharides Chemical class 0.000 abstract description 3
- 238000000855 fermentation Methods 0.000 abstract description 3
- 230000004151 fermentation Effects 0.000 abstract description 3
- 150000002772 monosaccharides Chemical class 0.000 abstract description 3
- 229920001542 oligosaccharide Polymers 0.000 abstract description 3
- 241000195493 Cryptophyta Species 0.000 abstract description 2
- 235000016709 nutrition Nutrition 0.000 abstract description 2
- 239000000047 product Substances 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 abstract 2
- 230000000593 degrading effect Effects 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 230000008014 freezing Effects 0.000 abstract 1
- 238000007710 freezing Methods 0.000 abstract 1
- 150000002482 oligosaccharides Chemical class 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 241000607598 Vibrio Species 0.000 description 4
- 238000009776 industrial production Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- -1 sulfate oligosaccharides Chemical class 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 239000005955 Ferric phosphate Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 102000012086 alpha-L-Fucosidase Human genes 0.000 description 1
- 108010061314 alpha-L-Fucosidase Proteins 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940032958 ferric phosphate Drugs 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- WBJZTOZJJYAKHQ-UHFFFAOYSA-K iron(3+) phosphate Chemical compound [Fe+3].[O-]P([O-])([O-])=O WBJZTOZJJYAKHQ-UHFFFAOYSA-K 0.000 description 1
- 229910000399 iron(III) phosphate Inorganic materials 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
技术领域technical field
本发明涉及一种用细菌生产岩藻聚糖硫酸酯酶的方法。The invention relates to a method for producing fucoidan sulfatase by bacteria.
背景技术Background technique
据本发明人所知，岩藻聚糖硫酸酯酶在制备具有不同分子量的岩藻聚糖硫酸酯低聚糖、双糖、单糖等方面显示出极其重要的作用，它对一些海洋药物的研制和工业化生产有决定性的意义。目前，已经有人用Vibrio sp，N-5得到外切型岩藻糖酶。另有人用Bacillus等其它微生物生产岩藻糖酶。但用这些菌种产酶时间长，产出的酶活性低，培养成分复杂，难适于工业化生产。As far as the inventors know, fucoidan sulfatase shows an extremely important effect in the preparation of fucoidan sulfate oligosaccharides, disaccharides, monosaccharides, etc. with different molecular weights. Development and industrial production have decisive significance. At present, some people have used Vibrio sp, N-5 to obtain exo-fucosidase. Others use Bacillus and other microorganisms to produce fucosidase. However, it takes a long time to produce enzymes with these strains, the enzyme activity of the output is low, and the culture components are complicated, so it is difficult to be suitable for industrial production.
发明内容Contents of the invention
本发明的目的是提供一种用河弧菌生产岩藻聚糖硫酸酯酶的方法，它能克服现有技术的上述缺点。The object of the present invention is to provide a method for producing fucoidan sulfatase by Vibrio riparianus, which can overcome the above-mentioned shortcomings of the prior art.
一种用细菌生产岩藻聚糖硫酸酯酶的方法，包括把细菌接入海藻粉发酵液中，在18-28℃的培养温度下培养18-24小时，然后投入提酶剂(NH4)2SO4，使产生的沉淀物下沉，再进行分离，最后冷冻干燥，其特征是所用的菌种为河弧菌Vibrio fluvialis。A method for producing fucoidan sulfatase by bacteria, comprising inserting bacteria into seaweed powder fermentation broth, cultivating at a culture temperature of 18-28°C for 18-24 hours, and then adding an enzyme extracting agent (NH 4 ) 2 SO 4 , to sink the produced sediment, then separate it, and finally freeze-dry it. It is characterized in that the strain used is Vibrio fluvialis.
本发明用的菌种具有营养要求范围广，容易培养和代时短的特点，特别是具有高的产酶活性，其发酵液酶活力为226U/ml。用本发明的方法生产的岩藻聚糖硫酸酯酶为内切和外切两类，降解岩藻聚糖硫酸酯可得到岩藻聚糖硫酸酯低聚糖、双糖、单糖。该酶产品的活性高，稳定性好，成本低，能够实现工业化生产。The bacterial species used in the present invention has the characteristics of wide range of nutritional requirements, easy cultivation and short generation time, especially high enzyme-producing activity, and the enzyme activity of the fermented liquid is 226U/ml. The fucoidan sulfatase produced by the method of the present invention is endocut and exocut, and the fucoidan sulfate can be degraded to obtain fucoidan sulfate oligosaccharides, disaccharides and monosaccharides. The enzyme product has high activity, good stability and low cost, and can realize industrial production.
具体实施方式Detailed ways
本发明用的河弧菌 Vibrio fluvialis菌株为弧形，端生单鞭毛，G-，菌落圆形，乳白色。培养该菌采用的培养基为海水细菌培养基2216E，培养基的组分为酵母膏1克，蛋白胨5克，磷酸高铁0.01克，琼脂15克，陈海水1000ml，其pH为7.6-7.8。本发明所用的产酶培养基为上述培养基，另外又加入0.8-1.5％(按重量，以下同)的海藻粉，培养20小时即可得到酶液。上述河弧菌的保藏单位为中国典型培养物保藏中心，简称CCTCC，地址：中国武汉、武汉大学，保藏日期为2000年6月5日。保藏号为M200015。The strain of Vibrio fluvialis used in the present invention is arc-shaped, end-growth single flagella, G- , colonies are round and milky white. The medium used for cultivating the bacteria is seawater bacterial medium 2216E, and the components of the medium are 1 gram of yeast extract, 5 grams of peptone, 0.01 gram of ferric phosphate, 15 grams of agar, 1000 ml of aged seawater, and its pH is 7.6-7.8. The enzyme-producing medium used in the present invention is the above-mentioned medium, and in addition, 0.8-1.5% (by weight, the same below) of seaweed powder is added, and the enzyme liquid can be obtained by culturing for 20 hours. The depository unit of the above-mentioned Vibrio river is China Center for Type Culture Collection, referred to as CCTCC, address: Wuhan, China, Wuhan University, and the date of deposit is June 5, 2000. The deposit number is M200015.
将河弧菌用上述2216E培养基活化后，以5％的接种量接入装有100ml含有1％藻粉培养基的500ml三角瓶中，26℃摇瓶培养18-24h后，将该培养液用冷冻离心机以4000rpm离心10分钟去除菌体，获得发酵酶液。将该酶液于4℃冰箱中加入(NH4)2SO4至80％的饱和度，4℃下沉淀24h，冷冻离心收集沉淀，该沉淀经冷冻干燥后制成粗酶制剂。所述的培养温度为18-28℃。After activating Vibrio riverina with the above-mentioned 2216E medium, insert 5% of the inoculum into a 500ml Erlenmeyer flask containing 100ml of 1% algae powder medium, and shake the flask at 26°C for 18-24h. Use a refrigerated centrifuge to centrifuge at 4000rpm for 10 minutes to remove the bacteria, and obtain the fermented enzyme liquid. Add (NH 4 ) 2 SO 4 to the enzyme liquid in a refrigerator at 4°C to a saturation of 80%, precipitate at 4°C for 24 hours, collect the precipitate by refrigerated centrifugation, and freeze-dry the precipitate to prepare a crude enzyme preparation. The culture temperature is 18-28°C.
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| CN 03138866 CN1206344C (en) | 2003-07-28 | 2003-07-28 | Method for producing fucan sulfatase by means of bacteria |
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| CN 03138866 CN1206344C (en) | 2003-07-28 | 2003-07-28 | Method for producing fucan sulfatase by means of bacteria |
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| CN1483814A CN1483814A (en) | 2004-03-24 |
| CN1206344C true CN1206344C (en) | 2005-06-15 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988109A (en) * | 2017-12-21 | 2018-05-04 | 青岛农业大学 | A kind of Flavobacterium mutant strain and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045512B (en) * | 2012-12-19 | 2014-07-16 | 青岛农业大学 | Flavobacterium and application thereof |
| CN103114063B (en) * | 2013-02-03 | 2014-03-19 | 中国海洋大学 | Strain for producing fucosan sulfatase and application thereof |
| IL312865B2 (en) | 2013-09-11 | 2025-06-01 | Eagle Biologics Inc | Liquid protein formulations containing viscosity-reducing agents |
| CN103834593B (en) * | 2014-03-05 | 2016-02-17 | 青岛农业大学 | A kind of secondary coccus and application thereof |
| CN106999510B (en) * | 2014-10-01 | 2021-04-30 | 伊格尔生物制品有限公司 | Polysaccharide and nucleic acid formulations containing viscosity reducing agents |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107988109A (en) * | 2017-12-21 | 2018-05-04 | 青岛农业大学 | A kind of Flavobacterium mutant strain and its application |
| CN107988109B (en) * | 2017-12-21 | 2019-12-10 | 青岛农业大学 | A kind of Flavobacterium mutant strain and its application |
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| CN1483814A (en) | 2004-03-24 |
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