CN106834386A - 一种几丁二糖的生产方法 - Google Patents

一种几丁二糖的生产方法 Download PDF

Info

Publication number
CN106834386A
CN106834386A CN201510883057.5A CN201510883057A CN106834386A CN 106834386 A CN106834386 A CN 106834386A CN 201510883057 A CN201510883057 A CN 201510883057A CN 106834386 A CN106834386 A CN 106834386A
Authority
CN
China
Prior art keywords
chitin
production method
chitobiose
monooxygenase
chitinase
Prior art date
Legal status (The legal status 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 status listed.)
Granted
Application number
CN201510883057.5A
Other languages
English (en)
Other versions
CN106834386B (zh
Inventor
赵勇
王文霞
尹恒
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian Institute of Chemical Physics of CAS
Original Assignee
Dalian Institute of Chemical Physics of CAS
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 Dalian Institute of Chemical Physics of CAS filed Critical Dalian Institute of Chemical Physics of CAS
Priority to CN201510883057.5A priority Critical patent/CN106834386B/zh
Publication of CN106834386A publication Critical patent/CN106834386A/zh
Application granted granted Critical
Publication of CN106834386B publication Critical patent/CN106834386B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

本发明公开了一种以几丁质为底物生产几丁二糖的方法，即通过一种来自苏云金芽孢杆菌的多糖裂解单加氧酶(BtLPMO10A)与几丁质酶的协同作用，将几丁质转化为几丁二糖。

Description

一种几丁二糖的生产方法
技术领域
本发明涉及一种以几丁质为底物生产几丁二糖的方法。
背景技术
几丁质是一种主要来源于海洋虾蟹壳、真菌细胞壁、昆虫外壳等的难溶性天然多糖，几丁质经过降解可得到水溶性的几丁二糖，几丁二糖可作为一种合成前体，在生物医药、绿色农业和材料化工等领域具有广泛的应用。
目前几丁二糖的制备主要采用生物酶降解的方法或酸水解的方法。由于生物酶解具有反应条件温和、反应过程可控等优点，近几年来生物酶法生产几丁二糖已成为趋势。自然界中存在大量的几丁质酶，主要来源于植物、微生物以及昆虫等，从几丁质酶的催化反应模式上又可分为三类，包括内切型几丁质酶、外切型几丁质酶和N-乙酰氨基葡萄糖苷酶，其中内切型几丁质酶主要作用是将几丁质随机切割成不同聚合度的几丁寡糖，外切型几丁质酶主要是将几丁质按照一定的顺序切割成几丁二糖，而N-乙酰氨基葡萄糖苷酶则是把几丁二糖转化为两个N-乙酰氨基葡萄糖。
虽然到目前为止已有大量的关于这些几丁质酶的报道，由于几丁质具有非常致密的晶体结构，特别是来源于虾蟹壳的几丁质，其内部分子层与层之间，链与链之间存在大量的氢键，使得生物酶在降解几丁质时效率非常低，这在很大程度上限制了几丁二糖的生产与应用。
发明内容
针对上述问题，本发明提供了一种以几丁质为底物生产几丁二糖的方法，即利用多糖裂解单加氧酶和外切型几丁质酶相结合，协同高效降解几丁质，生产几丁二糖的方法。其中，多糖裂解单加氧酶是一类新发现的酶，该酶可作用于晶体几丁质的表面，破坏几丁质的表面结构，从而提高几丁质酶对几丁质的酶解效率。
本发明提供的技术方案包括：
将几丁质与溶剂混合，几丁质的浓度为0.5-2g/100ml，所用的溶剂包括磷酸缓冲液、Tris-HCl缓冲液或醋酸缓冲液中一种。
将来自苏云金芽孢杆菌的多糖裂解单加氧酶与来自粘质沙雷氏菌的外切型几丁质酶添加到几丁质与溶剂的混合体系中，对几丁质进行酶催化降解反应，反应温度为20-40℃，处理持续时间为8-24h，反应体系中需要添加0.5-2mM的还原剂。
附图说明
图1A.利用多糖裂解单加氧酶和几丁质酶协同降解虾蟹壳来源的几丁质。
图1B.利用多糖裂解单加氧酶和几丁质酶协同降解鱿鱼来源的几丁质。
具体实施方式
实施例1利用多糖裂解单加氧酶(BtLPMO10A)和几丁质酶(SmChiA)协同降解不同来源的几丁质
1)虾蟹壳来源的几丁质。如图1A所示，两组反应均在1.0ml的几丁质-磷酸缓冲液(20mM，pH＝6.0)中进行，其中一组只添加5.0μM几丁质酶(黑色方块表示)，另一组同时添加了5.0μM几丁质酶、1.0μM的多糖裂解单加氧酶和1.0mM的抗坏血酸(黑色三角表示)，反应在37℃振荡条件下进行，分别在0,1,2,3,4,5,8,24h取样，样品中几丁二糖的含量通过HPLC的方法进行定量分析。结果如图1A所示，经过24h酶解，不含多糖裂解单加氧酶的一组几丁二糖的终浓度为8.1μg/ml,而含有多糖裂解单加氧酶的一组几丁二糖的终浓度为92μg/ml,即通过添加多糖裂解单加氧酶，可将虾蟹壳来源的几丁质的酶水解效率提高10倍以上。
2)鱿鱼来源的几丁质。如图1B所示，两组反应均在1.0ml的几丁质-磷酸缓冲液(20mM，pH＝6.0)中进行，其中一组只添加5.0μM几丁质酶(黑色方块表示)，另一组同时添加了5.0μM几丁质酶、1.0μM的多糖裂解单加氧酶和1.0mM的抗坏血酸(黑色三角表示)，反应在37℃振荡条件下进行，分别在0,0.5,1,2,3,4,5,8h取样，样品中几丁二糖的含量通过HPLC的方法进行定量分析。结果如图1B所示，几丁二糖的生成量随之酶解时间的延长而不断增加。经过24h酶解，不含多糖裂解单加氧酶的一组几丁二糖的终浓度为23.5μg/ml,而含有多糖裂解单加氧酶的一组几丁二糖的终浓度为73μg/ml,即通过添加多糖裂解单加氧酶，可将鱿鱼来源的几丁质的酶水解效率提高3.1倍。

Claims (6)

1.一种几丁二糖的生产方法，其特征在于：
利用一种多糖裂解单加氧酶与几丁质酶的协同作用，将几丁质转化为几丁二糖。
2.按照权利要求1所述的生产方法，其特征在于：几丁质来自于虾蟹壳、真菌细胞壁、昆虫外壳和鱿鱼中的一种或二种以上。
3.按照权利要求1所述的生产方法，其特征在于：反应于溶剂中进行，溶剂为磷酸盐缓冲液、醋酸盐缓冲液、Tris-HCl缓冲液中的一种。
4.按照权利要求3所述的生产方法，其特征在于：几丁质与溶剂的质量体积比0.5-2g/100ml。
5.按照权利要求1所述的生产方法，其特征在于：几丁质酶为来自于粘质沙雷氏菌中的外切型几丁质酶(SmChiA)；多糖裂解单加氧酶为来自于苏云金芽孢杆菌的多糖裂解单加氧酶(BtLPMO10A)；BtLPMO10A用量为1-5μM/g几丁质；SmChiA用量为1-5μM/g几丁质。
6.按照权利要求1所述的生产方法，其特征在于：酶处理体系中含0.5-2.0mM的抗坏血酸，在20-40℃，振荡条件下反应8-24h。
CN201510883057.5A 2015-12-04 2015-12-04 一种几丁二糖的生产方法 Active CN106834386B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510883057.5A CN106834386B (zh) 2015-12-04 2015-12-04 一种几丁二糖的生产方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510883057.5A CN106834386B (zh) 2015-12-04 2015-12-04 一种几丁二糖的生产方法

Publications (2)

Publication Number Publication Date
CN106834386A true CN106834386A (zh) 2017-06-13
CN106834386B CN106834386B (zh) 2021-03-02

Family

ID=59148834

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510883057.5A Active CN106834386B (zh) 2015-12-04 2015-12-04 一种几丁二糖的生产方法

Country Status (1)

Country Link
CN (1) CN106834386B (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1847267A (zh) * 2006-05-10 2006-10-18 国家***第三海洋研究所 几丁寡糖制备方法
CN102286414A (zh) * 2011-09-02 2011-12-21 海南大学 一株几丁质降解菌株及其制备几丁寡糖的方法
CN102573937A (zh) * 2009-10-07 2012-07-11 株式会社耐热性酵素研究所 含有几丁质或壳聚糖的可降解复合材料
CN104762307A (zh) * 2015-04-30 2015-07-08 大连大学 几丁质酶基因chiC及其编码蛋白和应用
WO2015165953A1 (en) * 2014-04-30 2015-11-05 Dsm Ip Assets B.V. Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1847267A (zh) * 2006-05-10 2006-10-18 国家***第三海洋研究所 几丁寡糖制备方法
CN102573937A (zh) * 2009-10-07 2012-07-11 株式会社耐热性酵素研究所 含有几丁质或壳聚糖的可降解复合材料
CN102286414A (zh) * 2011-09-02 2011-12-21 海南大学 一株几丁质降解菌株及其制备几丁寡糖的方法
WO2015165953A1 (en) * 2014-04-30 2015-11-05 Dsm Ip Assets B.V. Process for enzymatic hydrolysis of lignocellulosic material and fermentation of sugars
CN104762307A (zh) * 2015-04-30 2015-07-08 大连大学 几丁质酶基因chiC及其编码蛋白和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DAFN I K. PAS PALIARI 等: "Listeria mono cytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase", 《FEBS J.》 *
HUIYAN ZHANG 等: "Expression and characterization of a lytic polysaccharide monooxygenase from Bacillus thuringiensis", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 *
任春梅 等: "细菌几丁质酶基因转化番茄的研究", 《湖南农业大学学报》 *
施贤卫 等: "Actinosynnema mirum DSM43827 溶解性多糖单加氧酶的异源表达和酶学性质表征", 《中国生物工程杂志》 *

Also Published As

Publication number Publication date
CN106834386B (zh) 2021-03-02

Similar Documents

Publication Publication Date Title
Chauhan et al. Bacterial carrageenases: an overview of production and biotechnological applications
Zhu et al. Insight into carrageenases: major review of sources, category, property, purification method, structure, and applications
Arnold et al. Enzymatic modification of native chitin and conversion to specialty chemical products
Garron et al. Uronic polysaccharide degrading enzymes
Villa-Lerma et al. Ultrasonication and steam-explosion as chitin pretreatments for chitin oligosaccharide production by chitinases of Lecanicillium lecanii
Yu et al. Core-shell structured quorum quenching beads for more sustainable anti-biofouling in membrane bioreactors
TW200801195A (en) Treatment of cellulosic material and enzymes useful therein
CN104480161A (zh) 一种超微粉碎辅助酶法制备小麦麸皮低聚糖的方法
Tantayotai et al. Effect of cellulase-producing microbial consortium on biogas production from lignocellulosic biomass
Yuan et al. Enzymatic production of specifically distributed hyaluronan oligosaccharides
Fu et al. Bioconversion of chitin waste using a cold-adapted chitinase to produce chitin oligosaccharides
CN105218248A (zh) 一种生防活性液体海藻肥的制备方法
Doan et al. Production of thermophilic chitinase by Paenibacillus sp. TKU052 by bioprocessing of chitinous fishery wastes and its application in N-acetyl-d-glucosamine production
Doan et al. Microbial conversion of shrimp heads to proteases and chitin as an effective dye adsorbent
Nakagawa et al. Analysis of four chitin-active lytic polysaccharide monooxygenases from Streptomyces griseus reveals functional variation
CN101475969B (zh) 木瓜蛋白酶降解壳聚糖的方法
Hu et al. Current status of mining, modification, and application of cellulases in bioactive substance extraction
Song et al. Efficient digestion of chitosan using chitosanase immobilized on silica-gel for the production of multisize chitooligosaccharides
Allardyce et al. Purification and characterisation of endo-β-1, 4-glucanase and laminarinase enzymes from the gecarcinid land crab Gecarcoidea natalis and the aquatic crayfish Cherax destructor
Duarte et al. Use of residual biomass from the textile industry as carbon source for production of a low-molecular-weight xylanase from Aspergillus oryzae
Li et al. Co-immobilization of β-xylosidase and endoxylanase on zirconium based metal–organic frameworks for improving xylosidase activity at high temperature and in acetone
CN106834386A (zh) 一种几丁二糖的生产方法
Torpenholt et al. Activity of three β-1, 4-galactanases on small chromogenic substrates
CN102286414B (zh) 一株几丁质降解菌株及其制备几丁寡糖的方法
Zhu et al. Application of carboxylated multi-walled carbon nanotubes in bacteria-microalgae-fungi consortium for efficient antibiotics removal

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant