CN106834386A - 一种几丁二糖的生产方法 - Google Patents
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- QLTSDROPCWIKKY-PMCTYKHCSA-N beta-D-glucosaminyl-(1->4)-beta-D-glucosamine Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O)[C@@H](CO)O1 QLTSDROPCWIKKY-PMCTYKHCSA-N 0.000 title claims abstract description 20
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 13
- 229920002101 Chitin Polymers 0.000 claims abstract description 31
- 102000012286 Chitinases Human genes 0.000 claims abstract description 19
- 108010022172 Chitinases Proteins 0.000 claims abstract description 19
- 102000008109 Mixed Function Oxygenases Human genes 0.000 claims abstract description 18
- 108010074633 Mixed Function Oxygenases Proteins 0.000 claims abstract description 18
- 150000004676 glycans Chemical class 0.000 claims abstract description 11
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 11
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- 241000193388 Bacillus thuringiensis Species 0.000 claims abstract description 3
- 229940097012 bacillus thuringiensis Drugs 0.000 claims abstract description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 6
- 241000238557 Decapoda Species 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 241000238366 Cephalopoda Species 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 241000238631 Hexapoda Species 0.000 claims description 3
- 229960005070 ascorbic acid Drugs 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 3
- 239000011668 ascorbic acid Substances 0.000 claims description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 241000607715 Serratia marcescens Species 0.000 claims description 2
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- 238000006731 degradation reaction Methods 0.000 description 4
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- 230000015556 catabolic process Effects 0.000 description 3
- 230000002195 synergetic effect Effects 0.000 description 3
- 101000588395 Bacillus subtilis (strain 168) Beta-hexosaminidase Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
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- MJBPUQUGJNAPAZ-AWEZNQCLSA-N butin Chemical compound C1([C@@H]2CC(=O)C3=CC=C(C=C3O2)O)=CC=C(O)C(O)=C1 MJBPUQUGJNAPAZ-AWEZNQCLSA-N 0.000 description 2
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- MJBPUQUGJNAPAZ-UHFFFAOYSA-N Butine Natural products O1C2=CC(O)=CC=C2C(=O)CC1C1=CC=C(O)C(O)=C1 MJBPUQUGJNAPAZ-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
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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
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。
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Citations (5)
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| CN1847267A (zh) * | 2006-05-10 | 2006-10-18 | 国家海洋局第三海洋研究所 | 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 |
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| CN1847267A (zh) * | 2006-05-10 | 2006-10-18 | 国家海洋局第三海洋研究所 | 几丁寡糖制备方法 |
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