CN112708575B - 大洋中华微菌及在制备褐藻聚糖硫酸酯降解酶中的应用 - Google Patents
大洋中华微菌及在制备褐藻聚糖硫酸酯降解酶中的应用 Download PDFInfo
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Abstract
本发明公开一种大洋中华微菌（Sinomicrobium oceani）及在制备褐藻聚糖硫酸酯降解酶中的应用。本发明经分离、纯化、鉴定、优化筛选出一种大洋中华微菌（Sinomicrobium oceani），已保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC №18258。该菌株的菌液可提取用于降解褐藻聚糖硫酸酯的胞内和胞外酶且产酶活力高，达到1050U/mL，具有在工业生产中的应用价值。
Description
技术领域
本发明涉及一种菌及应用，尤其是一种大洋中华微菌（Sinomicrobium oceani，保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC №18258）及在制备褐藻聚糖硫酸酯降解酶中的应用。
背景技术
褐藻聚糖硫酸酯（FUC）具有包括抗氧化、降血脂、降血压、抗凝血及抗癌等多种生物活性，但因其分子量较大、结构复杂而不能被人体很好吸收，从而抑制了活性的发挥。可了更大程度地发挥褐藻聚糖硫酸酯的活性，人们采用将褐藻聚糖硫酸酯降解为利于人体吸收的分子量小的低聚糖。目前应用较多的降解方法主要包含化学法降解、物理法降解和酶解法降解等。
化学法降解主要是在反应体系中添加酸、碱等化学试剂导致糖苷键断裂，但是降解得到的糖结构具有损伤，对低聚糖或者寡糖的生物活性产生了负面影响；物理法降解主要是利用超声波、γ射线或微波等导致共价键发生断裂，但是最终降解产物与多糖的分子结构、作用时间和操作环境等多种因素有关，重复性较差；酶法降解主要是利用糖苷键酶使糖苷键发生断裂，具有专一性、高效性、反应条件温和等优点，能够对降解产物的相对分子量进行很好的掌握控制且没有副产物产生，但是目前微生物产糖苷键酶活力普遍较低，无法满足大规模生产的需要，故现有糖苷键酶大都是在海洋微生物和肝胰腺等海洋生物内脏中筛选得到，价格较高。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种大洋中华微菌及在制备褐藻聚糖硫酸酯降解酶中的应用。
本发明的技术解决方案是：一种大洋中华微菌（Sinomicrobium oceani），已保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC №18258。
一种上述大洋中华微菌（Sinomicrobium oceani）在制备褐藻聚糖硫酸酯降解酶中的应用。
本发明经分离、纯化、鉴定、优化筛选出一种菌株，命名为2S19，鉴定为大洋中华微菌（Sinomicrobium oceani），已保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC №18258。该菌株的菌液可提取用于降解褐藻聚糖硫酸酯的胞内和胞外酶且产酶活力高，达到1050U/mL，具有在工业生产中的应用价值。
附图说明
图1为本发明实施例酶解产物的洗脱曲线示意图。
大洋中华微菌菌株保藏日期：2019-7-22；
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心（地址：北京市朝阳区北辰西路1号院）；
保藏号：CGMCC №18258。
具体实施方式
1.大洋中华微菌（Sinomicrobium oceani）的筛选
对大连旅顺养殖场沿海海水采集样品。
1.1微生物富集培养：
采集样品在富集培养基中经过3天富集培养后，涂布在选择性培养基中进行培养，挑取不同形态的菌株进行划线纯化，作为复筛菌株；富集培养基：1L培养基中含FUC2g，NaNO33g，NaCl 20g，MgSO4・7H2O 0.2g，CaCl20.1g，FeSO4・7H2O 0.01g。
1.2产酶微生物初筛
经过三次纯化初筛，在不同NaCl质量浓度的选择性培养基平板中共初步筛选到多种株，分别编号，将初步筛选得到的菌株进行复筛，测定菌株的多糖利用率和酶活力，确定目标菌株。选择性培养基：1L培养基中含FUC2 g，NaNO33g，NaCl 20g，MgSO4・7H2O 0.2g，CaCl20.1g，FeSO4・7H2O 0.01g，琼脂20 g。
1.3 产酶微生物复筛：
将在选择性培养基纯化后的目标菌株分别在复筛培养基中培养进行菌株复筛，利用苯酚硫酸法测定菌株对多糖的利用情况，选取多糖利用率较高的菌株并进行酶活力的测定。复筛培养基：1L培养基中含FUC 2 g，蛋白胨 3 g，NaCl 20 g，MgSO4・7H2O 0.2 g，CaCl20.1 g，FeSO4・7H2O 0.01 g。
所选出菌株代号为HSKF-3，已保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC №18258。
2. 大洋中华微菌（Sinomicrobium oceani）在制备褐藻聚糖硫酸酯降解酶中的应用
2.1 将上述大洋中华微菌（Sinomicrobium oceani）接种于液体发酵培养基中，接种量1%，培养温度为25℃，培养时间为30h。1L培养基中含FUC2g，酵母粉质量12g，蛋白胨3g，NaCl20g，MgSO4・7H2O 0.2g，CaCl20.1g，FeSO4・7H2O 0.01g，pH6.0；
2.2 将2.1步骤的菌液经在4℃经超声波进行破碎，随后于4℃、10000g离心5~10分钟，弃沉淀，上清液即为酶液；
2.3取步骤2.2所得酶液0.25 mL与0.2%（w/v）FUC（用T-buffer配制，pH值8.00）等体积混合后，在30 ℃、150 r/min的水浴恒温振荡器中反应2 h后，在80 ℃水浴灭酶20min，用铁***法测定还原糖含量；空白对照是取步骤2.2所得酶液0.25 mL在沸水浴中灭酶15 min后再与0.2%（w/v）FUC（用T-buffer配制）等体积混合后，在30 ℃、150 r/min的水浴恒温振荡器中反应2 h后，在80 ℃水浴灭酶20 min，用铁***法测定还原糖含量。
酶活力单位定义（U/mL）：在30 ℃、pH值8.00条件下每小时释放1 nmoL岩藻糖需要的酶量。经测定，大洋中化微菌（Sinomicrobium oceani）培养30h后的菌液产酶活力达1050U/mL。
2.4所产酶为褐藻聚糖硫酸酯降解酶的鉴定实验
利用丙酮混合液，将步骤2.2所得酶液进行分离，利用提取的粗酶对岩藻聚糖硫酸酯（FUC）进行酶解：按照10 U/mg的酶添加量确定粗酶与FUC的量，酶解24 h，之后在沸水浴中加热15 min终止酶解反应，室温冷却后在10000 g离心10 min，取上清液冷冻干燥制得酶解产物。
分别将FUC（对照）及酶解产物经Sephacryl S-100洗脱，结果如图1所示。经过测定对比，发现酶解产物洗脱体积在136.5 mL后仍有洗脱峰，而FUC（对照）在此位置没有洗脱峰，说明此部分小分子物质是酶降解的产物，表明该酶可将FUC大分子物质降解为小分子的物质。根据标准曲线的比对，分别收集F1、F2、F3和F4四个组分，其分子量分别为10 KDa以上、5~10 KDa、1~5 KDa和1 KDa以下。
采用液相色谱法分析F1、F2、F3和F4四个组分的化学组成，结果见表1所示。
表1 酶解产物化学组成分析
Table1 The chemical comparision of enzymatic product
结果表明：原样（未酶解的FUC、对照）中葡萄糖和木糖的含量均高于20%；FUC经酶解后各个组分中的葡萄糖和木糖含量均下降，而岩藻糖和半乳糖的含量均上升，其余单糖成分变化不大。
Claims (2)
1.一种大洋中华微菌（Sinomicrobium oceani），已保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏号为CGMCC No.18258。
2. 一种如权利要求1所述大洋中化微菌（Sinomicrobium oceani）在制备褐藻聚糖硫酸酯降解酶中的应用。
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| CN104195080A (zh) * | 2014-08-23 | 2014-12-10 | 中国科学院天津工业生物技术研究所 | 一株产褐藻胶裂解酶的芽孢杆菌及其应用 |
| CN106635920A (zh) * | 2017-01-17 | 2017-05-10 | 中国海洋大学 | 一种高产岩藻多糖酶的海洋交替假单胞菌及其应用 |
| CN106701627A (zh) * | 2017-01-05 | 2017-05-24 | 中国海洋大学 | 一种高产褐藻胶裂解酶的海洋弧菌及其应用 |
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| CN104195080A (zh) * | 2014-08-23 | 2014-12-10 | 中国科学院天津工业生物技术研究所 | 一株产褐藻胶裂解酶的芽孢杆菌及其应用 |
| CN106701627A (zh) * | 2017-01-05 | 2017-05-24 | 中国海洋大学 | 一种高产褐藻胶裂解酶的海洋弧菌及其应用 |
| CN106635920A (zh) * | 2017-01-17 | 2017-05-10 | 中国海洋大学 | 一种高产岩藻多糖酶的海洋交替假单胞菌及其应用 |
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