这项研究是由基础科学研究计划通过韩国国家研究基金会（NRF）由教育，科学和技术（NRF-2013R1A1A2012159和NRF-2013R1A1A2007561），建国大学部提供资金，化学工程与MCubed部支持节目在密歇根大学。

1.全细胞生物催化剂制备方法注:重组E.大肠杆菌细胞窝藏pET28a- SpNox 31或pET28a- HjLAD 28以下简称为E.大肠杆菌SpNox和E.大肠杆菌HjLAD分别。 <ol><li>接种大肠杆菌的单个菌落大肠杆菌 HjLAD在3ml的Luria-BERTANI（LB）补充有卡那霉素（50微克/毫升）的培养基中并孵育在培养箱摇床O / N在37℃，250转。 </li><li>由1稀释培养:在200ml新鲜LB的含有50μg/ ml卡那霉素和孵育在37℃下100，250转，直到OD 600达到〜0.6。 </li><li>通过加入0.1mM异丙基β-D-硫代半乳糖苷（IPTG）到培养基中诱导HjLAD蛋白表达，并在16℃，180rpm下16小时孵育。 <ol><li>另外，在25℃，200 RP执行感应在同一天进行的m表示6小时，如果L-木酮糖的生物合成（下面的步骤2）。 </li></ol></li><li>收获诱导E.大肠杆菌 HjLAD细胞离心3,200 XG 20分钟，在4℃弃上清，进行第2步处理细胞沉淀。 </li><li>与此同时，执行步骤1.1 - 1.4 E.大肠杆菌 SpNox。 </li></ol>通过耦合E. 2.合成L-木酮糖大肠杆菌 HjLAD和E.大肠杆菌 SpNox的辅因子再生<ol><li>悬浮大肠杆菌的细胞沉淀大肠杆菌 HjLAD和E.大肠杆菌 SpNox分别在5.0克干细胞重（gDCW）/ L的细胞密度的50mM Tris-HCl缓冲液（pH8.0）中。 注意:gDCW和在600nm（OD 600）测量的光密度之间的相关性，可以建立以促进实验。中所使用的公式该协议是1 gDCW / L = 0.722 * 600 OD - 0.0965，可在不同的光谱仪各不相同。 </li><li>混合600微升5.0 gDCW / L E.大肠杆菌 HjLAD，600微升5.0 gDCW / L E.大肠杆菌 SpNox，100微升的20mM NAD +的，和150微升的2M -L-阿拉伯糖醇在14毫升的圆底管并通过加入550微升的50mM Tris-HCl中使反应体积为2毫升（pH8.0）中。 注意:两个全细胞生物催化剂量的比例可以进行优化，以提高产品的生物合成。对于所描述的系统， 大肠杆菌的比率大肠杆菌 SpNox:E.大肠杆菌 HjLAD = 1:1被发现是最佳的L-木酮糖的生物合成（ 图1B）。 </li><li>孵育在30℃，200rpm下进行8小时，将反应混合物。 </li><li>收集离心后取上清液在摄氏 4 度 3200 XG 10分钟次继续如在下面的步骤3中描述的量化的L-木酮糖的生产。 </li></ol> 3. MTT法检测L-木酮糖定量<ol><li>抽吸将100μl来自步骤2.4收集到1.5毫升管的反应上清液。 </li><li>加入50μl1.5％半胱氨酸，900微升70％硫酸和50微升0.1％的咔唑溶解在乙醇中，并通过反相管3倍轻轻混匀。 </li><li>在37℃，200rpm下20分钟，将反应混合物。 </li><li>测量在使用分光光度计560纳米（A 560）的反应混合物的光学吸光度。 <ol><li>稀释反应混合物，如果A 560读数高于1。 </li></ol></li></ol>在海藻酸钙珠重组全细胞催化剂4.固定<ol><li>溶解4克藻酸钠在100毫升蒸馏水中。通过添加S准备藻酸盐溶液海藻酸钠水，以避免团块的形成。如果需要，加热所述混合物。 </li><li>添加5.0 gDCW / L E. 600微升大肠杆菌 HjLAD和600微升5.0 gDCW / L E.大肠杆菌 SpNox 1.2毫升4-％海藻酸钠在步骤4.1编写，并轻柔吹打混合细胞和海藻酸钠，以避免泡沫形成。 </li><li>吸藻酸盐/细胞悬液到用针头的注射器，并逐滴添加该混合物成酰氯的0.3M钙在100毫升的烧杯中在连续搅拌下（ 氯化钙 ）的解决方案。 注意:用于藻酸盐珠形成的CaCl 2溶液的量应足以使藻酸盐的液滴被完全淹没。此外，注射器针头和氯化钙溶液的表面之间的距离必须在一个最佳范围内被维持，以确保形成均匀的球形珠。的最佳距离范围可被确定实验同盟并取决于针的内径。作为估计，约15±5厘米的距离被发现是最优的一个0.6厘米（内径）的注射器针头。 </li><li>留在2所述的CaCl 2溶液中的珠-在RT 3小时，不进行搅拌，以允许交联和凝胶珠的形成。 </li><li>倒出的CaCl 2溶液，而不通过仔细倾倒的CaCl 2溶液到50毫升锥形管扰乱珠，并在氯化钙溶液中的剩余的珠转移到另一个50ml锥形管中。 </li><li>用10毫升50毫米的Tris-HCl（pH值8.0）洗珠缓冲区三次，除去过量的氯化钙和未封装的细胞。 注意: 在任何给定的步骤，不要离心珠，因为这样做会破裂它们。从溶液中分离磁珠，使悬浮液静置3 - 5分钟。珠将沉降在底部和洗涤缓冲液可以滗到另一个容器。 <ol><li> 不要丢弃用过的洗涤液。池所使用的Tris-HCl洗涤缓冲液（30毫升）与来自步骤4.5中收集的用过的CaCl 2溶液。 </li></ol></li><li>颗粒未固定的E. 3,200×g离心20分钟从步骤4.6收集大肠杆菌细胞被汇集的CaCl 2和的Tris-HCl溶液的离心分离。为了确定固定化效率，如在步骤2.1中所述计算gDCW / L时沉淀未包封细胞的密度。 </li><li>从步骤4.6转移洗涤珠粒成管。按照步骤2.2 - 3.4评价使用所有的洗涤珠粒代替细胞沉淀的L木酮糖的生物合成。 </li></ol> 5.固定化生物催化剂的稳定性测定对L-木酮糖生产<ol><li>从步骤4.8收集珠和不离心，用10ml的50mM的Tris-HCl（pH为8.0）缓冲液洗两次（如步骤4.6中所述）。 </li><li>所有使用3.4 - 的洗涤珠粒如步骤2.2中所述进行反应。 </li><li>重复步骤5.1 - 5.2的生产周期所需数目并测量在每个周期中的反应上清液中产生的L-木酮糖的量。 </li></ol>

作者宣称没有竞争经济利益。本文针对报告的详细方法，生成固定在藻酸盐耦合全细胞生物催化系统。科学新奇已经报道在先前的研究16。

最近的技术进步已经使重组生物治疗的商业化的激增，导致其市场价值逐渐上升的生物技术产业。一个这样的进步是在重组微生物代谢工程，这表明在建立可扩展的工业系统38一诺大的来临。如同大多数工艺，通过遗传工程微生物生产的重组生物分子的成功的商业化是高度依赖于系统39的生产率。这样导致的"蛮力"遗传学39，其中，生物催化的酶的定向进化已经为了提高生物催化剂的活性3所实施的快速发展。然而，对于某些代谢途径，如氧化还原途径，光生物催化剂的改善是不充分的影响由于的OTH的显著效果生产节约儿基本反应的要求，如昂贵的辅助因子。向上扩展这样的系统，即使是具有改进的生物催化剂，只会增加生产成本。因此，需要对于涉及辅助因子的化学计量比生物反应的商品化附加战略方法。此外，生物催化剂的再利用性也需要改善生物过程的经济可行性。为了克服快速辅消费和全细胞生物催化剂的可重用性差的限制作用，我们以前开发的固定，加上全细胞生物催化的辅因子再生和稳定的再利用16系统。在本研究中所描述的所需的耦合，并共同固定，以提高生物转化产率和可重用两个完整细胞的生物催化剂，用代表性的结果沿实验程序。 要考虑用于耦合全细胞biocat一个关键因素alysts是超过对蛋白质的诱导和生物催化产物形成确定，以确保系统的再现条件的精确控制的维护。在像感应外径600，诱导时间，和IPTG浓度参数的变化，应避免以尽量减少批与批变异。此外，需要为不同的生物转化的细胞 - 细胞比率的重新评估。需要注意的是大肠杆菌大肠杆菌 HjLAD:E. 1 大肠杆菌 SpNox:1的比例在本研究报告是由实验优化而不是理论化学计量比来确定。对于在藻酸钙珠的重组全细胞生物催化剂的固定化，有几个关键步骤，以确保相对于胎圈的刚性和细胞分布均匀。首先，藻酸盐溶液必须慢慢加入藻酸钠来制备成水，而不是相反，以防止结块该共ULD影响本地藻酸盐浓度，从而交联的程度。其次，处理细胞 - 藻酸盐悬浮液，以避免产生气泡，这造成对封装的细胞的剪切应力的形成时温和移液应使用，妨碍有效传质和甚至可导致珠粒浮动。万一气泡引入悬浮液中，使细胞 - 藻酸盐悬浮液静置20 - 30分钟，让气泡逸出。第三，使珠时，细胞 - 藻酸盐悬浮液必须缓慢并小心地加入在一个逐滴方式成为均匀尺寸和形态氯化钙溶液的足够体积。在氯化钙不完全淹没珠子将具有不规则的形状和刚性差，可以向细胞泄漏。最后，为避免在从残留的洗涤缓冲液，反应体积可变性，在步骤4.8珠粒可以轻轻用滤纸吸干（不要风干）。 大肠杆菌的一个不同的策略大肠杆菌 SpNox和E.大肠杆菌 HjLAD细胞培养物，提供在维持两种酶之间的期望比率的更好的控制。此外，表达单个蛋白的细胞经受较小的应力比共表达多种蛋白质，这可能导致降低蛋白质错误折叠和提高的蛋白质表达40,41。因此，直接全细胞耦合呈现给调解多酶催化过程没有显著影响产品产量的能力。然而，建立了战略，如细 ​​菌启动精心挑选具有不同强度42,43和改进核糖体结合位点44可以雇工d，来减轻在靶酶的共培养或共表达系统的比例有限的控制。在众多的固定化技术，封装呈现超过替代方法，如交联和吸附，这是更适合于纯化的酶固定化全细胞生物催化剂的多个优点。细胞的包封也可以在各种像琼脂，脱乙酰壳多糖，树胶，角叉菜胶，明胶生物材料的执行，并且用于改进生物催化剂的可重用性藻酸盐45。然而，藻酸钙包封已被证明是相对于固定化效率和生物分子的生产46,47的最有效方法。 利用细菌的全细胞生物催化剂的一个主要限制是在野生型大肠杆菌的异源蛋白质的翻译后修饰的最小能力大肠杆菌 48。这限制了成功使用生物催化剂真核生物W的ithin这个系统。克服这一限制的替代可以使用生物如酵母被更好装有真核翻译后机械。所描述的系统的另一个限制是相关于使用固定化的改善可重用性。固定是用来抵消生物催化剂的稳定性差，并通过简化生物加工49提高他们的周转率共同成本效益的战略- 51。影响包封的全细胞生物催化剂的催化性能的主要参数包括藻酸钠的浓度， 氯化钙的浓度，小区负载和珠的大小。认识到，这些参数不是相互独立的是重要的。这样，各种因素的单个研究内全面分析非常费时，因而有必要选择进行优化的最关键的参数。评估，并在这里提出的的影响两个参数，藻酸钠和氯化钙浓度。虽然在这里没有详细讨论的那样，我们先前已经执行小区负载的优化，在固定化16细胞的重量比较产量的依赖性。在文献的报告表明珠尺寸的该变异也可以调节通过增加酶的封装，提高通过固定系统的增加的表面面积的活动或通过改变底物和产物的扩散生产率由于改变的传输特性。取决于所考虑的生物催化反应中，可能需要的交替关键因素选择，这可能导致一组不同的最佳条件的产生。设立的最佳条件，其他参数的详细研究提供范围在加上全细胞系统的性能进一步提高。 综上所述，我们报告了实验implemen固定在藻酸钙珠的耦合全细胞生物催化系统，示出了改进的生产L-木酮糖的塔季翁。通过表达促进其他辅助因子依赖性生物催化途径，许多生物相关分子的高产率不同的重组酶可以使用此处描述的协议来获得。此外，该系统可以扩展以容纳的产品52和应用比生物催化生产其它，诸如用于生化需氧量53的微生物生物传感器一锅多酶生物合成的两个以上的重组生物催化剂。采取这种制度的协同特征的优点，共生微生物群落的封装可用于应用如生物修复54无数- 56，全细胞生物处理生物燃料57中 ，植物生长促进由土壤生物强化58和biomining用于金属回收59 </SUP&gt;。两者合计，辅因子再生，多小区耦合和本文中所描述的生物催化剂固定化的策略可以用作用于改进生产的由他人在实地实行重要生物分子的有前途的平台。

用微生物还原全细胞生物转化已成为商业上和治疗上重要的生物分子1化疗酶合成的普遍做法- 3。它呈现比使用分离的酶的几个优点，特别是成本密集型的下游纯化过程的消除和延长寿命4的示范- 7。用于需要对产物形成辅因子的生物催化途径，全细胞系统具有通过加入廉价供电子共基质5,8,9 原位辅因子再生提供的潜力。然而，这种能力被减少为需要稀有或昂贵的共同基片10的化学计量浓度的反应- 13。与整个细胞的可重用性差在一起，这阻碍了建立一个可扩展的和连续的机生产线ction系统。这些辅助因子依赖性的生物转化的全细胞系统的战略的修改都需要克服上述的局限性。具体地，工作协同全细胞生物催化剂的组合已显示出显著增强窝藏酶14的生产率和稳定性。这些因素，这往往是用于实现的商业上可行的产品的大规模生产的关键，可以通过共固定生物催化的微生物15进一步优化。我们最近开发出一种简单和可重复使用的全细胞生物催化系统，使两个辅因子再生和生物催化剂的固定化用于L木酮糖生产16。在这项研究中，该系统被用作一个例子来说明应用这两种策略，以提高生物转化产率和生物催化剂可重用性的实验程序。 的L-木酮糖属于共轭亚油酸生物有用分子的SS命名罕见的糖。稀有糖是在自然界中很少出现单糖独特或糖衍生物，但起关键作用作为生物活性分子17,18识别元件。它们有各种各样的应用，从增甜剂，功能食品，以潜在治疗19。的L-木酮糖可用作多种α葡糖苷酶的潜在抑制剂，并且还可以被用作肝炎或肝硬化17,20的一个指标。木糖醇在全细胞系统的L-木酮糖高效率转换已经在泛菌属先前报道ananatis中 21,22， 产碱菌 701B 23， 苍白球芽孢杆菌 Y25 24,25和大肠杆菌 26。在E.大肠杆菌，然而，它只是实现使用低（&lt;67毫摩尔）的木糖醇浓度26由于初始木糖醇浓度高于1潜在抑制作用00毫米木糖醇4脱氢酶活性21,26。木酮糖和木糖醇之间热力学平衡已表明强烈支持木糖醇25,27的形成。此外，木酮糖产量是由具有在原位辅因子再生系统的情况下的要被提供昂贵的辅助因子的量的限制。总之，这些因素限制了用于缩放到可持续系统对L-木酮糖的生物合成的潜力。 为了克服这些限制，提高的L-木酮糖的生物转化产率，辅因子再生的策略在建立一个耦合全细胞生物催化系统首先使用。具体地说，L-阿拉伯糖醇4-脱氢酶（EC 1.1.1.12）从红褐肉座菌 （HjLAD），在真菌的L-阿拉伯糖分解代谢途径的酶，被选择催化L-阿拉伯糖醇转化成L-木酮糖28,29 。像许多生物合成酶的一大limitatioHjLAD的n是它需要价格昂贵的烟酰胺腺嘌呤二核苷酸辅因子（NAD + NADH的氧化形式）的化学计量的量来进行这种转换。在酿脓链球菌 （SpNox）发现的NADH氧化酶已经显示出，以显示高辅因子再生活性30,31。以SpNox， 大肠杆菌的这个属性的优势表达HjLAD用于生产L-木酮糖的大肠杆菌细胞偶合与E.表达SpNox为NAD +的再生的大肠杆菌细胞，以提高由图1A中所示的耦合反应中所描绘的的L-木酮糖的生产。在最佳条件下，用150毫-L-阿拉伯糖醇的初始浓度，最大的L-木酮糖收率达到96％，从而使该系统比在文献中报道的有效得多​​。 全细胞固定化的策略采用下，进一步提高了耦合biocatalyt的可重用性IC系统。对于全细胞固定化的常用方法包括吸附/共价连接到固体基质，在聚合物网络32交联/包封和封装。在这些方法中，对细胞固定化的最合适的方法是封装在藻酸钙珠。其温和凝胶化性能，惰性水性基质和高孔隙度有助于保持包封的生物制品33的生理特性和功能。因此，耦合的生物催化剂系统含有大肠杆菌大肠杆菌细胞窝藏HjLAD或SpNox被固定在藻酸钙珠，使L -木酮糖生产的多个循环（ 图2）.The固定的生物催化剂体系表现出良好的操作稳定性，保持了第一次循环的转化率的65％的7次循环后连续再利用，而将无细胞系统几乎完全失去其催化活性。

<table><tbody><tr><td>LB broth&amp;nbsp;</td><td>Sigma Aldrich</td><td>L3022-6X1KG</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher</td><td>BP906-5</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-thiogalactopyranoside (IPTG)</td><td>Sigma Aldrich</td><td>I6758-10G</td><td></td></tr><tr><td>Tris base</td><td>Fisher</td><td>BP1521</td><td></td></tr><tr><td>B-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma Aldrich</td><td>N7004-1G</td><td></td></tr><tr><td>L-Arabinitol</td><td>Sigma Aldrich</td><td>A3506-10G</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma Aldrich</td><td>168149</td><td></td></tr><tr><td>Sulfuric acid</td><td>Sigma Aldrich</td><td>320501-500ML</td><td></td></tr><tr><td>Carbazole</td><td>Sigma Aldrich</td><td>C5132</td><td></td></tr><tr><td>Ethanol&amp;nbsp;</td><td>Fisher</td><td>BP2818-4</td><td></td></tr><tr><td>Sodium alginate</td><td>Sigma Aldrich</td><td>W201502</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>223506-500G</td><td></td></tr><tr><td>Excella E24 shaker incubator</td><td>New Brunswick Scientific</td><td></td><td></td></tr><tr><td>Cary 60 UV-Vis Spectrophotometer</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendrof</td><td></td><td></td></tr><tr><td>Beakers</td><td>Fisher</td><td></td><td></td></tr><tr><td>Syringe</td><td>Fisher</td><td></td><td></td></tr><tr><td>Needle</td><td>Fisher</td><td></td><td></td></tr><tr><td>Pioneer Analytical and Precision Weighing Balance</td><td>Ohaus</td><td></td><td></td></tr></tbody></table>

immobilization of multi-biocatalysts in alginate beads for cofactor regeneration and improved reusability

我们最近开发出一种简单，可重复使用以及耦合全细胞生物催化系统，辅因子再生和生物催化剂固定化，以提高产量和持续的综合能力。描述兹为这样一个系统由两个大肠杆菌发展的实验程序大肠杆菌菌株表达功能互补的酶。一起，这两种酶可起到共可操作以介导昂贵辅因子的再生用于提高生物反应的产物产率。此外，据报道通过在海藻酸钙珠全细胞的包封合成耦合生物催化系统的固定形式的方法。作为一个例子，我们通过偶合大肠杆菌呈现的L-木酮糖的改进的生物合成从L-阿拉伯糖醇大肠杆菌细胞中表达的酶的L-阿拉伯糖醇脱氢酶或NADH氧化酶。在最佳条件下和使用的150的初始浓度毫-L-阿拉伯糖醇，最大的L-木酮糖收率达到96％，这是比在文献中报道的更高。耦合全细胞生物催化剂的固定化形式表现出良好的操作稳定性，保持连续重复使用的7个周期后的第一个周期中获得的产率的65％，而将无细胞系统几乎完全失去催化活性。因此，这里所报告的方法提供了两种策略可以帮助提高工业生产的L-木酮糖，以及需要在一般的使用辅因子的其他增值化合物。

启用辅因子再生，L-木酮糖合成在含有大肠杆菌耦合全细胞生物催化系统中进行大肠杆菌 HjLAD和E.大肠杆菌 SpNox细胞。以下的各种参数的最佳化，该系统的可重用性是通过固定它在海藻酸钙珠（ 图2）的改善。 L-木酮糖的生产与辅因子再生偶合 E.大肠杆菌 HjLAD 和 E.大肠杆菌 SpNox 细胞 。 提高L-阿拉伯糖醇的生物转化成L-木酮糖， 大肠杆菌大肠杆菌 HjLAD和E.大肠杆菌 SpNox细胞耦合来使能辅因子再生。为了说明在大肠杆菌中表达和辅可用性的差异大肠杆菌 SpNox和E.大肠杆菌 HjLAD细胞中，L木酮糖产量在各种大肠杆菌的比较研究大肠杆菌 SpNox -到- E.进行大肠杆菌 HjLAD比率。 如图1B中所示，产量增加的大肠杆菌大肠杆菌 SpNox -到- E.大肠杆菌 HjLAD比从0.13升高:1至1:1。 1和1.33:为比1获得的收益率分别为1和相当于最大为这个耦合系统。所有进一步的实验，与大肠杆菌进行大肠杆菌 SpNox -到- E. 1 大肠杆菌 HjLAD:1的比例。用150mM的L-阿拉伯糖醇的初始浓度，利用大肠杆菌的开始大肠杆菌 HjLAD生物催化剂生产的单独118毫米L木酮糖后8小时（饱和点对L-木酮糖浓度），占79％（ 图1C）的收率。相比较而言，使用的大肠杆菌的大肠杆菌 HjLAD和E.大肠杆菌 SpNox细胞以1:1的比率产生144毫摩尔L-木酮糖，提高了产率96％。 全细胞催化剂固定化的优化 在海藻酸钙珠的生物催化剂的固定化提供了许多优点，如改进的生存能力，作为保护细胞免受在生物反应器的物理应力的轻度凝胶环境的结果。这些海藻珠的性能在很大程度上是由配方和工艺参数确定34。为了通过上述的耦合全细胞生物催化剂的固定化形式进行优化的L-木酮糖的产率，两个主要的参数，即藻酸钠的浓度和氯化钙浓度，进行评价。这些是确定的藻酸钙珠，这反过来又影响生物催化剂固定化效率和分子扩散的完整性和刚度的主要参数。 如图3A中所示，生物催化剂固定化效率表现出增加趋势，在范围1增加褐藻酸钠浓度- 3％（重量/体积）。当在固定化基质中使用的藻酸钠的浓度为小于1％，所得到的珠脆弱易断由于机械稳定性低，释放大部分细胞向周围的CaCl 2溶液（未示出数据）。一旦海藻酸钠浓度提高至大于2％，所得到的珠硬化过度导致的问题中的分子扩散35（ 图4）。改变的CaCl 2浓度，得到的生物催化剂immobilizat有类似的趋势离子效率。为藻酸钠的固定浓度（2％重量/体积）并在0.1的范围内-的0.4M的CaCl 2，低级的CaCl 2浓度导致降低的珠粒刚度和增加的细胞渗漏到周围溶液中。这是由于钙离子的用于交联的藻酸盐聚合物链36（ 图3B）的古洛糖醛酸盐块有限。然而，增加从0.3至0.4M的氯化钙浓度提高了生物催化剂固定化效率只有轻微。由于高固定化效率（&gt; 90％）， 氯化钙的浓度超出0.4M的未被评估。当使用小于0.1M的氯化钙浓度，将含有生物催化剂的藻酸钠微滴在氯化钙溶液解体，形成没有球形珠粒（数据未示出）。 为了最大限度地没有显著放缓的海藻酸钙珠分子扩散，海藻酸钠和氯化钙2浓度的生物催化剂固定化效率下一步改进的L-木酮糖的生产进行了优化。使用相同浓度范围为固定化效率（1 - 3％w / v的褐藻酸钠和0.1 -的0.4M的CaCl 2），2％的最佳海藻酸钠浓度观察到达成的L-木酮糖生产的最大产率（ 图4A ）。超出此最佳浓度，产量呈下降趋势。这是意料之中的，因为与的海藻酸钙珠的过度刚性关联中扩散的问题。同样，最佳的CaCl 2浓度为0.3 M（ 图4B）。与在图3中 ，最佳海藻酸钠浓度（2％）和氯化钙2浓度（0.3 M），允许珠的形成与示出的数据相结合合适的刚性和渗透性的成立，使最小细胞渗漏和最大的L-木酮糖产量。 应当指出的是，这会影响固定的生物催化剂的总催化效率的另一因素是封装在细胞培养物的重量。随细胞接种到一个最佳的接种物重量，超过此它显示了减少的趋势37的催化效率增加。对于这种减少的一个可能的解释是蜂窝过分拥挤，阻碍分子扩散遍及海藻酸钙珠和降低了体系的催化效率。使用上述优化固定化条件下，具有3.75的细胞加载（gDCW），获得最高的L-木酮糖的生产/ L 16（数据未显示）。 固定化和自由全细胞生物催化剂的可重用性L-木酮糖生产。 使用1的既定最佳条件:1 E.大肠杆菌 SpNox到E.大肠杆菌 HjLAD比率，2％（重量/体积）藻酸钠和0.3M的氯化钙 ，固定耦合全细胞生物催化剂输出的64％的最大的L-木酮糖的产量相比，96％的游离高得多的产率耦合全细胞系统（ 图5的循环1）。需要注意的是固定的大肠杆菌的产量大肠杆菌 HjLAD细胞单独仅为32.5％（数据未显示），比该固定联接系统以及自由单一生物催化剂体系（79％， 图1C）的低。预期该还原为固定已知降低生物催化剂的活性，这可能是由于衬底扩散的限制，但由提高经固定化的生物催化剂的可重用的优点补偿。在生物催化剂的稳定性这一效果是通过在上反复对所述自由和固定系统，以间歇反应过程观察到的L-木酮糖产量的下降趋势，在图5所示证实。对自由系统中的产率显示出比该固定系统的更陡的下降，表明在免费的系统酶活性的更快速的损失。其结果是，这两个系统有对生产周期2的可比的L-木酮糖产量在连续重复使用的7个周期结束时，包封的生物催化剂保持它们的稳定性和保留了原始产率的65％，而自由全细胞系统保留了其生产L-木酮糖能力小于10​​％。这表明，固定化的生物催化剂可有效地用于生产L-木酮糖的和可重复使用改进的可重用性和稳定性的益处通过固定化带来的远远超过在生物催化剂的活性的降低，过赋予一个显著优势的生产过程一个自由全细胞系统。 <img alt="图1" src="/files/ftp_upload/53944/53944fig1.jpg" /> 图1.加之全细胞系统的L-木酮糖的生物催化生产 。 （ 一 ）示意图说明包括大肠杆菌耦合全细胞系统中发生的辅因子再生反应大肠杆菌细胞分别窝藏HjLAD或SpNox。期L木酮糖与不同大肠杆菌的产率（B）的变异大肠杆菌 SpNox到E.大肠杆菌 HjLAD比率。 （C）从全细胞生物催化剂的L-木酮糖产量只包括大肠杆菌的大肠杆菌 HjLAD相比，含有大肠杆菌的耦合全细胞系统大肠杆菌 HjLAD和E.大肠杆菌 SpNox细胞能够辅因子再生的。所示的图代表平均站三个独立实验ndard偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53944/53944fig2.jpg" /> 图2.固定藻酸盐珠通过封装全细胞生物催化剂 。示意图代表的统一大小的钙藻酸盐珠逐滴加入海藻酸钠，细胞悬液的氯化钙溶液的形成。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53944/53944fig3.jpg" /> 图3.参数影响的固定效率<html>在藻酸盐珠的全细胞生物催化剂。固定化效率（A）的藻酸钠的函数（％重量/体积）（B）中的CaCl 2（M）的浓度。所示的图代表三个独立实验的平均值和标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53944/53944fig4.jpg" /> 图4.评价从固定化耦合全细胞生物催化剂如（A）中藻酸钠的函数L木酮糖的产率的 （％重量/体积）（B）中的CaCl 2（M）的浓度。示出的曲线表示的三个独立实验的平均值和标准偏差。平直"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/53944/53944fig5.jpg" /> 图5.生物催化剂可重用。L-木酮糖生产固定化和自由耦合全细胞生物催化剂的再利用的连续7个循环的产率的评价是分别示出在暗和浅灰，。图中的曲线代表了三个独立实验的平均值和标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53944/53944fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability at JoVE.com

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

呈现为共固定化全细胞生物催化剂用于辅因子再生和改善的可重用性，使用生产L-木酮糖的作为例子的协议。辅因子再生是通过偶合表达功能上互补酶2 大肠杆菌菌株实现;全细胞生物催化剂固定化是通过细胞封装在藻酸钙珠来实现。

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

immobilization-multi-biocatalysts-alginate-beads-for-cofactor

Nanyang Technological University

Research

JoVE Journal

Chemistry

17.3K 次观看.  Konkuk University. 呈现为共固定化全细胞生物催化剂用于辅因子再生和改善的可重用性，使用生产L-木酮糖的作为例子的协议。辅因子再生是通过偶合表达功能上互补酶2 大肠杆菌菌株实现;全细胞生物催化剂固定化是通过细胞封装在藻酸钙珠来实现。 

视频: Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

微流控芯片上捕获环加成反应可逆地固定小分子或多组分结构的生物传感器应用

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

科研

化学

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

发育生物学

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.

This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.

固定化酶和稳定的疏水盐改性的Nafion

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

生物工程

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

医学

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造:一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

使用简单搅拌容器在藻酸盐珠中的哺乳动物细胞包封

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Fabrication of Amyloid-&#946;-Secreting Alginate Microbeads for Use in Modelling Alzheimer's Disease

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic amyloid-&#946; (A&#946;) fragments, eventually leading to the classical features of the disease: amyloid plaques, neurofibrillary tangles and synaptic and neuronal loss. The lack of relevant non-transgenic preclinical models reflective of disease progression is one of the main factors hindering the discovery of effective drug treatments. To this end, we have developed a protocol for the fabrication of alginate microbeads containing amyloid-secreting cells useful for the study of the effects of chronic A&#946; production.
Chinese hamster ovary cells previously transfected with a human APP gene, secreting A&#946; (i.e., 7PA2 cells), were used in this study. A three-dimensional (3D) in vitro model for the sustained release of A&#946; was fabricated by encapsulation of 7PA2 cells in alginate. The process was optimized to target a bead diameter of 500-600 &#956;m for further in vivo studies. Optimization of 7PA2 cell encapsulation in alginate was performed altering fabrication parameters, e.g., alginate concentration, gel flow rate, electrostatic potential, head vibration frequency, gelling solution. Levels of secreted A&#946; were analyzed over time and compared between alginate beads and standard cell culture methods (up to 96 h).
A concentration of 1.5 x 106 7PA2 cells/mL and an alginate concentration of 2% (w/v) buffered with HEPES and subsequent gelation in 0.5 M calcium chloride for 5 min were found to fabricate the most stable microbeads. Fabricated microbeads were 1) of uniform size, 2) with an average diameter of 550 &#956;m, 3) containing about 100-150 cells per microbead and 4) able to secrete A&#946;.
In conclusion, our optimized method for the production of stable alginate microbeads containing amyloid-producing 7PA2 cells might enable the modeling of important aspects of AD both in vitro and in vivo.

This protocol illustrates a cell encapsulation method by rapid physical gelation of alginate to immobilize cells. Obtained microbeads allow controlled and sustained secretion of amyloid-&#946; over time and can be used to study the effects of secreted amyloid-&#946; in in vitro and in vivo models.

制造淀粉样蛋白-β-分泌阿尔金酸微珠,用于模拟阿尔茨海默氏病

According to the amyloid cascade hypothesis, the earliest trigger in the development of Alzheimer&#8217;s disease (AD) is the accumulation of toxic ...

生物学

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...