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Summary
Abstract
Introduction
Protocol
Results
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자동 번역
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화학

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

Published: April 22, 2016
doi:
10.3791/53944
, , ,
1Department of Chemical Engineering,Konkuk University, 2Department of Chemical Engineering,University of Michigan

Summary

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。

Abstract

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。

Introduction

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（<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。

Protocol

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。 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。 </…

Representative Results

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 <strong…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

ImmobilizationMulti-biocatalystsAlginate BeadsCofactor RegenerationReusabilityWhole Cell Biocatalytic SystemBiocatalystsStabilityCofactor RegeneratingE. ColiSpNOxHjLADNADH OxidaseL-arabinitol DehydrogenaseRecombinantIPTGTris-HCl BufferNAD+L-arabinitolL-xylulose
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Gao, H., Khera, E., Lee, J., Wen, F. Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability. J. Vis. Exp. (110), e53944, doi:10.3791/53944 (2016).
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