Freeman A, Abramov S, Georgiou G
Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Green Building for Biotechnology, Room 222, Tel Aviv University, Tel Aviv 69978,
Biotechnol Bioeng. 1999 Jan 20;62(2):155-9. doi: 10.1002/(sici)1097-0290(19990120)62:2<155::aid-bit4>3.0.co;2-u.
Bacteria displaying heterologous receptors or enzymes on their surface hold great potential as whole-cell adsorbents and biocatalysts, respectively. For industrial applications, such surface-engineered cells need to be killed and chemically fixed to prevent disintegration and leakage of the displayed proteins under process conditions. It is also highly desirable to couple the chemically stabilized cells onto a solid support matrix for additional mechanical stability, flexibility in reactor choice, and easy separation from processed medium. Recently, we described the development of a readily scalable methodology for cell killing, fixation, and outer membrane stabilization via glutaraldehyde fixation followed by secondary crosslinking (Freeman, A., Abramov, S. and Georgiou, G. 1996. Biotechnol. Bioeng. 52: 625-630). Glutaraldehyde treatment was also found, however, to reduce the specific activity of a model enzyme, beta-lactamase displayed on the surface of E. coli. Here, we show that crosslinking carried out in the presence of beta-lactamase inhibitors, namely phenyl boronic acid or sodium borate, protects the active site from chemical modification resulting in up to threefold higher specific activities without affecting the cell-stabilizing effect of the glutaraldehyde treatment. To prepare an immobilized whole cell biocatalyst, residual unreacted surface aldehyde groups were employed to immobilize covalently the fixed bacteria onto chitosan-coated cellulose powder. The binding of the bacteria onto chitosan-coated cellulose was quantitative up to cell loading of 83 mg dry cell weight/g of support. Cell immobilization did not introduce mass transfer limitations and created only a modest reduction in Vmax. Thus, chemical crosslinking, affected in presence of reversible active-site inhibitors and coupled with cell immobilization on chitosan-coated cellulose represents a widely useful methodology for the process application of recombinant bacteria displaying surface-anchored heterologous proteins.
在其表面展示异源受体或酶的细菌，分别作为全细胞吸附剂和生物催化剂具有巨大潜力。对于工业应用而言，此类表面工程化细胞需要被杀死并进行化学固定，以防止在工艺条件下所展示蛋白质的分解和泄漏。将化学稳定的细胞偶联到固体支持基质上，以获得额外的机械稳定性、在反应器选择上的灵活性以及便于从处理后的培养基中分离，这也是非常理想的。最近，我们描述了一种易于扩展的方法，通过戊二醛固定随后进行二次交联来实现细胞杀死、固定和外膜稳定（弗里曼，A.，阿布拉莫夫，S.和乔治乌，G. 1996.《生物技术与生物工程》52: 625 - 630）。然而，还发现戊二醛处理会降低展示在大肠杆菌表面的模型酶β - 内酰胺酶的比活性。在此，我们表明在β - 内酰胺酶抑制剂（即苯硼酸或硼酸钠）存在下进行交联，可保护活性位点免受化学修饰，从而使比活性提高多达三倍，同时不影响戊二醛处理的细胞稳定效果。为制备固定化全细胞生物催化剂，利用残留的未反应表面醛基将固定化细菌共价偶联到壳聚糖包被的纤维素粉末上。在细胞负载量高达83毫克干细胞重量/克支持物时，细菌与壳聚糖包被纤维素的结合是定量的。细胞固定化未引入传质限制，仅使Vmax有适度降低。因此，在可逆活性位点抑制剂存在下进行化学交联，并与细胞在壳聚糖包被纤维素上的固定相结合，代表了一种广泛适用于展示表面锚定异源蛋白质的重组细菌工艺应用的方法。
