Institute for Biochemistry, Biotechnology and Bioinformatics, Technische Universität Braunschweig, Braunschweig, Germany.
Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Braunschweig, Germany.
Biotechnol Bioeng. 2023 Nov;120(11):3210-3223. doi: 10.1002/bit.28528. Epub 2023 Aug 18.
Halohydrin dehalogenase HheG is an industrially interesting biocatalyst for the preparation of different β-substituted alcohols starting from bulky internal epoxides. We previously demonstrated that the immobilization of different HheG variants in the form of cross-linked enzyme crystals (CLECs) yielded stable and reusable enzyme immobilizes with increased resistance regarding temperature, pH, and the presence of organic solvents. Now, to further establish their preparative applicability, HheG D114C CLECs cross-linked with bis-maleimidoethane have been successfully produced on a larger scale using a stirred crystallization approach, and their application in different chemical reactor types (stirred tank reactor, fluidized bed reactor, and packed bed reactor) was systematically studied and compared for the ring opening of cyclohexene oxide with azide. This revealed the highest obtained space-time yield of 23.9 kg g h L along with the highest achieved product enantiomeric excess [64%] for application in a packed-bed reactor. Additionally, lyophilization of those CLECs yielded a storage-stable HheG preparation that still retained 67% of initial activity (after lyophilization) after 6 months of storage at room temperature.
卤代醇脱卤酶 HheG 是一种在工业上很有前途的生物催化剂，可用于从大体积的内环氧出发制备不同的 β-取代醇。我们之前证明，以交联酶晶体（CLECs）的形式固定化不同的 HheG 变体可得到稳定且可重复使用的酶固定化剂，其对温度、pH 值和有机溶剂的存在具有更高的抗性。现在，为了进一步确定其制备适用性，使用搅拌结晶法成功地在更大规模上生产了 D114C HheG CLECs 与双马来酰亚胺乙烷交联，并在不同的化学反应器类型（搅拌釜反应器、流化床反应器和填充床反应器）中系统地研究和比较了它们在环氧化环己烯与叠氮化物开环反应中的应用。这揭示了在填充床反应器中应用时，获得的时空产率最高为 23.9kg g h L ，产物对映体过量最高为 64%。此外，这些 CLECs 的冻干产生了一种储存稳定的 HheG 制剂，在室温下储存 6 个月后，仍保留初始活性的 67%（冻干后）。
