固定化细胞催化合成6-氰基-(3R,5R)-二羟基己酸叔丁酯工艺研究

Biocatalytic Process of t-Butyl 6-cyano-(3R,5R)-dihydroxylhexanoate by Immobilized Cells

采用硅藻土作载体,聚乙烯亚胺和戊二醛作交联剂来固定化E.coli BL21(DE3)/pCDFDuet-gdh-cr共表达菌株,以此来催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯[(5R)-1]合成6-氰基-(3R,5R)-二羟基己酸叔丁酯[(3R,5R)-2],考察了硅藻土-聚乙烯亚胺-戊二醛固定化细胞的催化性能,进一步优化了固定化细胞催化合成(3R,5R)-2工艺。结果表明:在固定化细胞用量100g/L、葡萄糖与底物质量浓度比为1:1、转化温度30℃、pH7.0、流速12mL/min、100 g/L的(5R)-1转化完全的条件下,填充床式反应器可以连续反应五个批次,转化570 g/L的(5R)-1,较搅拌式反应器提高了107.3%,较游离细胞间歇催化操作提高了185%。

Chiral diol t-butyl6-cyano-(3R,5R)-dihydroxyhexanoate synthetic process was established byusing the celite-polyethyleneimine(PEI)-glutaraldehyde(GA)immobilized E.coli BL21(DE3)/pCDFDuet-gdh-cr cells in present work.The optimized batch bioconversion conditions for the celite-PEI-GA immobilized E.coli BL21(DE3)/pCDFDuet-gdh-cr were as follows:30℃,pH7.0,100g/L immobilized cell loading size,50g/L t-butyl6-cyano-(5R)-hydroxyl-3-oxolhexanoate,mass ratio of glucose to t-butyl6-cyano-(5R)-hydroxyl-3-oxolhexanoate at1:1(w/w).Results demonstrated that the celite-PEI-GA immobilized E.coli BL21(DE3)/pCDFDuet-gdh-cr cells can be reused for five intermittent batches.Further,its catalytic conditions were studied in the stirred tank bioreactor and packed bed reactor.Under the optimized biocatalytic conditions,570g/L t-butyl6-cyano-(5R)-hydroxyl-3-oxolhexanoate was completely converted by the celite-PEI-GA immobilized cells in72h in the packed-bed bioreactor,which was improved185%and107.3%in comparison with those of the free cells and the celite-PEI-GA immobilized cells in the stirred tank bioreactor,respectively.