Co-immobilization Of NADH Oxidase With Combined Cross-linked Enzyme Aggregates For In Situ NAD~+ Regeneration

Combined cross-linked enzyme aggregates?combi-CLEAs?are versatile and novel carrier-free biocatalysts for the co-immobilization of two or several enzymes.These enzymes are able to catalyze one-pot cascade or parallel reactions.Compared with the conventional immobilization methods,this method has several advantages,such as higher enzymatic activity and controllability,lower production cost and no requirement of purified enzymes.Oxidoreductase-catalyzed redox reaction has advantages of mild conditions,high specificity and stereo-selectivity,and plays an important role in practical applications.More than 80%oxidoreductase require the participation of NAD?P?⁺/NAD?P?H.These cofactors are necessary to regenerate because they are expensive.Although the regeneration method of NAD?P?H has been widely reported,the regeneration of NAD?P?⁺is still a bottleneck in the field of industrial production.Coupling enzymes that are parallel but catalytically opposite directions can be used to regenerate cofactors.This simple regeneration system has advantage such as high regeneration efficiency.However,for many systems involved free enzyme,the free enzymes have disadvantages such as difficulty in recovery and poor stability.The recovery and reuse of combi-CLEAs can compensate for these defects in the application of the free enzymes.Thus,this study intends to use combi-CLEAs to achieve in-situ NAD⁺regeneration,and it is expected to reduce costs through the cofactor regeneration.In the second chapter,combi-CLEAs of NADH oxidase?Nox?and glycerol dehydrogenase?GDH?were successfully prepared.The preparation process generally comprises two steps:First,the free enzyme is precipitated from the solution with a precipitator,and then immobilized by glutaraldehyde.To compare the efficiency of combi-CLEAs for in situ NAD⁺regeneration,Nox-CLEAs and GDH-CLEAs were also prepared,respectively.The effects of various factors on the immobilization including the type and concentration of precipitant,the ratio of GDH and Nox,the ratio of protein and glutaraldehyde,and the temperature were studied.The highest activity recovery of 2.14 and 1.08 U/mg of combi-CLEAs and CLEAs was obtained under the conditions:saturated ammonium sulfate,GDH/Nox 5/1 mol/mol,protein/glutaraldehyde 20/1 mol/mol,immobilization time 2 h,temperature 4 ^oC.When the concentration of NAD⁺was 0.05?M,the total turnover number?TTN?of combi-CLEAs,CLEAs mixture and mixed free enzyme were 2137,1849 and 1970,respectively,indicating that the efficiency of combi-CLEAs in regenerating NAD⁺was higher than free enzyme.After optimization,the combi-CLEAs increased 11%in t_1/2 value over free enzymes mixture at 55°C.And after 10 cycles,the combi-CLEAs and CLEAs mixture retained 65%and 56%initial activity,respectively.Finally,we also used the immobilized enzymes to produce DHA.After 30 h,the combi-CLEAs produced 9.1 mM DHA,2.5 and 2 times the CLEAs mixture and free enzymes mixture,respectively.In the third chapter,the composite combi-CLEAs of Nox and galactitol dehydrogenase?GatDH?,Nox-CLEAs and GatDH-CLEAs were prepared.The effects of factors such as Nox/GatDH,protein/glutaraldehyde and immobilization time were also explored.The highest enzyme activities of combi-CLEAs and CLEAs mixture were 0.19 U/mg and 0.17 U/mg,respectively when the GatDH/Nox is 1:5,glutaraldehyde/protein is 1:20,and immobilization time 8 h.When NAD⁺was 0.05?M,the TTN of combi-CLEAs was 134?1.6 times both of the CLEAs mixture and the free enzymes mixture?,indicating that the efficiency of combi-CLEAs in regenerating NAD⁺was higher than that of CLEAs mixture and free enzymes mixture.After 10 cycles,the combi-CLEAs and CLEAs mixtures maintained 40%and 32%initial activity,respectively.In conclusion,two combi-CLEAs were prepared,and the covalent immobilization of the enzyme was achieved by reacting glutaraldehyde with amino acid residues of the protein.In situ NAD⁺regeneration was achieved by these two combi-CLEAs.The results showed that combi-CLEAs owed better stability and reusability,and combi-CLEAs had better catalytic efficiency in bio-catalytic reactions.The research provides a new idea for further study of immobilization technology on the application of cofactor regeneration.