Selective monooxygenation of inert hydrocarbon bonds is a kind of chemical reaction with exploratory significance in organic synthesis. At present, chemical method and enzymatic method are mainly used for catalysis. Chemical catalysis method often has harsh reaction conditions, while enzymatic catalysis method is relatively mild and has high regional and enantioselectivity. Unspecific peroxidase (UPO, EC 1.11.2.1) from Agrocybe aegerita can carry out complex monooxygenation reaction with H2O2 as co substrate. However, unspecific peroxidase (AaeUPO) as a heme-dependent enzyme, is easy to inactivate under high concentration of H2O2. In situ H2O2 production catalyzed by enzyme can provide lower concentration H2O2 for AaeUPO continuously and stably, and the process is mild and efficient. It is one of the effective ways to avoid adding high concentration H2O2. Therefore, taking AaeUPO and glucose oxidase (GOx) as the research objects, a cross-linked enzyme aggregate (combi-CLEAs) was prepared, and a method for enzyme catalyzed in-situ production of H2O2 and chiral alcohols was established. In this cascade system, GOx oxidizes glucose while reducing O2 to H2O2, and then AaeUPO catalyzes hydroxylation reaction with H2O2 as co substrate. In order to obtain combi-CLEAs with good performance, the effect of preparation conditions on the recovery of combi-CLEAs enzyme activity was investigated. The results showed that the optimum preparation conditions of combi-CLEAs were 6:1 (U:U) of GOx:AaeUPO , the volume ratio of precipitant isopropanol to crude enzyme solution was 2:1, the volume fraction of crosslinker glucan aldehyde (200 kDa) was 15%, and the mass concentration of BSA was 40 μg/mL and the crosslinking time was 3 h. At this time, the recovery of enzyme activity of combi-CLEAs was 87.43% and the specific enzyme activity was 63.12 U/mg. When it is used to catalyze the hydroxylation of ethylbenzene, under the optimal catalytic conditions of pH 7.0 and 30 ℃, continue to optimize the concentration of glucose to make GOx use glucose to stably and continuously generate H2O2 required for AaeUPO. When the concentration of glucose is 6 mmol/L, the conversion of ethylbenzene can reach 99.9%, and the ee value of (R)-phenylethanol is greater than 99.9%. In the reusability experiment of ethylbenzene catalyzed by combi-CLEAs, it can still maintain 67.20% ethylbenzene conversion after 8 cycles. The hydroxylation of different substrates was catalyzed by combi-CLEAs. The results show that the catalytic performance of combi-CLEAs is better than AaeUPO-CLEAs with H2O2, and has a good application prospect.
[1] ZHAO L K, WANG C P, GAO X K, et al.Characterization of P450 monooxygenase gene family in the cotton aphid, Aphis gossypii Glover[J].Journal of Asia-Pacific Entomology, 2022, 25(2):101861.
[2] HOFRICHTER M, ULLRICH R.Oxidations catalyzed by fungal peroxygenases[J].Current Opinion in Chemical Biology, 2014, 19:116-125.
[3] AYALA M, BATISTA C V, VAZQUEZ-DUHALT R.Heme destruction, the main molecular event during the peroxide-mediated inactivation of chloroperoxidase from Caldariomyces fumago[J].JBIC Journal of Biological Inorganic Chemistry, 2011, 16(1):63-68.
[4] PESIC M, WILLOT S J P, FERNÁNDEZ-FUEYO E, et al.Multienzymatic in situ hydrogen peroxide generation cascade for peroxygenase-catalysed oxyfunctionalisation reactions[J].Zeitschrift Fur Naturforschung.C, Journal of Biosciences, 2019, 74(3-4):101-104.
[5] PEREIRA P C, ARENDS I W C E, SHELDON R A.Optimizing the chloroperoxidase-glucose oxidase system:The effect of glucose oxidase on activity and enantioselectivity[J].Process Biochemistry, 2015, 50(5):746-751.
[6] YANG K L. Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions[J]. Enzyme and Microbial Technology, 2017,100:52-59.
[7] ABD RAHMAN N H, JAAFAR N R, ABDUL MURAD A M, et al.Novel cross-linked enzyme aggregates of levanase from Bacillus lehensis G1 for short-chain fructooligosaccharides synthesis:Developmental, physicochemical, kinetic and thermodynamic properties[J].International Journal of Biological Macromolecules, 2020, 159:577-589.
[8] LUCENA G N, SANTOS C C D, PINTO G C, et al.Synthesis and characterization of magnetic cross-linked enzyme aggregate and its evaluation of the alternating magnetic field (AMF) effects in the catalytic activity[J].Journal of Magnetism and Magnetic Materials, 2020, 516:167326.
[9] 张鹏. 以ABTS为底物测定漆酶活力的方法[J].印染助剂, 2007, 24(1):43-45.
ZHANG P.Test method for the laccase activity with ABTS as the substrate[J].Textile Auxiliaries, 2007, 24(1):43-45.
[10] ÖZACAR M, MEHDE A A, MEHDI W A, et al.The novel multi cross-linked enzyme aggregates of protease, lipase, and catalase production from the sunflower seeds, characterization and application[J].Colloids and Surfaces B:Biointerfaces, 2019, 173:58-68.
[11] ARAYA E, URRUTIA P, ROMERO O, et al.Design of combined crosslinked enzyme aggregates (combi-CLEAs) of β-galactosidase and glucose isomerase for the one-pot production of fructose syrup from lactose[J].Food Chemistry, 2019, 288:102-107.
[12] ZHANG D Q, MU T H, SUN H N, et al.Comparative study of potato protein concentrates extracted using ammonium sulfate and isoelectric precipitation[J].International Journal of Food Properties, 2017, 20(9):2 113-2 127.
[13] MATEO C, PALOMO J M, VAN LANGEN L M, et al.A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates[J].Biotechnology and Bioengineering, 2004, 86(3):273-276.
[14] JUNG D H, JUNG J H, SEO D H, et al.One-pot bioconversion of sucrose to trehalose using enzymatic sequential reactions in combined cross-linked enzyme aggregates[J].Bioresource Technology, 2013, 130:801-804.
[15] CUI J D, LIU R L, LI L B.A facile technique to prepare cross-linked enzyme aggregates of bovine pancreatic lipase using bovine serum albumin as an additive[J].Korean Journal of Chemical Engineering, 2016, 33(2):610-615.
[16] GUAUQUE TORRES M P, FORESTI M L, FERREIRA M L.CLEAs of Candida Antarctica lipase B (CALB) with a bovine serum albumin (BSA) cofeeder core:Study of their catalytic activity[J].Biochemical Engineering Journal, 2014, 90:36-43.
[17] HILL A, KARBOUNE S, MATEO C.Investigating and optimizing the immobilization of levansucrase for increased transfructosylation activity and thermal stability[J].Process Biochemistry, 2017, 61:63-72.
[18] 陈海龙, 田耀旗, 李丹, 等.脂肪酶交联聚集体的制备及其催化合成月桂酸淀粉酯的研究[J].食品与发酵工业, 2017, 43(2):21-25.
CHEN H L, TIAN Y Q, LI D, et al.Preparation of cross linked lipase aggregates for synthesis of starch laurate[J].Food and Fermentation Industries, 2017, 43(2):21-25.
[19] 陈宁, 延文星, 路福平, 等.磷脂酶D交联聚集体的制备及其酶学性能研究[J].食品与发酵工业, 2022, 48(5):1-7.
CHEN N, YAN W X, LU F P, et al.Preparation and characterization of cross-linked enzyme aggregates of phospholipase D[J].Food and Fermentation Industries, 2022, 48(5):1-7.
[20] NGUYEN L T, YANG K L.Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions[J].Enzyme and Microbial Technology, 2017, 100:52-59.
[21] KARICH A, SCHEIBNER K, ULLRICH R, et al.Exploring the catalase activity of unspecific peroxygenases and the mechanism of peroxide-dependent heme destruction[J].Journal of Molecular Catalysis B:Enzymatic, 2016, 134:238-246.
[22] 姜艳军, 王旗, 王温琴,等.交联酶聚集体与仿生硅化技术结合制备固定化脂肪酶[J].催化学报, 2012, 33(5):857-862.
JIANG Y J, WANG Q, WANG W Q, et al.Preparation of immobilized lipase through combination of cross-linked enzyme aggregates and biomimetic silicification[J].Chinese Journal of Catalysis, 2012, 33(5):857-862.
