过氧化酶与葡萄糖氧化酶复合交联酶聚集体的制备与应用

Preparation and application of combi-CLEAs of unspecific peroxygenase and glucose oxidase

建立了一种酶催化原位产生H_(2)O_(2)和手性醇的方法,以非特异性过氧化酶(Aae UPO)和葡萄糖氧化酶(glucose oxidase,GO_(x))为研究对象,制备了双酶复合交联酶聚集体(combi-CLEAs)。考察了制备条件对combi-CLEAs酶活回收率的影响。结果表明,combi-CLEAs的最优制备条件为GO_(x)与Aae UPO的酶活力比为6∶1、沉淀剂-异丙醇与酶液的体积比为2∶1、交联剂-葡聚糖醛(200 kDa)的体积分数为15%、牛血清白蛋白(bovine serum albumin,BSA)质量浓度为40μg/mL,交联时间为3 h,此时,所得combi-CLEAs的酶活力回收率为87.43%,比酶活力为63.12 U/mg。将其用于催化乙苯的羟基化反应时,乙苯的转化率可达99.9%,(R)-苯乙醇的对映体过量值大于99.9%,经过8次循环后,combi-CLEAs仍能保持67.20%的乙苯转化率。对不同底物进行单加氧催化时,combi-CLEAs的性能优于外加H_(2)O_(2)时的Aae UPO-CLEAs,有良好的应用前景。

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 H_(2)O_(2) as co substrate.However,unspecific peroxidase(Aae UPO)as a heme-dependent enzyme,is easy to inactivate under high concentration of H_(2)O_(2).In situ H_(2)O_(2) production catalyzed by enzyme can provide lower concentration H_(2)O_(2) for Aae UPO continuously and stably,and the process is mild and efficient.It is one of the effective ways to avoid adding high concentration H_(2)O_(2).Therefore,taking Aae UPO and glucose oxidase(GO_(x))as the research objects,a cross-linked enzyme aggregate(combi-CLEAs)was prepared,and a method for enzyme catalyzed in-situ production of H_(2)O_(2) and chiral alcohols was established.In this cascade system,GO_(x) oxidizes glucose while reducing O_(2) to H_(2)O_(2),and then Aae UPO catalyzes hydroxylation reaction with H_(2)O_(2) 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 GO_(x)∶Aae UPO,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 40μg/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 GO_(x) use glucose to stably and continuously generate H_(2)O_(2) required for Aae UPO.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 Aae UPO-CLEAs with H_(2)O_(2),and has a good application prospect.