CN110964710B - 固定化酶、其制备方法及其应用 - Google Patents
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Abstract
本发明提供了一种固定化酶、其制备方法及其应用。该固定化酶包括酶及固定酶的氨基树脂载体，酶选自转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶中的任意一种，氨基树脂载体为交联剂修饰的氨基树脂载体，交联剂为经过高聚物处理过的交联剂。通过采用高聚物处理过的交联剂对氨基树脂载体进行修饰，有利于使固定于其上的酶形成网状交联，进而使这些酶的固定效果更加稳定，从而提高这些酶的循环利用效率。
Description
技术领域
本发明涉及固定化酶领域，具体而言，涉及一种固定化酶、其制备方法及其应用。
背景技术
微生物细胞或分离的酶或工程酶的应用使得生物催化取得了重大进展，且使得制造方式发生了转变。许多种类的酶如酰基转移酶、酰胺酶、转氨酶、酮还原酶、氧化酶、单加氧酶及水解酶等被用于生产涉及抗生素、除草剂、医药中间体和新时代治疗剂的反应中。
当游离酶用作生物催化剂时，会有很大的酶浪费，因为回收水溶性酶非常困难。而水不溶性固定化酶，在每个循环后，通过非常简单的过滤就可以容易地回收。
现有技术中已经有报道单一酶的固定化方法，但不同的酶所适合的固定化方法是不同的。比如Bolivar等(Biomacromol.2006,7,669-673)研究了来自假单胞菌SP101的FDH的共价固定化，包括共价固定于改性琼脂糖、CNBr活化的琼脂糖、Sepabeads(葡聚糖)及乙醛琼脂糖的各种载体上。其得出的结论是：固定化于溴化物，聚乙烯亚胺，戊二醛等活化的载体上并不会促进酶在热灭活下的任何稳定作用。然而，高度活化的乙二醛琼脂糖的优化的酶被证明是具有很高的热稳定性、pH稳定性并且在具有增强的稳定性情况下具有超过50％的活性。
Kim et al(J.Mol.Catal B:Enzy 97(2013)209–214)报道了使用交联酶聚集体(CLEA)的方法来固定来自Candida boidinii.的甲酸脱氢酶(formate dehydrogenase，FDH)，并认为葡聚糖多醛(dextran polyaldehyde)作为交联剂代替戊二醛(glutaraldehyde)对于固定化酶更好，经过10次重复使用后残留活性超过95％。此外，葡聚糖多醛形成的交联酶聚集体(Dex-CLEA)的热稳定性比游离酶的热稳定性提高了3.6倍。
Binay et al(Beilstein J.Org.Chem.2016,12,271–277)报道了高活性的来源于Candida methylica的FDH的固定化酶，FDH共价固定到环氧活化Immobead 150载体上。Immobead 150载体先经过乙二胺(ethylenediamin)修饰，然后依次经过戊二醛活化(FDHIGLU)及醛基官能化(FDHIALD)。当使用醛基官能化的Immobead 150作为载体时，分别获得最高的固定化产率和活性产率，分别为90％和132％。在35℃下，游离FDH、FDHI150、FDHIGLU和FDHIALD的半衰期(t1/2)分别计算为10.6、28.9、22.4和38.5小时。FDHI150、FDHIGLU和FDHIALD在10次重复使用后分别保留了其初始活动的69％，38％和51％。
Jackon等(Process Biochem.Vol.1,9,Sep 2016,1248-1255)报道了采用乙二醛-琼脂糖对LDH的固定化。与其可溶性对应物相比，固定化LDH获得的热稳定因子大1600倍。
综上可知，现有技术中仍存在大量的酶尚无有效的固定化酶形式，对这些酶的固定化以提高酶的可循环利用性便成了亟待解决的问题。
发明内容
本发明的主要目的在于提供一种固定化酶、其制备方法及其应用，以解决现有技术此类酶难以循环利用的问题。
为了实现上述目的，根据本发明的一个方面，提供了一种固定化酶，该固定化酶包括酶及固定酶的氨基树脂载体，酶选自转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶中的任意一种，氨基树脂载体为交联剂修饰的氨基树脂载体，交联剂为经过高聚物处理过的交联剂。
进一步地，转氨酶为来源于Chromobacterium violaceum DSM30191的转氨酶或来源于Aspergillus fumigatus的转氨酶或者来源于Arthrobacter citreus的转氨酶，优选地，来源于Chromobacterium violaceum DSM30191的转氨酶为具有SEQ ID NO：2或SEQ IDNO：3所示序列的突变体；来源于Arthrobacter citreus的转氨酶为具有SEQ ID NO：5或SEQID NO：6所示序列的突变体；
优选地，酮还原酶为来源于Acetobacter sp.CCTCC M209061或者Candidamacedoniensis AKU4588的酮还原酶，更优选来源于Acetobacter sp.CCTCC M209061的酮还原酶为具有SEQ ID NO：8或SEQ ID NO：9所示序列的突变体；
优选地，单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的单加氧酶，更优选地，来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEQ ID NO：11或SEQ ID NO：12所示序列的突变体；来源于Rhodococcus ruber-SD1的环己酮单加氧酶为具有SEQ ID NO：14或SEQ ID NO：15所示序列的突变体；
优选地，氨裂解酶为来源于photorhabdus luminescens或Solenostemonscutellarioides的氨裂解酶；优选地，烯还原酶为来源于Saccharomyces cerevisiae或Chryseobacterium sp.CA49的烯还原酶；优选地，亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；优选地，氨基酸脱氢酶为来源于Bacillus cereus的亮氨酸脱氢酶或者来源于Bacillus sphaericus的苯丙氨酸脱氢酶；优选地，腈水解酶为来源于Aspergillus niger CBS 513.88或Neurospora crassa OR74A的腈水解酶。
进一步地，氨基树脂载体为带有C2或C4连接臂的氨基树脂载体，优选地，氨基树脂载体选自如下任意一种：LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HM100D、ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、ESR-1、ESR-3、ESR-5及ESR-8。
进一步地，交联剂为戊二醛，高聚物为PEG或PEI；优选地，PEG选自PEG400～PEG6000中的任一种；优选地，PEI选自分子量为3～70KDa的PEI；更优选地，PEG与戊二醛的质量比为1:1～10:1，进一步优选为2：1～5:1；更优选地，PEI与戊二醛的质量比为3：1～1：5，进一步优选为1：1～1：2。
在本申请的第二个方面提，供了一种固定化酶的制备方法，该制备方法包括：采用高聚物对交联剂进行前处理，得到处理交联剂；利用处理交联剂对氨基树脂载体进行修饰，得到修饰载体；将酶固定于修饰载体上，得到固定化酶；其中，酶选自转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶中的任意一种。
进一步地，转氨酶为来源于Chromobacterium violaceum DSM30191的转氨酶或来源于Aspergillus fumigatus的转氨酶或者来源于Arthrobacter citreus的转氨酶，优选地，来源于Chromobacterium violaceum DSM30191的转氨酶为具有SEQ ID NO：2或SEQ IDNO：3所示序列的突变体；来源于Arthrobacter citreus的转氨酶为具有SEQ ID NO：5或SEQID NO：6所示序列的突变体；
优选地，酮还原酶为来源于Acetobacter sp.CCTCC M209061或者Candidamacedoniensis AKU4588的酮还原酶，更优选来源于Acetobacter sp.CCTCC M209061的酮还原酶为具有SEQ ID NO：8或SEQ ID NO：9所示序列的突变体；
优选地，单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的单加氧酶，更优选地，来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEQ ID NO：11或SEQ ID NO：12所示序列的突变体；来源于Rhodococcus ruber-SD1的环己酮单加氧酶为具有SEQ ID NO：14或SEQ ID NO：15所示序列的突变体；
优选地，氨裂解酶为来源于photorhabdus luminescens或Solenostemonscutellarioides的氨裂解酶；优选地，烯还原酶为来源于Saccharomyces cerevisiae或Chryseobacterium sp.CA49的烯还原酶；优选地，亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；优选地，氨基酸脱氢酶为来源于Bacillus cereus的亮氨酸脱氢酶或者来源于Bacillus sphaericus的苯丙氨酸脱氢酶；优选地，腈水解酶为来源于Aspergillus niger CBS 513.88或Neurospora crassa OR74A的腈水解酶。
进一步地，氨基树脂载体为带有C2或C4连接臂的氨基树脂载体，优选地，氨基树脂载体选自如下任意一种：LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HM100D、ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、ESR-1、ESR-3、ESR-5及ESR-8。
进一步地，交联剂为戊二醛，高聚物为PEG或PEI，优选地，PEG选自PEG400～PEG6000中的任一种；优选地，PEI选自分子量为3～70KDa的PEI。
进一步地，PEG与戊二醛的质量比为1:1～10:1，优选为2:1～5:1；优选地，PEI与戊二醛的质量比为3:1～1:5，更优选为1:1～1:2。
根据本申请的第三个方面，提供了上述任一种固定化酶，或者上述任一种制备方法制备而成的固定化酶在生物催化反应中的应用。
进一步地，生物催化反应为连续化的生物催化反应或批量反应，优选地，固定化酶在以水相或有机相反应条件下的循环利用次数为6～16次。
应用本发明的技术方案，通过采用高聚物处理过的交联剂对氨基树脂载体进行修饰，有利于使固定于其上的酶形成网状交联，进而使上述酶的固定效果更加稳定，从而提高这些酶的循环利用效率。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
氨基树脂：该树脂可以在用于酶固定之前，先用戊二醛预活化，然后使载体上的醛基与酶分子上的氨基发生反应形成Schiff碱，构建牢固的多点共价键合位点。具有长或短的氨基连接臂。
酶吸附树脂载体：该类型树脂载体以物理吸附的原理把酶固定在不溶于水的载体树脂表面，固定化方法温和，几乎不改变酶的构象，对酶活性中心也无损害，特别适用于有机溶剂或疏水性溶剂中的固定化，同时固定化过程不需要任何其他试剂。
离子吸附酶载体树脂，在较高的离子强度内可以与酶分子形成离子相互作用力，从而吸附固定酶。其吸附是可逆的，但吸附力强于范德华力，当酶活力消失后，载体可以回收重新利用。
如背景技术所提到的，现有技术中仍有许多酶未实现固定化，循环利用率有限，为改善这一现状，在本申请一种典型的实施方式中，提供了一种固定化酶，该固定化酶包括酶及固定酶的氨基树脂载体，酶选自转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶中的任意一种，氨基树脂载体为交联剂修饰的氨基树脂载体，交联剂为经过高聚物处理过的交联剂。
通过采用高聚物处理过的交联剂对氨基树脂载体进行修饰，有利于使固定于其上的酶形成网状交联，进而使这些酶的固定效果更加稳定，从而提高这些酶的循环利用效率。
上述固定化酶中，酶在氨基树脂载体上的固定形式不限，可以是共价固定的，也可以是非共价固定。共价固定的形式稳定性更强，因而在一种优选的实施例中，酶共价键固定于氨基树脂载体上。
上述固定化酶中酶的具体种类选自转氨酶(Transaminase，本申请中简称TA)、酮还原酶(Ketoreductase，本申请中简称KRED)、单加氧酶(Cyclohexanone monooxygenase，本申请中简称CHMO)、氨裂解酶(Phenylalanine Ammonia lyase，本申请中简称PLA)、烯还原酶(Ene Reductase，本申请中简称ERED)、亚胺还原酶(Imine Reductase，本申请中简称IRED)、氨基酸脱氢酶(Amino acid Dehydrogenase，本申请中简称AADH)及腈水解酶(Nitrilase，本申请中简称NIT)中的任意一种。
上述酶所参与反应的化学过程简述如下：
上述反应式中的R，R1和R2可以各自独立的选自H、取代或未取代的烷基、取代或未取代的环烷基、取代或未取代的芳烷基、取代或未取代的杂环基、取代或未取代的杂环烷基，或R1与其所连接的杂环形成稠环体系。
上述各种酶的具体种类或物种来源也无特殊限定，可以根据实际需要选择所用种类即可。在本申请一种优选的实施例中，转氨酶为来源于Chromobacterium violaceumDSM30191的转氨酶或来源于Aspergillus fumigatus的转氨酶或者来源于Arthrobactercitreus的转氨酶，优选地，来源于Chromobacterium violaceum DSM30191的转氨酶为具有SEQ ID NO：2或SEQ ID NO：3所示序列的突变体；来源于Arthrobacter citreus的转氨酶为具有SEQ ID NO：5或SEQ ID NO：6所示序列的突变体。
在本申请另一种优选的实施例中，酮还原酶为来源于Acetobacter sp.CCTCCM209061或者Candida macedoniensis AKU4588的酮还原酶，更优选来源于Acetobactersp.CCTCC M209061的酮还原酶为具有SEQ ID NO：8或SEQ ID NO：9所示序列的突变体。
在本申请另一种优选的实施例中，单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的单加氧酶，更优选地，来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEQ ID NO：11或SEQ ID NO：12所示序列的突变体；来源于Rhodococcusruber-SD1的环己酮单加氧酶为具有SEQ ID NO：14或SEQ ID NO：15所示序列的突变体。
在本申请另一种优选的实施例中，氨裂解酶为来源于photorhabdus luminescens的氨裂解酶或来源于Solenostemon scutellarioides的氨裂解酶；优选地，烯还原酶为来源于Saccharomyces cerevisiae的烯还原酶或来源于Chryseobacterium sp.CA49的烯还原酶；优选地，亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；优选地，氨基酸脱氢酶为来源于Bacillus cereus的亮氨酸脱氢酶或者来源于Bacillussphaericus的苯丙氨酸脱氢酶；优选地，腈水解酶为来源于Aspergillus niger CBS513.88的腈水解酶或来源于Neurospora crassa OR74A的腈水解酶。
需要说明的是，上述各种来源的酶，如无特殊标明为突变体的，均指野生型，具体的序列可以从NCBI上查询得到。
上述固定化酶中的氨基树脂载体可以是现有市售的种类。在本申请中优选氨基树脂载体为带有C2或C4连接臂的氨基树脂载体。同一载体对不同酶的固定效果也存在一定差异，具体的具有C2和C4连接臂的氨基树脂载体的种类可以根据实际酶种类的不同从现有种类中进行优化选择得到。在本申请一种优选的实施例中，氨基树脂载体选自如下任意一种LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HM100D、ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、ESR-1、ESR-3、ESR-5及ESR-8。
其中，LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HM100D为SUNRISE公司的产品，ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415为Purolite公司的产品，ESR-1、ESR-3、ESR-5及ESR-8为南开合成公司的产品。
上述种类中LX1000HA、ECR8409和LX1000EPN载体对转氨酶的固定化效果相对最好；LX1000HA和ESR-1载体对酮还原酶的固定化效果相对最好；LX1000HA和ECR8409载体对环己酮单加氧酶酶的固定化效果相对最好；LX1000HA、LX1000EA、ECR8309和ECR8409等载体对烯还原酶的固定化效果相对最好；LX1000HA载体对腈水解酶的固定化效果相对最好；ECR8409和LX1000EPN对亚胺还原酶的固定化效果相对最好；LX1000EPN和ECR8309对氨裂解酶的固定化效果相对最好；LX1000HA和ECR8409载体对氨基酸脱氢酶的固定化效果相对最好。
上述固定化酶中，交联剂为戊二醛，优选的高聚物为PEG或PEI；优选地，PEG选自PEG400～PEG6000中的任一种；优选地，PEI选自分子量为3～70KDa的PEI；更优选地，PEG与戊二醛的质量比为1:1～10:1，进一步优选为2：1～5:1；更优选地，PEI与戊二醛的质量比为3：1～1：5，进一步优选为1：1～1：2。
上述优选的实施例中，采用高聚物PEG或PEI对戊二醛进行处理，使得戊二醛的醛基与PEG的羟基或PEI的氨基发生共价结合，最后形成分散有醛基、氨基/羟基的网状结构，这种网状结构中的各官能团与酶蛋白间通过共价作用、氢键作用、离子作用、以及疏水作用等多种方式结合，而不是像戊二醛那样仅仅是共价结合，共价键作用极易破坏酶的活性。
PEG或PEI的具体分子量大小，可以根据所固定酶种类的不同进行合理优化选择，在上述分子量范围内，对现有酶类的固定化效果相对更好。PEG或PEI对戊二醛进行处理的质量比在上述范围内时，具有网状结构的交联剂-高聚物组合物中，醛基与氨基/羟基的分布比较均匀。高聚物比例过低，则游离的醛基较多，醛基之间间距小，在与酶蛋白的结合中共价结合方式占主导，导致酶活性较低。若高聚物比例过高，游离的醛基量太少，与酶蛋白结合时因共价结合变弱，固定化酶的稳定性降低。在更优的范围内，具有网状结构的交联剂-高聚物组合物中，醛基与氨基/羟基的比例及分布达到更优，在与酶的结合中，共价作用、氢键作用、离子作用、以及疏水作用等以更优的配比组合，使固定化酶的活性和稳定性进一步提高。其它具有羟基官能团的高聚物，如聚乙烯醇，也可以应用，但其在常温下水溶性差，应用效果受限，故优先推荐使用PEG。而其他具有氨基官能团的高聚物，如聚醚胺也有一定效果，但考虑到其在一定程度上容易引起酶蛋白的变性，故优先推荐使用PEI。
在本申请第二种典型的实施方式中，提供了一种固定化酶的制备方法，该制备方法包括：采用高聚物对交联剂进行前处理，得到处理交联剂；利用处理交联剂对氨基树脂载体进行修饰，得到修饰载体；将酶固定于修饰载体上，得到固定化酶；酶选自转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶中的任意一种。
本申请的制备方法通过采用高聚物对交联剂进行前处理后，使得交联剂具有更多网状结构，进而利用该具有更多网状结构的交联剂对氨基树脂载体进行修饰时，使得载体上也具有更多的网状结构，因而将酶固定于该修饰载体上时，使得对酶的固定化效果更稳定。
在本申请另一种优选的实施例中，转氨酶为来源于Chromobacterium violaceumDSM30191的转氨酶或来源于Aspergillus fumigatus的转氨酶或者来源于Arthrobactercitreus的转氨酶，优选地，来源于Chromobacterium violaceum DSM30191的转氨酶为具有SEQ ID NO：2或SEQ ID NO：3所示序列的突变体；来源于Arthrobacter citreus的转氨酶为具有SEQ ID NO：5或SEQ ID NO：6所示序列的突变体；
在本申请另一种优选的实施例中，酮还原酶为来源于Acetobacter sp.CCTCCM209061或者Candida macedoniensis AKU4588的酮还原酶，更优选来源于Acetobactersp.CCTCC M209061的酮还原酶为具有SEQ ID NO：8或SEQ ID NO：9所示序列的突变体。
在本申请另一种优选的实施例中，单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的单加氧酶，更优选地，来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEQ ID NO：11或SEQ ID NO：12所示序列的突变体；来源于Rhodococcusruber-SD1的环己酮单加氧酶为具有SEQ ID NO：14或SEQ ID NO：15所示序列的突变体。
在本申请另一种优选的实施例中，氨裂解酶为来源于photorhabdus luminescens的氨裂解酶或来源于Solenostemon scutellarioides的氨裂解酶；优选地，烯还原酶为来源于Saccharomyces cerevisiae的烯还原酶或来源于Chryseobacterium sp.CA49的烯还原酶；优选地，亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；优选地，氨基酸脱氢酶为来源于Bacillus cereus的亮氨酸脱氢酶或者来源于Bacillussphaericus的苯丙氨酸脱氢酶；优选地，腈水解酶为来源于Aspergillus niger CBS513.88的腈水解酶或来源于Neurospora crassa OR74A的腈水解酶。
上述物种来源的酶，固定于经过高聚物处理过的交联剂修饰后的氨基树脂载体上形成的固定化酶的活性及稳定性都相对较好，因而循环利用次数也较高。
上述制备方法中，氨基树脂载体可以根据酶类的不同选择合适的载体。在一种优选的实施例中，氨基树脂载体为带有C2或C4连接臂的氨基树脂载体，优选地，氨基树脂载体选自如下任意一种：LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HM100D、ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、ESR-1、ESR-3、ESR-5及ESR-8。选择上述种类的案件树脂载体有利于实现对多种酶类的固定化。
上述制备方法中，交联剂可以根据实际需要从现有的交联剂种类中进行优化选择。高聚物的主要作用是对交联剂中游离的醛基进行结合，从而形成具有网状结构的交联剂-高聚物组合物，以降低对待固定的酶活性的影响。
在一种优选的实施例中，交联剂为戊二醛，高聚物为PEG或PEI，优选地，PEG选自PEG400～PEG6000中的任一种；优选地，PEI选自分子量为3～70KDa的PEI。
上述优选的实施例中，PEG或PEI的具体分子量大小，可以根据所固定酶种类的不同进行合理优化选择，在上述分子量范围内，对现有酶类的固定化效果相对更好。
上述制备方法中，PEG与戊二醛的质量比为1:1～10:1，优选为2：1～5:1；优选地，PEI与戊二醛的质量比为3：1～1：5，更优选为1：1～1:2。
具有羟基或氨基的高聚物均适用于本申请，但上述实施例优选采用PGE或PEI。其它具有羟基官能团的高聚物，如聚乙烯醇，也可以应用，但其在常温下水溶性差，应用效果受限，故优先推荐使用PEG。而其他具有氨基官能团的高聚物，如聚醚胺也有一定效果，但考虑到其在一定程度上容易引起酶蛋白的变性，故优先推荐使用PEI。
PEG或PEI对戊二醛进行处理的质量比在上述范围内时，具有网状结构的交联剂-高聚物组合物中，醛基与氨基/羟基的分布比较均匀。高聚物比例过低，则游离的醛基较多，醛基之间间距小，在与酶蛋白的结合中共价结合方式占主导，导致酶活性较低。若高聚物比例过高，游离的醛基量太少，与酶蛋白结合时因共价结合变弱，固定化酶的稳定性降低。在更优的范围内，具有网状结构的交联剂-高聚物组合物中，醛基与氨基/羟基的比例及分布达到更优，在与酶的结合中，共价作用、氢键作用、离子作用、以及疏水作用等以更优的配比组合，使固定化酶的活性和稳定性进一步提高。
在本申请第三种典型的实施例中，还提供了上述任一种固定化酶，或者上述任一种制备方法制备而成的固定化酶在生物催化反应中的应用。该固定化酶具有稳定性高，循环利用效率高的优势，因而在生物催化反应中可多次重复使用。
在一种更优选的实施例中，上述固定化酶所应用的生物催化反应为连续化的生物催化反应或批次反应。固定化酶循环利用效率高，因而适合应用于连续化的生物催化反应，提高反应效率。
上述物种来源的酶，固定于经过高聚物处理过的交联剂修饰后的氨基树脂载体上形成的固定化酶的活性及稳定性都相对较好，因而循环利用次数也较高。在一种优选的实施例中，上述固定化酶在水相或有机相反应条件下的循环利用次数为6～16次。
下面将结合具体的实施例来进一步说明本申请的有益效果。
以下实施例中用到的酶及其来源见下表1。部分酶的序列见表2至表6。
表1：
表2：
表3：
表4：
表5：
表6：
下列实施例中的PB代表磷酸缓冲液的意思。
实施例1：固定化的转氨酶
取1g氨基树脂，用20mM的磷酸缓冲液(简称PB，pH7.0)冲洗后备用。
向4mL PB缓冲液(20mM,pH 7.0)中添加160μL戊二醛(50％水溶液)，并添加合适比例的PEG或PEI，室温孵育30-60min后，向溶液中添加上述氨基树脂，接着在20-25℃下孵育1-2h。过滤后得到修饰的氨基树脂，然后向修饰后的氨基树脂中添加4mL酶溶液(20-25mg/mL蛋白，包括主酶及其相应的辅酶)，然后置于20℃孵育16-24h。最后用20mM PB(pH为8.0包含0.5M NaCl)冲洗3次。
固定化转氨酶活性及重复使用性测试：
在10mL的反应瓶中，加入0.3mL MeOH，溶解0.1g主原料1或主原料2，并加入4eq异丙胺盐酸盐和1.0mg PLP(5’-磷酸吡哆醛)，补加0.1M PB 7.0至反应液终体积为1mL，再加入0.1g酶粉或由0.1g酶粉制备的交联酶聚集体湿酶或交联酶聚集体冻干粉，在30℃搅拌16-20h。体系经HPLC检测转化率，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。反应数据如下：
表7：
实施例2：固定化的酮还原酶(ketoreductases)的转化率及再利用性测试
固定化方式同实施例1
固定化酶活性及重复使用性检测：
在10mL的反应瓶中，加入0.5mL异丙醇(IPA)，溶解0.1g主原料3或4，并加入0.5mL0.1M PB 7.0和1-10mg NAD+，再加入0.05g酶粉或由0.1-0.3g酶粉制备的固定化酶，在30℃搅拌16-20h。体系经GC检测转化率，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。反应数据如下：
表8：
实施例3：固定化的CHMOs的转化率及再利用性测试
固定化方式同实施例1
CHMO氨基载体固定化酶的活性通过利用以下底物5进行反应来检测
0.3mL的异丙醇装入10ml的反应瓶中，随后加入500mg的底物5，3mL含有5mg NADP+的0.1M PB(pH 8.0)，然后加入50mg醇脱氢酶ADH-Tb游离酶及100-200mg的CHMO氨基载体共固定化酶(湿的，含有50～80％的水)。在30℃下反应16-20小时，测试转化率，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。结果见下表。
表9.
实施例4：固定化EREDs的转化率及再利用性测试
固定化方式同实施例1
ERED氨基载体固定化酶的活性通过利用以下底物6进行反应来检测
3mL的0.1M PB(pH7.0-8.0)装入10ml的反应瓶中，随后通过加入100mg的底物6，接着加入10mg NAD(P)+，80mg甲酸铵，20mg FDH及100mg的ERED固定化酶。在30℃下反应16-20小时，测试转化率，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。测试结果见下表。
表10.
实施例5：固定化的NITs的转化率及再利用性测试
固定化方式同实施例1
NIT氨基载体固定化酶的活性通过利用以下底物7进行反应来检测
将2mL 0.1M PB缓冲液(pH 7.0-8.0)加入10mL反应瓶中，并加入100mg上述底物9，然后加入含有200mg NIT的氨基载体固定化酶。在30℃下反应16小时后，检测转化率，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。测试结果见下表。
表11：
实施例6：固定化的IREDs的转化率及再利用性测试
固定化方式同实施例1
IRED氨基载体固定化酶的活性通过利用以下底物8进行反应来检测
将2mL 0.1M PB缓冲液(pH 7.0-8.0)加入10mL反应球中，然后加入100mg上述底物8、10mg NAD+、60mg甲酸铵、10mg FDH和含有400mg IRED氨基载体固定化酶。在30℃下反应20小时后，检测转化率，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。测试结果如下。
表12：
实施例7：固定化的PALs的转化率及再利用性测试
固定化方式同实施例1
PAL氨基载体固定化酶的活性通过利用以下底物9进行反应来检测
将8mL 4M氨基甲酸铵水溶液(pH 9.0～9.5)加入10mL反应瓶中，并加入100mg上述底物10，然后加入含有400mg NIT固定化酶。在30℃下反应16-20小时后，检测转化率，每一轮反应结束后将固定化酶分离出来，下一轮反应中重复使用，考察重复使用次数。测试结果见下表。
表13.
实施例8：固定化的AADHs的转化率及再利用性测试
在10mL的反应瓶中，加入5mL 0.1M Tris-Cl(pH 8.0-9.0)，随后加入100mg主原料10、11或12，108mg氯化铵(ammonium chloride)，调节pH值至7.5-8.0，接着加10-50mg NAD+、50mg GDH以及100mg AADH酶(或者由100-300mg游离酶制备的固定化的AADH)。在30℃下反应16-20h后用于转化率测试。测试结果见下表。
表14：
实施例9转氨酶氨基载体固定化酶在填充床连续反应中的应用
实施例1中转氨酶TA-Cv固定化至载体LX1000HA，交联剂GA经PEI(3KDa):GA＝1:1修饰，所得固定化酶填充至120mL柱体积的柱状反应器中，固定化酶用量72g。
500g底物1，用1.5L的甲醇溶解，并加入4eq的异丙胺盐酸盐(1.8L的6M异丙胺盐酸盐水溶液)和5g PLP，不加PB缓冲液(0.1M,pH8.0)定容至5L。
设置流速0.6mL/min，即保留时间200min，进行连续化反应，出口端流出液检测转化率，转化率＞98％，持续运行400h，转化率无降低，运行420h，转化率降低至89％。具体见下表。
表15：
实施例10转氨酶固定化酶在连续搅拌罐反应中的应用
使用同实施例1的固定化酶TA-Ac，载体为ECR8409,所用交联剂GA经PEG6000:GA＝5:2修饰。200mL反应器中加入50g转氨酶TA-Ac的固定化酶，加入150mL磷酸缓冲液。
500g底物1，加入3.2L PB(0.1M,pH 7.0)，1.8L异丙胺盐酸盐水溶液(6M)和5gPLP，打浆制成混悬液。
以0.4mL/min的速度向反应瓶中连续添加底物混悬液(即保留时间500min)，同时以同样的流速在出口抽出反应体系(管道末端加过滤头，防止将固定化酶抽出)。在该条件下，转化率可达90％以上，且连续运行400h，转化率基本无降低。结果如下表所示。
表16：
实施例11
实施例7制备的氨裂解酶PAL-Ss固定化酶，载体为LX1000EPN，交联剂经PEG6000:GA＝5:2修饰。所得固定化酶6g填充至10mL柱状反应器。
500g底物9，用4.5L的氨基甲酸铵水溶液(4M,pH9.0～9.5)溶解。
设置流速0.03mL/min，即保留时间330min，进行连续化反应，出口端流出液检测转化率，转化率80％，持续运行360h，转化率无降低，运行400h，转化率降低至72％。见下表。
表17.
实施例12
实施例2制备的酮还原酶KRED-Ac固定化酶，载体为LX1000HA，交联剂经PEI(3KDa:GA＝1:2修饰。所得固定化酶6g填充至10mL柱状反应器。。
100g底物3，用0.3L的异丙醇溶解，加入0.7LPB缓冲液(0.1M,pH7.0)溶解，然后加入0.1g NAD+。
设置流速0.05mL/min，即保留时间200min，进行连续化反应，出口端流出液检测转化率，转化率＞90％，持续运行200h，转化率无降低，运行220h，转化率降低至84％。见下表。
表18：KRED-Ac固定化酶在填充床连续反应中的反应结果
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：通过采用高聚物处理过的交联剂对氨基树脂载体进行修饰，有利于使固定于其上的酶形成网状交联，进而使酶的固定效果更加稳定，从而提高酶的循环利用效率。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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130 135 140
Glu His Asp Tyr His Gly Trp Thr Gly Gly Ala Ala Thr Val Thr Arg
145 150 155 160
Leu Arg Ser Leu Arg Ser Gly Leu Val Gly Glu Asn Ser Glu Ser Phe
165 170 175
Ser Leu Gln Val Pro Gly Ser Ser Cys Gly Ala Ala Val Leu Met Ala
180 185 190
Pro Ser Ser Asn Thr Phe Gln Asp Ser Asn Gly Asn Tyr Leu Lys Asp
195 200 205
Glu Asn Gly Glu Leu Leu Ser Val Lys Tyr Thr Arg Arg Met Ile Glu
210 215 220
Asn Tyr Gly Pro Glu Gln Val Ala Ala Val Ile Thr Glu Val Ser Gln
225 230 235 240
Gly Val Gly Ser Thr Met Pro Pro Tyr Glu Tyr Ile Pro Gln Ile Arg
245 250 255
Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Ser Asp Glu Val Leu
260 265 270
Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly
275 280 285
Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser
290 295 300
Leu Pro Ala Gly Ala Val Val Val Ser Lys Glu Ile Ala Ala Phe Met
305 310 315 320
Asp Lys His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val
325 330 335
Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn
340 345 350
Leu Val Glu Gln Ala Lys Asn Ser Gly Glu Tyr Ile Arg Ser Lys Leu
355 360 365
Glu Ala Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Phe
370 375 380
Gly Leu Leu Trp Phe Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro
385 390 395 400
Tyr Val Lys Leu Asp Arg Asn Phe Arg His Asp Met Asn Pro Asn Gln
405 410 415
Ile Pro Thr Gln Ile Ile Lys Gln Lys Ala Leu Glu Lys Gly Val Leu
420 425 430
Ile Gly Gly Ala Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn
435 440 445
Val Ser Arg Gly Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala
450 455 460
Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Gln Ser
465 470 475
<210> 7
<211> 253
<212> PRT
<213> 酮还原酶(Acetobacter sp. )
<220>
<221> SITE
<222> (1)..(253)
<223> KRED-Ac-母本
<400> 7
Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn
1 5 10 15
Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys
20 25 30
Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala
35 40 45
Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val
50 55 60
Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu
65 70 75 80
Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Ala Tyr Ser
85 90 95
Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser
100 105 110
Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala
115 120 125
Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Glu
130 135 140
Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Asn Ala Ser Lys Gly
145 150 155 160
Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser
165 170 175
Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr
180 185 190
Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys
195 200 205
Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile
210 215 220
Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr
225 230 235 240
Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 8
<211> 253
<212> PRT
<213> 酮还原酶(Acetobacter sp. )
<220>
<221> VARIANT
<222> (1)..(253)
<223> KRED-Ac-V1
<220>
<221> MUTAGEN
<222> (1)..(253)
<223> E144S+A94N+N156V
<400> 8
Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn
1 5 10 15
Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys
20 25 30
Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala
35 40 45
Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val
50 55 60
Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu
65 70 75 80
Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Asn Tyr Ser
85 90 95
Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser
100 105 110
Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala
115 120 125
Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Ser
130 135 140
Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Val Ala Ser Lys Gly
145 150 155 160
Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser
165 170 175
Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr
180 185 190
Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys
195 200 205
Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile
210 215 220
Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr
225 230 235 240
Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 9
<211> 253
<212> PRT
<213> 酮还原酶(Acetobacter sp. )
<220>
<221> VARIANT
<222> (1)..(253)
<223> KRED-Ac-V2
<220>
<221> MUTAGEN
<222> (1)..(253)
<223> E144S+A94T+N156T
<400> 9
Met Ala Arg Val Ala Gly Lys Val Ala Ile Val Ser Gly Ala Ala Asn
1 5 10 15
Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys
20 25 30
Val Val Ile Gly Asp Leu Lys Glu Glu Asp Gly Gln Lys Ala Val Ala
35 40 45
Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val
50 55 60
Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu
65 70 75 80
Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Thr Tyr Ser
85 90 95
Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser
100 105 110
Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala
115 120 125
Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Ser
130 135 140
Gly Leu Ile Gly Asp Pro Met Leu Ala Ala Tyr Thr Ala Ser Lys Gly
145 150 155 160
Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser
165 170 175
Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr
180 185 190
Pro Met Val Ala Gly Leu Thr Lys Glu Asp Ala Ala Ala Arg Gln Lys
195 200 205
Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile
210 215 220
Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr
225 230 235 240
Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln
245 250
<210> 10
<211> 541
<212> PRT
<213> 环己酮单加氧酶(Rhodococcus sp. Phi1)
<220>
<221> SITE
<222> (1)..(541)
<223> CHMO-Rs-母本
<400> 10
Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly
1 5 10 15
Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln
20 25 30
Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr
35 40 45
Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His
50 55 60
Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp
65 70 75 80
Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser
85 90 95
Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu
100 105 110
Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser
115 120 125
Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val
130 135 140
Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr
145 150 155 160
Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn
165 170 175
Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln
180 185 190
Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe
195 200 205
Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr
210 215 220
Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp
225 230 235 240
Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu
245 250 255
Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu
260 265 270
Ala Trp Asp His Gly Gly Gly Phe Arg Phe Met Phe Gly Thr Phe Gly
275 280 285
Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile
290 295 300
Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys
305 310 315 320
Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly
325 330 335
Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys
340 345 350
Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp
355 360 365
Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp
370 375 380
Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly
385 390 395 400
Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly
405 410 415
Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn
420 425 430
Gly Pro Phe Thr Asn Leu Pro Pro Ser Ile Glu Thr Gln Val Glu Trp
435 440 445
Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile
450 455 460
Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp
465 470 475 480
Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly
485 490 495
Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Phe Tyr Leu Gly Gly
500 505 510
Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr
515 520 525
Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala
530 535 540
<210> 11
<211> 541
<212> PRT
<213> 环己酮单加氧酶(Rhodococcus sp. Phi1)
<220>
<221> VARIANT
<222> (1)..(541)
<223> CHMO-Rs-V1
<220>
<221> MUTAGEN
<222> (1)..(541)
<223> F508Y+F435N+L438A+T436S+F280V+S441V
<400> 11
Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly
1 5 10 15
Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln
20 25 30
Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr
35 40 45
Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His
50 55 60
Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp
65 70 75 80
Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser
85 90 95
Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu
100 105 110
Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser
115 120 125
Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val
130 135 140
Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr
145 150 155 160
Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn
165 170 175
Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln
180 185 190
Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe
195 200 205
Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr
210 215 220
Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp
225 230 235 240
Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu
245 250 255
Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu
260 265 270
Ala Trp Asp His Gly Gly Gly Val Arg Phe Met Phe Gly Thr Phe Gly
275 280 285
Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile
290 295 300
Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys
305 310 315 320
Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly
325 330 335
Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys
340 345 350
Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp
355 360 365
Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp
370 375 380
Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly
385 390 395 400
Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly
405 410 415
Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn
420 425 430
Gly Pro Asn Ser Asn Ala Pro Pro Val Ile Glu Thr Gln Val Glu Trp
435 440 445
Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile
450 455 460
Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp
465 470 475 480
Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly
485 490 495
Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Tyr Tyr Leu Gly Gly
500 505 510
Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr
515 520 525
Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala
530 535 540
<210> 12
<211> 541
<212> PRT
<213> 环己酮单加氧酶(Rhodococcus sp. Phi1)
<220>
<221> VARIANT
<222> (1)..(541)
<223> CHMO-Rs-V2
<220>
<221> MUTAGEN
<222> (1)..(541)
<223> F508Y+F435N+L438A+T436S+F280V+S441V+L510V
<400> 12
Met Thr Ala Gln Ile Ser Pro Thr Val Val Asp Ala Val Val Ile Gly
1 5 10 15
Ala Gly Phe Gly Gly Ile Tyr Ala Val His Lys Leu His Asn Glu Gln
20 25 30
Gly Leu Thr Val Val Gly Phe Asp Lys Ala Asp Gly Pro Gly Gly Thr
35 40 45
Trp Tyr Trp Asn Arg Tyr Pro Gly Ala Leu Ser Asp Thr Glu Ser His
50 55 60
Leu Tyr Arg Phe Ser Phe Asp Arg Asp Leu Leu Gln Asp Gly Thr Trp
65 70 75 80
Lys Thr Thr Tyr Ile Thr Gln Pro Glu Ile Leu Glu Tyr Leu Glu Ser
85 90 95
Val Val Asp Arg Phe Asp Leu Arg Arg His Phe Arg Phe Gly Thr Glu
100 105 110
Val Thr Ser Ala Ile Tyr Leu Glu Asp Glu Asn Leu Trp Glu Val Ser
115 120 125
Thr Asp Lys Gly Glu Val Tyr Arg Ala Lys Tyr Val Val Asn Ala Val
130 135 140
Gly Leu Leu Ser Ala Ile Asn Phe Pro Asp Leu Pro Gly Leu Asp Thr
145 150 155 160
Phe Glu Gly Glu Thr Ile His Thr Ala Ala Trp Pro Glu Gly Lys Asn
165 170 175
Leu Ala Gly Lys Arg Val Gly Val Ile Gly Thr Gly Ser Thr Gly Gln
180 185 190
Gln Val Ile Thr Ala Leu Ala Pro Glu Val Glu His Leu Thr Val Phe
195 200 205
Val Arg Thr Pro Gln Tyr Ser Val Pro Val Gly Asn Arg Pro Val Thr
210 215 220
Lys Glu Gln Ile Asp Ala Ile Lys Ala Asp Tyr Asp Gly Ile Trp Asp
225 230 235 240
Ser Val Lys Lys Ser Ala Val Ala Phe Gly Phe Glu Glu Ser Thr Leu
245 250 255
Pro Ala Met Ser Val Ser Glu Glu Glu Arg Asn Arg Ile Phe Gln Glu
260 265 270
Ala Trp Asp His Gly Gly Gly Val Arg Phe Met Phe Gly Thr Phe Gly
275 280 285
Asp Ile Ala Thr Asp Glu Ala Ala Asn Glu Ala Ala Ala Ser Phe Ile
290 295 300
Arg Ser Lys Ile Ala Glu Ile Ile Glu Asp Pro Glu Thr Ala Arg Lys
305 310 315 320
Leu Met Pro Thr Gly Leu Tyr Ala Lys Arg Pro Leu Cys Asp Asn Gly
325 330 335
Tyr Tyr Glu Val Tyr Asn Arg Pro Asn Val Glu Ala Val Ala Ile Lys
340 345 350
Glu Asn Pro Ile Arg Glu Val Thr Ala Lys Gly Val Val Thr Glu Asp
355 360 365
Gly Val Leu His Glu Leu Asp Val Leu Val Phe Ala Thr Gly Phe Asp
370 375 380
Ala Val Asp Gly Asn Tyr Arg Arg Ile Glu Ile Arg Gly Arg Asn Gly
385 390 395 400
Leu His Ile Asn Asp His Trp Asp Gly Gln Pro Thr Ser Tyr Leu Gly
405 410 415
Val Thr Thr Ala Asn Phe Pro Asn Trp Phe Met Val Leu Gly Pro Asn
420 425 430
Gly Pro Asn Ser Asn Ala Pro Pro Val Ile Glu Thr Gln Val Glu Trp
435 440 445
Ile Ser Asp Thr Val Ala Tyr Ala Glu Arg Asn Glu Ile Arg Ala Ile
450 455 460
Glu Pro Thr Pro Glu Ala Glu Glu Glu Trp Thr Gln Thr Cys Thr Asp
465 470 475 480
Ile Ala Asn Ala Thr Leu Phe Thr Arg Gly Asp Ser Trp Ile Phe Gly
485 490 495
Ala Asn Val Pro Gly Lys Lys Pro Ser Val Leu Tyr Tyr Val Gly Gly
500 505 510
Leu Gly Asn Tyr Arg Asn Val Leu Ala Gly Val Val Ala Asp Ser Tyr
515 520 525
Arg Gly Phe Glu Leu Lys Ser Ala Val Pro Val Thr Ala
530 535 540
<210> 13
<211> 603
<212> PRT
<213> 环己酮单加氧酶(Rhodococcus ruber-SD1)
<220>
<221> SITE
<222> (1)..(603)
<223> CHMO-Rr-母本
<400> 13
Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu
1 5 10 15
Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg
20 25 30
Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Met Pro Ile Thr
35 40 45
Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly
50 55 60
Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val
65 70 75 80
Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp
85 90 95
Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val
100 105 110
Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr
115 120 125
Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His
130 135 140
Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu
145 150 155 160
Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly
165 170 175
Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Pro Leu His
180 185 190
Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys
195 200 205
Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala
210 215 220
Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile
225 230 235 240
Gly Thr Gly Ala Thr Ala Val Gln Cys Val Pro Glu Leu Ala Lys Tyr
245 250 255
Cys Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu
260 265 270
Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr
275 280 285
Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp
290 295 300
Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu
305 310 315 320
Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val
325 330 335
Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala
340 345 350
Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val
355 360 365
Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp
370 375 380
Phe Arg Gln Met Cys Lys Arg Pro Cys Phe His Asp Asp Tyr Leu Pro
385 390 395 400
Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly
405 410 415
Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr
420 425 430
Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr
435 440 445
Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val
450 455 460
Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met
465 470 475 480
His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly
485 490 495
Ala Ala Leu Gly Ser Asn Ile Pro His Asn Phe Val Glu Ala Ala Arg
500 505 510
Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser
515 520 525
Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu
530 535 540
Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Tyr Tyr
545 550 555 560
Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly
565 570 575
Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu
580 585 590
Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg
595 600
<210> 14
<211> 603
<212> PRT
<213> 环己酮单加氧酶(Rhodococcus ruber-SD1)
<220>
<221> VARIANT
<222> (1)..(603)
<223> CHMO-Rr-V1
<220>
<221> MUTAGEN
<222> (1)..(603)
<223> P190L + Y559M + C249V + C393V + C257A + M45T
<400> 14
Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu
1 5 10 15
Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg
20 25 30
Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Thr Pro Ile Thr
35 40 45
Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly
50 55 60
Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val
65 70 75 80
Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp
85 90 95
Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val
100 105 110
Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr
115 120 125
Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His
130 135 140
Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu
145 150 155 160
Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly
165 170 175
Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Leu Leu His
180 185 190
Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys
195 200 205
Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala
210 215 220
Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile
225 230 235 240
Gly Thr Gly Ala Thr Ala Val Gln Val Val Pro Glu Leu Ala Lys Tyr
245 250 255
Ala Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu
260 265 270
Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr
275 280 285
Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp
290 295 300
Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu
305 310 315 320
Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val
325 330 335
Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala
340 345 350
Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val
355 360 365
Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp
370 375 380
Phe Arg Gln Met Cys Lys Arg Pro Val Phe His Asp Asp Tyr Leu Pro
385 390 395 400
Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly
405 410 415
Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr
420 425 430
Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr
435 440 445
Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val
450 455 460
Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met
465 470 475 480
His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly
485 490 495
Ala Ala Leu Gly Ser Asn Ile Pro His Asn Phe Val Glu Ala Ala Arg
500 505 510
Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser
515 520 525
Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu
530 535 540
Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Met Tyr
545 550 555 560
Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly
565 570 575
Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu
580 585 590
Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg
595 600
<210> 15
<211> 603
<212> PRT
<213> 环己酮单加氧酶(Rhodococcus ruber-SD1)
<220>
<221> VARIANT
<222> (1)..(603)
<223> CHMO-Rr-V2
<220>
<221> MUTAGEN
<222> (1)..(603)
<223> Y559M+P190L+P504V
<400> 15
Met Thr Thr Ser Ile Asp Arg Glu Ala Leu Arg Arg Lys Tyr Ala Glu
1 5 10 15
Glu Arg Asp Lys Arg Ile Arg Pro Asp Gly Asn Asp Gln Tyr Ile Arg
20 25 30
Leu Asp His Val Asp Gly Trp Ser His Asp Pro Tyr Met Pro Ile Thr
35 40 45
Pro Arg Glu Pro Lys Leu Asp His Val Thr Phe Ala Phe Ile Gly Gly
50 55 60
Gly Phe Ser Gly Leu Val Thr Ala Ala Arg Leu Arg Glu Ser Gly Val
65 70 75 80
Glu Ser Val Arg Ile Ile Asp Lys Ala Gly Asp Phe Gly Gly Val Trp
85 90 95
Tyr Trp Asn Arg Tyr Pro Gly Ala Met Cys Asp Thr Ala Ala Met Val
100 105 110
Tyr Met Pro Leu Leu Glu Glu Thr Gly Tyr Met Pro Thr Glu Lys Tyr
115 120 125
Ala His Gly Pro Glu Ile Leu Glu His Cys Gln Arg Ile Gly Lys His
130 135 140
Tyr Asp Leu Tyr Asp Asp Ala Leu Phe His Thr Glu Val Thr Asp Leu
145 150 155 160
Val Trp Gln Glu His Asp Gln Arg Trp Arg Ile Ser Thr Asn Arg Gly
165 170 175
Asp His Phe Thr Ala Gln Phe Val Gly Met Gly Thr Gly Leu Leu His
180 185 190
Val Ala Gln Leu Pro Gly Ile Pro Gly Ile Glu Ser Phe Arg Gly Lys
195 200 205
Ser Phe His Thr Ser Arg Trp Asp Tyr Asp Tyr Thr Gly Gly Asp Ala
210 215 220
Leu Gly Ala Pro Met Asp Lys Leu Ala Asp Lys Arg Val Ala Val Ile
225 230 235 240
Gly Thr Gly Ala Thr Ala Val Gln Cys Val Pro Glu Leu Ala Lys Tyr
245 250 255
Cys Arg Glu Leu Tyr Val Val Gln Arg Thr Pro Ser Ala Val Asp Glu
260 265 270
Arg Gly Asn His Pro Ile Asp Glu Lys Trp Phe Ala Gln Ile Ala Thr
275 280 285
Pro Gly Trp Gln Lys Arg Trp Leu Asp Ser Phe Thr Ala Ile Trp Asp
290 295 300
Gly Val Leu Thr Asp Pro Ser Glu Leu Ala Ile Glu His Glu Asp Leu
305 310 315 320
Val Gln Asp Gly Trp Thr Ala Leu Gly Gln Arg Met Arg Ala Ala Val
325 330 335
Gly Ser Val Pro Ile Glu Gln Tyr Ser Pro Glu Asn Val Gln Arg Ala
340 345 350
Leu Glu Glu Ala Asp Asp Glu Gln Met Glu Arg Ile Arg Ala Arg Val
355 360 365
Asp Glu Ile Val Thr Asp Pro Ala Thr Ala Ala Gln Leu Lys Ala Trp
370 375 380
Phe Arg Gln Met Cys Lys Arg Pro Cys Phe His Asp Asp Tyr Leu Pro
385 390 395 400
Ala Phe Asn Arg Pro Asn Thr His Leu Val Asp Thr Gly Gly Lys Gly
405 410 415
Val Glu Arg Ile Thr Glu Asn Gly Val Val Val Ala Gly Val Glu Tyr
420 425 430
Glu Val Asp Cys Ile Val Tyr Ala Ser Gly Phe Glu Phe Leu Gly Thr
435 440 445
Gly Tyr Thr Asp Arg Ala Gly Phe Asp Pro Thr Gly Arg Asp Gly Val
450 455 460
Lys Leu Ser Glu His Trp Ala Gln Gly Thr Arg Thr Leu His Gly Met
465 470 475 480
His Thr Tyr Gly Phe Pro Asn Leu Phe Val Leu Gln Leu Met Gln Gly
485 490 495
Ala Ala Leu Gly Ser Asn Ile Val His Asn Phe Val Glu Ala Ala Arg
500 505 510
Val Val Ala Ala Ile Val Asp His Val Leu Ser Thr Gly Thr Ser Ser
515 520 525
Val Glu Thr Thr Lys Glu Ala Glu Gln Ala Trp Val Gln Leu Leu Leu
530 535 540
Asp His Gly Arg Pro Leu Gly Asn Pro Glu Cys Thr Pro Gly Met Tyr
545 550 555 560
Asn Asn Glu Gly Lys Pro Ala Glu Leu Lys Asp Arg Leu Asn Val Gly
565 570 575
Tyr Pro Ala Gly Ser Ala Ala Phe Phe Arg Met Met Asp His Trp Leu
580 585 590
Ala Ala Gly Ser Phe Asp Gly Leu Thr Phe Arg
595 600
Claims (14)
1.一种固定化酶，所述固定化酶包括酶及固定所述酶的氨基树脂载体，其特征在于，
所述酶选自转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶中的任意一种；
所述氨基树脂载体为交联剂修饰的氨基树脂载体，其中所述交联剂为经过高聚物处理过的交联剂；
所述氨基树脂载体为带有C2或C4连接臂的氨基树脂载体，所述交联剂为戊二醛，所述高聚物为PEG或PEI，所述PEG选自PEG400～PEG6000中的任一种，所述PEI选自分子量为3～70KDa的PEI，所述PEG与所述戊二醛的质量比1:1～10:1，所述PEI与所述戊二醛的质量比为3:1～1:5；
当所述转氨酶为Aspergillus fumigatus时，所述带有C2或C4连接臂的氨基树脂载体不包括LX1000EPN。
2.根据权利要求1所述的固定化酶，其特征在于，
所述转氨酶为来源于Chromobacterium violaceum DSM3019、Aspergillusfumigatus、Arthrobacter citreus的转氨酶；
所述酮还原酶为来源于Acetobacter sp.CCTCC M209061或者Candida macedoniensisAKU4588的酮还原酶；
所述单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的单加氧酶；
所述氨裂解酶为来源于photorhabdus luminescens或Solenostemonscutellarioides的氨裂解酶；
所述烯还原酶为来源于Saccharomyces cerevisiae或Chryseobacterium sp.CA49的烯还原酶；
所述亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；
所述氨基酸脱氢酶为来源于Bacillus cereus的亮氨酸脱氢酶或者来源于Bacillussphaericus的苯丙氨酸脱氢酶；
所述腈水解酶为来源于Aspergillus niger CBS 513.88或Neurospora crassa OR74A的腈水解酶。
3.根据权利要求2所述的固定化酶，其特征在于，
所述来源于Chromobacterium violaceum DSM30191的转氨酶为具有SEQ ID NO：2 或SEQ ID NO：3所示序列的突变体；
所述来源于Arthrobacter citreus的转氨酶为具有SEQ ID NO：5或SEQ ID NO：6所示序列的突变体；
所述来源于Acetobacter sp.CCTCC M209061的酮还原酶为具有SEQ ID NO：8或SEQ IDNO：9所示序列的突变体；
所述来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEQ ID NO：11或SEQ IDNO：12所示序列的突变体；
所述来源于Rhodococcus ruber-SD1的环己酮单加氧酶为具有SEQ ID NO：14或SEQ IDNO：15所示序列的突变体。
4.根据权利要求1至3中任一项所述的固定化酶，其特征在于，所述带有C2或C4连接臂的氨基树脂载体选自如下任意一种：LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HM100D、ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、ESR-1、ESR-3、ESR-5及ESR-8。
5.根据权利要求1所述的固定化酶，其特征在于，所述PEG与所述戊二醛的质量比为2：1～5:1；所述PEI与所述戊二醛的质量比为1：1～1：2。
6.一种固定化酶的制备方法，其特征在于，所述制备方法包括：
采用高聚物对交联剂进行前处理，得到处理交联剂；
利用所述处理交联剂对氨基树脂载体进行修饰，得到修饰载体；
将酶固定于所述修饰载体上，得到所述固定化酶；
其中，所述酶选自转氨酶、酮还原酶、单加氧酶、氨裂解酶、烯还原酶、亚胺还原酶、氨基酸脱氢酶及腈水解酶中的任意一种；
所述交联剂为戊二醛，所述高聚物为PEG或PEI，所述PEG选自PEG400～PEG6000中的任一种，所述PEI选自分子量为3～70KDa的PEI，所述PEG与所述戊二醛的质量比1:1～10:1，所述PEI与所述戊二醛的质量比为3:1～1:5；所述氨基树脂载体为带有C2或C4连接臂的氨基树脂载体；
当所述转氨酶为Aspergillus fumigatus时，所述带有C2或C4连接臂的氨基树脂载体不包括LX1000EPN。
7.根据权利要求6所述的制备方法，其特征在于，
所述转氨酶为来源于Chromobacterium violaceum DSM30191、Aspergillusfumigatus、或者Arthrobacter citreus的转氨酶；
所述酮还原酶为来源于Acetobacter sp.CCTCC M209061或者Candida macedoniensisAKU4588的酮还原酶；
所述单加氧酶为来源于Rhodococcus sp.Phi1的环己酮单加氧酶，或者来源于Brachymonas petroleovorans的环己酮单加氧酶，或来源于Rhodococcus ruber-SD1的单加氧酶；
所述氨裂解酶为来源于photorhabdus luminescens或Solenostemonscutellarioides的氨裂解酶；
所述烯还原酶为来源于Saccharomyces cerevisiae或Chryseobacterium sp.CA49的烯还原酶；
所述亚胺还原酶为来源于Streptomyces sp或Bacillus cereus的亚胺还原酶；
所述氨基酸脱氢酶为来源于Bacillus cereus的亮氨酸脱氢酶或者来源于Bacillussphaericus的苯丙氨酸脱氢酶；
所述腈水解酶为来源于Aspergillus niger CBS 513.88或Neurospora crassa OR74A的腈水解酶。
8.根据权利要求7所述的制备方法，其特征在于，
所述来源于Chromobacterium violaceum DSM30191的转氨酶为具有SEQ ID NO：2或SEQ ID NO：3所示序列的突变体；
所述来源于Arthrobacter citreus的转氨酶为具有SEQ ID NO：5或SEQ ID NO：6所示序列的突变体；
所述来源于Acetobacter sp.CCTCC M209061的酮还原酶为具有SEQ ID NO：8或SEQ IDNO：9所示序列的突变体；
所述来源于Rhodococcus sp.Phi1的环己酮单加氧酶为具有SEQ ID NO：11或SEQ IDNO：12所示序列的突变体；
所述来源于Rhodococcus ruber-SD1的环己酮单加氧酶为具有SEQ ID NO：14或SEQ IDNO：15所示序列的突变体。
9.根据权利要求6所述的制备方法，其特征在于，所述带有C2或C4连接臂的氨基树脂载体选自如下任意一种：LX1000EA、LX1000HA、LX1000NH、LX1000EPN、HM100D、ECR8309、ECR8409、ECR8305、ECR8404、ECR8315、ECR8415、ESR-1、ESR-3、ESR-5及ESR-8。
10.根据权利要求6所述的制备方法，其特征在于，所述PEG与所述戊二醛的质量比为2:1～5:1；所述PEI与所述戊二醛的质量比为3:1～1:5。
11.根据权利要求6或10所述的制备方法，其特征在于，所述PEG与所述戊二醛的质量比为1:1～1:2。
12.权利要求1至5中任一项所述的固定化酶，或者权利要求6至11中任一项所述的制备方法制备而成的固定化酶在生物催化反应中的应用。
13.根据权利要求12所述的应用，其特征在于，所述生物催化反应为连续化的生物催化反应或批量反应。
14.根据权利要求13所述的应用，其特征在于，所述固定化酶在水相或有机相的反应条件下循环利用6-16次。
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