中文摘要：酶在磁性可回收载体上的表面固定化对于大分子生物催化具有重要意义。本研究用富含氨基的纳米级丝状多功能的生物支架M13病毒，作为脂肪酶固定化的独特软骨架。以M13噬菌体的结构和衣壳蛋白为基础，设计了两种由M13水凝胶和磁性颗粒组成的磁性可回收模拟游离脂肪酶（MFL）。第一种设计中，纳米级野生M13噬菌体通过pVIII肽的N端交联成噬菌体水凝胶，而NH2-Fe3O4磁性纳米颗粒（MNP）通过戊二醛连接到M13病毒，形成M13-（NH2-Fe3O4）磁性噬菌体水凝胶。第二种设计中，对具有Fe3O4亲和性pIII肽的特殊M13（FAP-M13）进行生物退火，通过“钩状”pIII肽与纯Fe3O4强结合。TEM观察证实FAP-M13直接抓住Fe3O4，形成磁性（FAP-M13）-Fe3O4病毒水凝胶。通过与pVIII肽N-端交联，将脂肪酶均匀固定在纳米级噬菌体表面，构建脂肪酶@M13-(NH2-Fe3O4)和脂肪酶@(FAP-M13)-Fe3O4 MFLs。研究发现，这两种MFL，活性回收率高（>95%）且具有高效磁选的特点。与传统的NH2-Fe3O4载体相比，MNP使用量显著减少，脂肪酶负载量增加约40倍。定量的K-m和V-max/K-m值几乎与游离脂肪酶相同，验证了MFL的游离酶模拟特征。两种MFL均具有较高的pH耐受性、较宽的温度适应性、较强的热稳定性和稳定的磁选能力。特别是(FAP-M13)-Fe3O4磁性病毒水凝胶，仅使用纯Fe3O4 MNPs，可以更方便、更经济地进行规模化生物催化。
外文摘要：Surface immobilization of enzymes on magnetic-recoverable carriers is of great interest and importance for the biocatalysis of relatively large molecules. In this work, the nanosized amino-rich filamentous M13 virus, a versatile biological scaffold, was applied as the unique soft backbone for lipase immobilization. Based on the structure and capsid proteins of M13 phages, the magnetic-recoverable mimic-free-lipases (MFLs) composed of the M13 hydrogels and magnetic particles were developed in two designs. In the first design, nanosized wild M13 phages were crosslinked into a phage hydrogel through the N-terminals of pVIII peptides while NH2-Fe3O4 magnetic nanoparticles (MNPs) were attached to the M13 virus through glutaraldehyde, forming the M13-(NH2-Fe3O4) magnetic phage hydrogel. In the second design, special M13 with Fe3O4 affinity pIII-peptide (FAP-M13) was biopanned for strongly binding towards bare Fe3O4 with the "hook"-like pIII-peptide (N-LPLSTQH-C). TEM observation confirmed the direct grasp of FAP-M13 on bare Fe3O4, forming the magnetic (FAP-M13)-Fe3O4 virus hydrogel. Lipases were uniformly anchored on the phage surface of nanoscale by crosslinking with the N-terminals of pVIII peptides, and then lipase@M13-(NH2-Fe3O4) and lipase@(FAP-M13)-Fe3O4 MFLs were constructed. For both MFLs, high activity recovery yield (>95%) and efficient magnetic separation were characterized. Significantly reduced MNP-usage-amount and enhanced lipase-loading-amount both by about 40 folds were obtained, compared with the conventional NH2-Fe3O4 carriers. The quantified K-m and V-max/K-m values were almost equal to those of the free lipases, verifying free-enzyme-mimicking features of the MFLs. High pH-tolerance, wide temperature adaptability, enhanced thermal stability and stable magnetic separation capability of both MFLs were also observed. In particular, the (FAP-M13)-Fe3O4 magnetic virus hydrogel simply using bare Fe3O4 MNPs would be more convenient and economical in the scaled-up biocatalysis.
外文关键词：METAL-ORGANIC FRAMEWORK；OPTIMUM AFFINITY TAGS；PHAGE DISPLAY；BIODIESEL PRODUCTION；PEPTIDE LIBRARY；ENZYME；NANOPARTICLES；EFFICIENT；M13；NANOFIBERS
作者：Qi, WJ；Yu, HM
作者单位：Tsinghua Univ
期刊名称：NANOSCALE
期刊影响因子：6.895
出版年份：2021
出版刊次：
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