【摘要】：提出了一種采用羧基磁性納米粒子制備雜化磁響應(yīng)交聯(lián)酶聚集體(M-CLEAs)的方法。表面羧基修飾的約10 nm的磁性納米粒子與酶分子表面的氨基位點(diǎn)通過靜電相互作用,形成復(fù)合物,在磁場作用下可將磁性納米粒子-酶復(fù)合物從溶液中分離,經(jīng)戊二醛交聯(lián)即形成M-CLEAs。傳統(tǒng)的表面氨基修飾的磁性納米粒子與酶需在沉淀劑作用下,從溶液中分離,而后采用戊二醛共交聯(lián),而本方法無須沉淀劑,過程更為簡化。以糖化酶為對象,對該過程的影響因素(交聯(lián)時(shí)間、pH、酶濃度、戊二醛濃度等條件)進(jìn)行了探索,并對制得的M-CLEAs的酶學(xué)性質(zhì)進(jìn)行了較為詳細(xì)考察。結(jié)果表明,最優(yōu)制備條件為:酶濃度1 mg·ml-1,磁流體濃度10 mg·ml-1,戊二醛濃度0.25%(質(zhì)量體積比),在pH 6.0下交聯(lián)反應(yīng)6 h,最終載酶量可達(dá)80 mg·g~(-1)、比活為50 U·mg~(-1)。制得的固定化酶pH穩(wěn)定性、熱穩(wěn)定性和儲(chǔ)存穩(wěn)定性均顯著改善,可實(shí)現(xiàn)糖化酶重復(fù)使用10次,仍保留接近60%的酶活。
[Abstract]:A method for preparing hybrid magnetic response crosslinking enzyme aggregates (M-CLEAs) using carboxyl magnetic nanoparticles was proposed. The magnetic nanoparticles modified by carboxyl group on the surface interact with the amino sites on the surface of enzyme molecules by electrostatic interaction to form a complex, which can separate the magnetic nanoparticles and enzyme complexes from the solution under the action of magnetic field. M-CLEAs were formed by crosslinking with glutaraldehyde. The traditional surface modified magnetic nanoparticles and enzymes need to be separated from solution under the action of precipitator and then cross-linked by glutaraldehyde. The influencing factors of Glucoamylase (such as crosslinking time pH, enzyme concentration, glutaraldehyde concentration, etc.) were explored and the enzymatic properties of the prepared M-CLEAs were investigated in detail. The results showed that the optimal preparation conditions were as follows: enzyme concentration 1 mg ml -1, magnetic fluid concentration 10 mg ml -1, glutaraldehyde concentration 0.25% (mass / volume ratio), crosslinking reaction at pH 6.0 for 6 h, the final enzyme loading capacity was 80 mg g ~ (-1) and the specific activity was 50 U mg ~ (-1). The pH stability, thermal stability and storage stability of the immobilized enzyme were improved significantly. The enzyme activity could be reused for 10 times, and the enzyme activity was about 60%.
【作者單位】： 華僑大學(xué)化工學(xué)院生物工程與技術(shù)系;福建省生物化工技術(shù)重點(diǎn)實(shí)驗(yàn)室(華僑大學(xué));華僑大學(xué)分析測試中心;
【基金】：國家自然科學(xué)基金面上基金項(xiàng)目(21576108) 福建省新世紀(jì)優(yōu)秀人才項(xiàng)目 華僑大學(xué)研究生科研創(chuàng)新能力培育計(jì)劃資助項(xiàng)目~~
【分類號】：Q814.2;TB383.1

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