Immobilization and Properties of Lipase from Candida rugosa on Electrospun Nanofibrous Membranes with Biomimetic Phospholipid Moities

Reported here is a protocol to fabricate a biocatalyst with high enzyme loading and activity retention, from the conjugation of electrospun nanofibrous membrane having biomimetic phospholipid moiety and lipase. To improve the catalytic efficiency and activity of the immobilized enzyme, poly（acrylonitrile-co-2-methacryloyloxyethyl phosphorylcholine）s（PANCMPCs） were, respectively, electrospun into nanofibrous membranes with a mean diameter of 90 nm, as a support for enzyme immobilization. Lipase from Candida rugosa was immobilized on these nanofibrous membranes by adsorption. Properties of immobilized lipase on PANCMPC nanofibrous membranes were compared with those of the lipase immobilized on the polyacrylonitrile（PAN） nanofibrous and sheet membranes, respectively. Effective enzyme loading on the nanofibrous membranes was achieved up to 22.0 mg/g, which was over 10 times that on the sheet membrane. The activity retention of immobilized lipase increased from 56.4% to 76.8% with an increase in phospholipid moiety from 0 to 9.6%（molar fraction） in the nanofibrous membrane. Kinetic parameter Km was also determined for free and immobilized lipase. The Km value of the immobilized lipase on the nanofibrous membrane was obviously lower than that on the sheet membrane. The optimum pH was 7.7 for free lipase, but shifted to 8.3-8.5 for immobilized lipases. The optimum temperature was determined to be 35 ℃ for the free enzyme, but 42-44℃ for the immobilized ones, respectively. In addition, the thermal stability, reusability, and storage stability of the immobilized lipase were obviously improved compared to the free one.     

脂肪酶催化乳酸与乙醇合成乳酸乙酯的反应动力学

对脂肪酶催化乳酸与乙醇合成乳酸乙酯反应的动力学进行了研究,根据乒乓机制和双底物抑制的特性建立了反应速率方程.反应时间常数(tR)和扩散时间常数(tD)的计算结果表明,酯化反应速率未受到明显的限制.反应速率方程可以很好地预测实验结果,由非线性拟合得到的动力学参数中,乳酸(A)和乙醇(B)的抑制常数分别为KiA=10.7mmol/L和KiB=275.0mmol/L.这说明乳酸作为短链极性脂肪酸,对酶的失活作用远大于乙醇.乳酸在微液层中聚集并产生了使酶失活的低pH值环境,同时在酯化反应中存在竞争性抑制作用.     

少量离子液体对有机相R.miehei脂肪酶催化合成辛酸戊酯的影响

研究了疏水性离子液体[BMIM][PF6]对脂肪酶R. miehei有机相催化合成辛酸戊酯反应的影响,并结合荧光发射光谱分析R. miehei构象变化与酶活性表达的关联。结果表明,该酯化反应在饱和烷烃中的初速度比在芳烃中的大,且当溶剂logP为1.5～3.5时,反应初速度随logP的增加而降低。在不同有机溶剂中,加入占辛酸质量1%的[BMIM][PF6]均导致反应初速度降低15%左右,降低幅度几乎不与溶剂种类有关。表明[BMIM][PF6]对该反应的影响与所在溶剂系统无关,而只与离子液体的加入有关。[BMIM][PF6]加入量为辛酸质量1%～3%时,以壬烷为溶剂时的反应初速度随[BMIM][PF6]加入量的增加而降低,进一步表明离子液体的加入量是影响反应初速度的重要因素。荧光光谱分析表明,酶活性的表现与酶分子的构象变化有一定的相关性。     

双重包埋激活脂肪酶内花凝胶微球的制备与表征

首先利用聚乙二醇8000(PEG8000)将脂肪酶激活为开放构象(PEG-Lip),再将PEG-Lip与Ca₃(PO₄)₂共结晶制得Ca₃(PO₄)₂@PEG-Lip晶体花,然后将晶体花包覆于海藻酸钙(CA)凝胶(Gel)微球中,制得一种双重包埋激活脂肪酶的内花凝胶微球CA-Gel@Ca₃(PO₄)₂@PEG-Lip.该微球呈白色椭圆状,直径约2 mm.利用扫描电子显微镜(SEM)、X射线光电子能谱(EDS)、共聚焦显微镜(CLSM)和傅里叶变换红外光谱(FTIR)表征了其微观形貌和组成,发现脂肪酶被包埋在Ca₃(PO₄)₂晶体中,形成的Ca₃(PO₄)₂@PEG-Lip晶体花被包覆于海藻酸钙凝胶微球内的网络中.CA-Gel@Ca₃(PO<...