Industrial enzymes are often immobilized via chemical cross-linking onto solid supports to enhance stability and facilitate repeated use in bioreactors. For starch-degrading enzymes, immobilization usually places constraints on enzymatic conversion due to the limited diffusion of the macromolecular substrate through available supports. This study describes the one-step immobilization of a highly thermostable α-amylase (BLA) from Bacillus licheniformis and its functional display on the surface of polyester beads inside engineered Escherichia coli. An optimized BLA variant (Termamyl) was N-terminally fused to the polyester granule-forming enzyme PhaC of Cupriavidus necator. The fusion protein lacking the signal sequence mediated formation of stable polyester beads exhibiting α-amylase activity. The α-amylase beads were assessed with respect to α-amylase activity, which was demonstrated qualitatively and quantitatively. The immobilized α-amylase showed Michaelis-Menten enzyme kinetics exerting a Vmax of about 506 mU/mg of bead protein with a Km of about 5 μM, consistent with that of free α-amylase. The stability of the enzyme at 85°C and the capacity for repeated usage in a starch liquefaction process were also demonstrated. In addition, structural integrity and functionality of the beads at extremes of pH and temperature, demonstrating their suitability for industrial use, were confirmed by electron microscopy and protein/enzyme analysis. This study proposes a novel, cost-effective method for the production of immobilized α-amylase in a single step by using the polyester granules forming protein PhaC as a fusion partner in engineered E. coli.
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机译:工业用酶通常通过化学交联固定在固体支持物上,以增强稳定性并促进在生物反应器中的重复使用。对于淀粉降解酶,由于大分子底物通过可用载体的有限扩散,固定化通常对酶促转化施加限制。这项研究描述了一步固定化地衣芽孢杆菌的高度稳定的α-淀粉酶(BLA)及其在工程大肠杆菌内部聚酯珠表面上的功能展示。将优化的BLA变体(Termamyl)N-末端融合到Cupriavidus necator的聚酯颗粒形成酶PhaC上。融合蛋白缺乏信号序列介导的稳定的显示α-淀粉酶活性的聚酯珠的形成。对α-淀粉酶珠的α-淀粉酶活性进行了评估,这在定性和定量方面得到了证明。固定化的α-淀粉酶显示了Michaelis-Menten酶动力学,其动力学最大值为约506 mU / mg珠蛋白,Km为约5μM,与游离α-淀粉酶一致。还证明了该酶在85℃下的稳定性以及在淀粉液化过程中重复使用的能力。另外,通过电子显微镜和蛋白质/酶分析证实了在极端的pH和温度下珠的结构完整性和功能性,证明了其在工业上的适用性。这项研究提出了一种新颖,经济高效的方法,可通过使用形成蛋白质PhaC的聚酯颗粒作为工程大肠杆菌中的融合伴侣,一步一步生产固定化α-淀粉酶。
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