Department of Microbiology, Panjab University, Chandigarh, India.
Department of Biotechnology, Panjab University, Chandigarh, India.
Int J Biol Macromol. 2019 Feb 15;123:1052-1061. doi: 10.1016/j.ijbiomac.2018.11.174. Epub 2018 Nov 19.
A bacterial laccase having potential to work in industry without mediator is of special interest. In this work, gene (1.83kb) encoding a novel laccase from Rheinheimera sp., having potential to deink waste paper without mediator, was cloned, over-expressed and the induced 69kDa protein (RhLacc) was purified and characterized. rhlacc gene was mutated by error prone PCR and mutants were sequenced. One mutant showed protein truncation resulting in the absence of domain 3 that contains T1 copper center. It is known that redox potential of T1 is the key parameter for substrate oxidation. Overexpression of this mutant gene showed induced 41.1kDa protein (∆RhLacc) that exhibited laccase activity but in the presence of added copper, compared to RhLacc which showed activity without added copper ions. Optimum temperature for both was 55°C. However, optimum pH varied with substrates. Kinetic studies showed ∆RhLacc had lower affinity for substrates except for guaiacol and reduced k in comparison to RhLacc. Both were able to deink old newspaper and degrade indigo carmine without mediator. The study suggests that the novel property to deink waste paper without mediator may not depend on the redox potential of T1 but other mechanisms using domains 1 and 2 may be involved.
一种无需介体即可在工业中发挥作用的细菌漆酶特别受到关注。在这项工作中，从莱茵海默氏菌（Rheinheimera sp.）克隆、过表达并纯化了一种新型漆酶基因（1.83kb），该基因具有无需介体脱墨废纸的潜力。rhlacc 基因通过易错 PCR 发生突变，并对突变体进行了测序。一个突变体显示出蛋白质截断，导致缺少包含 T1 铜中心的结构域 3。众所周知，T1 的氧化还原电位是底物氧化的关键参数。该突变基因的过表达显示出诱导的 41.1kDa 蛋白质（∆RhLacc），它表现出漆酶活性，但在添加铜的情况下，与 RhLacc 相比，RhLacc 无需添加铜离子即可显示活性。两者的最适温度均为 55°C。然而，最适 pH 值随底物而变化。动力学研究表明，与 RhLacc 相比，除了愈创木酚和降低的 k 外，∆RhLacc 对底物的亲和力较低。两者都能够在没有介体的情况下脱墨旧报纸并降解靛蓝胭脂红。研究表明，无需介体即可脱墨废纸的新特性可能不依赖于 T1 的氧化还原电位，而是可能涉及使用结构域 1 和 2 的其他机制。
