基于定向进化提高漆酶的催化活性及其在造纸制浆中的应用

Directed evolution improves the catalytic activity of laccase in papermaking

摘要:

在造纸工业中，漆酶作为一种生物催化剂，已经被广泛研究和应用。然而，天然漆酶在工业应用中存在着催化效率低、稳定性差等问题，限制了其在制浆工艺中的应用。为了进一步开发出酶活性高，耐受性强的漆酶，本研究对短小芽孢杆菌属来源漆酶进行定向进化改造，利用高通量筛选方法从随机突变体文库中筛选得到突变体F282L/F306L、Q275P，其比酶活分别为280.87 U/mg、453.94 U/mg，是野生型漆酶的1.42倍、2.30倍；突变体Q275P的温度稳定性有明显提升，其在40、50、70℃孵育4 h后的剩余酶活与野生型漆酶相比均提高了20%以上；突变体F282L/F306L和Q275P相较野生型漆酶对多种金属离子和有机溶剂的耐受性增强。野生型漆酶的K_m值为374.97 μmol/L，突变体F282L/F306L和Q275P的K_m值分别减小至318.96 μmo/L和360.71 μmo/L，底物亲和性有所提高；F282L/F306L和Q275P对底物2,2'-联氨-双(3-乙基苯并噻唑啉-6-磺酸)二胺盐[2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid),ABTS]的k_cat值分别为574.00 s^–1、898.03 s^–1，分别是野生型漆酶的1.1倍和1.7倍，催化效率较高。在处理纸浆应用中，突变体Q275P处理纸浆效果最好，相比于野生型漆酶，卡伯值降低了0.82，白度提高了2.00% ISO，抗张指数和裂断长分别提高7.8%和7.2%。本研究为漆酶更好地适应造纸工业环境奠定了基础。

关键词:漆酶;生物漂白;定向进化;酶学特性;纸浆

Abstract:

As a biocatalyst, laccase has been widely studied and applied in the papermaking industry. However, the low catalytic efficiency and poor stability of natural laccase limit its application in the pulping process. To develop the laccase with high activity and strong tolerance, we carried out directed evolution for modification of the laccase derived from Bacillus pumilus and screened out the mutants F282L/F306L and Q275P from the random mutant library by high-throughput screening. The specific activities of F282L/F306L and Q275P were 280.87 U/mg and 453.94 U/mg, respectively, which were 1.42 times and 2.30 times that of the wild-type laccase. Q275P demonstrated significantly improved thermal stability, with the relative activity 20% higher than that of the wild-type laccase after incubation at 40 ℃, 50 ℃, and 70 ℃ for 4 h. F282L/F306L and Q275P showed greater tolerance to metal ions and organic solvents than the wild-type laccase. The K_m value of the wild-type laccase was 374.97 μmo/L, and those of F282L/F306L and Q275P were reduced to 318.96 μmo/L and 360.71 μmo/L, respectively, which suggested that the substrate affinity of laccase was improved after mutation. The k_cat values of F282L/F306L and Q275P for the substrate ABTS were 574.00 s^–1 and 898.03 s^–1, respectively, which were 1.1 times and 1.7 times that of the wild-type laccase, indicating the improved catalytic efficiency. Q275P demonstrated better performance than the wild-type laccase in pulping, as manifested by the reduction of 0.82 in the Kappa number and the increases of 2.00% ISO, 7.8%, and 7.2% in whiteness, tensile index, and breaking length, respectively. This work lays a foundation for improving the adaptation of laccase to the environment of the papermaking industry.

Key words:laccase;biobleaching;directed evolution;enzymatic characteristics;paper pulp