Abstract
BackgroundEffective and simple methods that lead to higher enzymatic efficiencies are highly sough. Here we proposed a foldon-triggered trimerization of the target enzymes with significantly improved catalytic performances by fusing a foldon domain at the C-terminus of the enzymes via elastin-like polypeptides (ELPs). The foldon domain comprises 27 residues and can forms trimers with high stability.ResultsLichenase and xylanase can hydrolyze lichenan and xylan to produce value added products and biofuels, and they have great potentials as biotechnological tools in various industrial applications. We took them as the examples and compared the kinetic parameters of the engineered trimeric enzymes to those of the monomeric and wild type ones. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the trimeric lichenase and xylanase increased 4.2- and 3.9- fold. The catalytic constant (kcat) of the trimeric lichenase and xylanase increased 1.8- fold and 5.0- fold than their corresponding wild-type counterparts. Also, the specific activities of trimeric lichenase and xylanase increased by 149% and 94% than those of the monomeric ones. Besides, the recovery of the lichenase and xylanase activities increased by 12.4% and 6.1% during the purification process using ELPs as the non-chromatographic tag. The possible reason is the foldon domain can reduce the transition temperature of the ELPs.ConclusionThe trimeric lichenase and xylanase induced by foldon have advantages in the catalytic performances. Besides, they were easier to purify with increased purification fold and decreased the loss of activities compared to their corresponding monomeric ones. Trimerizing of the target enzymes triggered by the foldon domain could improve their activities and facilitate the purification, which represents a simple and effective enzyme-engineering tool. It should have exciting potentials both in industrial and laboratory scales.
Highlights
Effective and simple methods that lead to higher enzymatic efficiencies are highly sough
The precise molecular weights (MWs) of purified B-E and X-E were further determined by MALDI-TOF mass spectrometry (MS)
They presented with MWs of 42,696.3 Da and 39,562.6 Da respectively (Fig. 1b,d), which matched their theoretical values at 42262.5 Da and 39,509.3 Da calculated by ProtParam
Summary
Effective and simple methods that lead to higher enzymatic efficiencies are highly sough. Enzymatic catalysis played a significant role in industry and laboratory, especially in enzymatic hydrolysis of lignocellulose to produce fuel-grade ethanol. It was an attractive opportunity for producing renewable and environmentally friendly biofuels [1]. Within this context, since the limited catalytic performance in the reaction process, many studies on the functional characteristics such as substrate affinity, high catalytic properties received extensive attention [2]. Zhang [4] and coworkers revealed a series of xylanases mutants which displayed 35–45% decrease in Km and 75–105% increase in kcat and leading to an approximately 200% increase in catalytic efficiencies by directed evolution.
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