Stabilization ofBacillus circulansxylanase by combinatorial insertional fusion to a thermophilic host protein

Abstract

High thermostability of an enzyme is critical for its industrial application. While many engineering approaches such as mutagenesis have enhanced enzyme thermostability, they often suffer from reduced enzymatic activity. A thermally stabilized enzyme with unchanged amino acids is preferable for subsequent functional evolution necessary to address other important industrial needs. In the research presented here, we applied insertional fusion to a thermophilic maltodextrin-binding protein from Pyrococcus furiosus (PfMBP) in order to improve the thermal stability of Bacillus circulans xylanase (BCX). Specifically, we used an engineered transposon to construct a combinatorial library of randomly inserted BCX into PfMBP. The library was then subjected to functional screening to identify successful PfMBP-BCX insertion complexes, PfMBP-BCX161 and PfMBP-BCX165, displaying substantially improved kinetic stability at elevated temperatures compared to unfused BCX and other controls. Results from subsequent characterizations were consistent with the view that lowered aggregation of BCX and reduced conformational flexibility at the termini was responsible for increased thermal stability. Our stabilizing approach neither sacrificed xylanase activity nor required changes in the BCX amino acid sequence. Overall, the current study demonstrated the benefit of combinatorial insertional fusion to PfMBP as a systematic tool for the creation of enzymatically active and thermostable BCX variants.

Amyloid Aggregation of Bacillus circulans Xylanase under Native Conditions and its Modulation by β‐Amyloid‐Derived Peptide Fragments

“…Kinetic stability is related to the timescale over which an enzyme remains active . Wild‐type BCX was found to lose xylanase activity during incubation (Figure A) . Less than 5 % of the original xylanase activity of wild‐type BCX was detected after 14 days′ incubation.…”

“…The DNA coding for the resulting BCX variant was referred to as bcx‐klvfwak . For the PCR, a plasmid pET23b‐BCX, in which DNA encoding BCX was cloned, was used as a template. The PCR was carried out with 5′‐TATCT TCATA TGCAC CACCA CCACC ACCAC GCCTC CACAG ACTAC TGGCA AAA‐3′ and 5′‐AATGT ATAAC TAGTT TATCA TTTCG CCCAA AACAC CAGTT TGCCA CCGCC CCACA CTGTA ACGTT GGAAG AACCA G‐3′ as forward and reverse primers, respectively; the NdeI restriction enzyme site at the 5′‐end and the SpeI restriction enzyme site at the 3′‐end of bcx‐klvfwak are underlined.…”

“…Enzymatic hydrolysis of ONPX2 was measured under constant stirring at 40 °C in PBSA, as described previously . The ratio of k cat / K m was determined from the initial rates, as described previously . For the enzyme activity assay, the concentrations of proteins and ONPX2 were 1 and 148 μ m , respectively, unless otherwise mentioned.…”

“…Reversible thermal unfolding was monitored at 222 nm while the temperature was increased from 20 to 75 °C. To prevent irreversible aggregation during the thermal unfolding while allowing a clear CD transition, the ellipticity of protein samples (with and without 4 μ m peptides) was measured at a protein concentration of 2 μ m in the presence of urea (2.5 m ), as described previously . For CD temperature scans, a scanning rate of 0.2 °C min −1 and a response time of 16 s were chosen to minimize the scan‐rate dependencies for thermal unfolding of BCX and its variant .…”

Thermophilic Proteins as Versatile Scaffolds for Protein Engineering

“…More recently, a series of studies performed by Kim and coworkers demonstrated that domain insertion of a guest protein may benefit from high thermostability of a host protein [38,39,40,41]. Insertional fusion has recently been highlighted as a novel means of creating multi-domain protein complexes, where functionalities are often integrated and coupled with each other.…”