HaloTag™: Evaluation of a covalent one-step immobilization for biocatalysis

Rapid, selective and stable HaloTag-LbADH immobilization directly from crude cell extract for the continuous biocatalytic production of chiral alcohols and epoxides

“…In this case the size per subunit of the enzyme was more than doubled by the HaloTag-fusion from 252 amino acids (26.76 KDa) for the native enzyme to 581 amino acids (63.58 KDa) for the HaloTag-LbADH fusion enzyme. By contrast, the previously immobilized tetrameric HaloTag-PfBAL fusion enzyme 5 revealed a residual activity of 65% indicating that the influence of the tag cannot be predicted and seems to depend on the relative sizes and overall conformation of the particular fusion partners. Then, we investigated the influence of the sacrificial co-substrate 2-propanol on the activity of the free LbADH and the immobilized HaloTag-LbADH (Fig.…”

“…Recently, we reported on an innovative immobilization strategy based on the commercially available HaloTag™ tech-nology (Promega). 5 The HaloTag™ is a modified dehalogenase enzyme from Rhodococcus species 6 that can be genetically fused to an enzyme to effect rapid binding from a crude cell extract. This fusion tag recognizes terminal chloroalkane substituents exposed on the surface of the HaloLink™ resin and affords a stable covalent ester bond via a selfterminating mechanism thereby immobilizing the target enzyme ( Fig.…”

“…We applied this concept successfully for the one-step purification and immobilization of the benzaldehyde lyase from Pseudomonas fluorescence (PfBAL) and could prove its usefulness for the efficient recycling of PfBAL in repetitive batch reactions. 5 Here we extend the HaloTag™ method to a continuous biocatalytic production process involving the alcohol dehydrogenase from Lactobacillus brevis (LbADH). 7 LbADH is a complex biocatalyst system consisting of four subunits forming a tetramer to achieve the active conformation.…”

Covalent and non-covalent strategies for the immobilization of Tobacco Etch Virus protease (TEVp) on superparamagnetic nanoparticles

“…Following confirmation of TEVp-STRPT fusion expression and activity, we investigated the ability of the STRPT tag to serve as an immobilizing anchor on solid support. Using commercially available biotin-labelled superparamagnetic iron oxide nanoparticles (SPIONs), soluble TEVp-STRPT fusion protein was isolated directly from bacterial lysates [16]. Following thorough washing, the resulting SPIONs were then assayed for TEVp activity using an MBP-GFP protein fusion with a TEV recognition site between the MBP and GFP domains.…”

“…In this strategy, we anticipated that the HALOtag would provide a highly selective means of protein fusion immobilization, while eliminating the solubility issues posed by STRPT and TRPT [21]. In addition, prior literature demonstrates that enzymes generally tolerate HALOtag fusion with good retention of activity [16,22]. Following protein expression, the TEVp-HALO fusion was captured from bacterial lysate using commercially available chloroalkane functionalized SPIONs, followed by extensive washing.…”