| 1. |
- Cassimjee, Karim Engelmark, et al.
(author)
-
One-step enzyme extraction and immobilization for biocatalysis applications
- 2011
-
In: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 6:4, s. 463-469
-
Journal article (peer-reviewed)abstract
- An extraction/immobilization method for HIs(6) -tagged enzymes for use in synthesis applications is presented. By modifying silica oxide beads to be able to accommodate metal ions, the enzyme was tethered to the beads after adsorption of Co(II). The beads were successfully used for direct extraction of C. antarctica lipase B (CalB) from a periplasmic preparation with a minimum of 58% activity yield, creating a quick one-step extraction-immobilization protocol. This method, named HisSi Immobilization, was evaluated with five different enzymes [Candida antarctica lipase B (CalB), Bacillus subtilis lipase A (BslA), Bacillus subtilis esterase (BS2), Pseudomonas fluorescence esterase (PFE), and Solanum tuberosum epoxide hydrolase 1 (StEH1)]. Immobilized CalB was effectively employed in organic solvent (cyclohexane and acetonitrile) in a transacylation reaction and in aqueous buffer for ester hydrolysis. For the remaining enzymes some activity in organic solvent could be shown, whereas the non-immobilized enzymes were found inactive. The protocol presented in this work provides a facile immobilization method by utilization of the common His(6) -tag, offering specific and defined means of binding a protein in a specific location, which is applicable for a wide range of enzymes.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
- Irani, Mehdi, et al.
(author)
-
Amino Acid Oxidation of Candida antarctica Lipase B Studied by Molecular Dynamics Simulations and Site-Directed Mutagenesis
- 2013
-
In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:7, s. 1280-1289
-
Journal article (peer-reviewed)abstract
- Molecular dynamics simulations have been performed on lipase B from Candida antarctica (CalB) in its native form and with one or two oxidized residues, either methionine oxidized to methionine sulfoxide, tryptophan oxidized to 5-hydroxytryptophan, or cystine oxidized to a pair of cysteic acid residues. We have analyzed how these oxidations affect the general structure of the protein as well as the local structure around the oxidized amino acid and the active site. The results indicate that the methionine and tryptophan oxidations led to rather restricted changes in the structure, whereas the oxidation of cystines, which also caused cleavage of the cystine S-S linkage, gave rise to larger changes in the protein structure. Only two oxidized residues caused significant changes in the structure of the active site, viz., those of the Cys-22/64 and Cys-216/258 pairs. Site-directed mutagenesis studies were also performed. Two variants showed a behavior similar to that of native CalB,(M83I and M129L), whereas W155Q and M72S had severely decreased specific activity. M83I had a slightly higher thermostability than native CalB. No significant increase in stability toward hydrogen peroxide was observed. The same mutants were also studied by molecular dynamics. Even though no significant increase in stability toward hydrogen peroxide was observed, the results from simulations and site-directed mutagenesis give some clues about the direction of further work on stabilization.
|
|
| 5. |
- Larsen, Marianne Wittrup, et al.
(author)
-
Suppression of Water as a Nucleophile in Candida antarctica Lipase B Catalysis
- 2010
-
In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 11:6, s. 796-801
-
Journal article (peer-reviewed)abstract
- A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mm butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild-type lipase. Mutants with a blocked, tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water.
|
|
| 6. |
|
|
| 7. |
|
|
| 8. |
|
|
| 9. |
|
|
| 10. |
- Takwa, Mohamad, et al.
(author)
-
Rational redesign of Candida antarctica lipase B for the ring opening polymerization of D,D-lactide
- 2011
-
In: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; 47:26, s. 7392-7394
-
Journal article (peer-reviewed)abstract
- Based on molecular modelling, the enzyme Candida antarctica lipase B was redesigned as a catalyst for the ring opening polymerization of D, D-lactide. Two mutants with 90-fold increased activity as compared to the wild-type enzyme were created. In a preparative synthesis of poly(D,D-lactide) the mutants greatly improved the rate and the degree of polymerization.
|
|
