Dataset Information

Strep-tag II fusion technology for the modification and immobilization of lipase B from Candida antarctica (CALB).

ABSTRACT: Fusion tags - amino acid sequences that are genetically coded to be expressed as attached moieties to a protein - have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from Candida antarctica (CALB). The gene encoding the mature portion of CALB was codon-optimized and cloned in pASG-IBA2 plasmid for expression in E. coli. Purified recombinant Strep-tag II CALB was immobilized to Strep-Tactin based support through affinity binding, and the immobilized and free Strep-tag II CALB were compared to a commercial CALB. Following modification, the enzyme could be selectively purified from culture media with no observable non-specific binding. The catalytic efficiency of the purified fusion-tagged enzyme was significantly greater than that of the commercial CALB in its free form. Immobilization of the fusion-tagged enzyme to Strep-Tactin modified crosslinked agarose support yielded a catalytically active enzyme; however, the kcat of the immobilized enzyme was significantly reduced compared to the free tagged enzyme. This work indicates that a C-terminus Strep-tag II fusion tag may be employed to improve the catalytic efficiency of free CALB, but may not be suitable for immobilized applications that employ binding of the enzyme to a Strep-Tactin-modified support.

SUBMITTER: Johar SS

PROVIDER: S-EPMC6296563 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Strep-tag II fusion technology for the modification and immobilization of lipase B from <i>Candida antarctica</i> (CALB).

Johar Sumreet Singh SS Talbert Joey N JN

Journal, genetic engineering & biotechnology 20170708 2

Fusion tags - amino acid sequences that are genetically coded to be expressed as attached moieties to a protein - have the potential to enhance the activity of native enzyme, enable specific purification of the enzyme, and promote simple and efficient immobilization of enzymes onto material supports. In this work, we demonstrate the effect of a Strep-tag II fusion tag on the properties of free and immobilized lipase B from <i>Candida antarctica</i> (CALB). The gene encoding the mature portion of ...[more]

PMID: 30647674

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Project description:A strategy for the bioconjugation of the enzyme Candida antarctica lipase B onto titania ceramic membranes with varied pore sizes (15, 50, 150, and 300 kDa) was successfully performed. The relationship between the membrane morphology, i.e.,the pore size of the ceramic support, and bioconjugation performance was considered. Owing to the dimension of the enzyme (~33 kDa), the morphology of the ceramics allowed (50, 150, and 300 kDa) or did not allow (15 kDa) the entrance of the enzyme molecules into the porous structure. Such a strategy made it possible to better understand the changes in the material (morphology) and physicochemical features (wettability, adhesiveness, and surface charge) of the samples, which were systematically examined. The silane functionalization and enzyme immobilization were accomplished via the covalent route. The samples were characterized after each stage of the modification, which was very informative from the material point of view. As a consequence of the modification, significant changes in the contact angle, roughness, adhesion, and zeta potential were observed. For instance, for the 50 kDa membrane, the contact angle increased from 29.1 ± 1.5° for the pristine sample to 72.3 ± 1.5° after silane attachment; subsequently, it was reduced to 57.2 ± 1.5° after the enzyme immobilization. Finally, the contact angle of the bioconjugated membrane used in the enzymatic process rose to 92.9 ± 1.5°. By roughness (Sq) controlling, the following amendments were noticed: for the pristine 50 kDa membrane, Sq = 1.87 ± 0.21 µm; after silanization, Sq = 2.33 ± 0.30 µm; after enzyme immobilization, Sq = 2.74 ± 0.26 µm; and eventually, after the enzymatic process, Sq = 2.37 ± 0.27 µm. The adhesion work of the 50 kDa samples was equal to 136.41 ± 2.20 mN m-1 (pristine membrane), 94.93 ± 2.00 mN m-1 (with silane), 112.24 ± 1.90 mN m-1 (with silane and enzyme), and finally, 69.12 ± 1.40 mN m-1 (after the enzymatic process). The materials and physicochemical features changed substantially, particularly after the application of the membrane in the enzymatic process. Moreover, the impact of ceramic material morphology on the zeta potential value is here presented for the first time. With an increase in the ceramic support cut-off, the amount of immobilized lipase rose, but the specific productivity was higher for membranes possessing smaller pores, owing to the higher grafting density. For the enzymatic process, two modes of accomplishment were selected, i.e., stirred-tank and cross-flow. The latter method was characterized by a much higher effectiveness, with a resulting productivity equal to 99.7 and 60.3 µmol h-1 for the 300 and 15 kD membranes, respectively.

Project description:BackgroundThe Lipase Engineering Database (LED) integrates information on sequence, structure and function of lipases, esterases and related proteins with the alpha/beta hydrolase fold. A new superfamily for Candida antarctica lipase A (CALA) was introduced including the recently published crystal structure of CALA. Since CALA has a highly divergent sequence in comparison to other alpha/beta hydrolases, the Lipase Engineering Database was used to classify CALA in the frame of the already established classification system. This involved the comparison of CALA to similar structures as well as sequence-based comparisons against the content of the LED.ResultsThe new release 3.0 (December 2009) of the Lipase Engineering Database contains 24783 sequence entries for 18585 proteins as well as 656 experimentally determined protein structures, including the structure of CALA. In comparison to the previous release 1 with 4322 protein and 167 structure entries this update represents a significant increase in data volume. By comparing CALA to representative structures from all superfamilies, a structure from the deacetylase superfamily was found to be most similar to the structure of CALA. While the alpha/beta hydrolase fold is conserved in both proteins, the major difference is found in the cap region. Sequence alignments between both proteins show a sequence similarity of only 15%. A multisequence alignment of both protein families was used to create hidden Markov models for the cap region of CALA and showed that the cap region of CALA is unique among all other proteins of the alpha/beta hydrolase fold. By specifically comparing the substrate binding pocket of CALA to other binding pockets of alpha/beta hydrolases, the binding pocket of Candida rugosa lipase was identified as being highly similar. This similarity also applied to the lid of Candida rugosa lipase in comparison to the potential lid of CALA.ConclusionThe LED serves as a valuable tool for the systematic analysis of single proteins or protein families. The updated release 3.0 was used for the evaluation of alpha/beta hydrolases. The HTML version of the database with new features is available at http://www.led.uni-stuttgart.de and provides sequences, structures and a set of analysis tools including phylogenetic trees and HMM profiles.

Dataset Information

Strep-tag II fusion technology for the modification and immobilization of lipase B from Candida antarctica (CALB).

Publications

Strep-tag II fusion technology for the modification and immobilization of lipase B from <i>Candida antarctica</i> (CALB).

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