Dataset Information

Characterization of Full-Length and Truncated Recombinant ?-Carrageenase Expressed in Pichia pastoris.

ABSTRACT: ?-Carrageenase belongs to glycoside hydrolase family 16 and cleaves the ?-(1?4) linkages of ?-carrageenan. In this study, genes encoding the full-length (cgkZ), Por secretion tail-truncated (cgkZ?Pst) and carbohydrate binding domain-truncated (cgkZ?CBM) ?-carrageenase proteins were expressed in Pichia pastoris. The copy numbers of gene cgkZ, cgkZ?Pst and cgkZ?CBM were 7, 7 and 6, respectively. The enzymatic activities of recombinant enzymes cgkZ, cgkZ?Pst and cgkZ?CBM reached 4.68, 5.70, and 3.02 U/mL, respectively, after 120 h of shake flask fermentation at 22°C and pH 6 in the presence of 1 % (v/v) methanol. The molecular weights of recombinant cgkZ, cgkZ?Pst, and cgkZ?CBM were approximately 65, 45, and 40 kDa; their Km values were 2.07, 1.85, and 1.04 mg/mL; and they exhibited optimal activity at 45-50°C and pH 6-7. All the recombinant enzymes were stimulated by Na+, Mg2+, Ca2+, and dithiothreitol. The end-products of enzymatic hydrolysis were mainly composed of ?-carrageenan tetrasaccharide and hexasaccharide. The removal of the Por secretion tail of ?-carrageenase promoted the transcription of ?-carrageenase gene, enhancing the specific activity of ?-carrageenase without significantly changing its catalytic properties. Although the transcription level of ?-carrageenase gene after the removal of the carbohydrate binding domain was relatively high, the specific activity of the recombinant enzyme significantly decreased. The comprehensive application of the P. pastoris expression system combined with the rational modification of genes may provide a novel approach for the heterologous expression of various marine enzymes with high activities.

SUBMITTER: Yu Y

PROVIDER: S-EPMC5561669 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Characterization of Full-Length and Truncated Recombinant κ-Carrageenase Expressed in Pichia pastoris.

Yu Yuan Y Liu Zhemin Z Yang Min M Chen Meng M Wei Zhihan Z Shi Lixia L Li Li L Mou Haijin H

Frontiers in microbiology 20170815

κ-Carrageenase belongs to glycoside hydrolase family 16 and cleaves the β-(1→4) linkages of κ-carrageenan. In this study, genes encoding the full-length (cgkZ), Por secretion tail-truncated (cgkZΔPst) and carbohydrate binding domain-truncated (cgkZΔCBM) κ-carrageenase proteins were expressed in Pichia pastoris. The copy numbers of gene cgkZ, cgkZΔPst and cgkZΔCBM were 7, 7 and 6, respectively. The enzymatic activities o ...[more]

PMID: 28861059

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Project description:BACKGROUND: ?-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing ?-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of ?-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel ?-1,3-glucanases with high catalytic efficiency and stability is of particular interest. RESULTS: A ?-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the ?-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes ?-1,3-glucan. Insoluble ?-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains. CONCLUSIONS: This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of ?-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble ?-1,3-glucan, and could be classified into CBM families.

Project description:The type 4 phosphodiesterase (PDE) family comprises four enzymes (4A, 4B, 4C and 4D) that are characterized by their specificity for cAMP and selective inhibition by the anti-depressant drug rolipram (4-[3-(cyclopentoxyl)-4-methoxyphenyl]2-pyrrolidone). In common with other PDEs, they consist of a central conserved domain associated with catalytic activity in addition to two N-terminal upstream conserved regions (UCR1 and UCR2) that are unique to the type 4 enzymes. We have isolated a 2 kb cDNA encoding a full-length type 4A PDE{HSPDE4A4B[Bolger, Michaeli, Martins, St.John, Steiner, Rodgers, Riggs, Wigler and Ferguson (1993) Mol. Cell. Biol. 13, 6558-6571]} from a human frontal cortex cDNA library. Northern blot analysis showed that the major PDE4A mRNA of 4.5 kb was widely distributed in different human tissues. The recombinant PDE4A expressed in COS cells had a molecular mass of approx. 117 kDa as revealed by SDS/PAGE/Western blotting with a PDE4A-specific antibody and was specific for cAMP with a Km of 4.8 microM. The enzyme activity was potently inhibited by R-rolipram (IC50 204 nM) and showed a 2.7-fold stereoselectivity over the S enantiomer. Analysis of the kinetics of inhibition indicated that R-rolipram did not behave as a simple competitive inhibitor. Dixon replots suggested that there was more than one mode of interaction consistent with the detection in the enzyme of a high-affinity binding site for R-rolipram with a Kd of 2.3 nM. Truncation of the PDE4A enzyme by deletion mutagenesis showed that neither of the UCRs was required for catalytic activity and identified an approx. 71 kDa core enzyme with a K(m) for cAMP of 3.3 microM. In contrast with the full-length PDE4A, R-rolipram behaved as a simple competitive inhibitor of this form of the enzyme with decreased potency (IC50 1022 nM) and no stereoselectivity. In addition, no high-affinity rolipram-binding site was detected in the truncated enzyme, indicating that this interaction involves sequences upstream of the catalytic domain of the enzyme.

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Dataset Information

Characterization of Full-Length and Truncated Recombinant ?-Carrageenase Expressed in Pichia pastoris.

Publications

Characterization of Full-Length and Truncated Recombinant κ-Carrageenase Expressed in <i>Pichia pastoris</i>.

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