Efficient production of a recombinant ι-carrageenase in Brevibacillus choshinensis using a new integrative vector for the preparation of ι-carrageenan oligosaccharides

Xu, Yanyan; Mao, Weian; Gao, Wenxuan; Chi, Zhe; Chi, Zhen‐Ming; Liu, Guanglei

doi:10.1016/j.procbio.2018.09.023

Cited by 11 publications

(6 citation statements)

References 40 publications

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“…23 To investigate the role of PpBig_2 in enzyme properties, the intact enzyme PpCgk (Q 28 −N 407 ), its mutant PpCgkCD (Q 28 −V 296 ) where the Big_2 domain was truncated, and PpBig_2 (G 297 − N 407 ) were cloned into pNCMO2 and expressed in B. choshinensis for protein characterization, considering that the B. choshinensis expression system has been regarded as an attractive heterologous expression platform and is demonstrated to be suitable for the extracellular production of carrageenases. 7,30 All extracellular recombinant proteins were purified by Ni-affinity chromatography followed by gelfiltration chromatography. Based on SDS−PAGE analysis (Figure 1C), the indicated molecular masses of PpCgk, PpCgkCD, and PpBig_2 were 45, 32, and 18 kDa, respectively, confirming the absence of the PpBig_2 domain in PpCgkCD.…”

Section: Resultsmentioning

confidence: 99%

C-Terminal Bacterial Immunoglobulin-like Domain of κ-Carrageenase Serves as a Multifunctional Module to Promote κ-Carrageenan Hydrolysis

Xing

Wang

Zhao

et al. 2022

J. Agric. Food Chem.

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κ-Carrageenase is an important component for κ-carrageenan oligosaccharide production. Generally, noncatalytic domains are appended to carbohydrate-active domains and potentiate catalytic activity. However, studies devoted to κ-carrageenase are relatively few. Here, a C-terminal bacterial immunoglobulin-like domain (Big_2) was identified in κ-carrageenase (PpCgk) from Pseudoalteromonas porphyrae. Biochemical characterization of native PpCgk and its two truncations, PpCgkCD (catalytic domain) and PpBig_2 (Big_2 domain), revealed that the specific activity, catalytic efficiency (k cat /K m(app) ), specific κ-carrageenan-binding capacity, and thermostability of PpCgk were significantly higher than those of PpCgkCD, suggesting that the noncatalytic PpBig_2 domain is a multifunctional module and essential for maintaining the activity and thermostability of PpCgk. Furthermore, it was found that the mode of action of PpCgk was more processive on both the dissolved and gelled substrates than that of PpCgkCD, indicating that PpBig_2 contributes to the processivity of PpCgk. Interestingly, PpBig_2 can be used as an independent module to enhance the hydrolysis of κ-carrageenan through its disruptive function. In addition, sequence analysis suggests that Big_2 domains are highly conserved in bacterial κ-carrageenases, implying the universality of their noncatalytic functions. These findings reveal the multifunctional role of the noncatalytic PpBig_2 and will guide future functional analyses and biotechnology applications of Big_2 domains.

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Section: Resultsmentioning

confidence: 99%

C-Terminal Bacterial Immunoglobulin-like Domain of κ-Carrageenase Serves as a Multifunctional Module to Promote κ-Carrageenan Hydrolysis

Xing

Wang

Zhao

et al. 2022

J. Agric. Food Chem.

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“…ι‐carrageenase, for which degradation product is DP8, has not been reported previously. Degradation products of ι‐carrageenase have been reported to be oligomers, including those with DP2, DP4, and DP6 36 . The ι‐carrageenases from Alteromonas sp.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of ι‐carrageenase from macroalgae‐associated bacterium Microbulbifer sp. YNDZ01

et al. 2023

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BACKGROUND In the present study, the ι‐carrageenase gene, Car1293, was obtained from the genome of Microbulbifer sp. YNDZ01, which was isolated from the surface of macroalgae. To date, there are few studies on ι‐carrageenase and the anti‐inflammatory activity of ι‐carrageenan oligosaccharides (CGOS). To enhance our perspective on ι‐carrageenase and ι‐carrageen oligosaccharides, the sequence, protein structure, enzymatic properties, enzymatic digestion products and anti‐inflammatory activity of the gene were investigated. RESULTS The gene length of Car1293 is 2,589 bp, encoding an enzyme with 862 amino acids, which shares 34% similarity with any previously reported ι‐carrageenase. The spatial structure of Car1293 consists of many α‐helices with a β‐fold binding module located at its terminus, and eight binding sites were found in the binding module as a result of docking with CGOS‐DP4 ligand. The optimum temperature and pH for the activity of recombinant Car1293 toward ι‐carrageenan were 50 °C and 6.0, respectively. The hydrolysates of Car1293 are mainly degree of polymerization (DP)8, with minor products showing DP2, DP4, and DP6. The enzymatic hydrolysates CGOS‐DP8 showed prominent anti‐inflammatory activity, which was greater than that of the positive control l‐monomethylarginine in lipopolysaccharide‐induced RAW264.7 macrophages. It inhibited nitric oxide production, as well as significantly inhibited tumor necrosis factor‐α and interleukin‐6 secretion. CONCLUSION The ι‐carrageenase sequence encoded by Car1293 is novel and can hydrolyze carrageenan into CGOS‐DP8 that has a significant anti‐inflammatory effect. The present study fills a gap in the research on the biological activity of oligosaccharides in ι‐carrageenan and provides promising data for the development of natural anti‐inflammatory agent. © 2023 Society of Chemical Industry.

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“…The gene copy numbers of KmGCR1 , KmGCR1ΔQ , and KlGCR1 in KM‐0 and its derivatives were analyzed by RT‐PCR using genomic DNA as templates according to a previous study (Xu et al, 2019). The INU1 gene, which was a single‐copy gene based on the genomic data of K. marxianus (accession no.…”

Section: Methodsmentioning

confidence: 99%

“…The gene copy numbers of KmGCR1, KmGCR1ΔQ, and KlGCR1 in KM-0 and its derivatives were analyzed by RT-PCR using genomic DNA as templates according to a previous study (Xu et al, 2019).…”

Section: Gene Copy Number Analysismentioning

confidence: 99%

A novel transcriptional activation mechanism of inulinase gene in Kluyveromyces marxianus involving a glycolysis regulator KmGcr1p with unique and functional Q‐rich repeats

Wang

Chi

et al. 2022

Molecular Microbiology

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Kluyveromyces marxianus is the most suitable fungus for inulinase industrial production. However, the underlying transcriptional activation mechanism of the inulinase gene (INU1) is hitherto unclear. Here, we undertook genetic and biochemical analyses to elucidate that a glycolysis regulator KmGcr1p with unique Q‐rich repeats is the key transcriptional activator of INU1. We determined that INU1 and glycolytic genes share similar transcriptional activation patterns and that inulinase activity is induced by fermentable carbon sources including the hydrolysis products of inulin (fructose and glucose), which suggests a novel model of product feedback activation. Furthermore, all four CT‐boxes in the INU1 promoter are important for KmGcr1p DNA‐binding in vitro, but the most downstream CT‐box 1 primarily confers upstream activating sequence activity in vivo. More intriguingly, the use of artificial and natural GCR1 mutants suggests that the Q‐rich repeats act as a functional module to maintain KmGcr1p transcriptional activity by contributing to its solubility and DNA‐binding affinity. Altogether, this study uncovers a novel transcriptional activation mechanism for the inulinase gene, that is different from the previous understanding for filamentous fungi, but might have universal significance among inulinase‐producing yeasts, thereby leading to a better understanding of the regulation mechanism of yeast inulinase genes.

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Efficient production of a recombinant ι-carrageenase in Brevibacillus choshinensis using a new integrative vector for the preparation of ι-carrageenan oligosaccharides

Cited by 11 publications

References 40 publications

C-Terminal Bacterial Immunoglobulin-like Domain of κ-Carrageenase Serves as a Multifunctional Module to Promote κ-Carrageenan Hydrolysis

C-Terminal Bacterial Immunoglobulin-like Domain of κ-Carrageenase Serves as a Multifunctional Module to Promote κ-Carrageenan Hydrolysis

Identification and characterization of ι‐carrageenase from macroalgae‐associated bacterium Microbulbifer sp. YNDZ01

A novel transcriptional activation mechanism of inulinase gene in Kluyveromyces marxianus involving a glycolysis regulator KmGcr1p with unique and functional Q‐rich repeats

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