Abstract
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 of recombinant enzymes cgkZ, cgkZΔPst and cgkZΔCBM 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, cgkZΔPst, and cgkZΔCBM 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 κ-carrageenan tetrasaccharide and hexasaccharide. The removal of the Por secretion tail of κ-carrageenase promoted the transcription of κ-carrageenase gene, enhancing the specific activity of κ-carrageenase without significantly changing its catalytic properties. Although the transcription level of κ-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.
Highlights
Reviewed by: Luciano Takeshi Kishi, Univ Estadual Paulista UNESP/FCAV, Brazil Jose M
The structure of κ-carrageenase from Pseudoalteromonas carrageenovora was previously described by X-ray diffraction experiments. κ-Carrageenase molecules are mainly composed of β sheets and random coils, with each β sheet containing 6–7 β chains arranged in a “tunnel-like” three-dimensional structure
Based on the functional annotations of the cgkZ gene in the NCBI Genbank database, the Pst was truncated in cgkZ Pst and the CBM was truncated in cgkZ CBM, whereas their catalytic domains were retained
Summary
Reviewed by: Luciano Takeshi Kishi, Univ Estadual Paulista UNESP/FCAV, Brazil Jose M. Acid hydrolysis and enzymatic hydrolysis methods are typically used to prepare κ-carrageenan oligosaccharides. Enzymatic hydrolysis is considered a mild, environmentally friendly, and sustainable method for preparing κ-carrageenan oligosaccharides. Κ-Carrageenase molecules are mainly composed of β sheets and random coils, with each β sheet containing 6–7 β chains arranged in a “tunnel-like” three-dimensional structure. ZM-2 (cgkZ) are 39◦C and 6.0, respectively This enzyme contains 12 active sites and 3 catalytic sites. The functional site of cgkZ includes the following regions (Figure 1A): signal peptide (1–29 aa), CH-16 κ-carrageenase catalytic domain (35–315 aa), carbohydrate binding domain (CBM, 320–399 aa), and C-terminal Por secretion tail (Pst, 470-545 aa) (NCBI).1 Liu et al (2013) expressed the cgkZ gene in Escherichia coli, and found the total enzymatic activity of κ-carrageenase to be 2.98 U/mL
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
