AUTHOR=Yu Yuan , Liu Zhemin , Yang Min , Chen Meng , Wei Zhihan , Shi Lixia , Li Li , Mou Haijin 

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

JOURNAL=Frontiers in Microbiology

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.01544

DOI=10.3389/fmicb.2017.01544

ISSN=1664-302X

ABSTRACT=<p>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 (<italic>cgkZ</italic>), Por secretion tail-truncated (<italic>cgkZ</italic>Δ<italic>Pst</italic>) and carbohydrate binding domain-truncated (<italic>cgkZ</italic>Δ<italic>CBM</italic>) κ-carrageenase proteins were expressed in <italic>Pichia pastoris</italic>. The copy numbers of gene <italic>cgkZ</italic>, <italic>cgkZ</italic>Δ<italic>Pst</italic> and <italic>cgkZ</italic>Δ<italic>CBM</italic> 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 K<sub>m</sub> 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<sup>+</sup>, Mg<sup>2+</sup>, Ca<sup>2+</sup>, 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 <italic>P. pastoris</italic> 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.</p>