Purification, Cloning, Characterization and Essential Amino Acid Residues Analysis of a New ι-Carrageenase from Cellulophaga sp. QY3

Abstract

ι-Carrageenases belong to family 82 of glycoside hydrolases that degrade sulfated galactans in the red algae known as ι-carrageenans. The catalytic mechanism and some substrate-binding residues of family GH82 have been studied but the substrate recognition and binding mechanism of this family have not been fully elucidated. We report here the purification, cloning and characterization of a new ι-carrageenase CgiA_Ce from the marine bacterium Cellulophaga sp. QY3. CgiA_Ce was the most thermostable carrageenase described so far. It was most active at 50°C and pH 7.0 and retained more than 70% of the original activity after incubation at 50°C for 1 h at pH 7.0 or at pH 5.0–10.6 for 24 h. CgiA_Ce was an endo-type ι-carrageenase; it cleaved ι-carrageenan yielding neo-ι-carrabiose and neo-ι-carratetraose as the main end products, and neo-ι-carrahexaose was the minimum substrate. Sequence analysis and structure modeling showed that CgiA_Ce is indeed a new member of family GH82. Moreover, sequence analysis of ι-carrageenases revealed that the amino acid residues at subsites −1 and +1 were more conserved than those at other subsites. Site-directed mutagenesis followed by kinetic analysis identified three strictly conserved residues at subsites −1 and +1 of ι-carrageenases, G228, Y229 and R254 in CgiA_Ce, which played important roles for substrate binding. Furthermore, our results suggested that Y229 and R254 in CgiA_Ce interacted specifically with the sulfate groups of the sugar moieties located at subsites −1 and +1, shedding light on the mechanism of ι-carrageenan recognition in the family GH82.

Efficient enzymatic degradation process for hydrolysis activity of the Carrageenan from red algae in marine biomass

“…Conversely, -carrageenase activity had not been described for this study because -carrageenase is similar tocarrageenase. Both the -carrageenases and -carrageenase are endohydrolases, breaking internal linkages randomly rather than hydrolyzing units from the ends however, -carrageenase is processive, hydrolyzing several units in succession (Ma et al, 2013). Therefore, we selected -type and -type carrageenase for synergistic activity and the miniCbpA for an attractive framework for the development of functional carrageenolytic complexes.…”

Bacterial carrageenases: an overview of production and biotechnological applications

“…Temperature and pH, giving maximum yield, correspond with the optimal conditions for the growth of the organism. Similar to other metabolic enzymes, optimum carrageenase production in bacteria is in the neutral temperature (Zhou et al 2008; Feixue et al 2010; Ma et al 2013) with the exception of Bacillus sp. Lc50-1 which require 55 °C for production (Li et al 2014a, b).…”

“…LPLM f /30 °C/48 h/pH 7.53550 %/35 °C/36 h6.0>50 %/pH 5.0–9.0/35 °C/1 h75.5 kDaLi et al (2015)7 Cellulosimicrobium cellulans Kappa-carrageenan/37 °C/250 rpm/24 h/pH 7.530NR n 6.0NR n NR n Beltagy et al (2012)8 Cellulosimicrobium cellulans ZM g /37 °C/250 rpm/24 h/pH 7.537NR n 7.5NR n NR n Youssef et al (2012)9 Cellulophaga lytica strain N5-2Kappa-carrageenan/35 °C/20 h35>85 %/40 °C/pH 7.0/150 min7.0>80 %/pH 7.0/360 min/35 °C40.8Yao et al (2013)10 Cellulophaga sp. QY3Iota-carrageenan/25 °C/150 rpm/20 h/pH 7.050>80 %/50 °C/pH 7.0/60 min7.0>70 %/pH 5.0–10.6/720 min/4 °C48.3 kDaMa et al (2013)11 Cytophaga lk-C783ZM g having carrageenan/25 °C/150 rpm/72 h25 °C50 %/50 °C/pH 7.0/10 min7.6NR n 100 kDaSarwar et al (1983a, b, 1987)12 Cytophaga MCA-2ZM g having carrageenan/32 °C/150 rpm/36 h/pH 7.5280 %/55 °C/30 min7.232 %/pH 10.83/24 h30 kDaMou et al (2004)13Dsij strainZM g /22 °C/250 rpm/24–40 h40...…”

“…The inhibition by mercuric ions may indicate the importance of thiol containing amino acid residues in the carrageenase function (Li et al 2015). However, metals like Na + , K + , Ca 2+ , Mg 2+ are known to increase the carrageenase activity (Ma et al 2010; Shangyong et al 2013; Li et al 2014b) especially Na 1+ at high concentration (up to 500 mmol L −1 ) significantly increase the activity (Ma et al 2013). It was hypothesized that at high concentration it changes the physical condition of carrageenan would have an effect on enzyme reaction.…”

Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications

“…Additional iota-carrageenase genes have been cloned from Cellulophaga sp. QY3, a flavobacterium isolated from the red alga Grateloupia livida (Ma et al 2013),and from Microbulbifer thermotolerans JAMB-A94T, a deep-sea bacterium (Hatada et al 2011). The iotacarrageenase CgiA of A. fortis adopts a right-handed β-helix fold with two additional domains (A and B) in the C-terminal region (Michel et al 2001b).…”