Carrageenase

Madhav KumarGoa university

INTRODUCTION

▪ Carrageenan, one of the phycocolloids is a sulfated galactanmade up of linear chains of galactose and 3,6-anhydrogalactose with alternating α-(1→3) and β-(1→4) linkages and further classified based on the number and the position of sulfated ester(s); κ-, ι- and λ-carrageenan.

▪ They are a family of linear sulphated polysaccharides that are extracted from red seaweeds.

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▪ They are widely used in the food industry, for their gelling, thickening, and stabilizing properties. Their main application is in dairy and meat products, due to their strong binding to food proteins. There are three main varieties of carrageenan, which differ in their degree of sulphation.

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▪ There are three main commercial classes of carrageenan:

▪ Kappa forms strong, rigid gels in the presence of potassium ions; it reacts with dairy proteins. It is sourced mainly from Kappaphycus alvarezzi.

Kappa-carrageenan has one sulphate group per disaccharide

▪ Iota forms soft gels in the presence of calcium ions. It is produced mainly from Eucheuma denticulatum.

Iota-carrageenan has two sulphates per disaccharide.

▪ gth gels, or contribute to gel inhibition (lambda carrageenan). 4

▪ Lambda does not gel, and is used to thicken dairy products. The most common source is Gigartina.

Lambda-carrageenan has three sulphates per disaccharide.

▪ The primary differences that influence the properties of kappa, iota, and lambda carrageenan are the number and position of the ester sulfate groups on the repeating galactose units. Higher levels of ester sulfate lower the solubility temperature of the carrageenan and produce lower stren

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▪ Microbial enzymes which hydrolyse phycocolloids have drawn considerable interest. The biotechnology of enzymes and enzyme degraded products of carrageenan is still in infancy compared to that of agar, alginate, starch or pectin. Carrageenases which hydrolyse 1,4 linkages in carrageenan to a series of homologous, even numbered oligosaccharides are useful tool for the structural analysis of the cell walls and protoplast isolation from red algae. The sulfated carrageeno-oligosaccharides have also drawn considerable interest owing to their diverse biological and physiological activities including anticoagulation, anti-inflammation , anti-thrombosis, antitumor activity and viral inactivation , which depend on structural parameters such as carbohydrate structure, molecular mass, degree of sulfateesterification, and the linking position of sulpho groups.

▪ Several bacterial genera including Pseudomonas carrageenovora , Cytophaga , Alteromonas carrageenovora, Vibrio sp. and Zobelliagalactanovorans have been reported carrageenase production.

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▪ Types & properties of carrageenase:

▪ Kappa-carrageenase (EC 3.2.1.83) is an enzyme with system name kappa-carrageenan 4-beta-D-glycanohydrolase This enzyme catalyses the following chemical reaction.

▪ Endohydrolysis of (1->4)-beta-D-linkages between D-galactose 4-sulfate and 3,6-anhydro-D-galactose in kappa-carrageenans.

▪ The main products of hydrolysis are neocarrabiose-sulfate and neocarratetraose-sulfate.

▪ Iota-carrageenase (EC 3.2.1.157) is an enzyme with system name iota-carrageenan 4-beta-D-glycanohydrolase. This enzyme catalyses the following chemical reaction.

▪ Endohydrolysis of (1->4)-beta-D-linkages between D-galactose 4-sulfate and 3,6-anhydro-D-galactose-2-sulfate in iota-carrageenans

▪ The main products of hydrolysis are iota-neocarratetraose sulfate and iota-neocarrahexaose sulfate.

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▪ Lambda-carrageenase (EC 3.2.1.162, endo-beta-1,4-carrageenose 2,6,2'-trisulfate-hydrolase) is an enzyme which breaks down a polysaccharide found in red seaweeds, lambda-carrageenan.

▪ Lambda-carrageenase cleaves the beta 1-4 glycosidic bonds in the linear backbone of lambda-carrageenan. This results in the formation of a tetrasaccharide: alpha-D-Galp2,6S(2)-(1->3)-beta-D-Galp2S-(1->4)-alpha-D-Galp2,6S(2)-(1->3)-D-Galp2S.

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▪ K-carrageenase is a monomer and stable in broad range of temperature(20˚c to 60˚c) and 35˚c to 40˚c was optimum temperature. When the temperature was above 40˚c, the enzyme activity decreased significantaly as time prolonging.

▪ The optimum pH for k-carrageenasse is 7.0

▪ Iota-carrageenase is a monomer and has temperature range in(40˚c to 60˚c) and optimum is found to be 50˚c and pH range from 7.0 to 10.0 and its optimum pH is found to be 7.5

▪ Lambda-carrageenase is found as a monomer. Its optimum pH for activity is 7.0, and optimum temperature is 35°C.

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Production

▪ Isolation of bacteria.

▪ Stored at 4 °C on minimal mineral salts (MMS) agar slants.

▪ The bacterium was routinely grown on MMS medium with agar as energy source at 37 °C.

▪ Seed culture was prepared from 24h grown cultures on MMS liquid broth at 180 rpm, 37 °C.

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▪ Fermentation medium composed g.L-1; K2HPO4, 0.38; MgSO4, 0.20; FeCl3, 0.05; NH4NO3, 1.0; pH 8.0 was supplemented with carrageenan (0.3 %) as the only source of carbon.

▪ Seed culture used for inoculation.

▪ Provide favourable condition pH 8, temp. 28 °C.

▪ Incubate for 24 hrs.

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Purification

▪ Culture media was centrifuged at 4 °C , 10000g for 20 min.

▪ Supernatant was fractionated at 40% and 80% ammonium sulphate solution.

▪ 80% ammonium sulphate saturation was resusepanded in D/W and dialyzed in diasysis bag.

▪ Obtained enzyme powder was dissolved in 5ml Tris-HCL buffer (pH 7.0).

▪ Enzyme solution was applied to sephadex G200 column chromatography and eluted.

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▪ Elutes were monitored continuously at 280 nm for protein and fractions were assayed for activity against k-carrageenan.

▪ All the fractions of the first peak from Sephadex G-200 column chromatography containing carrageenase activity were gathered, concentrated and applied to another column of Sephadex G-75, furthermore equilibrated with the same eluent.

▪ Fractions were collected and monitored for the presence of carrageenase.

▪ The purity of the fractions was assessed by SDS-PAGE. Pure fractions with activity were stored at −20 °C.

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▪ The peaks from Sephadex G-200 contained three protein bands (1); Only one protein band, with an apparent molecular mass of 40.8 kDa, was detected in the activity peak from Sephadex G-75 (2); The sample of the activity peak from Sephadex G-75 was applied to zymography experiment, stained with alcian blue. A degradated band was detected at the same position, just as pointed by the arrow (3). Line M was protein molecular mass marker.

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Properties of K-Carrageenase

▪ Stable at broad range of temperature (20-60 °C ) and 35 °C is optimum temperature.

▪ Good thermal stability at 40 °C .

▪ with the prolongation of time the enzyme activity remained stable at least for 2.5 h.

▪ When the temperature was above 40 °C, the enzyme activity decreased significantly as time prolonging.

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Effect of temperature...

▪ The enzyme solution was incubated at each temperature (20–70 °C) for 0.5 to 2.5 h and then the residual enzyme activity was measured.

▪ The activity of untreated enzyme was regarded as 100% and relative activity was determined.

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Effect of pH...

▪ The optimum pH of the κ-carrageenaseis 7.0.

▪ Pre-incubating the enzyme solution at each pH (4.0–10.0) at 35 °C for 6 h and then the enzyme activity was determined in the same pH buffer. The activity of untreated enzyme was regarded as 100% and relative activity was determined.

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Effect of NaCl...

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Effect of incubation time...

▪ Effect of incubation time on the growth of Pseudomonas aeruginosaZSL-2 and the production of extracellular carrageenase . 19

▪ Thin layer chromatography (TLC) of the κ-carrageenase degradation products.

▪ Enzymatic hydrolysis of κ-carrageenan was conducted under standard condition with 0.5% κ-carrageenan as substrate and reaction for 5 min (1); 30 min (2); 1 h (3); 2 h (4); 3 h (5); 6 h (6); 9 h (7); 12 h (8); 24 h (9) and 48 h (10), respectively. M, control (galactose).

▪ Combined with HPLC and mass spectrometry results, a, b and c were e-neocarraoctaose, κ-neocarrabiose and κ-neocarrabioserespectively.

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Degradation mode...

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Enzyme assay

Stock solution of enzyme(6mg/ml) were diluted with 100mM TrisHCl,200mM NaCl and 5mM EDTA(pH 7.2)

100μl of this soution was incubated with 200μl of substrate solution(0.125% carragenase,50mM Tris HCL and 100mM NaCl, pH 7.2) for 15 mins at 420C

200μl of this reaction mixture was assayed for reducing sugar.

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Application

▪ Used in degrading the cell walls of seaweeds to obtain protoplasts, and in hydrolyzing κ-carrageenan to produce oligosaccharides.

▪ κ-Carrageenases can be applied to study the composition and structure of carrageenans from different red alga, and to study the bacterial κ-carrageenan metabolism.

▪ To investigate the structure-function relationship of the hydrolases that degrade self-associating sulfated polysaccharides.

▪ To study cell-cell cohesion and exchange boundary, signaling (cell-cell recognition)

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References…

▪ Potin, P., Sanseau, A., Le Gall, Y., Rochas, C. and Kloareg, B. (1991). "Purification and characterization of a new κ-carrageenase from a marine Cytophaga-like bacterium". Eur. J. Biochem. 201: 241–247. doi:10.1111/j.1432-1033.1991.tb16280.x. PMID 1915370.

▪ Tristan Barbeyron, Anita Gerard,Philippe Potin,Bernard Henrissat, and Bernard Kloareg” The Kappa-Carrageenase of the Marine Bacterium Cytophagadrobachiensis. Structural and Phylogenetic Relationships Within Family-16 Glycoside Hydrolases.” Mol. Biol. Evol. 15(5):528–537. 1998

▪ Weigl, J. and Yashe, W. (1966). "The enzymic hydrolysis of carrageenan by Pseudomonas carrageenovora: purification of a κ-carrageenase". Can. J. Microbiol. 12: 939–947.doi:10.1139/m66-127. PMID 5972647.

▪ Ziang Yao 1, Feifei Wang 1, Zheng Gao 1, Liming Jin 2 and Haige Wu 1 (2013).” Characterization of a κ-Carrageenase from Marine Cellulophaga lyticastrain N5-2 and Analysis of Its Degradation Products “. 2 Int. J. Mol. Sci. 2013, 14, 24592-24602; doi:10.3390/ijms141224592

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▪ en.wikipedia.org/wiki/Lambda-carrageenase

▪ en.wikipedia.org/wiki/Kappa-carrageenase

▪ en.wikipedia.org/wiki/Iota-carrageenase

▪ omicsonline.org/orthogonal-array-approach-for-optimization-of-carrageenase

▪ www.brenda-enzymes.info/php/result_flat.php3?ecno=3.2.1.83

▪ www.ncbi.nlm.nih.gov/pubmed/11435116

▪ www.brenda-enzymes.org/php/result_flat.php4?ecno=3.2.1.162

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