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Agarase


Official Full Name
Agarase
Background
Agarase is an enzyme with system name agarose 4-glycanohydrolase. It found in agarolytic bacteria and is the first enzyme in the agar catabolic pathway. It is responsible for allowing them to use agar as their primary source of Carbon and enables their ability to thrive in the ocean. Agarases are classified as either α-agarases or β-agarases based upon whether they degrade αor β linkages in agarose, breaking them into oligosaccharides. When secreted, α-agarases yield oligosaccharides with 3.6 anhydro-L-galactose at the reducing end whereas β-agarases result in D-galactose residues.
Synonyms
agarase; AgaA; AgaB; endo-β-agarase; agarose 3-glycanohydrolase; EC 3.2.1.81; 37288-57-6

Catalog
Product Name
EC No.
CAS No.
Source
Price
CatalogEXWM-3945
ProductNameβ-agarase
EC No.EC 3.2.1.81
CAS No.37288-57-6
Source
CatalogNATE-1293
EC No.EC 3.2.1.81
CAS No.37288-57-6
SourceE. coli
CatalogNATE-1282
EC No.
CAS No.
SourceE. coli
CatalogNATE-0040
EC No.EC 3.2.1.81
CAS No.37288-57-6
SourcePseudomonas atl...
Related Services
Related Protocols
AGARASE -Enzymatic Assay Protocol
Related Reading

Agarase (EC 3.2.1.81) belongs to hydrolases, is a glycosylated hydrolase which catalyze the hydrolysis of agar. The systematic name of agarase is agarose 4-glycanohydrolase. Other names in common use include AgaA, AgaB, endo-β-agarase, and agarose 3-glycanohydrolase. It was firstly identified in 1992 as an agar-degrading pseudomonas isolated from seawater. Agarase can be isolated from seawater, marine sediments, marine algae, marine mollusks, and soil. According to the degradation pattern of agar, agarase were divided into two categories: α-agarase (E.C. 3.2.1.158) and β-agarase (E.C. 3.2.1.81).

Classification

Agarases are classified into α-agarases and β-agarases. In the indentified agarases, β-agarases are more abundant than α-agarases. There are only two α-agarases have been reported, produced by Alteromonas agarlyticus GJ1B and Thalassomonas sp. JAMB-A33. α-agarases belong to the glycoside hydrolase (GH) family 96. β-agarases have been classified into three GH family. Most β-agarases belong to GH16 family, six β-agarases from Vibrio sp. JT0107 belong to GH50 family and only three β-agarases belong to GH86 family. GH16 family include agarase, glucanase, carrageenase, galactosidase, etc., while GH50 and GH86 family only contain agarases.

Structure

There is no publication about the structure of α-agarases, but the domain of β-agarases have been well studied. β-agarases contain conserved glycoside hydrolase modules that function in catalysis and some contain carbohydrate binding modules (CBM). GH16 family agarases contain a signal peptide in the N-terminal, followed by the GH16 module, and the CBM-6 module in the C-terminal. GH50 family agarases carry partially conserved GH50 modules in the C-terminal, contrary to the GH-16 family module. GH86 family agarases are modular proteins consist of CBM-6 module and GH86 catalytic module.

Agarase Figure 1. Structure of β-Agarase A (top) and β-Agarase B (bottom). (Allouch J. 2003)

The threedimensional crystal structure of β-Agarase A and β-Agarase B showed that both β-agarases fold into globular ellipsoids. β-agarases consist of β-sheets and surface loops, each β-sheet consists of seven β-strands. The shape of the molecule is slightly elongated, with a cleft on one side. The cleft is at least 35 Å long.

Physicochemical properties

Most agarases are purified from marine microorganisms derived from seawater, marine sediments, marine algae, marine mollusks, and soil. Their molecular weights vary widely from 20 kDa to 360 kDa. The smallest agarase is produced by Vibrio sp. AP-2, while the largest agarase is produced by Alteromonas agarlyticus GJ1B. The specific activities ranging from 6.3 to 292 U/mg. Except agarase produced from Alteromonas agarlyticus GJ1B, most agarases are composed of a polypeptide.The optimal temperature for their activity is about 38 °C, and the optimal PH is neutral. In alkaline PH, their activity only last for one week. The products are different between the two categories: α-agarases produce agarobioses while β-agarases produce neoagarobioses.

Catalytic Mechanism

α-agarases cleavage α-1,3 linkages of agarose to produce agarobiose, β-agarases cleavage β-1,4 linkages to produce neoagarobiose. The catalytic glycosidic bond cleavage reaction that retains glycosidase mainly includes two steps. The first step: glycosylation. In this step, a carboxyl acts as a general acid, protonated the glycosidic oxygen, which promotes the separation of aglycone. At the same time, other catalytic residues act as a nucleophile to attack anomeric carbon and form covalent glycosylase intermediates. The second step: deglycosylation. The general acid now acts as a general base, deprotonates a water molecule, hydrolyzes glycosylase intermediates and releases the product. Catalytic nucleophiles and acid residues have been identified in the GH16 glycoside hydrolases family. On the β9 strand, Glu147 and Glu184 as the nucleophilic residues, Glu152 and Glu189 as the acid residues.

Applications

Agarase are widely used in industry, scientific research, etc. As one of the main components of seaweed lyase, agarases have been used as a tool enzyme for enzymatic hydrolysis of seaweed, and has important applications in the preparation of seaweed protoplasts and the preparation of seaweed single cells. In molecular biology, the use of agarase to recover DNA and RNA from agarose gels has proven to be one of the best methods. Besides, it is an effective method to hydrolyze the polysaccharide of certain seaweed by agarases, determine the structure of the hydrolyzate, and then infer the structure of the polysaccharide. In addition, agarases can be used to hydrolyze agar to produce oligosaccharides, which play an important role in food and chemical applications.

References

  1. Fu X.T., Kim S.M. Agarase: Review of major sources, categories, purification method, enzyme characteristics and applications. Marine Drugs, 2010, 8: 200-218.
  2. Allouch J., Jam M., Helbert, W., Barbeyron D., Kloareg B., Henrissat B., Czjzek M. The three-dimensional structures of two β-agarases. The Journal of Biological Chemistry, 2003, 278(47): 47171-47180.

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