Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the common sea hare Aplysia kurodai.
Two alginate lyase isozymes, AkAly28 and AkAly33, with approximate molecular masses of 28 and 33kDa, respectively, were isolated from the digestive fluid of the common sea hare, Aplysia kurodai. Both of AkAly28 and AkAly33 were regarded as the endolytic polymannuronate (poly(M)) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate producing unsaturated tri- and disaccharides and rapidly decreased the viscosity of sodium alginate solution in the initial phase of degradation. Optimal pH and temperature of the two enzymes were similarly observed at pH 6.7 and 40°C, respectively. Temperature that caused a half inactivation of the two enzymes during 20-min incubation was also similar to each other, i.e., 38°C. However, NaCl requirement and activity toward oligosaccharide substrates of the two enzymes were significantly different from each other. Namely, AkAly28 showed practically no activity in the absence of NaCl and the maximal activity at NaCl concentrations higher than 0.2 M, whereas AkAly33 showed ~20% of maximal activity despite the absence of NaCl and the maximal activity at around 0.1 M NaCl. AkAly28 hardly degraded oligosaccharides smaller than tetrasaccharide, while AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and 2-keto-3-deoxy-gluconaldehyde (an open chain form of unsaturated monosaccharide). Analysis of the N-terminal and internal amino-acid sequences of AkAly28 and AkAly33 indicated that both of the two enzymes belong to polysaccharide lyase family 14.
Rahman
Mohammad Matiur
MM
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido, Japan.
Inoue
Akira
A
Tanaka
Hiroyuki
H
Ojima
Takao
T
D000464:Alginates / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001048:Aplysia / Q000201:enzymology*; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011108:Polymers / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature
D000464:Alginates; D006603:Hexuronic Acids; D007527:Isoenzymes; D011108:Polymers; D012965:Sodium Chloride; D020723:Glucuronic Acid; D011133:Polysaccharide-Lyases; C016158:poly(beta-D-mannuronate) lyase