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KINETIC CHARACTERIZATION, EXPRESSION AND MOLECULAR MODELING OF A CHITINASE FROM THE PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEI
Author(s) -
SOTELOMUNDO R.R.,
MORÁNPALACIO E.F.,
GARCÍAOROZCO K.D.,
FIGUEROASOTO C.,
ROMOFIGUEROA M.G.,
VALENZUELASOTO E.M.,
YEPIZPLASCENCIA G.
Publication year - 2009
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.2009.00215.x
Subject(s) - chitinase , shrimp , litopenaeus , molecular mass , biochemistry , isoelectric point , complementary dna , chemistry , biology , enzyme kinetics , chromatography , amino acid , enzyme , microbiology and biotechnology , active site , fishery , gene
We purified and characterized a chitinase from the digestive gland of the white shrimp, Litopenaeus vannamei . The enzyme has a molecular mass of 50 kDa and a pI of 4.7, similar to the values calculated from the cDNA‐deduced amino acid sequence. L. vannamei chitinase was stable at pH 5.0 to 8.0 and NaCl concentrations of 0–1 M, presenting an acidic pH‐optimum (pH 5.5–6.0) and maximum activity at 0.45–0.6 M NaCl. Steady‐state kinetics was obtained using the fluorescent substrate analog 4‐methylumbelliferyll‐β‐D‐N,N ′ N ″ ‐triacetylchitotrioside. The Michaelis‐Menten constant was K m = 165 µM and V max = 0.59 µmol/min/mL at pH 6.0 and 25C, with a marginal effect of NaCl concentration on the kinetic parameters. Its molecular mass and isoelectric point indicate similarity to chitinase 3 from Penaeus japonicus . Molecular modeling revealed the presence of a hydrophobic cavity at the substrate binding site. The mRNA was detected through reverse transcription‐polymerase chain reaction in the digestive gland but not in muscle, pleopods or gills, suggesting a role in food digestion.PRACTICAL APPLICATIONS The shrimp processing industry generates large amounts of shell and heads waste. Shells are chemically treated, requiring hard acid and alkali, to obtain chitosan. Shrimp heads waste also contains a large amount of hydrolytic enzymes, such as proteases and chitinases. We characterized the kinetics and the expression of a shrimp chitinase. This enzyme can be obtained as a subproduct from heads waste or produced as recombinant protein, since the cDNA sequence is available, to be used potentially for less‐contaminating chitosan production.