Purification and characterization of (1 to 3, 1 to 4)-beta-glucan endohydrolases from germinated wheat (Triticum aestivum)

• Published in: Plant molecular biology 1993-08, Vol.22 (5), p.847-859
• Main Authors: Lai, D.M.L. (Trobe Univ., Bundoora, Vic. (Australia). Dept. of Biochemistry), Hoej, P.B, Fincher, G.B
• Format: Article
• Language: English
• Subjects: ACIDE AMINE; ADN; aleurone layer; Amino Acid Sequence; amino acid sequences; AMINO ACIDS; AMINOACIDOS; Base Sequence; Biological and medical sciences; Biotechnology; Blotting, Western; Chromatography, Thin Layer; Codon; codon usage; complementary DNA; DNA; Electrophoresis, Polyacrylamide Gel; enzyme activity; Enzymes; Fundamental and applied biological sciences. Psychology; genbank/z22873; genbank/z22874; genetic code; GLUCANE; GLUCANO; GLUCANS; Glycoside Hydrolases - genetics; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; LIASAS; licheninase; LYASE; LYASES; Metabolism; Molecular Sequence Data; NUCLEOTIDE SEQUENCE; nucleotide sequences; Plant physiology and development; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; purification; SECUENCIA NUCLEICA; Sequence Homology, Amino Acid; SEQUENCE NUCLEIQUE; Substrate Specificity; Triticum - enzymology; Triticum - genetics; Triticum - physiology; TRITICUM AESTIVUM
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/bf00027370

A (1 leads to 3, 1 leads to 4)-beta-glucan 4-glucanohydrolase [(1 leads to 3, 1 leads to 4)-beta-glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (1 leads to 3, 1 leads to 4)-beta-glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (1 leads to 3, 1 leads to 4)-beta-glucanase isoenzyme EI from barley. The complete primary structure of the wheat (1 leads to 3, 1 leads to 4)-beta-glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)+ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated lambda LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH2-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated lambda LW1, carries a 109 nucleotide pair sequence at its 5' end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3'-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.