Purification, characterization and amino‐acid sequence analysis of a thermostable, low molecular mass endo‐β‐1,4‐glucanase from blue mussel, Mytilus edulis

Abstract

A cellulase (endo-b-1,4-d-glucanase, EC 3.2.1.4) from blue mussel (Mytilus edulis) was purified to homogeneity using a combination of acid precipitation, heat precipitation, immobilized metal ion affinity chromatography, size-exclusion chromatography and ion-exchange chromatography. Purity was analyzed by SDS/PAGE, IEF and RP-HPLC. The cellulase (endoglucanase) was characterized with regard to enzymatic properties, isoelectric point, molecular mass and amino-acid sequence. It is a single polypeptide chain of 181 amino acids cross-linked with six disulfide bridges. Its molecular mass, as measured by MALDI-MS, is 19 702 Da; a value of 19 710.57 Da was calculated from amino-acid composition. The isoelectric point of the enzyme was estimated by isoelectric focusing in a polyacrylamide gel to a value of 7.6. According to amino-acid composition, the theoretical pI is 7.011. The effect of temperature on the endoglucanase activity, with carboxymethyl cellulose and amorphous cellulose as substrates, respectively, was studied at pH 5.5 and displayed an unusually broad optimum activity temperature range between 30 and 50 8C. Another unusual feature is that the enzyme retains 55±60% of its maximum activity at 0 8C. The enzyme readily degrades amorphous cellulose and carboxymethyl cellulose but displays no hydrolytic activity towards crystalline cellulose (Avicel) and shows no cross-specificity for xylan; there is no binding to Avicel. The enzyme can withstand 10 min at 100 8C without irreversible loss of enzymatic activity. Amino-acid sequence-based classification has revealed that the enzyme belongs to the glycoside hydrolase family 45, subfamily 2 (B. Henrissat, Centre de Recherches sur les Macromolecules Ve Âge Âtales, CNRS, Joseph Fourier Universite Â, Grenoble, France, personal communication).

Purification and biochemical characterization of a cellulase from the digestive organs of the short-spined sea urchin Strongylocentrotus intermedius

“…In our study, short-spined sea urchin cellulase was purified using a combination of ionexchange chromatography and gel filtration together with CMCase activity assays. The molecular weights of cellulases have been reported from various organisms, such as plants (50- [15][16][17][18]) and sea urchin (54 kDa [20]), and the range has been found to be quite wide. The molecular weight of purified short-spined sea urchin cellulase was estimated to be 59 kDa, a value which falls well within the range of previously reported cellulases from marine invertebrates, including abalone [18] and Northern sea urchin [20].…”

“…Complete depolymerization of cellulose to glucose requires another sort of cello-oligosaccharide-degrading enzyme, 1,4-b-glucosidase (EC 3.2.1.21). Thus far, cellulolytic enzymes have been isolated and characterized from bacteria [2], fungi [2], plants [3], molds [4], microbes [5], arthropods [6][7][8][9][10][11], nematodes [12], mollusks [13][14][15][16][17][18][19] and sea urchin [20].…”

Purification and Characterization of Recombinant Endoglucanases from the Pine Wood NematodeBursaphelenchus xylophilus

“…This is also the case for other cell-wall enzymes such as -1,3-glucanase 12) and pectate lyase 13) from B. xylophilus and several invertebrate GHF45 endoglucanases. [14][15][16] Bx-ENGs displayed the highest activity toward lichenan and did not hydrolyze -1,3-1,6 linked glucan (pustran), -1,3 linked glucan (laminarin), galactomannan, xylan, or xyloglucan as substrates. Similar results (strict specificity toward -(1,4)-glucoside linkage) have been obtained for other GHF45 enzymes.…”

“…Similar results (strict specificity toward -(1,4)-glucoside linkage) have been obtained for other GHF45 enzymes. [16][17][18][19] The lack of activity toward xyloglucan suggests that the high level of branching interfered with access to the catalytic cleft of the GHF45 enzyme.…”

The genome of the mustard leaf beetle encodes two active xylanases originally acquired from bacteria through horizontal gene transfer

“…gh11-1 and gh11-2 ORFs were amplified from cDNAs using genespecific primers (see the electronic supplementary material, table rspb.royalsocietypublishing.org Proc R Soc B 280: 20131021 S1) and were cloned into the pMIB/V5-His vector B, in frame with a V5-(His) 6 epitope at the carboxyl-terminus. Positive constructs were then transfected in High Five cells (Invitrogen) using Insect GeneJuice (Novagen) as a transfection reagent.…”