Characterization and action patterns of two beta-1,4-glucanases purified from cellulomonas uda CS1-1.
Two beta-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were 1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr15- for DI and 1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys15- for DIII. The apparent sequences exhibited high sequence similarities with other bacterial beta-1,4-glucanases as well as beta-1,4-xylanases.
Yoon
Min-Ho
MH
Department of Bio-Environmental Chemistry, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 305-764, Korea. mhyoon@cnu.ac.kr
Choi
Woo-Young
WY
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D040143:Cellulomonas / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002482:Cellulose / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D044643:Glucan 1,4-beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014990:Xylans / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D014990:Xylans; D002482:Cellulose; D002480:Cellulase; D044643:Glucan 1,4-beta-Glucosidase; D002266:Carboxymethylcellulose Sodium