Shelby N. Freer

Purification and characterization of an extracellular endoglucanase from the marine shipworm bacterium

Bacterial cultures isolated from the gland of Deshayes of marine shipworm (Psiloteredo healdi) produced extracellular endoglucanase activity when cultured with 1% cellulose. An endoglucanase of subunit relative molecular mass 58,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to homogeneity from cell-free culture medium. Similarly, the relative molecular mass of the native enzyme was 60,100 as determined by gel permeation chromatography. No carbohydrate appeared to be associated with the purified protein. The action of the purified enzyme on various cellodextrins was also studied. Only interior glucosyl linkages of cellodextrin chains larger than cellotriose were cleaved by the enzyme and the centermost bond of cellohexaose was preferentially cleaved. The Km values of the purified endoglucanase were 0.12 mM for cellotetraose, 0.05 mM for cellopentaose, and 0.11 mM for cellohexaose. Glucose, cellobiose, and cellotriose did not inhibit enzymatic activity.

Direct fermentation of cellodextrins to ethanol byCandida wickerhamii andC. Lusitaniae

SummaryProduction of ethanol from cellodextrins, as large as cellohexose, byCandida lusitaniae andC. wickerhamii was studied.C. lusitaniae fermented only glucose and cellobiose, whereasC. wickerhamii efficiently fermented cellodextrins. Maximum ethanol yields of 29.2 g/liter from 54 g/liter cellodextrins were achieved byC. wickerhamii in 3–4 days.

Purification and characterization of the extracellular β-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.

Extracellular endoglucanase activity by a novel bacterium isolated from marine shipworm

An extracellular enzyme preparation from shipworm bacterium cultures dramatically increased reducing sugar content of carboxymethylcellulose (CMC3), but did not solubilize sugar from particulate cellulose. The preparation degraded cellodextrins larger than cellotriose (G3). Only interior cellodextrin chain linkages were cleaved and the center-most bond of cellohexaose (G6) was preferentially cleaved. Activity maxima were observed at 60 degrees C and between pH 5.0 and 7.0. The activity was resistant to protease treatment and little loss of activity was observed after 14 d at 25 degrees C.