Biomedical subjects

Sarah B Batt

Publications and source records attributed to Sarah B Batt.

4 recordsLinked to original sources

PubMed2005

Characterization of active Lentinula edodes glucoamylase expressed and secreted by Saccharomyces cerevisiae.

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH4)2SO4 fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4-6.4 stability range. The temperature optimum was 50 degrees C with stability <or=50 degrees C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn++ activated the enzyme by 45%, while Li+, Zn++, Mg++, Cu+, Ca++, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10x the amount of GLA. Apparent Km and Vmax for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40 degrees C, pH 5.3), with an apparent kcat of 2.9 x 10(5) min(-1).

Amino Acid Sequence↗

PubMed2004

High-activity barley alpha-amylase by directed evolution.

Barley alpha-amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to directed evolution. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of directed evolution, one mutant (Mu322) showed 1000 times the total activity and 20 times the specific activity of the wild-type enzyme produced by the same yeast expression system. Comparison of the amino acid sequence of this mutant with the wild type revealed five substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations. Q44H and R303K, result in amino acids highly conserved in cereal alpha-amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.

Amino Acid Sequence↗

PubMed2003

Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch hydrolysis.

High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of reducing agents, particularly the glucose used in yeast growth media. The present investigation employed colorimetric and chemiluminescent detection systems that enable direct and rapid screening of activities on raw starch substrate. Active clones could be separated into two groups, based on high total activity or high specific activity.

Base Sequence↗

PubMed2002

Increased expression and secretion of recombinant alpha-amylase in Saccharomyces cerevisiae by using glycerol as the carbon source.

Saccharomyces cerevisiae transformed with plasmids containing the barley alpha-amylase gene was cultured, and enzyme activity and cell density were monitored at various time intervals. Proteins in yeast extract and culture medium were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blots of intra- and extracellular proteins were sequentially probed with anti-amylase antibody and anti-rabbit horseradish peroxidase conjugate, followed by chemiluminescent detection. The enzyme activity of recombinant barley alpha-amylase secreted by the yeast clone DY150[pYEX-Amyl] showed a significant increase when the culture medium included glycerol as the carbon source. The enhancement reached a 4.5-fold increase at 120 hr, and the effect was strain-nonspecific. Intra- and extracellular proteins increased significantly with time in both the yeast clone and the control grown in YEPG (2% yeast extract, 1% bacto-peptone, 2% glycerol). Proteins in YEPD (2% yeast extract,1% bacto-peptone, 2% glucose) and YEPG cultures showed very different band patterns, indicating that the metabolic pathway was altered. Western blot analysis indicated that the recombinant amylase accumulated inside yeast cells, at a relatively low level, compared with that in the culture medium. The transcript level of the alpha-amylase gene was significantly increased in the clone cultured in YEPG. This investigation demonstrates that the use of glycerol as a carbon source for S. cerevisiae enhances the synthesis and secretion of the recombinant enzyme while suppressing cell growth.

Blotting, Western↗