Sarah B. Batt

Increased Expression and Secretion of Recombinant α-Amylase in Saccharomyces cerevisiae by Using Glycerol as the Carbon Source

Saccharomyces cerevisiae transformed with plasmids containing the barley α-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).4 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 α-amylase secreted by the yeast clone DY150[pYEX-Amy1] 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 α-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.

Characterization of Active Barley α-Amylase 1 Expressed and Secreted by Saccharomyces cerevisiae

Recombinant barley α-amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified α-amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The kcat/Km was 2.7 × 102 mM−1.s−1, consistent with those of α-amylases from plants and other sources.

Cloning and Characterization of an Exo-Xylogucanase from Rumenal Microbial Metagenome

A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (a/b)(8) fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards only xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding K(m) and V(max) of 2.12+/-0.13 mg/ml and 0.17+/-0.01 mg/ml/min (37 degrees C, pH 6.0), respectively.

Direct Screening Of Libraries Of Yeast Clones For α-Amylase Activity On Raw Starch Hydrolysis

High-throughput screening for high-activity barley a-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.

Isolation of a raw starch-binding fragment from barley alpha-amylase.

Barley α-amylase was purified by ammonium sulfate fraction, ion-exchange, ultrafiltration, and gel filtration to homogeneity. The purified enzyme was partially digested with trypsin, and the reaction mixture was applied to a cyclohepta-amylose epoxy Sepharose 6B column. Bound fragments were eluted by free cyclohepta-amylose, lyophilized, and separated on Tricine gels. Four fragments were shown to interact with β-cyclodextrin. The fragment that could be identified on the gel with the lowest molecular weight (11 kDa) was electroblotted onto PVDF membrane for sequencing. The N-terminal sequence of this fragment was determined with the N-terminal amino acid corresponding to Ala283 in the whole protein. The trypsin cleavage was at Lys282/Ala283 and the C-terminal cleavage occurred at Lys354/Ile355 to give a fragment size of 11 kDa as estimated by SDS-PAGE. The fragment would be located at the C-terminal region, forming a majority of the antiparallel β-sheets in domain C and the α7-and α8-helices of the (α/β)8 domain.