George H. Robertson

Purification of limonoid glucosyltransferase from navel orange albedo tissues

Abstract UDP- d -glucose: limonoid glucosyltransferase was purified from albedo tissues of navel orange (Citrus sinensis) cultivars, Frost and Newhall, by a combination of (NH4)2SO4 fractionation, UDP-glucuronic acid affinity chromatography and DEAE ion exchange HPLC. This procedure resulted in a 452-fold increase in enzyme purification. This enzyme catalysed the glucosylation of both nomilin and limonin. SDS-PAGE showed a Mr, of 56–58 k for the enzyme. The enzyme displayed a peak of activity between pH 6.5 and 9.0 with an optimum at 8.0. Mn2+ stimulated enzyme activity by 66% over basal activity observed with EDTA. Activity was lost when the purified enzyme was frozen and stored in Tris-HCl buffer at pH 8.0. Published by Elsevier Science Ltd

A Functional Raw Starch-Binding Domain of Barley α-Amylase Expressed in Escherichia coli

The mature form of barley seed low-pI α-amylase (BAA1) possesses a raw starch-binding site in addition to the catalytic site. A truncated cDNA encoding the C-terminal region (aa 281–414) and containing the proposed raw starch-binding domain (SBD) but lacking Trp278/Trp279, a previously proposed starch granule-binding site, was synthesized via PCR and expressed in Escherichia coli as an N-terminal His-Tag fusion protein. SBD was produced in the form of insoluble inclusion bodies that were extracted with urea and successfully refolded into a soluble form via dialysis. To determine binding, SBD was purified by affinity chromatography with cycloheptaamylose as ligand cross-linked to Sepharose. This work demonstrates that a SBD is located in the C-terminal region and retains sufficient function in the absence of the N-terminal, catalytic, and Trp278/279 regions.

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.

An α-Glucuronidase Enzyme Activity Assay Adaptable for Solid Phase Screening

Glucuronic acid is a common chemical moiety that decorates the xylan polymer of hemicellulose. This chemical substituent impairs both enzymatic and acidic hydrolysis of xylosidic bonds. The α-glucuronidase enzyme hydrolyzes the 1,2-linked glucuronic acid from the terminal, non-reducing xylose of xylo-oligosaccharides. There are relatively few α-glucuronidase genes in the public databases. We have developed an assay with commercially available reagents that can be used to search DNA libraries for α-glucuronidase genes in a high-throughput, solid phase activity screen.

An E. coli Expression System for the Extracellular Secretion of Barley α-Amylase

Libraries of modified genes are often screened during the process of genetically engineering enzymes with specifically tailored activities. It is important, therefore, to create expression systems which allow for the rapid screening of many clones. We developed an Escherichia coli expression system which will secrete enzymes into the growth medium. We describe the first reported expression of barley α-amylase in E. coli. The enzyme is secreted onto solid media containing starch to produce easily visualized halos. In addition, the enzyme is secreted into liquid media in an intact, active form.

Combinatorial Chemistry and its Applications in Agriculture and Food

Combinatorial chemistry has become a major focus of research activity in the pharmaceutical industry for development new therapeutic compounds. The same techniques could be potentially applied to benefit agricultural and food research. This article reviews the various procedures used in combinatorial chemistry, outlines some of the strengths and limitations of the various methods, and proposes potential areas in agriculture and food that could be benefited by this technology. These areas include developing new antimicrobial agents, antioxidants, and other additives, creating antigen-binding molecules for the detection or removal of food pathogens or toxicants, engineering food proteins and enzymes for specific functions, and modifying biosynthetic pathways for the production of novel natural products.

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.