Purification, characterization, and partial nucleotide sequencing of soybean alpha-galactosidase

Jill Elizabeth Porter, Purdue University

Abstract

Soybeans (Glycine max) contain an $\alpha$-galactosidase that makes up 0.05% of the total protein of the seed. $\alpha$-Galactosidase hydrolyzes stachyose and raffinose into galactose and sucrose, the initial energy sources in the germinating seed. This work demonstrates the feasibility of a scale-up based on a laboratory scale purification of a process for recovery of a plant protein which comprised a minor part of the total plant protein. In carrying out this work, corn starch was examined as a desalting media and was characterized as a chromatographic support. It separates sugars based on molecular weight, and separates BSA from salt and may be a useful tool for desalting proteins such as $\alpha$-galactosidase on a large scale. Corn starch, which has narrow particle size of 24 $\mu$m, could be a useful tool for desalting proteins such as $\alpha$-galactosidase on a large scale. Another purification method considered for large scale use was selective precipitation of $\alpha$-galactosidase contaminants by heat treatment. Galactose, a competitive produce inhibitor of $\alpha$-galactosidase, strongly promotes the heat stability of the tetrameric form of the enzyme at temperatures of up to 70$\sp\circ$C for 60 minutes. Stachyose and raffinose also protect $\alpha$-galactosidase from denaturation at pH 4.0 although to a lesser extent. Glucose and mannose have little effect. The protective effect may be used to purify $\alpha$-galactosidase from other proteins, particularly in a recombinant microorganism fermentation. Purified protein from the large scale extraction was used to make polyclonal antibodies to $\alpha$-galactosidase. The polyclonal antibodies were used to show that $\alpha$-galactosidase polyA RNA is present in both the developing and germinating seed. A segment of the soybean $\alpha$-galactosidase cDNA has been isolated using homologue oligonucleotide probes in the polymerase chain reaction. This segment is very similar to guar cDNA and shares about 45% sequence identity with $\alpha$-galactosidase cDNA from human and yeast and N-acetylgalactosaminidase cDNA from human. The partial soybean sequence is a first step towards expressing $\alpha$-galactosidase in a recombinant microorganism. The partial cDNA sequence may be used in designing primers for further probing of a cDNA library to deduce the remainder of the sequence. (Abstract shortened with permission of author.)

Degree

Ph.D.

Advisors

Herrmann, Purdue University.

Subject Area

Agricultural engineering

COinS