Chapter 38
Xylose—Glucose Isomerases Structure, Homology, and Function
Downloaded by UNIV OF QUEENSLAND on October 30, 2015 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch038
Stanley M. Lastick and C. Thomas Spencer Applied Biological Sciences Section, Biotechnology Research Branch, Solar Fuels Research Division, Solar Energy Research Institute, 1617 Cole Boulevard, Golden, CO 80401
Interest in the bacterial enzyme xylose/glucose isomerase has been driven by its use in the isomerization of glucose to fructose to produce high-fructose corn syrups, and in the isomerization of xylose to xylulose for the conversion of the more fermentable xylulose to ethanol. In this work, a brief historical perspective is presented, followed by a summary of the current understanding of the enzyme's major features. Also, a useful compilation of available xylose isomerase DNA sequences is presented with annotation of some of the major areas identified as being of functional significance. The extent of homology between the xylose isomerases is discussed with reference to differences in their function. Xylose isomerases (EC 5.3.1.5), often referred to as glucose isomerase, have been studied extensively, in large part because of their use in the conversion of glucose to fructose for high-fructose corn syrup (HFCS). The world market for HFCS is expected to reach a total of 7.9 million metric tons in 1990 which, at a cost of $0.20/LB, would amount to $3.2 billion (1), and sales of xylose isomerase is expected to be about $15 million (T. Wallace, International Biosynthetics, personal communication). Research on xylose isomerase has produced DNA sequences of the gene from a number of bacterial strains, including the detailed structure of the xylose operon (2-7). In addition, x-ray crystallographic studies (8), kinetic measurements (9), and the use of inhibitors (10,11) have led to descriptions of the location of the active site and mechanistic models of its activity. The enzyme is also being studied for use in converting of biomass to ethanol for fuel usage. Prospects for the conversion of cellulolytic biomass to ethanol for fuel or as a fuel additive have improved within the last decade because of the development of methods for the fermentation of xylose, which can comprise as much as 50% of the fermentable sugars in these feedstocks . One of these methods uses xylose isomerase to convert xylose, which is difficult to ferment by ethanol-tolerant yeasts, to the fermentable sugar xylulose (12,13). Initial Discoveries. Xylose isomerase activity was initially found in 1953 in extracts of Lactobacillus pentosus (14), followed by similar activities in extracts of Pseudomonas hydrophila and Pasteurella pestis in the mid-1950s (15-17). An enzyme activity that was found to convert glucose to fructose was discovered in 1957 (18). This activity, found in sonicated extracts from Pseudomonas hydrophila, was enhanced in the presence of 0097-6156/91/0460-0486$06.00/0 © 1991 American Chemical Society In Enzymes in Biomass Conversion; Leatham, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
Downloaded by UNIV OF QUEENSLAND on October 30, 2015 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0460.ch038
38.
LASTICK AND SPENCER
Xylose-Glucose Isomerases
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arsenate and had a substrate specificity that was 100 fold higher on xylose than glucose. The requirement for arsenate and low affinity toward glucose makes the enzyme unsuitable for HFCS production, but the result showed that enzyme-mediated conversion of glucose to fructose was feasible. Other arsenate-requiring enzymes, isolated from an Aerobacter sp. and Escherichia freundii, were found to isomerize glucose to fructose (19,20), but, at least in some cases, the enzymes required arsenate only when glucose or fructose, and not when glucose-6phosphate or fructose-6-phosphate were used as substrates. Purification of the arsenatedependent activity from Escherichia intermedia allowed the conclusion that an arsenateglucose complex was formed during the reaction that allowed the enzyme, now identified as phospho-glucose isomerase (EC 5.3.1.9), to isomerize the sugar (21,22). True xylose isomerase activity was distinguished by constant glucose-to-xylose conversion ratios through purification and in the presence of inhibitors. A true xylose isomerase, that did not require arsenate for its activity, was found in strains of Lactobacillus, especially L. brevis (23). In this study, it was found that the activity of isomerization of glucose and xylose were essentially equal, and thatribosewas isomerized to ribulose at a reduced rate (24). The xylose isomerases from this strain, like those from other species, requires divalent cations for activity. In this case, Mn** and Co** were found to be required for activity. In other studies, Co** has been found to increase xylose isomerase stability (25,26,27). Since these early discoveries, xylose isomerases have been isolated from many bacterial species, and these enzymes have been intensely investigated, especially those of the genera Streptomyces, Lactobacillus, and Bacillus. The characteristics of substrate specificity (xylose > glucose > ribose), divalent metal cation activation (Mg**, Mn** or Co**), and activity at alkaline pH are properties that most of the enzymes share to a certain extent, but significant variations exist. Some of these enzymes have been immobilized and patented for commercial use. There are many good reviews in the literature that describe the enzymatic characteristics of the xylose isomerases (9,28,29). Xylose isomerases with higher thermostability were found in the strains of Streptomyces and related Actinoplanaceae (which includes the genera Ampullariella and Actinoplanes). High thermo-tolerance is desirable for production of HFCS because at equilibrium, as the temperature of the enzyme reaction is increased, the ketose/aldose ratio increases proportionately (30). In addition, reactors running at higher temperatures have less risk of microbial contamination, allowing for less frequent and less costly enzyme replacement. Another factor contributing to enzyme cost is the need for xylose, an expensive substrate, to induce the biosynthesis of the enzyme. This consideration has led to the discovery of strains having constitutive production of the enzyme (31,32) and work that significantly increased enzyme titres through recombinant DNA procedures have been successful (33,34). Mechanism of Isomerization. The overall reaction for xylose isomerase in the isomerization of aldose to ketose sugars is essentially the intramolecular transfer of a hydrogen from C2 to CI of the sugar, accompanied by loss of a proton from the C2hydroxyl and proton addition to the carbonyl group at CI. Although there is some controversy over the detailed mechanism of activity, it is generally agreed that there are two cation-binding sites (35), and that the primary substrate of the forward reaction is an a-D-pyranose (8). The first step of the reaction is the enzyme-mediated opening of the ring structure, followed by the hydrogen transfers. Studies using specific chemical modification of amino acids identified the presence of a single histidine residue that is essential for enzyme activity (10). Two models for the mechanism of the isomerization have been proposed. The first model was originally proposed because of similarities between enzymes involved in the activation of C-H bonds next to a carbonyl function (Fig. la). Stereochemical considerations were used to suggest that the ring structure of the sugar is opened to
In Enzymes in Biomass Conversion; Leatham, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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ENZYMES IN BIOMASS CONVERSION
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