Why do we study b-glucosidases?
b-Glycosidases occurs in all three domains of living organisms and play a key role in many biological processes, which make them a suitable target for protein engineering to address the problems of biomass production in agriculture and forestry, as well as biomass conversion in biotechnology. The following selected cases illustrate the importance of enzyme in plants.

Defense. Plants are anchored to the soil and generally cannot hide or escape from pests and environmental stresses. Consequently, plants have evolved defense mechanisms against pests based on storing and releasing toxic chemicals. These defense chemicals are typically b-glucosides in monocots and dicots and b-glucosinolates in certain dicots. b-glucosidic substrates and b-glucosidases are stored in different subcellular or tissue compartments (1,2). Damage to cells and tissues by pests brings the enzyme and substrate together, leading to the hydrolysis of substrate and release of bitter and toxic aglycones and their breakdown products (e.g., thiocyanates, isothiocyanates, nitriles, terpenoid alkaloids, saponins, hydroxymates, benzaldehydes, HCN). These substances then deter herbivores and inhibit the entry, growth and spread of phytopathogens, serving as a built-in pest control system. For example, it has been shown that environmental stresses ranging from chewing insects (3), nematodes (4) and phosphate starvation (5) to cold (6) as well as specific treatments (e.g., jasmonic acid) (3) induces b-glucosidase genes in Arabidopsis thaliana while NaCl suppresses (7). In a recent study of the transcriptional profile of Arabidopsis thaliana during systemic acquired resistance (SAR), Macek et al. (8) showed that transcription of the b-glucosidase gene psr3.1 was elevated nearly 8-fold within 48 hours after infection with the oomycete Peronospora parasitica. Another Arabidopsis thaliana b-glucosidase gene (T209.120) has been shown to be associated with growth arrest and senescence in cell cultures and mature plants (9) and is also reported to be cold inducible (6). Although the precise role of these genes in stress, defense and senescence response is not known, two (psr3.1 and F19K6.15) encode b-glucosidases with a C-terminal ER retention signal and may be involved in signal transduction by activating another protein or nonprotein component in the ER via deglycosylation. These genes are potential targets for engineering enhanced crop protection and reducing or eliminating the need for costly and environmentally undesirable pesticides.

Food Processing and Quality Enhancement. There are several hundred different b-glucosidic flavor precursor identified from plants whose aglycones are products of mevalonate or shikimate pathways. Obviously, there are b-glucosidases in source plant tissues that hydrolyze these flavor precursors. Thus, in each case, there is need for isolating and characterrizing the specific enzyme that hydrolyzes a b-glucoside whose aglycone moiety is of interest to food quality and processing. Such biochemical data are crucial to making practical decisions as to whether or not enzymes from host plants or other sources should be added to drinks and beverages before, during or after processing to enhance flavor, aroma and other quality factors. Likewise, such data are essential for targeting enzymes with desirable properties for overproduction in transgenic microbial or plant hosts and improvement of their catalytic properties and stability for specific uses by genetic engineering.
Another aspect of b-glucosidases that pertain to food processing and quality is that edible portions of some plants contain compartmentalized b-glucosidase-b-glucoside systems that produce toxic aglycones and/or HCN when tissue is macerated during preparation or by chewing. This is exemplified by cassava roots and leaves, lima beans and flax seed. Of these, cassava is a food staple in tropical regions of Africa, Asia and South America, consumption reaches about 1 kg/per capita/day in some parts of Africa (e.g., Congo). It contains the cyanogenic b-glucoside linamarin and the corresponding b-glucosidase linamarase. When consumed raw, cyanide poisoning can occur depending on the amount ingested, where symptoms are difficulty in breathing, paralysis, convulsion, coma and even death. Cooking inactivates the enzyme and eliminates the possibility of cyanogenesis. Similar symptoms can arise when bitter almonds are eaten and ingested without roasting.
The myrosinase-glucosinolate (or b-thioglucosidase-b-thioglucoside) system, which occurs in cruciferous vegetables (e.g., mustard, cabbage, kale, broccoli, rapeseed, horseradish, etc.), has also importance for food quality and processing because the aglycone moiety and its breakdown products from enzymatic hydrolysis of glucosinolates are responsible for bitter, pungent taste and aroma associated with these vegetables, as well as the processed foods and relishes that include them (10). The distinct flavor associated with glucosinolates comes primarily from isothiocyanates and is believed to have evolved to serve as a repellent against microorganisms and herbivores. Glucosinolates and their breakdown products may impart undesirable flavors to milk, meat and eggs when farm animals graze on cruciferous plants or when their feed includes seed meals from such plants. Besides off-odors and flavors in foods of animal origin associated with glcosinolates, direct ingestion of large amount of cruciferous vegetables is thought to cause endemic goiter in man, as well as toxicity in laboratory animals. Similarly, claims have been made on anti-carcinogenic effects of glucosinolates and their breakdown products in humans. Although the precise mechanism of action is not clear, studies on rodents showed that raw or cooked cruciferous vegetables (e.g., cabbage, broccoli, cauliflower and turnip) increased aryl hydrocarbon hydroxylase activity (11).

Biomass Conversion. Polysaccharides, specifically cellulose, are the most abundant substances in the biosphere (~5x1010 tons produced/year) and are potential renewable sources of chemicals and fuels. Moreover, about 40% of typical municipal garbage includes newspaper and other paper products. Hydrolysis of cellulose using inorganic acids and high temperature is not ecologically sound and economically feasible. An enzyme (cellulase) complex, secreted by cellulolytic organisms, can hydrolyze cellulose to glucose, thus presenting itself as a suitable model for industrial processes that need to be developed (12). The complex includes three enzymes: an endoglucanase, an exoglucanase (cellobiohydrolase) and a b-glucosidase. The rate-limiting step in cellulose degradation is the one that is catalyzed by b-glucosidase, which hydrolyzes cellobiose and other small cellodextrins to glucose. Therefore, cellulosic biomass degradation and cellulose conversion to glucose at industrial scale by using microorganisms or isolated cellulase complex hinge upon increasing the rate of enzymatic reactions and overcoming product inhibition. Some plant b-glucosidases are specifically implicated in cellulose or cell wall metabolism during germination and growth as they hydrolyze cellobiose, as well as other disaccharides and oligosaccharides resulting from cell wall catabolism (13,14). Such b-glucosidases are potential targets for engineering to use in the degradation of cellulosic biomass by the cellulase complex.

Lignin Biosynthesis and Paper Quality. Lignin is the second most abundant substance in the biosphere and its major precursor, coniferyl alcohol, is derived from coniferin (4-O-coniferyl glucoside) after hydrolysis by b-glucosidase (15), suggesting that some plant b-glucosidase isoforms are involved in lignin biosynthesis. This makes the enzyme a suitable target for improving wood strength and quality for paper production.

Growth and Development. There is circumstantial evidence that b-glucosidases are involved in growth and development by releasing active hormones from phytohormone glucoconjugates (16,17,18), another potential function for some plant b-glucosidases. If indeed this function could be unequivocally shown, it opens the door to engineering the enzyme for regulating and improving plant growth and development to enhance productivity.

Secondary Plant Metabolism. Many secondary plant biochemical pathways (e.g., phenyl propanoid metabolism) use b-glucosides as precursors, intermediates or synthesize them as end products. Some of these substances may be glycosylated to enhance solubility or may be deglycosylated as part of degradation pathway. Very little is known about the fate and function of many b-glucosides. These compounds must have a function and be hydrolyzed by b-glucosidases exhibiting unique substrate specificities and tissue and cellular localization, providing further clues to the existence of a 44-member multigene family encoding b-glucosidase in Arabidopsis thaliana.

References.