In today's world, there is an increasing trend towards the use of renewable, cheap and readily available biomass in the production of a wide variety of fine and bulk chemicals in different biorefineries. Biorefineries utilize the activities of microbial cells and their enzymes to convert biomass into target products. Many of these processes require enzymes which are operationally stable at high temperature thus allowing e.g. easy mixing, better substrate solubility, high mass transfer rate, and lowered risk of contamination. Thermophiles have often been proposed as sources of industrially relevant thermostable enzymes. Here we discuss existing and potential applications of thermophiles and thermostable enzymes with focus on conversion of carbohydrate containing raw materials. Their importance in biorefineries is explained using examples of lignocellulose and starch conversions to desired products. Strategies that enhance thermostablity of enzymes both in vivo and in vitro are also assessed. Moreover, this review deals with efforts made on developing vectors for expressing recombinant enzymes in thermophilic hosts.

Onion waste is a renewable raw material, rich in different molecular species of the antioxidant quercetin. To utilize this resource, an environmentally sustainable procedure has been developed, using pressurized hot water to extract the quercetin species, followed by biocatalytic conversion of the quercetin glycosides to quercetin and carbohydrates. Two different recombinantly expressed thermostable b-glucosidases, Thermotoga neapolitana b-glucosidase A and B, were utilized as catalysts. These enzymes maintain activity at temperatures around 90 uC, and are therefore ideal to use in combination with hot water extraction. Our results, based on experimental design, showed that they converted quercetin glycosides to active quercetin in less than 10 min reaction time in water at 90 uC, pH 5.0. Experimental design showed that the optimal extraction conditions included three 5 min extraction cycles with water at 120 uC and 50 bars, giving a total extraction time of 15 min. Several different types of quercetin and isorhamnetin glycosides as well as kaempferol were detected in onion waste using LC-MS/MS analysis. After converting the different glycosidic compounds to their respective aglycones, the quercetin content was 10 to 50 mg g 21 dry weight of onion waste (RSD 8%). In summary, our research demonstrates that subcritical water extraction followed by b-glucosidase-catalyzed hydrolysis is a rapid method to determine the content of quercetin and isorhamnetin in onion samples, and is environmentally sustainable as it only uses water as solvent and enzymes as catalysts.

Previous work [Lorand, L., Dailey, J. E., & Turner, P. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1057-1059] showed that fibronectin might serve as a specific carrier for transglutaminases accidentally discharged from erythrocytes or other cells into plasma. In the present study we examined the association of these proteins in purified systems. Complexation was readily demonstrable by nondenaturing electrophoresis, using dansylcadaverine-dependent activity staining as well as immunoblotting procedures, and also by HPLC gel filtration. The results indicate a stoichiometry of 2:1 for the binding of the human erythrocyte transglutaminase (80K) to human plasma fibronectin (440K). The attachment is noncovalent in nature and does not involve cross-linking of the proteins either to themselves or to each other. Binding occurs in the absence of Ca2+, suggesting that a domain on the transglutaminase molecule other than the catalytic site is needed for complexation with fibronectin. Limited proteolysis with chymotrypsin for delineating the relevant region in fibronectin yielded two gelatin- (collagen) binding fragments (56K and 46K), each displaying affinity for transglutaminase. Moreover, these fragments--like intact fibronectin--bound erythrocyte transglutaminase and gelatin simultaneously in ternary complexes.

Sphingobium chlorophenolicum Strain L-1 can mineralize the toxic pesticide pentachlorophenol (PCP). We have sequenced the genome of S. chlorophenolicum Strain L-1. The genome consists of a primary chromosome that encodes most of the genes for core processes, a secondary chromosome that encodes primarily genes that appear to be involved in environmental adaptation, and a small plasmid. The genes responsible for degradation of PCP are found on chromosome 2. We have compared the genomes of S. chlorophenolicum Strain L-1 and Sphingobium japonicum, a closely related Sphingomonad that degrades lindane. Our analysis suggests that the genes encoding the first three enzymes in the PCP degradation pathway were acquired via two different horizontal gene transfer events, and the genes encoding the final two enzymes in the pathway were acquired from the most recent common ancestor of these two bacteria.

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