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Purpose Calcium has beneficial effects in preventing osteoporosis and fractures among older adults but supplements may have adverse effects if calcium accumulates in the arteries. Aim was to examine total calcium intake from food and supplements of older residents in service houses and assess whether the use of supplements was directed appropriately. Subjects and methods Residents in service houses in the metropolitan region of Helsinki, Finland. Residents' calcium intake was calculated from one-day food diaries. Use of calcium supplements was retrieved from medical records. Results In total, 350 service house residents participated. Their mean age was 83 years; 82% were females. According to the MNA, 21% suffered from malnutrition. The mean dietary calcium intake of females was 1113mg per day. Including food and supplements, their mean intake was 1441mg. The corresponding figures for males were 1224mg and 1528mg. Of all residents, 13% had a total daily calcium intake from food and supplements that was<800mg, 29% 800-1200mg, and 59%>1200mg. More than one-third (37%) of the residents used calcium supplements daily. Of those having calcium intake from food<800mg, only 32% were administered calcium supplements. The respective proportions among groups in which calcium intake from food was 800-1200mg, 1200-1500mg, or>1500mg were 37%, 43%, and 36%. Conclusions Dietary intake of calcium should be evaluated and administration of calcium supplements should be directed to those whose intake of calcium from food is low. [ABSTRACT FROM AUTHOR]

Purpose: We evaluated oral frailty (OFr) and its association with health-related quality of life (HRQoL), energy and protein intake, and survival among older long-term care residents., Methods: This cross-sectional study with a 3-year follow-up for survival assessed 349 residents in long-term care facilities (73% female, mean age 82 years). We defined OFr with six signs (dry mouth, food residue on oral surfaces, unclear speech, inability to keep mouth open or pain expression during the clinical oral examination, diet pureed/soft) and OFr severity was categorized as Group 1, (mild) = 0-1 signs, Group 2 (moderate) = 2-4 signs, and Group 3 (severe) = 5-6 signs. We measured HRQoL with 15D instrument, and energy and protein intake by a 1- to 2-day food record. Mortality was retrieved from central registers on March 2021., Results: Of the residents, 15% had 0-1, 67% 2-4 and 18% 5-6 OFr signs. HRQoL decreased linearly from Group 1 to Group 3. OFr correlated with such dimensions of HRQoL as mobility, eating, speech, excretion, usual activities, mental function, and vitality. We found no association between OFr categories and energy and protein intake. Survival decreased linearly from Group 1 to Group 3., Conclusions: OFr was common among older long-term care residents and OFr severity predicts poorer outcomes. The six oral signs denoting OFr may be used at the bedside to screen residents at risk for OFr., (© 2023. The Author(s).)

Most new small drug molecules in pharmaceutical development require improvement of solubility. The controlled expansion of supercritical solutions (CESS®) process is a nanoparticle production technology, dedicated to enhancing the dissolution rate of active pharmaceutical ingredients (APIs) suffering from poor solubility and enabling novel drug delivery opportunities. In this process, the API is dissolved in supercritical carbon dioxide (scCO 2 ) and nanoparticles are formed through controlled pressure reduction. To improve process visibility and control, ultraviolet-visible (UV-Vis) spectroscopy was incorporated into CESS® process as a process analytical technology (PAT) tool. The tool quantifies the amount of API dissolved in scCO 2 during the solubilization phase of the process. Sample interfacing of the UV-Vis spectrometer was done with a custom-made pressure and temperature rated transmission flow-through cell. In-process calibration was developed to correlate the UV-Vis absorption spectra to the API concentration. Due to the density-dependent molar absorption coefficient of API in scCO 2 , the calibration was done for each combination of temperature and pressure. The developed PAT tool provides insight into the process enabling real-time API quantity estimation. It also facilitates process development through Quality by Design (QbD) and offers a system for enhanced process control and troubleshooting. For instance, the in-line API concentration data allows one to study the solubilization behavior of the API in the process and to optimize the process parameters in order to maximize throughput., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ari Kauppinen reports a relationship with Nanoform Finland Plc that includes: employment. Petteri Helander reports a relationship with Nanoform Finland Plc that includes: employment. Mikael Viitala reports a relationship with Nanoform Finland Plc that includes: employment. Tuomas Puranen reports a relationship with Nanoform Finland Plc that includes: employment. Tuomas Vainikka reports a relationship with Nanoform Finland Plc that includes: employment. Ilkka Lassila reports a relationship with Nanoform Finland Plc that includes: employment and equity or stocks. Edward Hæggström reports a relationship with Nanoform Finland Plc that includes: employment and equity or stocks. Niklas Sandler reports a relationship with Nanoform Finland Plc that includes: employment and equity or stocks. Edward Hæggström has patent A method and a device for producing nanoparticles issued to Nanoform Finland Oy. Ilkka Lassila has patent A method and a device for producing nanoparticles issued to Nanoform Finland Oy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Despite the ubiquitous use over the past 150 years, the functions of the current medical needle are facilitated only by mechanical shear and cutting by the needle tip, i.e. the lancet. In this study, we demonstrate how nonlinear ultrasonics (NLU) extends the functionality of the medical needle far beyond its present capability. The NLU actions were found to be localized to the proximity of the needle tip, the SonoLancet, but the effects extend to several millimeters from the physical needle boundary. The observed nonlinear phenomena, transient cavitation, fluid streams, translation of micro- and nanoparticles and atomization, were quantitatively characterized. In the fine-needle biopsy application, the SonoLancet contributed to obtaining tissue cores with an increase in tissue yield by 3-6× in different tissue types compared to conventional needle biopsy technique using the same 21G needle. In conclusion, the SonoLancet could be of interest to several other medical applications, including drug or gene delivery, cell modulation, and minimally invasive surgical procedures.

Oleogels are lipid-based soft materials composed of large fractions of oil (> 85%) developed as saturated and hydrogenated fat substitutes to reduce cardiovascular diseases caused by obesity. Promising oleogels are unstable during storage, and to improve their stability careful control of the crystalline network is necessary. However, this is unattainable with state-of-the-art technologies. We employ ultrasonic standing wave (USSW) fields to modify oleogel structure. During crystallization, the growing crystals move towards the US-SW nodal planes. Homogeneous, dense bands of microcrystals form independently of oleogelator type, concentration, and cooling rate. The thickness of these bands is proportional to the USSW wavelength. These new structures act as physical barriers in reducing the migration kinetics of a liposoluble colorant compared to statically crystallized oleogels. These results may extend beyond oleogels to potentially be used wherever careful control of the crystallization process and final structure of a system is needed, such as in the cosmetics, pharmaceutical, chemical, and food industries.

Studies in optics and acoustics have employed metamaterial lenses to achieve sub-wavelength localization, e.g. a recently introduced concept called 'acoustojet' which in simulations localizes acoustic energy to a spot smaller than λ/2. However previous experimental results on the acoustojet have barely reached λ/2-wide localization. Here we show, by simulations and experiments, that a sub-λ/2 wide localization can be achieved by translating the concept of a photonic jet into the acoustic realm. We performed nano- to macroscale molecular dynamics (MD) and finite element method (FEM) simulations as well as macroscale experiments. We demonstrated that by choosing a suitable size cylindrical lens, and by selecting the speed-of-sound ratio between the lens material(s) and the surrounding medium, an acoustic jet ('acoustic sheet') is formed with a full width at half maximum (FWHM) less than λ/2. The results show, that the acoustojet approach can be experimentally realized with easy-to-manufacture acoustic lenses at the macroscale. MD simulations demonstrate that the concept can be extended to coherent phonons at nanoscale. Finally, our FEM simulations identify some micrometer size structures that could be realized in practice. Our results may contribute to starting a new era of super resolution acoustic imaging: We foresee that jet generating constructs can be readily manufactured, since suitable material combinations can be found from nanoscale to macroscale. Tight focusing of mechanical energy is highly desirable in e.g. electronics, materials science, medicine, biosciences, and energy harvesting.

Introduction: The Mini Nutritional Assessment (MNA) is a well-validated instrument examining the nutritional status of older people. The aim of this study was to examine how older people's energy and nutrient intakes are associated with the MNA and to determine how sensitive and specific MNA is in identifying those having low energy and protein intakes., Materials and Methods: This cross-sectional study combined data from five nutritional studies (N=900): both home-dwelling and institutionalized older people without and with disabilities. Their nutritional status was assessed with MNA, and nutrient intakes were retrieved from 1 to 3day food diaries. Nutrient intakes were divided according to MNA status (normal nutritional status, at-risk of malnutrition, malnourished). Sensitivity, specificity, and likelihood ratios of MNA of various cut-off points were tested with recommended protein and energy intakes. ROC curves was constructed., Results: Energy, protein and most nutrient intakes showed logical linear trends according to MNA classes. However, more than three-fourths of the participants with MNA>23.5 had lower than recommended protein intakes. Sensitivity of MNA ranged from 0.32 to 0.82 for recommended energy (F:1570kcal/d/M:2070kcal/d) and protein intakes (1.0g/kg BW or 1.2g/kgBW) cut-off points, and specificity from 0.75 to 0.25, respectively. AUC values were low (0.52-0.53)., Conclusions: MNA status was consistently associated with nutrient intakes and diet quality. However, a high proportion of older people even with normal nutritional status had poor energy and protein intakes. Thus, MNA does not identify all those with poor nutrient intakes who may be at risk of developing malnutrition., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Malnutrition is associated with comorbidities and functional decline among older people. Less is known about nutrient intakes across heterogeneous older populations., Objective: We examined nutritional status and nutrient intakes in different samples of older people representing broad spectrum of healthy and frail populations. We evaluated adequacy of their energy, protein and micronutrient intakes in comparison to recommendations., Design and Participants: Cross-sectional study combined five datasets: home-dwelling older people participating in nutrition education and cooking classes (NC) [n=54], participants from Helsinki Businessmen Study [n=68], home-dwelling people with Alzheimer disease (AD) [n=99] and their spousal caregivers (n=97), participants from Porvoo Sarcopenia and Nutrition Trial (n=208), and residents of Helsinki assisted living facilities (ALF) [n=374]. Nutritional status was assessed using Mini Nutritional Assessment and nutrient intakes retrieved from 1 to 3 day food records., Results: Those suffering most from mobility limitation and cognitive decline had the poorest nutritional status (p<0.001; adjusted for age, sex, comorbidities). However, low intakes of energy, protein, and micronutrients were observed in high proportion in all groups, inadequate intakes of vitamins D, E, folate, and thiamine being most common. Protein intakes did not differ between the groups, but 77% of all participants had lower than recommended protein intake. In general, the NC group had highest micronutrient intakes and the ALF group the lowest. However, AD females had the lowest energy, protein, and vitamin C intakes., Conclusions: Our study provides a detailed picture of risks related to nutrient intakes in various groups of older people. These findings could be used in planning tailored nutrition interventions., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Optimal enzyme mixtures of six Trichoderma reesei enzymes and five thermostable enzyme components were developed for the hydrolysis of hydrothermally pretreated wheat straw, alkaline oxidised sugar cane bagasse and steam-exploded bagasse by statistically designed experiments. Preliminary studies to narrow down the optimization parameters showed that a cellobiohydrolase/endoglucanase (CBH/EG) ratio of 4:1 or higher of thermostable enzymes gave the maximal CBH-EG synergy in the hydrolysis of hydrothermally pretreated wheat straw. The composition of optimal enzyme mixtures depended clearly on the substrate and on the enzyme system studied. The optimal enzyme mixture of thermostable enzymes was dominated by Cel7A and required a relatively high amount of xylanase, whereas with T. reesei enzymes, the high proportion of Cel7B appeared to provide the required xylanase activity. The main effect of the pretreatment method was that the required proportion of xylanase was higher and the proportion of Cel7A lower in the optimized mixture for hydrolysis of alkaline oxidised bagasse than steam-exploded bagasse. In prolonged hydrolyses, less Cel7A was generally required in the optimal mixture. Five-component mixtures of thermostable enzymes showed comparable hydrolysis yields to those of commercial enzyme mixtures.

Background: Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis., Results: High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency., Conclusions: The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail.

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme-lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase-lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM-cellulose interaction were also shown to contribute to lignin-binding., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The role of CBM in two fungal model cellulase systems, consisting of Cel7A and Cel5A, from Trichoderma reesei and Thermoascus aurantiacus, were compared in the hydrolysis of various substrates. For comparison, family-1 CBM's were introduced to the T. aurantiacus and removed from the T. reesei enzymes. Especially at high dry matter consistencies of lignocellulosic substrates, pretreated wheat straw and spruce, the T. aurantiacus enzymes lacking CBM outperformed the enzymes carrying the CBM. In these conditions, the CBM-less enzymes from both organisms obviously recognized and bound to the substrate at higher probability than in dilute systems. Avoiding the unproductive binding to lignin caused by the CBMs obviously enhanced the hydrolytic performance. The lignin binding effect was, however, not entirely caused by the CBM, but also by the different structures and affinities of the core enzymes to lignin. Due to decreased binding, the CBM-less enzymes would allow reuse, potentially decreasing hydrolysis costs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Nutritional status often deteriorates in Alzheimer's disease (AD). Less is known about whether nutritional care reverses malnutrition and its harmful consequences in AD. The aim of this study is to examine whether individualized nutritional care has an effect on weight, nutrition, health, physical functioning, and quality of life in older individuals with AD and their spouses living at home., Methods: AD patients and their spouses (aged > 65 years) living at home (n = 202, 102 AD patients) were recruited using central AD registers in Finland. The couples were randomized into intervention and control groups. A trained nutritionist visited intervention couples 4-8 times at their homes and the couples received tailored nutritional care. When necessary, the couples were given protein and nutrient-enriched complementary drinks. All intervention couples were advised to take vitamin D 20 μg/day. The intervention lasted for one year. The couples of the control group received a written guide on nutrition of older people. Participants in the intervention group were assessed every three months. The primary outcome measure is weight change. Secondary measures are the intake of energy, protein, and other nutrients, nutritional status, cognition, caregiver's burden, depression, health related quality of life and grip strength., Discussion: This study provides data on whether tailored nutritional care is beneficial to home-dwelling AD patients and their spouses., Trial Registration: ACTRN 12611000018910.

Background: In the hydrolysis of lignocellulosic materials, thermostable enzymes decrease the amount of enzyme needed due to higher specific activity and elongate the hydrolysis time due to improved stability. For cost-efficient use of enzymes in large-scale industrial applications, high-level expression of enzymes in recombinant hosts is usually a prerequisite. The main aim of the present study was to compare the biochemical and hydrolytic properties of two thermostable recombinant glycosyl hydrolase families 10 and 11 (GH10 and GH11, respectively) xylanases with respect to their potential application in the hydrolysis of lignocellulosic substrates., Results: The xylanases from Nonomuraea flexuosa (Nf Xyn11A) and from Thermoascus aurantiacus (Ta Xyn10A) were purified by heat treatment and gel permeation chromatography. Ta Xyn10A exhibited higher hydrolytic efficiency than Nf Xyn11A toward birchwood glucuronoxylan, insoluble oat spelt arabinoxylan and hydrothermally pretreated wheat straw, and it produced more reducing sugars. Oligosaccharides from xylobiose to xylopentaose as well as higher degree of polymerization (DP) xylooligosaccharides (XOSs), but not xylose, were released during the initial hydrolysis of xylans by Nf Xyn11A, indicating its potential for the production of XOS. The mode of action of Nf Xyn11A and Ta Xyn10A on glucuronoxylan and arabinoxylan showed typical production patterns of endoxylanases belonging to GH11 and GH10, respectively., Conclusions: Because of its high catalytic activity and good thermostability, T. aurantiacus xylanase shows great potential for applications aimed at total hydrolysis of lignocellulosic materials for platform sugars, whereas N. flexuosa xylanase shows more significant potential for the production of XOSs.

Background: Thermostable enzymes have several benefits in lignocellulose processing. In particular, they potentially allow the use of increased substrate concentrations (because the substrate viscosity decreases as the temperature increases), resulting in improved product yields and reduced capital and processing costs. A short pre-hydrolysis step at an elevated temperature using thermostable enzymes aimed at rapid liquefaction of the feedstock is seen as an attractive way to overcome the technical problems (such as poor mixing and mass transfer properties) connected with high initial solid loadings in the lignocellulose to ethanol process., Results: The capability of novel thermostable enzymes to reduce the viscosity of high-solid biomass suspensions using a real-time viscometric measurement method was investigated. Heterologously expressed enzymes from various thermophilic organisms were compared for their ability to liquefy the lignocellulosic substrate, hydrothermally pretreated wheat straw. Once the best enzymes were identified, the optimal temperatures for these enzymes to decrease substrate viscosity were compared. The combined hydrolytic properties of the thermostable preparations were tested in hydrolysis experiments. The studied mixtures were primarily designed to have good liquefaction potential, and therefore contained an enhanced proportion of the key liquefying enzyme, EGII/Cel5A., Conclusions: Endoglucanases were shown to have a superior ability to rapidly reduce the viscosity of the 15% (w/w; dry matter) hydrothermally pretreated wheat straw. Based on temperature profiling studies, Thermoascus aurantiacus EGII/Cel5A was the most promising enzyme for biomass liquefaction. Even though they were not optimized for saccharification, many of the thermostable enzyme mixtures had superior hydrolytic properties compared with the commercial reference enzymes at 55°C.

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes., ((c) 2008 Wiley Periodicals, Inc.)

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter-1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.

The extracellular bga1-encoded beta-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 +/- 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-beta-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-beta-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is approximately 5.0, and the optimum temperature was found to be 60 degrees C. Bga1 is also capable of releasing D-galactose from beta-galactans and is thus actually a galacto-beta-D-galactanase. beta-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.

To develop functional enzymes in cellulose hydrolysis at or above 70 degrees C the cellobiohydrolase (CBHI/Cel7A) of Thermoascus aurantiacus was cloned and expressed in Trichoderma reesei Rut-C30 under the strong cbh1 promoter. Cellulase production of the parental strain and the novel strain (RF6026) was examined in submerged fermentation experiments using various carbon sources, which were lactose, Solka Floc 200 cellulose powder, and steam pretreated corn stover. An industrially feasible production medium was used containing only distiller's spent grain, KH(2)PO(4), and (NH(4))(2)SO(4). Enzyme production was followed by measurements of protein concentration, total cellulase enzyme activity (filter paper activity), beta-glucosidase activity, CBHI activity, and endogenase I (EGI) activity. The Thermoascus CBHI/Cel7A activity was taken as an indication of the heterologous gene expression under the cbh1 promoter.

Thermostable enzymes offer potential benefits in the hydrolysis of lignocellulosic substrates; higher specific activity decreasing the amount of enzymes, enhanced stability allowing improved hydrolysis performance and increased flexibility with respect to process configurations, all leading to improvement of the overall economy of the process. New thermostable cellulase mixtures were composed of cloned fungal enzymes for hydrolysis experiments. Three thermostable cellulases, identified as the most promising enzymes in their categories (cellobiohydrolase, endoglucanase and beta-glucosidase), were cloned and produced in Trichoderma reesei and mixed to compose a novel mixture of thermostable cellulases. Thermostable xylanase was added to enzyme preparations used on substrates containing residual hemicellulose. The new optimised thermostable enzyme mixtures were evaluated in high temperature hydrolysis experiments on technical steam pretreated raw materials: spruce and corn stover. The hydrolysis temperature could be increased by about 10-15 degrees C, as compared with present commercial Trichoderma enzymes. The same degree of hydrolysis, about 90% of theoretical, measured as individual sugars, could be obtained with the thermostable enzymes at 60 degrees C as with the commercial enzymes at 45 degrees C. Clearly more efficient hydrolysis per assayed FPU unit or per amount of cellobiohydrolase I protein used was obtained. The maximum FPU activity of the novel enzyme mixture was about 25% higher at the optimum temperature at 65 degrees C, as compared with the highest activity of the commercial reference enzyme at 60 degrees C. The results provide a promising basis to produce and formulate improved enzyme products. These products can have high temperature stability in process conditions in the range of 55-60 degrees C (with present industrial products at 45-50 degrees C) and clearly improved specific activity, essentially decreasing the protein dosage required for an efficient hydrolysis of lignocellulosic substrates. New types of process configurations based on thermostable enzymes are discussed.

Excess 17beta-estradiol (E2), the most potent of human estrogens, is known to act as a stimulus for the growth of breast tumors. Human estrogenic 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1), which catalyzes the reduction of inactive estrone (E1) to the active 17beta-estradiol in breast tissues, is a key enzyme responsible for elevated levels of E2 in breast tumor tissues. We present here the structure of the ternary complex of 17beta-HSD1 with the cofactor NADP+ and 3-hydroxyestra-1,3,5,7-tetraen-17-one (equilin), an equine estrogen used in estrogen replacement therapy. The ternary complex has been crystallized with a homodimer, the active form of the enzyme, in the asymmetric unit. Structural and kinetic data presented here show that the 17beta-HSD1-catalyzed reduction of E1 to E2 in vitro is specifically inhibited by equilin. The crystal structure determined at 3.0-A resolution reveals that the equilin molecule is bound at the active site in a mode similar to the binding of substrate. The orientation of the 17-keto group with respect to the nicotinamide ring of NADP+ and catalytic residues Tyr-155 and Ser-142 is different from that of E2 in the 17beta-HSD1-E2 complex. The ligand and substrate-entry loop densities are well defined in one subunit. The substrate-entry loop adopts a closed conformation in this subunit. The result demonstrates that binding of equilin at the active site of 17beta-HSD1 is the basis for inhibition of E1-to-E2 reduction by this equine estrogen in vitro. One possible outcome of estrogen replacement therapy in vivo could be reduction of E2 levels in breast tissues and hence the reduced risk of estrogen-dependent breast cancer.