Quality of life and socioeconomic and educational status in patients with congenital hypothyroidism.

BACKGROUND: The aim of this study was to investigate the influence of primary congenital hypothyroidism (CH) on quality of life, level of education and socioeconomic status (SES). METHODS: Two independent study cohorts, a national and a regional, were collected from Finnish national registers and patient records. Data on social security benefits, SES, marital status, and education were collected from Statistics Finland. Health-related quality of life (HRQoL) was studied in the regional patient cohort with the standardized 15D and 16D instruments. RESULTS: There were no statistically significant differences in education level, marital status, or SES between CH patients (n = 40) and their matched controls at the age of 25 years. The mean 15D score was both statistically significantly and clinically importantly lower in CH patients (n = 29) than controls (0.904 vs. 0.953, p = 0.008). CH patients reported significantly lower scores across various dimensions of physical and mental HRQoL, including breathing, sleeping, speech, excretion, mental function, distress, and vitality. The mean 16D score was lower in CH patients compared to controls (0.917, vs. 0.947), but without statistical significance. CONCLUSION: SES of CH patients did not differ from matched controls. Thus, most CH patients integrate well into society, but their HRQoL is impaired. IMPACT: Most patients with primary congenital hypothyroidism integrate well into society. In the current study, their socioeconomic and marital status did not differ from matched controls at the age of 25 years. However, health-related quality of life measured using 15D instrument was impaired. Every fourth patient reported that congenital hypothyroidism influenced everyday life.

Incidence of primary congenital hypothyroidism over 24 years in Finland.

BACKGROUND: A rise in the incidence of congenital hypothyroidism (CH) has been reported worldwide. This nationwide study aimed to describe the secular trends and current incidence of CH in Finland. METHODS: Two independent study cohorts, a national and a regional, were collected from national registers and patient records. The national cohort represents all CH cases born in Finland between 1994 and 2017. Birth data, results of the screening test, and the incidence of CH were reviewed. RESULTS: Between 1994 and 2017, 1,400,028 children were born in Finland. Of these children, 503 were diagnosed with primary CH (incidence 1:2783). Male-to-female sex ratio was 1:2.0. The nationwide incidence was 33 cases per 100,000 live births between 1994 and 1999, 38 cases per 100,000 live births between 2000 and 2005, 40 cases per 100,000 live births between 2006 and 2011, and 33 cases per 100,000 live births between 2012 and 2017. In the regional cohort (n = 139), the incidence of transient CH was 3.6%. The incidence of mild, moderate, and severe CH remained constant. CONCLUSIONS: In Finland, the incidence of CH has not changed during the 24-year study period. IMPACT: As opposed to recent reports worldwide, the incidence of congenital hypothyroidism has not changed between 1994 and 2017 in Finland. The proportions of mild, moderate, and severe congenital hypothyroidism did not change significantly over the study period. Lowering the TSH cut-off limit or increasing immigration did not affect the incidence rate of primary congenital hypothyroidism in Finland.

Carbohydrate-binding modules of fungal cellulases: occurrence in nature, function, and relevance in industrial biomass conversion.

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature.

Treatment of Congenital Hypothyroidism: Impact of Secular Changes in Levothyroxine Initial Dose on Early Growth.

INTRODUCTION: Newborn screening of congenital hypothyroidism (CH) has enabled early treatment with levothyroxine (LT4), ensuring normal growth and development. The initial LT4 dose recommendation has increased over decades. We evaluated whether the increased LT4 dosing influenced thyroid-stimulating hormone (TSH) and thyroxine (fT4) concentrations, growth, or treatment-related symptoms. METHODS: LT4 doses, TSH, fT4, anthropometrics, and treatment-related symptoms until age 2 years were evaluated in 172 Finnish CH patients born between 1980 and 2018. The patients were grouped according to birth decade: 1980s (n = 19, mean LT4 starting dose 6.8 µg/kg/day), 1990s (n = 50, 7.4 µg/kg/day), 2000s (n = 59, 9.7 µg/kg/day), and 2010s (n = 44, 10.8 µg/kg/day). RESULTS: TSH concentrations were higher during the first 2 years of life in children born in the 1980s compared to children born later. TSH concentrations were often subnormal in children receiving higher LT4 doses (children born in the 2000s and 2010s). However, symptoms of overtreatment were uncommon. Linear or head growth showed no differences between the groups during the first 2 years of life. Although growth was within the normal spectrum, children in all groups were shorter than their target length at 2 years and their weight-for-length was above the mean through the first 2 years of life. DISCUSSION: Current treatment practice with higher LT4 dose normalizes TSH rapidly without significant increase in side effects. However, irrespective of initial LT4 dose, children were shorter than expected at 2 years of age. Effects of different initial LT4 dose on cognitive development urges further investigation.

Comorbidity in Congenital Hypothyroidism-A Nationwide, Population-based Cohort Study.

CONTEXT: Patients with congenital hypothyroidism (CH) are affected more often than the general population by other chronic diseases and neurological difficulties. OBJECTIVE: The aim of this nationwide population-based register study was to investigate the incidence of congenital malformations, comorbidities, and the use of prescribed drugs in patients with primary CH. METHODS: The study cohort and matched controls were identified from national population-based registers in Finland. All diagnoses from birth until the end of 2018 were collected from the Care Register, and subject-specific prescription drug purchases were identified from The Prescription Register from birth until the end of 2017. RESULTS: Diagnoses of neonatal and chronic diseases were collected for 438 full-term patients and 835 controls (median follow-up time 11.6 years; range, 0-23 years). Newborns with CH were more often found to have neonatal jaundice (11.2% and 2.0%; P < .001), hypoglycemia (8.9% and 2.8%; P < .001), metabolic acidemia (3.2% and 1.1%; P = .007), and respiratory distress (3.9% and 1.3%; P < .003) as compared to their matched controls.Congenital malformations were diagnosed in 66 of 438 (15.1%) CH patients and in 62 of 835 (7.4%) controls (P < .001). The most commonly affected extrathyroidal systems were the circulatory and musculoskeletal systems. The cumulative incidence of hearing loss and specific developmental disorders was higher among CH patients than controls. The use of antidepressant and antipsychotic drugs was similar in CH patients and their controls. CONCLUSION: CH patients have more neonatal morbidity and congenital malformations than their matched controls. The cumulative incidence of neurological disorders is higher in CH patients. However, our results do not support the existence of severe psychiatric comorbidity.

Inhibition of enzymatic hydrolysis by residual lignins from softwood--study of enzyme binding and inactivation on lignin-rich surface.

Lignin-derived inhibition is a major obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides especially with softwood lignocellulosics. Enzyme adsorption on lignin is suggested to contribute to the inhibitory effect of lignin. The interaction of cellulases with softwood lignin was studied in the present work with commercial Trichoderma reesei cellulases (Celluclast) and lignin-rich residues isolated from steam pretreated softwood (SPS) by enzymatic and acid hydrolysis. Both lignin preparations inhibited the hydrolysis of microcrystalline cellulose (Avicel) and adsorbed the major cellulases present in the commercial cellulase mixture. The adsorption phenomenon was studied at low temperature (4°C) and at the typical hydrolysis temperature (45°C) by following activities of free and lignin-bound enzymes. Severe inactivation of the lignin-bound enzymes was observed at 45°C, however at 4°C the enzymes retained well their activity. Furthermore, SDS-PAGE analysis of the lignin-bound enzymes indicated that very strong interactions form between the residue and the enzymes at 45°C, because the enzymes were not released from the residue in the electrophoresis. These results suggest that heat-induced denaturation may take place on the surface of softwood lignin at the hydrolysis temperature.

Screening for Mutations in Isolated Central Hypothyroidism Reveals a Novel Mutation in Insulin Receptor Substrate 4.

Background: Central hypothyroidism (CeH) is a rare condition affecting approximately 1:16 000- 100 000 individuals. Congenital forms can harm normal development if not detected and treated promptly. Clinical and biochemical diagnosis, especially of isolated CeH, can be challenging. Cases are not usually detected in neonatal screening, which, in most countries, is focused on detection of the more prevalent primary hypothyroidism. Until now, five genetic causes for isolated CeH have been identified. Here we aimed to identify the genetic cause in two brothers with impaired growth diagnosed with CeH at the age of 5 years. We further evaluated the candidate gene variants in a large genetic database. Methods: Clinical and biochemical characterization together with targeted next-generation sequencing (NGS) was used to identify the genetic cause in a family of two brothers presenting with CeH. Screening of insulin receptor substrate 4 (IRS4) variants was carried out in the FinnGen database. Results: A novel monoallelic frameshift mutation c.1712_1713insT, p.Gly572Trp fs*32 in the X-linked IRS4 gene was identified by NGS analysis in both affected males and confirmed using Sanger sequencing. Their mother was an unaffected carrier. In addition to the declined growth at presentation, central hypothyroidism and blunted TRH test, no other phenotypic alterations were found. Diagnostic tests included head MRI, thyroid imaging, bone age, and laboratory tests for thyroid autoantibodies, glucose, insulin and glycosylated hemoglobin levels. Examination of the IRS4 locus in FinnGen (R5) database revealed the strongest associations to a rare Finnish haplotype associated with thyroid disorders (p = 1.3e-7) and hypothyroidism (p = 8.3e-7). Conclusions: Here, we identified a novel frameshift mutation in an X-linked IRS4 gene in two brothers with isolated CeH. Furthermore, we demonstrate an association of IRS4 gene locus to a general thyroid disease risk in the FinnGen database. Our findings confirm the role of IRS4 in isolated central hypothyroidism.

Changes in submicrometer structure of enzymatically hydrolyzed microcrystalline cellulose.

To understand the limitations occurring during enzymatic hydrolysis of cellulosic materials in renewable energy production, we used wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), X-ray microtomography, and transmission electron microscopy (TEM) to characterize submicrometer changes in the structure of microcrystalline cellulose (Avicel) digested with the Trichoderma reesei enzyme system. The microtomography measurements showed a clear decrease in particle size in scale of tens of micrometers. In all the TEM pictures, similar elongated and partly ramified structures were observed, independent of the hydrolysis time. The SAXS results of rewetted samples suggested a slight change in the structure in scale of 10-20 nm, whereas the WAXS results confirmed that the degree of crystallinity and the crystal sizes remained unchanged. This indicates that the enzymes act on the surface of cellulose bundles and are unable to penetrate into the nanopores of wet cellulose.

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases.

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.

Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces.

Three thermostable neutral cellulases from Melanocarpus albomyces, a 20-kDa endoglucanase (Cel45A), a 50-kDa endoglucanase (Cel7A), and a 50-kDa cellobiohydrolase (Cel7B) heterologously produced in a recombinant Trichoderma reesei were purified and studied in hydrolysis (50 degrees C, pH 6.0) of crystalline and amorphous cellulose. To improve their efficiency, M. albomyces cellulases naturally harboring no cellulose-binding module (CBM) were genetically modified to carry the CBM of T. reesei CBHI/Cel7A, and were studied under similar experimental conditions. Hydrolysis performance and product profiles were used to evaluate hydrolytic features of the investigated enzymes. Each cellulase proved to be active against the tested substrates; the cellobiohydrolase Cel7B had greater activity than the endoglucanases Cel45A and Cel7A against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence of synergism was observed when mixtures of the novel enzymes were applied in a constant total protein dosage. Presence of the CBM improved the hydrolytic potential of each enzyme in all experimental configurations; it had a greater effect on the endoglucanases Cel45A and Cel7A than the cellobiohydrolase Cel7B, especially against crystalline substrate. The novel cellobiohydrolase performed comparably to the major cellobiohydrolase of T. reesei (CBHI/Cel7A) under the applied experimental conditions.

Relationship between leptin and C-reactive protein in young Finnish adults.

CONTEXT: Leptin and C-reactive protein (CRP) concentrations are increased in inflammation, and both have been linked to increased risk for cardiovascular diseases. OBJECTIVE: The objective of the study was to explore in a population-based sample whether the relation between leptin and CRP is independent of obesity level and whether genetic causes of CRP elevation contribute to leptin levels. DESIGN: This was a population-based study including 1862 young adults (971 women; 891 men) aged 24-39 yr. SETTING: The study was conducted at five centers in Finland. MAIN OUTCOME MEASURES: Associations between leptin and CRP adjusted for obesity indices, risk factors, genetic variables, and lifestyle variables were measured. RESULTS: Women had 3.0-fold higher median concentrations of leptin (12.5 vs. 4.1 ng/ml) and 1.3-fold higher median concentrations of CRP (0.75 vs. 0.56 mg/liter) than men (P < 0.0001 in both comparisons). In univariate analyses, CRP and leptin were significantly intercorrelated (r = 0.47, P < 0.0001 for women; r = 0.46, P < 0.0001 for men). In multiple regression analysis including age, body mass index, waist circumference, insulin, lipids, systolic and diastolic blood pressures, smoking status, and use of oral contraceptives in women, leptin was the main determinant of CRP in men (P < 0.0001) and the second most important determinant in women (P < 0.0001). A Mendelian randomization test based on genetic variants in the CRP gene (five single nucleotide polymorphisms) provided no support for CRP as a causal agent for leptin. CONCLUSIONS: Leptin, obesity, and oral contraceptive use in women were the main factors related to CRP. The relation between leptin and CRP was independent of obesity and cardiovascular risk factors.

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides.

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and ß-glucosidase played an important role in mannan hydrolysis. Addition of 10mg/ml mannan reduced the glucose yield of Avicel (at 20g/l) from 40.1 to 24.3%. No inhibition of ß-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on ß-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.

Laccase from Melanocarpus albomyces binds effectively to cellulose.

A laccase from the thermophilic fungus Melanocarpus albomyces was shown to bind to softwood and pure microcrystalline cellulose. The binding isotherm fitted well the Langmuir type one-site binding model. The adsorption parameters indicated that M. albomyces laccase binds with high affinity to cellulose with a relatively low maximum binding capacity, as compared to the values for various cellulases. The binding was shown to be reversible and not influenced by non-specific protein or 0.1-0.5 M Na2SO4. No binding was detected with laccases from Trametes hirsuta or Mauginiella sp., which suggests that binding to cellulose is typical for only some laccases.

Cloning, characterisation and identification of several polymorphisms in the promoter region of the human alpha2B-adrenergic receptor gene.

Screening of a foetal brain genomic DNA library allowed to isolate a 10-kb fragment of the gene encoding the human alpha2B-adrenergic receptor, that contained 5.5 kb of the 5'-flanking region, the open reading frame and 2.9 kb of the 3'-flanking region. The 1-kb fragment upstream from the start codon was rich in GC, lacked consensus TATA or CAAT box, but contained several Sp1-binding sites. Other potential cis-regulatory elements found in the 5'-flanking region included AP2, USF, Stat-6, NFkappaB and Olf-1. A single canonical polyadenylation signal (AATAAA) was found at position +3252/+3257 and the polyadenylation site was 3274 nucleotides downstream from ATG. Transfection experiments with chimeric luciferase constructs containing various truncated fragments of the 5'-region showed that the fragment -3160/+3 exhibited promoter activity in all tested cell lines and permitted the definition of a minimal 200-bp promoter (-603/-411) containing three putative Sp1-binding sites and two initiator elements. Transcriptional activity of this region was inhibited by the addition of mithramycin, a specific inhibitor of Sp1 binding to GC-rich sequences. The search for sequence variants within a fragment covering 1.7 kb of 5'-flanking region and the coding region allowed us to identify five novel single nucleotide polymorphisms. Interestingly, the G/C substitution at position -98 relative to the start codon was common and in complete linkage with a previously identified insertion/deletion polymorphism in the coding region which was showed to affect alpha2B-adrenergic receptor function. Based on transfection data and computer-assisted sequence analysis, the -98 G/C single nucleotide polymorphism was located within a portion of the 5'-UTR (-127/+3) affecting luciferase activity and it created additional putative binding site for Sp1. However, G/C substitution had no significant incidence on promoter activity in BHK-21 or HeLa cells.

Effects of bacterial treatments on wood extractives.

Bacterial strains were isolated from spruce wood chips and their ability to reduce the content of wood extractives was studied. Strains were screened by cultivation on liquid media containing wood extractives as the major nutrient. Some bacterial species could decrease remarkably the amount of extractives in the liquid media and reduced the amount of triglycerides, steryl esters and total extractives by 100, 20 and 39%, respectively. Spruce wood chips were treated in controlled conditions with selected bacteria to test their effects on the chips. All the bacteria grew well on wood chips. The effect of bacterial metabolism on wood extractives was significant. Bacterial treatments reduced the amount of lipophilic extractives by 16-38% in 1 week of treatment and up to 67% in 2 weeks. The most efficient strain removed 90, 66 and 50% of triglycerides, steryl esters and resin acids, respectively, in 2 weeks. These results indicate that bacteria may be promising agents for the removal of extractives for improved pulping and papermaking processes.

Regioselective deacetylation of cellulose acetates by acetyl xylan esterases of different CE-families.

Cellulose acetate (CA) was found to be a substrate of several acetyl xylan esterases (AXE). Eight AXE from different carbohydrate esterase (CE) families were tested on their activity against CA with a degree of substitution of 0.7 and 1.4. The classification of the AXEs into CE families according to their structure by hydrophobic cluster analysis followed clearly their activity against CA. Within the same CE family similar, and between the CE families different deacetylation behaviours could be observed. Furthermore, each esterase family showed a distinct regioselective mode of action. The CE 1 family enzymes regioselectively cleaved the substituents in C2- and C3-position, while CE 5 family enzymes only cleaved the acetyl groups in C2-position. CE 4 family enzymes seemed to interact only with the substituents in C3-position. Evidence was found that the deacetylation reaction of the CE 1 family enzymes proceeded faster in C2- than in C3-position of CA. The enzymes were able to cleave acetyl groups from fully substituted anhydroglucose units.

Evaluation of wet oxidation pretreatment for enzymatic hydrolysis of softwood.

The wet oxidation pretreatment (water, oxygen, elevated temperature, and pressure) of softwood (Picea abies) was investigated for enhancing enzymatic hydrolysis. The pretreatment was preliminarily optimized. Six different combinations of reaction time, temperature, and pH were applied, and the compositions of solid and liquid fractions were analyzed. The solid fraction after wet oxidation contained 58-64% cellulose, 2-16% hemicellulose, and 24-30% lignin. The pretreatment series gave information about the roles of lignin and hemicellulose in the enzymatic hydrolysis. The temperature of the pretreatment, the residual hemicellulose content of the substrate, and the type of the commercial cellulase preparation used were the most important factors affecting the enzymatic hydrolysis. The highest sugar yield in a 72-h hydrolysis, 79% of theoretical, was obtained using a pretreatment of 200 degrees C for 10 min at neutral pH.

Impact of xylan on synergistic effects of xylanases and cellulases in enzymatic hydrolysis of lignocelluloses.

Supplementation of xylanase (XYL) has been found to synergistically improve the performance of cellulases (CEL) in the hydrolysis of lignocelluloses. However, the effect of xylan on the synergistic effects of XYL and CEL is still unclear. In this work, the effect of xylan on the synergy between CEL and XYL was investigated. Xylan content in corn stover was generally a good indicator of the degree of the synergism between CEL and XYL. Strongest synergism was observed in the hydrolysis of cellulose in corn stover with the highest xylan contents. A more evident synergistic effect of CEL in xylan hydrolysis was observed in the substrates with low original xylan content. It was also found that the ratio of cellulose to xylan in substrates correlated to the synergism between the two types of enzymes. The results indicated that supplementation of XYL with CEL was most effective in the hydrolysis of corn stover with the highest xylan content. For efficient enzymatic hydrolysis of lignocelluloses, both cellulases and xylanase were important because cellulose and xylan coved each other and these enzymes could improve their performance each other in the hydrolysis of cellulose and xylan in lignocelluloses.

Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce.

BACKGROUND: The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs). RESULTS: LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis. CONCLUSIONS: LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes.

Cellulases without carbohydrate-binding modules in high consistency ethanol production process.

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.