Potential and utilization of thermophiles and thermostable enzymes in biorefining

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

Differences and similarities in enzymes from the neopullulanase subfamily isolated from thermophilic species

Six glycoside hydrolase (GH) family 13 members, classified under the polyspecific neopullulanase subfamily GH13_20 (also termed cyclomaltodextrinase) were analysed. They originate from thermophilic bacterial strains (Anoxybacillus flavithermus, Laceyella sacchari, and Geobacillus thermoleovorans) or from environmental DNA, collected after in situ enrichments in Icelandic hot springs. The genes were isolated following the CODEHOP consensus primer strategy, utilizing the first two of the four conserved sequence regions in GH13. The typical domain structure of GH13_20, including an N-terminal domain (classified as CBM34), the catalytic module composed of the A-and B-domains, and a C-terminal domain, was found in five of the encoded enzymes (abbreviated Amy1, 89, 92, 98 and 132). These five enzymes degraded cyclomaltodextrins (CDs) and starch, while only three, Amy92 (L. sacchari), Amy98 (A. flavithermus) and Amy132 (environmental DNA), also harboured neopullulanase activity. The L. sacchari enzyme was monomeric, but with CD as the preferred substrate, which is an unusual combination. The sixth enzyme (Amy29 from environmental DNA), was composed of the ABC-domains only. Preferred substrate for Amy29 was pullulan, which was degraded to panose, and the enzyme had no detectable activity on CDs. In addition to its different activity profile and domain composition, Amy29 also displayed a different conservation (LPKF) in the fifth conserved region (MPKL) proposed to identify the subfamily. All enzymes had apparent temperature optima in the range 50–65°C, while thermostability varied, and was highest for Amy29 with a half-life of 480 min at 80°C. Calcium dependent activity or stability was monitored in four enzymes, but could not be detected for Amy29 or 98. Tightly bound calcium can, however, not be ruled out, and putative calcium ligands were conserved in Amy98

The Whole Genome Sequence of Sphingobium chlorophenolicum L-1: Insights into the Evolution of the Pentachlorophenol Degradation Pathway

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

A three-group study, internet-based, face-to-face based and standard- management after acute whiplash associated disorders (WAD) – choosing the most efficient and cost-effective treatment: study protocol of a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>The management of Whiplash Associated Disorders is one of the most complicated challenges with high expenses for the health care system and society. There are still no general guidelines or scientific documentation to unequivocally support any single treatment for acute care following whiplash injury.</p> <p>The main purpose of this study is to try a new behavioural medicine intervention strategy at acute phase aimed to reduce the number of patients who have persistent problems after the whiplash injury. The goal is also to identify which of three different interventions that is most cost-effective for patients with Whiplash Associated Disorders. In this study we are controlling for two factors. First, the effect of behavioural medicine approach is compared with standard care. Second, the manner in which the behavioural medicine treatment is administered, Internet or face-to-face, is evaluated in it's effectiveness and cost-effectiveness.</p> <p>Methods/Design</p> <p>The study is a randomized, prospective, experimental three-group study with analyses of cost-effectiveness up to two-years follow-up. <it>Internet – based programme </it>and <it>face-to-face group treatment programme </it>are compared to <it>standard-treatment </it>only. Patient follow-ups take place three, six, twelve and 24 months, that is, short-term as well as long-term effects are evaluated. Patients will be enrolled via the emergency ward during the first week after the accident.</p> <p>Discussion</p> <p>This new self-help management will concentrate to those psychosocial factors that are shown to be predictive in long-term problems in Whiplash Associated Disorders, i.e. the importance of self-efficacy, fear of movement, and the significance of catastrophizing as a coping strategy for restoring and sustaining activities of daily life. Within the framework of this project, we will develop, broaden and evaluate current physical therapy treatment methods for acute Whiplash Associated Disorders. The project will contribute to the creation of a cost-effective behavioural medicine approach to management of acute Whiplash Associated Disorders. The results of this study will answer an important question; on what extent and how should these patients be treated at acute stage and how much does the best management cost.</p> <p>Trial registration number</p> <p>Current Controlled Trials ISRCTN61531337</p

Exploring thermostable glycoside hydrolases: Amylases and beta-glucosidases

Two putative cyclodextrinases (CDases) were amplified from two moderate thermophiles isolated in Iceland: Anoxybacillus flavithermus and Laceyella sacchari. They were cloned, expressed and characterized. The expression of the former was optimized in E. coli by using a solubility-enhancing tag, NusA, and tuning of inducer concentration in the expression strain Tuner(DE3). The CDases were optimally active at around 57 ºC and had a poor thermal stability. AfCda13 had significantly higher activity for cyclodextrins and formed a dimer in solution, whereas LsCda13 existed as a monomer, which likely lowered the activity for cyclodextrins. Two beta-glucosidases, members of glycoside hydrolase families 1 and 3 and originating from the extreme thermophile Thermotoga neapolitana were cloned and expressed in E. coli. The apparent thermal unfolding temperatures were around 90 ºC and 102 ºC for TnBgl3B and TnBgl1A, respectively. TnBgl3B hydrolyzed para-nitrophenyl-glucoside with a Vmax at 90 ºC of 93 ± 13 U/mg and a Km of 0.11 ± 0.03 mM. TnBgl3B was tested for its transglycosylating ability by forming hexyl- and octyl-beta-D-glucoside. The selectivity of alcoholysis compared to hydrolysis was higher than for other reported enzymes. Both beta-glucosidases were also tested in reactions cleaving off glucose from quercetin-glycosides extracted from onion by subcritical water. They both performed better than the commercially available almond beta-glucosidase. Predicted catalytic amino acids of TnBgl3B were replaced with non-catalytic glycines. The aspartate nucleophile is highly conserved in GH3 and the D242G mutant lost all activity. The acid/base residue is less conserved and two residues were mutated, Glu458 and Asp461. Mutant E458G had low but significant activity, while D461G lost almost all activity, which points to the latter residue as the catalytic acid/base of TnBgl3B. However, further analyses including structural information should confirm this. TnBgl3B was crystallized and the structure was solved by multiple wavelength anomalous diffraction using selenomethionyl crystals in addition to native crystals. X-ray diffraction data was collected to 2.4 Å resolution and the structure has been solved to 2.7 Å. Manual model building and refinement is in progress

Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration

Cyclomaltodextrinases are multidomain and often dimeric proteins from the alpha-amylase family (glycoside hydrolase family 13) which frequently have been very difficult to express in active form in Escherichia coli. To express the soluble form of this type of proteins in larger quantities the expression has to be optimized. We have used and combined two strategies to increase the yield of soluble recombinant cyclomaltodextrinase expressed from a gene originating from the thermophilic Gram-positive bacterium Anoxybacillus flavithermus. One strategy involved tuning of the inducer concentration while the other involved fusion of the gene encoding the target protein to the gene encoding the solubility-enhancing protein NusA. The enzyme activity could be increased 6-7 times solely by finely tuning the IPTG concentration, but the activity level was very sensitive to the amount of inducer applied. Hence, the IPTG concentration may have to be optimized for every protein under the conditions used. The fusion protein-strategy gave a slightly lower total activity but the level of soluble recombinant protein obtained was in this case significantly less sensitive to the inducer concentration applied. Moreover, the activity could be increased about 2-fold by cleaving off the solubility-tag (NusA) by enterokinase. (C) 2004 Elsevier Inc. All rights reserved

A novel variant of Thermotoga neapolitana beta-glucosidase B is an efficient catalyst for the synthesis of alkyl glucosides by transglycosylation

Alkyl glycosides are surfactants with good biodegradability and low toxicity, attractive to produce by an enzymatic method to get a well-defined product. In this paper, we report a novel thermostable variant of a family 3 β-glucosidase to be an efficient catalyst in alkyl-glucoside forming reactions using transglycosylation with hexanol or octanol as the acceptor molecule. The enzyme has an apparent optimum for hydrolysis at 90 °C, which coincides with its unfolding temperature. The total activity is lower at lower temperature (60 °C), but the ratio of alcoholysis/hydrolysis is slightly more favourable. This ratio is however more heavily influenced by the water content and the pH. Optimal reaction conditions for hexyl glucoside synthesis from p-nitrophenyl-β-glucopyranoside were a water/hexanol two-phase system containing 16% (v/v) water, pH 5.8, and a temperature of 60 °C. Under these conditions, the total initial reaction rate was 153 μmol min−1 mg−1 and the alcoholysis/hydrolysis ratio was 5.1. Comparing with alcoholysis/hydrolysis ratios of other β-glycosidases, TnBgl3B can be considered to be a very promising catalyst for alkyl glucoside production

Subcritical water extraction and beta-glucosidase-catalyzed hydrolysis of quercetin glycosides in onion waste

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 beta-glucosidases, Thermotoga neapolitana beta-glucosidase A and B, were utilized as catalysts. These enzymes maintain activity at temperatures around 90 degrees C, 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 degrees C, pH 5.0. Experimental design showed that the optimal extraction conditions included three 5 min extraction cycles with water at 120 degrees C 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(-1) dry weight of onion waste (RSD 8%). In summary, our research demonstrates that subcritical water extraction followed by beta-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

Monomeric and dimeric cyclomaltodextrinases reveal different modes of substrate degradation

Two cyclomaltodextrinases (CDase) of thermophilic origin were investigated for their action on cyclodextrins. Although most CDases known today are made up of at least homodimers, one of the enzymes studied was shown to be a monomer in solution, while the other one was a dimer. Interestingly, the dimeric enzyme had a much superior selectivity for a cyclodextrin substrate compared to its monomeric homologue, with a specific activity on α-cyclodextrin around 100 times higher than for the polymeric substrates starch and pullulan. Moreover, the monomeric CDase had a 10 times higher activity on those polymers than the dimer. The degradation pattern on cyclodextrins was examined by high-performance anion-exchange chromatography in combination with microdialysis. The final products were almost exclusively maltose and glucose in an approximate molar ratio of 2:1. However, the intermediate product ratios were quite different for the two enzymes, revealing that the monomeric CDase had a more random distribution of transitional products. Moreover, the dimeric CDase accumulated maltotriose, which is believed to be due to transglycosylation. The oligomeric state of the enzymes is thought to be a key factor for exhibiting high cyclodextrinase as well as transglycosylation activity