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

Preparation of two glycoside hydrolases for use in micro-aqueous media

Enzymatic synthesis of alkyl glycosides using glycoside hydrolases is well studied, but has yet to reach industrial scale, primarily due to limited yields. Reduced water content should increase yields by limiting the unwanted hydrolytic side reaction. However, previous studies have shown that a reduction in water content surprisingly favors hydrolysis over transglycosylation. In addition, glycoside hydrolases normally require a high degree of hydration to function efficiently. This study compares six enzyme preparation methods to improve resilience and activity of two glycoside hydrolases from Thermotoga neapolitana (TnBgl3B and TnBgl1A) in micro-aqueous hexanol. Indeed, when adsorbed onto Accurel MP-1000 both enzymes increasingly favored transglycosylation over hydrolysis at low hydration, in contrast to freeze-dried or untreated enzyme. Additionally, they displayed 17–70× higher reaction rates compared to freeze-dried enzyme at low water activity, while displaying comparable or lower activity for fully hydrated systems. These results provide valuable information for use of enzymes under micro-aqueous conditions and build toward utilizing the full synthetic potential of glycoside hydrolases

Thermostable glycoside hydrolases in Biorefining

Glycoside hydrolases, which are responsible for the degradation of the major fraction of biomass, the polymeric carbohydrates in starch and lignocellulose, are predicted to gain increasing roles as catalysts in biorefining applications in the future bioeconomy. In this context, thermostable variants will be important, as the recalcitrance of these biomass-components to degradation often motivates thermal treatments. The traditional focus on degradation is also predicted to be changed into more versatile roles of the enzymes, also involving specific conversions to defined products. In addition, integration of genes encoding interesting target activities opens the possibilities for whole cell applications, in organisms allowing processing at elevated temperatures for production of defined metabolic products. In this review, we overview the application of glycoside hydrolases related to the biorefining context (for production of food, chemicals, and fuels). Use of thermostable enzymes in processing of biomass is highlighted, moving from the activities required to act on different types of polymers, to specific examples in today’s processing. Examples given involve (i) monosaccharide production for food applications as well as use as carbon source for microbial conversions (to metabolites such as fuels and chemical intermediates), (ii) oligosaccharide production for prebiotics applications (iii) treatment for plant metabolite product release, and (iv) production of surfactants of the alkyl glycoside class. Finally future possibilities in whole cell biorefining are shown

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background: Derivatized celluloses, such as methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC), are of pharmaceutical importance and extensively employed in tablet matrices. Each batch of derivatized cellulose is thoroughly characterized before utilized in tablet formulations as batch-to-batch differences can affect drug release. The substitution pattern of the derivatized cellulose polymers, i.e. the mode on which the substituent groups are dispersed along the cellulose backbone, can vary from batch-to-batch and is a factor that can influence drug release. Results: In the present study an analytical approach for the characterization of the substitution pattern of derivatized celluloses is presented, which is based on the use of carbohydrate-binding modules (CBMs) and affinity electrophoresis. CBM4-2 from Rhodothermus marinus xylanase 10A is capable of distinguishing between batches of derivatized cellulose with different substitution patterns. This is demonstrated by a higher migration retardation of the CBM in acrylamide gels containing batches of MC and HPMC with a more heterogeneous distribution pattern. Conclusions: We conclude that CBMs have the potential to characterize the substitution pattern of cellulose derivatives and anticipate that with use of CBMs with a very selective recognition capacity it will be possible to more extensively characterize and standardize important carbohydrates used for instance in tablet formulation

Glycoside hydrolases for extraction and modification of polyphenolic antioxidants

Antioxidants are important molecules that are widely used by humans, both as dietary supplements and as additives to different types of products. In this chapter, we review how flavonoids, a class of polyphenolic antioxidants that are often found in glycosylated forms in many natural resources, can be extracted and modified using glycoside hydrolases (GHs). Glycosylation is a fundamental enzymatic process in nature, affecting function of many types of molecules (glycans, proteins, lipids as well as other organic molecules such as the flavonoids). Possibilities to control glycosylation thus mean possibilities to control or modify the function of the molecule. For the flavonoids, glycosylation affect both the antioxidative power and solubility. In this chapter we overview results on in vitro deglycosylation and glycosylation of flavonoids by selected GHs. For optimal enzymatic performance, desired features include a correct specificity for the target, combined with high stability. Poor specificity towards a specific substituent is thus a major drawback for enzymes in particular applications. Efforts to develop the enzymes as conversion tools are reviewed

Exploration of high-pressure processing (HPP) for preservation of the Swedish grown brown macroalgae Saccharina latissima

Introducing seaweed to new food markets entails new challenges concerning efficient preservation. Hence, this study explores high-pressure processing (HPP) as an alternative technique to conventional methods by evaluating its effects on the composition, quality, and microbial safety of the Swedish grown macroalgae Saccharina latissima. The results from the physicochemical analysis showed that after high-pressure treatment the color was retained, while the algal texture was altered by up to an 87.7% reduction in hardness and a 60.0% reduction in compression. Biochemical analysis demonstrated some variations in the algal samples, but the nutritional content was overall retained after treatment. The microbial analysis showed a low microbial load of untreated fresh material, which was confirmed by a lack of amplification in polymerase chain reaction attempts and low growth during attempts on spontaneous proliferation using fresh and frozen algae. Additionally, shelf-life studies showed inconsistent growth, but overall, a low increase in unspecific bacteria, an increasing load of Enterobacteriaceae, no growth of Lactobacilli, and low fouling by mold and yeast. The results from this study can be useful in the continued attempts of introducing seaweed to new markets, with different prerequisites for post-harvest treatment

Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis

Background: The thermostable beta-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e. g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-beta-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3. Results: Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on beta-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as T-m by differential scanning calorimetry (101.9 degrees C for wt), was kept in the mutated variants and significant decrease (Delta T of 5 -10 degrees C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in K-M and turnover. The K-M-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 x increase for pNPGlc, while the K-M decreased a corresponding extent for Q3. Turnover was only significantly changed using pNPGlc, and was decreased 2-3 x in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 x compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in K-M for this substrate. Conclusion: These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both K-M and turnover. An affinity change, leading to a decreased K-M, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site

Exploring codon adjustment strategies towards Escherichia coli-based production of viral proteins encoded by HTH1, a novel prophage of the marine bacterium Hypnocyclicus thermotrophus

Marine viral sequence space is immense and presents a promising resource for the discovery of new enzymes interesting for research and biotechnology. However, bottlenecks in the functional annotation of viral genes and soluble heterologous production of proteins hinder access to downstream characterization, subsequently impeding the discovery process. While commonly utilized for the heterologous expression of prokaryotic genes, codon adjustment approaches have not been fully explored for viral genes. Herein, the sequence-based identification of a putative prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, moderately thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. A prophage-associated gene cluster, consisting of 46 protein coding genes, was identified and given the proposed name Hypnocyclicus thermotrophus phage H1 (HTH1). HTH1 was taxonomically assigned to the viral family Siphoviridae, by lowest common ancestor analysis of its genome and phylogeny analyses based on proteins predicted as holin and DNA polymerase. The gene neighbourhood around the HTH1 lytic cassette was found most similar to viruses infecting Gram-positive bacteria. In the HTH1 lytic cassette, an N-acetylmuramoyl-L-alanine amidase (Amidase_2) with a peptidoglycan binding motif (LysM) was identified. A total of nine genes coding for enzymes putatively related to lysis, nucleic acid modification and of unknown function were subjected to heterologous expression in Escherichia coli. Codon optimization and codon harmonization approaches were applied in parallel to compare their effects on produced proteins. Comparison of protein yields and thermostability demonstrated that codon optimization yielded higher levels of soluble protein, but codon harmonization led to proteins with higher thermostability, implying a higher folding quality. Altogether, our study suggests that both codon optimization and codon harmonization are valuable approaches for successful heterologous expression of viral genes in E. coli, but codon harmonization may be preferable in obtaining recombinant viral proteins of higher folding quality.publishedVersio

Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus

AbstractThe thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that the EPS were produced both in the exponential and stationary growth phase and that production in the exponential phase was boosted by maltose supplementation, while stationary phase production was boosted by lactose. The latter was higher, resulting in 8.8 (DSM4252T) and 13.7mg EPS/g cell dry weight (MAT493) in cultures in marine broth supplemented with 10g/L lactose. The EPSs were heteropolymeric with an average molecular weight of 8×104Da and different monosaccharides, including arabinose and xylose. FT-IR spectroscopy revealed presence of hydroxyl, carboxyl, N-acetyl, amine, and sulfate ester groups, showing that R. marinus produces unusual sulfated EPS with high arabinose and xylose content

Life-cycle assessment of yeast-based single-cell protein production with oat processing side-stream

Production of fish meal and plant-based feed proteins continues to increase to meet the growing demand for seafood, leading to impacts on marine and terrestrial ecosystems. Microbial proteins such as single-cell proteins (SCPs) have been introduced as feed alternatives since they can replace current fish feed ingredients, e.g., soybean, which are associated with negative environmental impacts. Microbial protein production also enables utilization of grain processing side-streams as feedstock sources. This study assesses the environmental impacts of yeast-based SCP using oat side-stream as feedstock (OS-SCP). Life-cycle assessment with a cradle-to-gate approach was used to quantify global warming, freshwater eutrophication, marine eutrophication, terrestrial acidification, land use, and water consumption of OS-SCP production in Finland. Dried and wet side-streams of oat were compared with each other to identify differences in energy consumption and transportation effects. Sensitivity analysis was performed to examine the difference in impacts at various locations and fermentation times. Benchmarking was used to evaluate the environmental impacts of OS-SCP and other feed products, including both conventional and novel protein products. Results highlight the importance of energy sources in quantifying the environmental performance of OS-SCP production. OS-SCP produced with dried side-streams resulted in higher global warming (16.3 %) and water consumption (7.5 %) than OS-SCP produced from wet side-streams, reflecting the energy and water requirements for the drying process. Compared with conventional products, such as soy protein concentrates, OS-SCP resulted in 61 % less land use, while exacerbating the environmental impacts in all the other categories. OS-SCP had more impact on global warming (205–754 %), water consumption (166–1401 %), freshwater eutrophication (118–333 %), and terrestrial acidification (85–340 %) than other novel products, including yeast protein concentrate, methanotrophic bacterial SCP, and insect meal, while lowering global warming (11 %) and freshwater eutrophication (20 %) compared with dry microalgae biomass.Peer reviewe