Alkaliphiles: The Versatile Tools in Biotechnology.

The extreme environments within the biosphere are inhabited by organisms known as extremophiles. Lately, these organisms are attracting a great deal of interest from researchers and industrialists. The motive behind this attraction is mainly related to the desire for new and efficient products of biotechnological importance and human curiosity of understanding nature. Organisms living in common "human-friendly" environments have served humanity for a very long time, and this has led to exhaustion of the low-hanging "fruits," a phenomenon witnessed by the diminishing rate of new discoveries. For example, acquiring novel products such as drugs from the traditional sources has become difficult and expensive. Such challenges together with the basic research interest have brought the exploration of previously neglected or unknown groups of organisms. Extremophiles are among these groups which have been brought to focus and garnering a growing importance in biotechnology. In the last few decades, numerous extremophiles and their products have got their ways into industrial, agricultural, environmental, pharmaceutical, and other biotechnological applications.Alkaliphiles, organisms which thrive optimally at or above pH 9, are one of the most important classes of extremophiles. To flourish in their extreme habitats, alkaliphiles evolved impressive structural and functional adaptations. The high pH adaptation gave unique biocatalysts that are operationally stable at elevated pH and several other novel products with immense biotechnological application potential. Advances in the cultivation techniques, success in gene cloning and expression, metabolic engineering, metagenomics, and other related techniques are significantly contributing to expand the application horizon of these remarkable organisms of the 'bizarre' world. Studies have shown the enormous potential of alkaliphiles in numerous biotechnological applications. Although it seems just the beginning, some fantastic strides are already made in tapping this potential. This work tries to review some of the prominent applications of alkaliphiles by focusing such as on their enzymes, metabolites, exopolysaccharides, and biosurfactants. Moreover, the chapter strives to assesses the whole-cell applications of alkaliphiles including in biomining, food and feed supplementation, bioconstruction, microbial fuel cell, biofuel production, and bioremediation.

Challenges and Adaptations of Life in Alkaline Habitats.

A vast array of organisms is known thriving in high pH environments. The biotechnological, medical, and environmental importance of this remarkable group of organisms has attracted a great deal of interest among researchers and industrialists. One of the most intriguing phenomena of alkaliphiles that engrossed researchers' attention is their adaptation to high pH and ability to thrive in the "extreme" condition which is often lethal to other organisms. Studies made in this line revealed that alkaliphiles deployed a range of adaptive strategies to overcome the various challenges of life in high pH environments. This chapter highlights some of the challenges and the most important structural and functional adaptations that alkaliphiles evolved to circumvent the hurdles and flourish in alkaline habitats. The fascinating alkaliphiles' pH homeostasis that effectively maintains a lower cytoplasmic pH than its extracellular environment and the remarkable bioenergetics that produce ATP much faster than non-alkaliphiles systems are reviewed in detail. Moreover, the adaptive mechanisms that alkaliphiles employ to keep the structural and functional integrity of their biomolecules at elevated pH are assessed.It is undeniable that our understanding of alkaliphiles adaptation mechanisms to high pH is expanding with time. However, considering that little is known so far about the adaptation of life in alkaline milieu, it seems that this is just the beginning. Probably, there is a lot more waiting for discovery, and some of these issues are raised in the chapter, which not only summarizes the relevant literature but also forwards new insights regarding high pH adaptation. Moreover, an effort is made to include the largely neglected eukaryotic organisms' adaptation to high pH habitats.

Alkaliphiles: The Emerging Biological Tools Enhancing Concrete Durability.

Concrete is one of the most commonly used building materials ever used. Despite it is a very important and common construction material, concrete is very sensitive to crack formation and requires repair. A variety of chemical-based techniques and materials have been developed to repair concrete cracks. Although the use of these chemical-based repair systems are the best commercially available choices, there have also been concerns related to their use. These repair agents suffer from inefficiency and unsustainability. Most of the products are expensive and susceptible to degradation, exhibit poor bonding to the cracked concrete surfaces, and are characterized by different physical properties such as thermal expansion coefficients which are different to that of concrete. Moreover, many of these repair agents contain chemicals that pose environmental and health hazards. Thus, there has been interest in developing concrete crack repair agents that are efficient, long lasting, safe, and benign to the environment and exhibit physical properties which resemble that of the concrete. The search initiated by these desires brought the use of biomineralization processes as tools in mending concrete cracks. Among biomineralization processes, microbially initiated calcite precipitation has emerged as an interesting alternative to the existing chemical-based concrete crack repairing system. Indeed, results of several studies on the use of microbial-based concrete repair agents revealed the remarkable potential of this approach in the fight against concrete deterioration. In addition to repairing existing concrete cracks, microorganisms have also been considered to make protective surface coating (biodeposition) on concrete structures and in making self-healing concrete.Even though a wide variety of microorganisms can precipitate calcite, the nature of concrete determines their applicability. One of the important factors that determine the applicability of microbes in concrete is pH. Concrete is highly alkaline in nature, and hence the microbes envisioned for this application are alkaliphilic or alkali-tolerant. This work reviews the available information on applications of microbes in concrete: repairing existing cracks, biodeposition, and self-healing. Moreover, an effort is made to discuss biomineralization processes that are relevant to extend the durability of concrete structures. Graphical Abstract.

Alkaline Active Hemicellulases.

Xylan and mannan are the two most abundant hemicelluloses, and enzymes that modify these polysaccharides are prominent hemicellulases with immense biotechnological importance. Among these enzymes, xylanases and mannanases which play the vital role in the hydrolysis of xylan and mannan, respectively, attracted a great deal of interest. These hemicellulases have got applications in food, feed, bioethanol, pulp and paper, chemical, and beverage producing industries as well as in biorefineries and environmental biotechnology. The great majority of the enzymes used in these applications are optimally active in mildly acidic to neutral range. However, in recent years, alkaline active enzymes have also become increasingly important. This is mainly due to some benefits of utilizing alkaline active hemicellulases over that of neutral or acid active enzymes. One of the advantages is that the alkaline active enzymes are most suitable to applications that require high pH such as Kraft pulp delignification, detergent formulation, and cotton bioscouring. The other benefit is related to the better solubility of hemicelluloses at high pH. Since the efficiency of enzymatic hydrolysis is often positively correlated to substrate solubility, the hydrolysis of hemicelluloses can be more efficient if performed at high pH. High pH hydrolysis requires the use of alkaline active enzymes. Moreover, alkaline extraction is the most common hemicellulose extraction method, and direct hydrolysis of the alkali-extracted hemicellulose could be of great interest in the valorization of hemicellulose. Direct hydrolysis avoids the time-consuming extensive washing, and neutralization processes required if non-alkaline active enzymes are opted to be used. Furthermore, most alkaline active enzymes are relatively active in a wide range of pH, and at least some of them are significantly or even optimally active in slightly acidic to neutral pH range. Such enzymes can be eligible for non-alkaline applications such as in feed, food, and beverage industries.This chapter largely focuses on the most important alkaline active hemicellulases, endo-ß-1,4-xylanases and ß-mannanases. It summarizes the relevant catalytic properties, structural features, as well as the real and potential applications of these remarkable hemicellulases in textile, paper and pulp, detergent, feed, food, and prebiotic producing industries. In addition, the chapter depicts the role of these extremozymes in valorization of hemicelluloses to platform chemicals and alike in biorefineries. It also reviews hemicelluloses and discusses their biotechnological importance.

Synergistic effect of haloduracin and chloramphenicol against clinically important Gram-positive bacteria.

The emergence of drug-resistant pathogens has triggered the search for more efficient antimicrobial agents and formulations for treatment of infections. In recent years, combination therapy has become one of the effective clinical practices in treating infections. The present study deals with the effect of haloduracin, a lantibiotic bateriocin and chloramphenicol against clinically important bacteria. The combined use of haloduracin and chloramphenicol resulted in remarkable synergy against a spectrum of microorganisms including strains of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis and different groups of Streptococcus. The synergy allowed using these antimicrobial agents at substantially reduced concentrations without compromising their efficiency. Use of lower doses of chloramphenicol can avoid the severity of its side effects. In addition to minimizing undesirable side effects of some drugs, this approach brings the possibility of using antibiotics that are no longer effective due to drug resistance. Furthermore, the observed synergy between haloduracin and chloramphenicol opens a new window of using bacteriocins and antibiotics in combination therapy of infections.

Antimicrobial Protein Candidates from the Thermophilic Geobacillus sp. Strain ZGt-1: Production, Proteomics, and Bioinformatics Analysis.

A thermophilic bacterial strain, Geobacillus sp. ZGt-1, isolated from Zara hot spring in Jordan, was capable of inhibiting the growth of the thermophilic G. stearothermophilus and the mesophilic Bacillus subtilis and Salmonella typhimurium on a solid cultivation medium. Antibacterial activity was not observed when ZGt-1 was cultivated in a liquid medium; however, immobilization of the cells in agar beads that were subjected to sequential batch cultivation in the liquid medium at 60 °C showed increasing antibacterial activity up to 14 cycles. The antibacterial activity was lost on protease treatment of the culture supernatant. Concentration of the protein fraction by ammonium sulphate precipitation followed by denaturing polyacrylamide gel electrophoresis separation and analysis of the gel for antibacterial activity against G. stearothermophilus showed a distinct inhibition zone in 15-20 kDa range, suggesting that the active molecule(s) are resistant to denaturation by SDS. Mass spectrometric analysis of the protein bands around the active region resulted in identification of 22 proteins with molecular weight in the range of interest, three of which were new and are here proposed as potential antimicrobial protein candidates by in silico analysis of their amino acid sequences. Mass spectrometric analysis also indicated the presence of partial sequences of antimicrobial enzymes, amidase, and dd-carboxypeptidase.

Anaerobes as Sources of Bioactive Compounds and Health Promoting Tools.

Aerobic microorganisms have been sources of medicinal agents for several decades and an impressive variety of drugs have been isolated from their cultures, studied and formulated to treat or prevent diseases. On the other hand, anaerobes, which are believed to be the oldest life forms on earth and evolved remarkably diverse physiological functions, have largely been neglected as sources of bioactive compounds. However, results obtained from the limited research done so far show that anaerobes are capable of producing a range of interesting bioactive compounds that can promote human health. In fact, some of these bioactive compounds are found to be novel in their structure and/or mode of action.Anaerobes play health-promoting roles through their bioactive products as well as application of whole cells. The bioactive compounds produced by these microorganisms include antimicrobial agents and substances such as immunomodulators and vitamins. Bacteriocins produced by anaerobes have been in use as preservatives for about 40 years. Because these substances are effective at low concentrations, encounter relatively less resistance from bacteria and are safe to use, there is a growing interest in these antimicrobial agents. Moreover, several antibiotics have been reported from the cultures of anaerobes. Closthioamide and andrimid produced by Clostridium cellulolyticum and Pantoea agglomerans, respectively, are examples of novel antibiotics of anaerobe origin. The discovery of such novel bioactive compounds is expected to encourage further studies which can potentially lead to tapping of the antibiotic production potential of this fascinating group of microorganisms.Anaerobes are widely used in preparation of fermented foods and beverages. During the fermentation processes, these organisms produce a number of bioactive compounds including anticancer, antihypertensive and antioxidant substances. The well-known health promoting effect of fermented food is mostly due to these bioactive compounds. In addition to their products, whole cell anaerobes have very interesting applications for enhancing the quality of life. Probiotic anaerobes have been on the market for many years and are receiving growing acceptance as health promoters. Gut anaerobes have been used to treat patients suffering from severe Clostridium difficile infection syndromes including diarrhoea and colitis which cannot be treated by other means. Whole cell anaerobes are also studied to detect and cure cancer. In recent years, evidence is emerging that anaerobes constituting the microbiome are linked to our overall health. A dysfunctional microbiome is believed to be the cause of many diseases including cancer, allergy, infection, obesity, diabetes and several other disorders. Maintaining normal microflora is believed to alleviate some of these serious health problems. Indeed, the use of probiotics and prebiotics which favourably change the number and composition of the gut microflora is known to render a health promoting effect. Our interaction with the microbiome anaerobes is complex. In fact, not only our lives but also our identities are more closely linked to the anaerobic microbial world than we may possibly imagine. We are just at the beginning of unravelling the secret of association between the microbiome and human body, and a clear understanding of the association may bring a paradigm shift in the way we diagnose and treat diseases and disorders. This chapter highlights some of the work done on bioactive compounds and whole cell applications of the anaerobes that foster human health and improve the quality of life.

Synthesis and use of protein G imprinted cryogel as affinity matrix to purify protein G from cell lyaste.

Monolithic macroporous cryogel imprinted with protein G was prepared using a functional co-monomer of N-methacryloyl-l-phenylalanine and 2-hydroxyethyl methacrylate. The chemical structure of the cryogel prepared was studied by FTIR-spectroscopy and its porosity was analysed using scanning electron microscopy. The cryogel was used to purify protein G from recombinant Escherichia coli cell lysate and the effect of pH, temperature, ionic strength, flow rate, etc on the adsorption of protein G to the monolithic column have been investigated. The selectivity of the imprinted cryogel was studied using protein A and myoglobin. It was possible to capture about 9mg of Protein G per g of the cryogel.

Enzymatic monitoring of lignin and lignin derivatives biooxidation.

Lignin oxidation was enzymatically monitored by measuring methanol released during the reaction. The methanol was oxidized to formaldehyde and hydrogen peroxide, and the latter used to oxidize ABTS to a product measured spectrophotometrically. The efficiency was comparable to the commonly used gas chromatography method. The assay was fast and inexpensive.

Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of inedible wild cassava flour to bioethanol.

The major bottlenecks in achieving competitive bioethanol fuel are the high cost of feedstock, energy and enzymes employed in pretreatment prior to fermentation. Lignocellulosic biomass has been proposed as an alternative feedstock, but because of its complexity, economic viability is yet to be realized. Therefore, research around non-conventional feedstocks and deployment of bioconversion approaches that downsize the cost of energy and enzymes is justified. In this study, a non-conventional feedstock, inedible wild cassava was used for bioethanol production. Bioconversion of raw starch from the wild cassava to bioethanol at low temperature was investigated using both a co-culture of Aspergillus sp. and Saccharomyces cerevisiae, and a monoculture of the later with enzyme preparation from the former. A newly isolated strain of Aspergillus sp. MZA-3 produced raw starch-degrading enzyme which displayed highest activity of 3.3 U/mL towards raw starch from wild cassava at 50°C, pH 5.5. A co-culture of MZA-3 and S. cerevisiae; and a monoculture of S. cerevisiae and MZA-3 enzyme (both supplemented with glucoamylase) resulted into bioethanol yield (percentage of the theoretical yield) of 91 and 95 at efficiency (percentage) of 84 and 96, respectively. Direct bioconversion of raw starch to bioethanol was achieved at 30°C through the co-culture approach. This could be attractive since it may significantly downsize energy expenses.

Cloning, expression and characterization of a versatile Baeyer-Villiger monooxygenase from Dietzia sp. D5.

A novel BVMO encoding gene was identified from a draft genome sequence of a newly isolated strain of Dietzia. Analysis of the protein sequence revealed that it belongs to a group of BVMOs whose most characterized member is cyclopentadecanone monooxygenase (CPDMO). The gene was PCR amplified, cloned and successfully expressed in E. coli. The expressed recombinant enzyme was purified using metal affinity chromatography. Characterization of the purified enzyme revealed that it has a broad substrate scope and oxidized different compounds including substituted and unsubstituted alicyclic, bicyclic-, aliphatic-ketones, ketones with an aromatic moiety, and sulfides. The highest activities were measured for 2- and 3-methylcyclohexanone, phenylacetone, bicyclo-[3.2.0]-hept-2-en-6-one and menthone. The enzyme was optimally active at pH 7.5 and 35°C, a temperature at which its half-life was about 20 hours. The stability studies have shown that this enzyme is more stable than all other reported BVMOs except the phenylacetone monooxygenase from the thermophilic organism Thermobifida fusca.

Ectoine-mediated protection of enzyme from the effect of pH and temperature stress: a study using Bacillus halodurans xylanase as a model.

Compatible solutes are small, soluble organic compounds that have the ability to stabilise proteins against various stress conditions. In this study, the protective effect of ectoines against pH stress is examined using a recombinant xylanase from Bacillus halodurans as a model. Ectoines improved the enzyme stability at low (4.5 and 5.0) and high pH (11 and 12); stabilisation effect of hydroxyectoine was superior to that of ectoine and trehalose. In the presence of hydroxyectoine, residual activity (after 10 h heating at 50 °C) increased from about 45 to 86 % at pH 5 and from 33 to 89 % at pH 12. When the xylanase was incubated at 65 °C for 5 h with 50 mM hydroxyectoine at pH 10, about 40 % of the original activity was retained while no residual activity was detected in the absence of additives or in the presence of ectoine or trehalose. The xylanase activity was slightly stimulated in the presence of 25 mM ectoines and then gradually decreased with increase in ectoines concentration. The thermal unfolding of the enzyme in the presence of the compatible solutes showed a modest increase in denaturation temperature but a larger increase in calorimetric enthalpy.

A method for rapid screening of ketone biotransformations: detection of whole cell Baeyer-Villiger monooxygenase activity.

A method for screening of ketone biotransformations was developed and applied to the identification of Baeyer-Villiger monooxygenase (BVMO) activity. The method was based on the formation of a purple coloured product on reaction between an enolizable ketone and 3,5-dinitrobenzoic acid in an alkaline solution. Absorbance of the colour decreased with the size of the cycloketone ring. Stoichiometric ratio between cycloketone and 3,5-dinitrobenzoic acid was 1:1 at maximum absorbance. The method was applied for monitoring the consumption of cyclohexanone by bacteria under aerobic conditions, and was found to be potentially useful for both screening assays and quantitative measurements of BVMO activity. Compared to other existing methods, this method is faster, cheaper and amenable for whole cell assays.

Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu.

Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.

Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability.

Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca(2+) on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca(2+) with Ba(2+), Mn(2+), Mg(2+) and Sr(2+) in the phytase resulted in the expression of >90% of the maximal activity with calcium-phytate as the substrate, while Fe(2+) and Zn(2+) rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg(2+), Sn(2+) and Sr(2+), respectively. On replacing Ca(2+) on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg(2+) and Sr(2+), respectively. Only Ca(2+) resulted in a marked increase in the melting temperature (T(m)) of the enzyme by 12-21°C, while Ba(2+), Mn(2+), Sr(2+) or Cu(2+) resulted in a modest (2-3.5°C) increase in T(m). In the presence of 1-5mM Ca(2+), the optimum temperature of the phytase activity was increased from 40°C to 70°C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region.

Production of haloduracin by Bacillus halodurans using solid-state fermentation.

Bacillus halodurans was cultivated on wheat bran as a solid-state substrate and produced haloduracin, a bacteriocin, at about 245 AU per wheat bran. Supplementation of the bran with Lauria-Bertani broth decreased haloduracin production. However, production was stimulated by addition of Mg(2)SO(4) and K(2)HPO(4). The highest production was achieved at a wheat bran/moisture ratio of 1:1.8 and in the presence of 10% (w/w) Na(2)CO(3). Under optimum conditions, the organism produced about 3,000 AU per gram dry bran.

A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli.

Phytase is used as a feed additive for degradation of antinutritional phytate, and the enzyme is desired to be highly thermostable for it to withstand feed formulation conditions. A Bacillus sp. MD2 showing phytase activity was isolated, and the phytase encoding gene was cloned and expressed in Escherichia coli. The recombinant phytase exhibited high stability at temperatures up to 100 degrees C. A higher enzyme activity was obtained when the gene expression was done in the presence of calcium chloride. Production of the enzyme by batch- and fed-batch cultivation in a bioreactor was studied. In batch cultivation, maintaining dissolved oxygen at 20-30% saturation and depleting inorganic phosphate below 1 mM prior to induction by IPTG resulted in over 10 U/ml phytase activity. For fed-batch cultivation, glucose concentration was maintained at 2-3 g/l, and the phytase expression was increased to 327 U/ml. Induction using lactose during fed-batch cultivation showed a lag phase of 4 h prior to an increase in the phytase activity to 71 U/ml during the same period as IPTG-induced production. Up to 90% of the total amount of expressed phytase leaked out from the E. coli cells in both IPTG- and lactose-induced fed-batch cultivations.

An alkaline active xylanase: insights into mechanisms of high pH catalytic adaptation.

The alkaliphilic bacterium, Bacillus halodurans S7, produces an alkaline active xylanase (EC 3.2.1.8), which differs from many other xylanases in being operationally stable under alkaline conditions as well as at elevated temperature. Compared to non-alkaline active xylanases, this enzyme has a high percent composition of acidic amino acids which results in high ratio of negatively to positively charged residues. A positive correlation was observed between the charge ratio and the pH optima of xylanases. The recombinant xylanase was crystallized using a hanging drop diffusion method. The crystals belong to the space group P2(1)2(1)2(1) and the structure was determined at a resolution of 2.1 A. The enzyme has the common eight-fold TIM-barrel structure of family 10 xylanases; however, unlike non-alkaline active xylanases, it has a highly negatively charged surface and a deeper active site cleft. Mutational analysis of non-conserved amino acids which are close to the acid/base residue has shown that Val169, Ile170 and Asp171 are important to hydrolyze xylan at high pH. Unlike the wild type xylanase which has optimum pH at 9-9.5, the triple mutant xylanase (V169A, I170F and D171N), which was constructed using sequence information of alkaline sensitive xylanses was optimally active around pH 7. Compared to non-alkaline active xylanases, the alkaline active xylanases have highly acidic surfaces and fewer solvent exposed alkali labile residues. Based on these results obtained from sequence, structural and mutational analysis, the possible mechanisms of high pH stability and catalysis are discussed. This will provide useful information to understand the mechanism of high pH adaptation and engineering of enzymes that can be operationally stable at high pH.

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.

Optimizing refolding and recovery of active recombinant Bacillus halodurans xylanase in polymer-salt aqueous two-phase system using surface response analysis.

An experimental design was used to determine optimal conditions for refolding of a recombinant thermostable and alkaline active xylanase from Bacillus halodurans in PEG-phosphate two-phase system. The influence of different experimental variables on the enzyme recovery has been evaluated. To build the mathematical model and minimize the number of experiments for the design parameters, response surface methodology with a face-centered central composite design (CCF) was defined based on the conditions found by preliminary tests that resulted in the highest refolding yield. The adequacy of the calculated model for the response was confirmed by means of variance analysis and additional experiments. Analysis of contours of constant response as a function of pH, polyethylene glycol (PEG) molecular weight and concentration, and salt concentration for different enzyme loads revealed different effects of these five factors on the studied parameters. Recovery of more than 92% active xylanase was predicted for a system with 18.3% (w/w) PEG 1000, 14.4% (w/w) phosphate at pH 8.5, and enzyme load corresponding to a protein concentration of about 0.05 mg/g system. The yield of the refolded enzyme was found to be optimal at 22 degrees C. The validity of the response model was verified by a good agreement between predicted and experimental results.