Facile syntheses of <Emphasis Type="SmallCaps">l</Emphasis>-β-haloalanine derivatives from <Emphasis Type="SmallCaps">l</Emphasis>-cysteine or <Emphasis Type="SmallCaps">l</Emphasis>-cystine

l-β-Haloalanines are physiologically active unnatural amino acids and they are useful intermediates for the synthesis of natural and unnatural amino acids, S-linked glycopeptides, and lanthionines. In general l-β-haloalanines were prepared predominantly from l-serine via functional group transformation. Here we reported an alternative approach for the preparation of l-β-haloalanines via halogenation of protected l-cysteine esters which was obtained from l-cysteine or l-cystine, respectively. The mercapto group of protected l-cysteine esters was efficiently transformed to halo groups by triphenylphosphine/N-halosuccinimides. It has been proved to be a versatile desulfurization strategy via this functional group transformation.     

The α-<Emphasis Type="SmallCaps">l</Emphasis>-fucosidase from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis>

Glycoside hydrolases form hyperthermophilic archaea are interesting model systems for the study of catalysis at high temperatures and, at the moment, their detailed enzymological characterization is the only approach to define their role in vivo. Family 29 of glycoside hydrolases classification groups α-l-fucosidases involved in a variety of biological events in Bacteria and Eukarya. In Archaea the first α-l-fucosidase was identified in Sulfolobus solfataricus as interrupted gene expressed by programmed −1 frameshifting. In this review, we describe the identification of the catalytic residues of the archaeal enzyme, by means of the chemical rescue strategy. The intrinsic stability of the hyperthermophilic enzyme allowed the use of this method, which resulted of general applicability for β and α glycoside hydrolases. In addition, the presence in the active site of the archaeal enzyme of a triad of catalytic residues is a rather uncommon feature among the glycoside hydrolases and suggested that in family 29 slightly different catalytic machineries coexist.     

Endogeneous β-<Emphasis Type="SmallCaps">d</Emphasis>-xylosidase and α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase activity in flax seed mucilage

Flax seed mucilage (FM) contains a mixture of highly doubly substituted arabinoxylan as well as rhamnogalacturonan I with unusual side group substitutions. Treatment of FM with a GH11 Bacillus subtilis XynA endo 1,4-β-xylanase (BsX) gave limited formation of reducing ends but when BsX and FM were incubated together on different wheat arabinoxylan substrates and birchwood xylan, significant amounts of xylose were released. Moreover, arabinose was released from both water-extractable and water-unextractable wheat arabinoxylan. Since no xylose or arabinose was released by BsX addition alone on these substrates, nor without FM or BsX addition, the results indicate the presence of endogenous β-d-xylosidase and α-l-arabinofuranosidase activities in FM. FM also exhibited activity on both p-nitrophenyl α-l-arabinofuranoside (pNPA) and p-nitrophenyl β-d-xylopyranoside (pNPX). Based on K _M values, the FM enzyme activities had a higher affinity for pNPX (K _M 2 mM) than for pNPA (K _M 20 mM).     

Biosynthesis of nearly monodispersed poly(ε-<Emphasis Type="SmallCaps">l</Emphasis>-lysine) in <Emphasis Type="Italic">Streptomyces</Emphasis> species

Poly(ε-l-lysine) (ε-PL) is a naturally occurring poly(amino acid) characterized by a unique structure linking ε-amino and carboxyl groups of l-lysine. Due to its various functions and its biodegradability and non-toxicity, the ε-PL polymer has attracted increasing attention in recent years. ε-PL is frequently found in various strains of Streptomyces sp. This review gives an up-to-date overview regarding the biosynthesis of ε-PL focussing mainly on results obtained from ten newly isolated producer strains, using the two-stage culture method of cell growth and ε-PL production cultures. The production of nearly monodispersed ε-PL is covered together with the development of ε-PL specific hydrolases and the release of synthesized ε-PL into the culture broth. From these results, coupled with the termination of polymerization through nucleophilic chain transfer, the biosynthetic mechanism of the polymer is discussed.     

α-<Emphasis Type="SmallCaps">l</Emphasis>-Rhamnosidase of <Emphasis Type="Italic">Aspergillus terreus</Emphasis> immobilized on ferromagnetic supports

α-l-Rhamnosidase from Aspergillus terreus was covalently immobilized on the following ferromagnetic supports: polyethylene terephthalate (Dacron-hydrazide), polysiloxane/polyvinyl alcohol (POS/PVA), and chitosan. The powdered supports were magnetized by thermal coprecipitation method using ferric and ferrous chlorides, and the immobilization was carried out via glutaraldehyde. The activity of the Dacron-hydrazide (0.53 nkat/μg of protein) and POS/PVA (0.59 nkat/μg of protein) immobilized enzyme was significantly higher than that found for the chitosan derivative (0.06 nkat/μg of protein). The activity–pH and activity–temperature profiles for all immobilized enzymes did not show difference compared to the free enzyme, except the chitosan derivative that presented higher maximum temperature at 65 °C. The Dacron-hydrazide derivative thermal stability showed a similar behavior of the free enzyme in the temperature range of 40–70 °C. The POS/PVA and chitosan derivatives were stable up to 60 °C, but were completely inactivated at 70 °C. The activity of the preparations did not appreciably decrease after ten successive reuses. Apparent K _m of α-l-rhamnosidase immobilized on magnetized Dacron-hydrazide (1.05 ± 0.22 mM), POS/PVA (0.57 ± 0.09 mM), and chitosan (1.78 ± 0.24 mM) were higher than that estimated for the soluble enzyme (0.30 ± 0.03 mM). The Dacron-hydrazide enzyme derivative showed better performance than the free enzyme to hydrolyze 0.3% narigin (91% and 73% after 1 h, respectively) and synthesize rhamnosides (0.116 and 0.014 mg narirutin after 1 h, respectively).     

Purification and partial characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase produced by<Emphasis Type="Italic">Thermomonospora fusca</Emphasis>

Thermomonospora fusca produced a relatively high level of alpha-L-arabinofuranosidase when growing on oat spelt xylan as the main carbon and energy source. The enzyme exhibited maximum relative activity (0.136 U/g protein) at pH 9.0 with 54 and 55% activity remaining at pH of 4.5 and 11.0, respectively. The apparent Km value for the crude alpha-L-arabinofuranosidase preparation was 180 mumol/L 4-nitrophenyl alpha-L-arabinofuranoside; the upsilon lim value was the release of 40 mumol/L 4-nitrophenol per min. Enzyme activity was eluted as a single peak (HPLC gel filtration chromatography) corresponding to molar mass of approximately 92 kDa. Native electrophoresis of crude cell lysate confirmed the presence of a single active intracellular alpha-L-arabinofuranosidase component. SDS-PAGE of this enzyme, developed as zymogram, did not demonstrate any activity; denaturing gel was stained and a protein band of relative molar mass of 46 kDa was revealed. Isoelectric focusing of a purified alpha-L-arabinofuranosidase yielded a single protein band for the corresponding activity zone with pI 7.9. The enzyme was purified approximately 21-fold the mean overall yield was about 16%.     

Characterization of a thermostable endo-1,5-α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinanase from <Emphasis Type="Italic">Caldicellulorsiruptor saccharolyticus</Emphasis>

A recombinant putative glycoside hydrolase from Caldicellulosiruptor saccharolyticus was purified with a specific activity of 12 U mg⁻¹ by heat treatment and His-Trap affinity chromatography, and identified as a single 56 kDa band upon SDS-PAGE. The native enzyme is a dimer with a molecular mass of 112 kDa as determined by gel filtration. The enzyme exhibited its highest activity when debranched arabinan (1,5-α-l-arabinan) was used as the substrate, demonstrating that the enzyme was an endo-1,5-α-l-arabinanase. The K _m, k _cat, and k _cat/K _m values were 18 mg ml⁻¹, 50 s⁻¹, and a 2.8 mg ml⁻¹ s⁻¹, respectively. Maximum enzyme activity was at pH 6.5 and 75°C. The half-lives of the enzyme at 65, 70 and 75°C were 2440, 254 and 93 h, respectively, indicating that it is the most thermostable of the known endo-1,5-α-l-arabinanases.     

Characterization of a family 54 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

A glycosyl hydrolase family 54 (GH54) α-l-arabinofuranosidase gene (abfA) of Aureobasidium pullulans was amplified by polymerase chain reaction from genomic DNA and a 498-amino-acid open reading frame deduced from the DNA sequence. Modeling of the highly conserved A. pullulans AbfA protein sequence on the crystal structure of Aspergillus kawachii AkabfB showed that the catalytic amino acid arrangement and overall structure were highly similar including the N-terminal catalytic and C-terminal arabinose binding domains. The abfA gene was expressed in Saccharomyces cerevisiae, and the heterologous enzyme was purified. The protein was monomeric, migrating at 49 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and eluting at 36 kDa upon gel filtration. AbfA showed maximal activity at 55°C and between pH 3.5 and pH 4. The enzyme had a K _m value for p-nitrophenyl-α-l-arabinofuranoside of 3.7 mM and a V _max of 34.8 μmol min⁻¹ mg protein⁻¹. Arabinose acted as a noncompetitive inhibitor with a K _i of 38.4 mM. The enzyme released arabinose from maize fiber, oat spelt arabinoxylan, and wheat arabinoxylan, but not from larch wood arabinogalactan or α-1,5-debranched arabinan. AbfA displayed low activity against α-1,5-l-arabino-oligosaccharides. The enzyme acted synergistically with endo-β-1,4-xylanase in the breakdown of wheat arabinoxylan. Binding of AbfA to xylan from several sources confirmed the presence of a functional carbohydrate-binding module. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Effect of Exogenous β-<Emphasis Type="Italic">N-</Emphasis>Methylamino-<Emphasis Type="SmallCaps">l</Emphasis>-alanine on the Growth of <Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803

β-N-Methylamino-l-alanine (BMAA), a non-proteinogenic amino acid, has been detected in a range of cyanobacteria, including terrestrial, aquatic, free living and endosymbiotic species. The widespread occurrence of cyanobacteria in the environment raises concerns regarding the ecological and toxicological impact of BMAA, and consequently, studies have focussed extensively on the toxicity and environmental impact of BMAA, while no research has addressed the ecophysiological or metabolic role of the compound in cyanobacteria. In this study, both the uptake of exogenous BMAA by and the effect of exogenous BMAA on the growth of Synechocystis PCC6803 were investigated. BMAA was rapidly taken up by the non-diazotrophic cyanobacterium Synechocystis PCC6803 in a concentration dependent manner. The presence of exogenous BMAA resulted in a substantial and concentration-dependent decrease in cell growth and the substantial loss of photosynthetic pigmentation. Similar effects were seen in the presence of the non-proteinogenic amino acid, 2,4-diaminobutyric acid but to a lesser degree than that of BMAA. The effects were reversed when light was decreased from 16 to 10 μmol m⁻² s⁻¹. Control cultures grown in the presence of l-arginine, l-asparagine, l-glutamate and glycine showed normal or slightly increased growth with no change in pigmentation. The decrease in growth rate coupled to bleaching indicates that BMAA may induce chlorosis in the presence of adequate photosynthetic radiation suggesting a connection between BMAA and the induction of conditions, such as nitrogen or sulphur depletion, that result in growth arrest and the induction of chlorosis.     

ɛ-Poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine producer, <Emphasis Type="Italic">Streptomyces albulus</Emphasis>, has feedback-inhibition resistant aspartokinase

Streptomyces albulus NBRC14147 produces ɛ-poly-l-lysine (ɛ-PL), which is an amino acid homopolymer antibiotic. Despite the commercial importance of ɛ-PL, limited information is available regarding its biosynthesis; the l-lysine molecule is directly utilized for ɛ-PL biosynthesis. In most bacteria, l-lysine is biosynthesized by an aspartate pathway. Aspartokinase (Ask), which is the first enzyme in this pathway, is subject to complex regulation such as through feedback inhibition by the end-product amino acids such as l-lysine and/or l-threonine. S. albulus NBRC14147 can produce a large amount of ɛ-PL (1–3 g/l). We therefore suspected that Ask(s) of S. albulus could be resistant to feedback inhibition to provide sufficient l-lysine for ɛ-PL biosynthesis. To address this hypothesis, in this study, we cloned the ask gene from S. albulus and investigated the feedback inhibition of its gene product. As predicted, we revealed the feedback resistance of the Ask; more than 20% relative activity of Ask was detected in the assay mixture even with extremely high concentrations of l-lysine and l-threonine (100 mM each). We further constructed a mutated ask gene for which the gene product Ask (M68V) is almost fully resistant to feedback inhibition. The homologous expression of Ask (M68V) further demonstrated the increase in ɛ-PL productivity.     

Poly(β-<Emphasis Type="SmallCaps">l</Emphasis>-malic acid) (PMLA) from <Emphasis Type="Italic">Aureobasidium</Emphasis> spp. and its current proceedings

Poly(β-l-malic acid) is one natural biopolymer that has the outstanding features of biocompatibility, biodegradability, water solubility, and non-immunogenicity, and it is easily chemically modified. So poly(β-l-malic acid) (PMLA) and its derivatives may have a great potential application as a novel drug delivery system and in the production of advanced biomaterials which have attracted so much research attention. The fungi of Aureobasidium spp. have been discovered to be the most suitable candidates for PMLA production in large quantities which satisfy the demand of either research or industry. In this review, we will give an overall summary about the PMLA produced by Aureobasidium spp. based on related research in the last decades and the elaboration of this PMLA producer will also be accomplished. More importantly, the latest proceedings will be specified and some suggestions to the elucidation of a PMLA biosynthesis pathway which remains undefined up to date will be proposed. Finally, through this review, the further exploitation for the application of PMLA from Aureobasidium spp. can be emphasized and promoted.     

High-level production of poly (β-<Emphasis Type="SmallCaps">l</Emphasis>-malic acid) with a new isolated <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> strain

Poly (β-l-malic acid) (PMLA) is a water-soluble polyester with many attractive properties in chemical industry and medicine development. However, the low titer of PMLA in the available producer strains limits further industrialization efforts and restricts its many potential applications. In order to solve this problem, a new strain with the distinguished high productivity of PMLA was isolated from fresh plants samples. It was characterized as the candidate of Aureobasidium pullulans based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences. After the optimization of culture conditions, the highest PMLA concentration (62.27 g l⁻¹) could be achieved in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO₃ in the medium. This high-level fermentation process was further scaled up in the 10 l benchtop fermentor with a high PMLA concentration (57.2 g l⁻¹) and productivity (0.35 g l⁻¹ h⁻¹), which are the highest level in all the literature. Finally, the suitable acid hydrolysis conditions of PMLA were also investigated with regard to the production of l-malic acid, and the kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. The present work paved the road to produce this multifunctional biomaterial (PMLA) at industrial scale and promised one alternative method to produce l-malic acid in the future.     

Production,partial purification and characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Penicillium ulaiense</Emphasis>

Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification, including (NH₄)₂SO₄ precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V _max = 26 ± 4 IU ml⁻¹ and K _m  = 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co²⁺ affected positively the activity; EDTA, Mn²⁺, Mg²⁺, dithiotreitol and Cu²⁺ reduced the activity by different amounts, and Hg²⁺ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries since P. ulaiense does not produce mycotoxins.     

<Emphasis Type="Italic">Streptomyces albulus</Emphasis> yields ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine and other products from salt-contaminated glycerol waste

Actinomycetes are the most important microorganisms for the industrial production of secondary metabolites with antimicrobial and anticancer properties. However, they have not been implicated in biorefineries. Here, we study the ability of the ε-poly-l-lysine producing Streptomyces albulus BCRC 11814 to utilize biodiesel-derived crude glycerol. S. albulus was cultured in a mineral medium supplemented with up to 10% w/v sodium chloride or potassium chloride, and with crude glycerol as the sole carbohydrate source. Under these conditions, the strain produced 0.1 g ε-poly-l-lysine per 1 g of biomass. RNA sequencing revealed upregulation of the ectoine biosynthetic pathway of S. albulus, which provides proof of halotolerance. S. albulus has several silent secondary metabolite biosynthetic clusters predicted within the genome. Based on the results, we conclude that S. albulus BCRC 11814 is a halotolerant microorganism capable of utilizing biodiesel-derived crude glycerol better than other actinomycetes included in the present study. S. albulus has the potential to be established as microbial platform production host for a range of high-value biological products.     

Efficient bioconversion of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate to γ-aminobutyric acid by <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> resting cells

This work investigated the efficient bioconversion process of l-glutamate to GABA by Lactobacillus brevis TCCC 13007 resting cells. The optimal bioconversion system was composed of 50 g/L 48 h cultivated wet resting cells, 0.1 mM pyridoxal phosphate in glutamate-containing 0.6 M citrate buffer (pH 4.5) and performed at 45 °C and 180 rpm. By 10 h bioconversion at the ratio of 80 g/L l-glutamic acid to 240 g/L monosodium glutamate, the final titer of GABA reached 201.18 g/L at the molar bioconversion ratio of 99.4 %. This process presents a potential for industrial and commercial applications and also offers a promising feasibility of continuous GABA production coupled with fermentation. Besides, the built kinetics model revealed that the optimum operating conditions were 45 °C and pH 4.5, and the bioconversion kinetics at low ranges of substrate concentration (0 < S < 80 g/L) was assumed to follow the classical Michaelis–Menten equation.     

Molecular identification of <Emphasis Type="Italic">Staphylococcus xylosus</Emphasis> MAK2, a new α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase producer

A bacterial strain, MAK-2, was isolated as a producer of α-l-rhamnosidase from a soil sample of Dehradoon, India. The strain was identified based on morphology, physiological tests and 16S rDNA analysis. The phylogenetic analysis based on the 16S rDNA sequence, identified the isolate as Staphylococcus xylosus, a non-pathogenic member of CNS (coagulase-negative staphylococci) family. The strain was capable of producing α-l-rhamnosidase by hydrolysing flavonoids thus confirming potential application in the citrus-processing industry.     

Isolation and characterization of a novel ε-poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine producing strain: <Emphasis Type="Italic">Streptomyces griseofuscus</Emphasis>

ε-poly-l-lysine (ε-PL) is a homo-poly-amino acid of l-lysine which is used as a safe food preservative. The productivity of ε-PL in currently reported wild type strains is low. This study was aimed at finding novel ε-PL producing strains with higher productivity and new fermentative characters. An improved detection method was employed using methylene blue as an ε-PL secretion indicator. 137 strains forming transparent circles were isolated. The best one was identified as Streptomyces griseofuscus according to the morphological characteristics and the comparison of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences. The fermentative behavior of S. griseofuscus was investigated, and the ε-PL production was enhanced to 2.3 g/L in 5-L bioreactor by a pH control strategy. The yield of ε-PL reached 7.5 g/L in the fed-batch process. Compared with the reported wild type strains, S. griseofuscus produced relatively higher amounts of ε-PL, and might be a promising Streptomyces for ε-PL production.     

Identification of aryl-phospho-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidases in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

Four aryl-phospho--d-glucosidases were identified in Bacillus subtilis by using 4-methylumbelliferyl-phospho--d-glucopyranoside as a substrate. Two of these enzymes are the products of the bglA and bglH genes, previously suggested to encode aryl-phospho--d-glucosidases, while the other enzymes are encoded by the yckE and ydhP genes. Together, these four genes account for >99.9% of the glucosidase activity in B. subtilis on aryl-phospho--d-glucosides. yckE was expressed at a low and constant level during growth, sporulation, and spore germination, and was not induced by aryl--d-glucosides. ydhP was also not induced by aryl--d-glucosides. However, while ydhP was expressed at only a very low level in exponential-phase cells and germinating spores, this gene was expressed at a higher levels upon entry into the stationary phase of growth. Strains lacking yckE or ydhP exhibited no defects in growth, sporulation, or spore germination or in growth on aryl--d-glucosides. However, a strain lacking bglA, bglH and yckE grew poorly if at all on aryl--d-glucosides as the sole carbon source.Abbreviations MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--d-glucopyranoside - MUGal 4-Methylumbelliferyl--d-galactopyranoside - MUG-P 4-Methylumbelliferyl--d-glucopyranoside-6-phosphate     

Purification and characterization of a highly thermostable α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Geobacillus caldoxylolyticus</Emphasis> TK4

The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-l-arabinofuranoside, with apparent K _m and V _max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K _m and V _max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.     

Functional and biophysical characterization of a hyperthermostable GH51 α-<Emphasis Type="SmallCaps">l</Emphasis>-arabinofuranosidase from <Emphasis Type="Italic">Thermotoga petrophila</Emphasis>

A hyperthermostable glycoside hydrolase family 51 (GH51) α-l-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/β barrel proteins analogously observed for other GH51 α-l-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.