High-level expression and biochemical characterization of a novel cold-active lipase from <Emphasis Type="Italic">Rhizomucor endophyticus</Emphasis>

Objectives

To identify novel cold-active lipases from fungal sources and improve their production by heterologous expression in Pichia pastoris.

Results

A novel cold-active lipase gene (ReLipB) from Rhizomucor endophyticus was cloned. ReLipB was expressed at a high level in Pichia pastoris using high cell-density fermentation in a 5-l fermentor with the highest lipase activity of 1395 U/ml. The recombinant lipase (RelipB) was purified and biochemically characterized. ReLipB was most active at pH 7.5 and 25 °C. It was stable from pH 4.5–9.0. It exhibited broad substrate specificity towards p-nitrophenyl (pNP) esters (C₂–C₁₆) and triacylglycerols (C₂–C₁₂), showing the highest specific activities towards pNP laurate (231 U/mg) and tricaprylin (1840 U/mg), respectively. In addition, the enzyme displayed excellent stability with high concentrations of organic solvents including cyclohexane, n-hexane, n-heptane, isooctane and petroleum ester and surfactants.

Conclusions

A novel cold-active lipase from Rhizomucor endophyticus was identified, expressed at a high level and biochemically characterized. The high yield and unique enzymatic properties make this lipase of some potential for industrial applications.

     

A glimpse of the diversity of complex polysaccharide-degrading culturable bacteria from Kongsfjorden,Arctic Ocean

Cold-adapted, complex polysaccharide-degrading marine bacteria have important implications in biogeochemical processes and biotechnological applications. Bacteria capable of degrading complex polysaccharide substrates, mainly starch, have been isolated from various cold environments, such as sea ice, glaciers, subglacial lakes, and marine sediments. However, the total diversity of polysaccharide-degrading culturable bacteria in Kongsfjorden, Arctic Ocean, remains unexplored. In the study reported here, we tested 215 cold-adapted heterotrophic bacterial cultures (incubated at 4 and 20 °C, respectively) isolated from Kongsfjorden, for the production of cold-active extracellular polysaccharide-degrading enzymes, including amylase, pectinase, alginase, xylanase, and carboxymethyl (CM)-cellulase. Our results show that 52 and 41% of the bacterial isolates tested positive for extracellular enzyme activities at 4 and 20 °C, respectively. A large fraction of the bacterial isolates (37% of the positive isolates) showed multiple extracellular enzyme activities. Alginase and pectinase were the most predominantly active enzymes, followed by amylase, xylanase, and CM-cellulase. All isolates which tested positive for extracellular enzyme activities were affiliated to microbial class Gammaproteobacteria. The four genera with the highest number of isolates were Pseudomonas, followed by Psychrobacter, Pseudoalteromonas, and Shewanella. The prevalence of complex polysaccharide-degrading enzymes among the isolates indicates the availability of complex polysaccharide substrates in the Kongsfjorden, likely as a result of glacial melting and/or macroalgal load. In addition, the observed high functional/phenotypic diversity in terms of extracellular enzyme activities within the bacterial genera indicates a role in regulating carbon/carbohydrate turnover in the Kongsfjorden, especially by reducing recalcitrance.     

A novel thermostable and organic solvent-tolerant lipase from <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> YB103: screening,purification and characterization

Thermostable lipases offer major biotechnological advantages over mesophilic lipases. In this study, an intracellular thermostable and organic solvent-tolerant lipase-producing strain YB103 was isolated from soil samples and identified taxonomically as Xanthomonas oryzae pv. oryzae. The lipase from X. oryzae pv. oryzae YB103 (LipXO) was purified 101.1-fold to homogeneity with a specific activity of 373.9 U/mg. The purified lipase showed excellent thermostability, exhibiting 51.1 % of its residual activity after incubation for 3 days at 70 °C. The enzyme showed optimal activity at 70 °C, suggesting it is a thermostable lipase. LipXO retained 75.1–154.1 % of its original activity after incubation in 20 % (v/v) hydrophobic organic solvents at 70 °C for 24 h. Furthermore, LipXO displayed excellent stereoselectivity (e.e._p >99 %) toward (S)-1-phenethyl alcohol in n-hexane. These unique properties of LipXO make it promising as a biocatalyst for industrial processes.     

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium <Emphasis Type="Italic">Thalassospira</Emphasis> sp.

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.     

Degradation properties of various macromolecules of cultivable psychrophilic bacteria from the deep-sea water of the South Pacific Gyre

The deep-sea water of the South Pacific Gyre (SPG, 20°S–45°S) is a cold and ultra-oligotrophic environment that is the source of cold-adapted enzymes. However, the characteristic features of psychrophilic enzymes derived from culturable microbes in the SPG remained largely unknown. In this study, the degradation properties of 174 cultures from the deep water of the SPG were used to determine the diversity of cold-adapted enzymes. Thus, the abilities to degrade polysaccharides, proteins, lipids, and DNA at 4, 16, and 28 °C were investigated. Most of the isolates showed one or more extracellular enzyme activities, including amylase, chitinase, cellulase, lipase, lecithinase, caseinase, gelatinase, and DNase at 4, 16, and 28 °C. Moreover, nearly 85.6 % of the isolates produced cold-adapted enzymes at 4 °C. The psychrophilic enzyme-producing isolates distributed primarily in Alteromonas and Pseudoalteromonas genera of the Gammaproteobacteria. Pseudoalteromonas degraded 9 types of macromolecules but not cellulose, Alteromonas secreted 8 enzymes except for cellulase and chitinase. Interestingly, the enzymatic activities of Gammaproteobacteria isolates at 4 °C were higher than those observed at 16 or 28 °C. In addition, we cloned and expressed a gene encoding an α-amylase (Amy2235) from Luteimonas abyssi XH031^T, and examined the properties of the recombinant protein. These cold-active enzymes may have huge potential for academic research and industrial applications. In addition, the capacity of the isolates to degrade various types of organic matter may indicate their unique ecological roles in the elemental biogeochemical cycling of the deep biosphere.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Agarolytic culturable bacteria associated with three antarctic subtidal macroalgae

Bacterial communities of Antarctic marine macroalgae remain largely underexplored in terms of diversity and biotechnological applications. In this study, three Antarctic subtidal macroalgae (Himantothallus grandifolius, Pantoneura plocamioides and Plocamium cartilagineum), two of them endemic of Antarctica, were investigated as a source for isolation of agar-degrading bacteria. A total of 21 epiphytic isolates showed agarolytic activity at low temperature on agar plates containing agar as the sole carbon source. 16S rRNA identification showed that the agar-degrading bacteria belonged to the genera Cellulophaga, Colwellia, Lacinutrix, Olleya, Paraglaciecola, Pseudoalteromonas and Winogradskyella. The agarase enzyme from a potential new species of the genus Olleya was selected for further purification. The enzyme was purified from the culture supernatant of Olleya sp. HG G5.3 by ammonium sulfate precipitation and ion-exchange chromatography. Molecular weight of the agarase was estimated to be 38 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme exhibited activity at 4 °C, retaining >?50% of its maximum activity at this temperature. This is the first study reporting the phylogeny of agar-degrading bacteria isolated from Antarctic subtidal macroalgae and the results suggest the huge potential of Antarctic algae-associated bacteria as a source of cold-active hydrolytic enzymes of biotechnological interest.     

Nitric oxide: a novel inducer for enhancement of microbial lipase production

The purpose of this study was to elucidate whether exogenous nitric oxide (NO) has a potential beneficial effect on lipase production capacity of some microorganisms. Sodium nitroprusside (SNP) was used as an exogenous NO donor in production medium. In comparison with the control (0 nM SNP), SNP concentrations from 10 to 100 nM induced lipase production in mesophilic bacterium Bacillus subtilis and cold-adapted yeast Yarrowia lipolytica. Especially, the maximum lipase activities for Y. lipolytica (81.2 U/L) and B. subtilis (74.5 U/L) were attained at 30 and 50 nM SNP concentrations, respectively. When compared to the control, the optimal SNP concentrations resulted in about 5.14 and 2.27-fold increases in lipase activities of B. subtilis and Y. lipolytica, respectively. Besides, it was found that the optimal SNP concentrations provided shorter incubation periods for lipase production. Conversely, no significant positive effect of exogenous NO on lipase production was determined for thermophilic bacterium Geobacillus stearothermophilus. This study showed for the first time that exogenous NO could be used as an inducer in the production of microbial lipases.     

Synthesis of 4-nitrophenyl acetate using molecular sieve-immobilized lipase from <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

Extracellular lipase from Bacillus coagulans BTS-3 was immobilized on (3 Å × 1.5 mm) molecular sieve. The molecular sieve showed approximately 68.48% binding efficiency for lipase (specific activity 55 IU mg^?1). The immobilized enzyme achieved approx 90% conversion of acetic acid and 4-nitrophenol (100 mM each) into 4-nitrophenyl acetate in n-heptane at 65°C in 3 h. When alkane of C-chain length other than n-heptane was used as the organic solvent, the conversion of 4-nitrophenol and acetic acid was found to decrease. About 88.6% conversion of the reactants into ester was achieved when reactants were used at molar ratio of 1:1. The immobilized lipase brought about conversion of approximately 58% for esterification of 4-nitrophenol and acetic acid into 4-nitrophenyl acetate at a temperature of 65°C after reuse for 5 cycles.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

A new recombinant <Emphasis Type="Italic">endo-</Emphasis>1,3-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from the marine bacterium <Emphasis Type="Italic">Formosa algae</Emphasis> KMM 3553: enzyme characteristics and transglycosylation products analysis

A specific endo-1,3-β-d-glucanase (GFA) gene was found in genome of marine bacterium Formosa algae KMM 3553. For today this is the only characterized endo-1,3-β-d-glucanase (EC 3.2.1.39) in Formosa genus and the only bacterial EC 3.2.1.39 GH16 endo-1,3-β-d-glucanase with described transglycosylation activity. It was expressed in E. coli and isolated in homogeneous state. Investigating the products of polysaccharides digestion with GFA allowed to establish it’s substrate specificity and classify this enzyme as glucan endo-1,3-β-d-glucosidase (EC 3.2.1.39). The amino-acid sequence of GFA consists of 556 residues and shows sequence similarity of 45–85% to β-1,3-glucanases of bacteria belonging to the CAZy 16th structural family of glycoside hydrolases GH16. Enzyme has molecular weight 61 kDa, exhibits maximum of catalytic activity at 45?°C, pH 5.5. Half-life period at 45 °С is 20 min, complete inactivation happens at 55?°C within 10 min. K_m for hydrolysis of laminarin is 0.388 mM. GFA glucanase from marine bacteria F. algae is one of rare enzymes capable to catalyze reactions of transglycosylation. It catalyzed transfer of glyconic part of substrate molecule on methyl-β-d-xylopyranoside, glycerol and methyl-α-d-glucopyranoside. The enzyme can be used in structure determination of β-1,3-glucans (or mixed 1,3;1,4- and 1,3;1,6-β-d-glucans) and enzymatic synthesis of new carbohydrate-containing compounds.     

A cold-adapted tyrosinase with an abnormally high monophenolase/diphenolase activity ratio originating from the marine archaeon <Emphasis Type="Italic">Candidatus Nitrosopumilus koreensis</Emphasis>

Objectives

To obtain an acidic and cold-active tyrosinase, which potentially minimizes unwanted self-oxidation of tyrosinase-catalyzed catechols, including 3,4-dihydroxyphenylalanine at elevated pH and high temperature.

Results

A putative psychrophilic tyrosinase (named as tyrosinase-CNK) was identified from the genome information of the marine archaeon Candidatus Nitrosopumilus koreensis. This protein contains key tyrosinase domains, such as copper-binding domains and an O₂-binding motif, and phylogenetic analysis revealed that it was distinct from other known bacterial tyrosinases. Functional tyrosinase-CNK was produced by applying a co-expression strategy together with chaperone proteins in Escherichia coli with a yield of approx. 30 mg l^?1 and a purity >95 %. The purified enzyme showed optimal activity at pH 6 and 20 °C and still had 50 % activity at 0 °C. Surprisingly, the enzyme exhibited an abnormally high monophenolase/diphenolase activity ratio.

Conclusions

The acidic and cold-adapted tyrosinase-CNK, as a new type of tyrosinase, could expand potential applications of tyrosinases including the production of catechols through minimizing unwanted self-oxidation and the modification of existing materials at low temperature.

     

Cloning,expression and characterization of a novel cold-active and organic solvent-tolerant esterase from <Emphasis Type="Italic">Monascus ruber</Emphasis> M7

Cold active esterases are a class of important biocatalysts that exhibit high activity at low temperatures. In this study, a search for putative cold-active esterase encoding genes from Monascus ruber M7 was performed. A cold-active esterase, named Lip10, was isolated, cloned, purified, and characterized. Amino acid sequence analysis reveals that Lip10 contained a conserved sequence motif Gly¹⁷³-Xaa-Ser¹⁷⁵-Xaa-Gly¹⁷⁷ that is also present in the majority of esterases and lipases. Phylogenetic analysis indicated that Lip10 was a novel microbial esterase. The lip10 gene was cloned and heterologously expressed in Escherichia coli BL21(DE3), resulting in the expression of an active and soluble protein that constituted 40 % of the total cell protein content. Lip10 maintained almost 50 % of its maximal activity at 4–10 °C, with optimal activity at 40 °C. Furthermore, Lip10 retained 184–216 % of its original activity, after incubation in 50 % (v/v) hydrophobic organic solvents for 24 h. The enzyme also exhibited high activity under alkaline conditions and good tolerance to metal ions in the reaction mixture. These results indicate that Lip10 may have potential uses in chemical synthesis and food processing industrial applications as an esterase.     

Characterization of bacteriocins produced by strains of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> isolated from Minas cheese

Interest in obtaining bacteriocin-producing strains of lactic acid bacteria (LAB) from different sources has been increasing in recent years due to their multiple applications in health and food industries. This study focused on the isolation and characterization of metabolically active populations of bacteriocinogenic LAB and the evaluation of their antimicrobial substances as well as of some nutritional requirements of them. One hundred and fifty colonies of LAB from artisanal cheeses produced in Minas Gerais state (Brazil) were isolated and screened for their antimicrobial activity. According to their activity against Listeria monocytogenes, ten strains were selected and subsequently identified using biochemical and molecular techniques including 16s rRNA amplification and sequencing as Enterococcus faecalis, Lactobacillus spp., and Pediococcus pentosaceus. Antimicrobial substances produced by four of the selected strains, P. pentosaceus 63, P. pentosaceus 145, P. pentosaceus 146, and P. pentosaceus 147, were biochemically characterized, and presented sensitivity to proteolytic enzymes (suggesting their proteinaceous nature) and to extreme pH. Antimicrobial activity showed stability after treatment with lipase, catalase, α-amylase, and chemicals. Growth kinetics of the P. pentosaceus selected showed maximal bacteriocin production at 37 °C during the end of the exponential growth phase (25,600 AU/mL) and stable production during 24 h of incubation. Dextrose, maltose, and a mixture of peptone, meat extract, and yeast extract increased bacteriocin production. This study demonstrated that dairy products provide a good alternative for obtaining LAB, with the ability to produce antimicrobial substances such as bacteriocins that have potential use as biopreservatives in food.     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

Cloning,expression, and characterization of a thermostable glucose-6-phosphate dehydrogenase from <Emphasis Type="Italic">Thermoanaerobacter tengcongensis</Emphasis>

Glucose-6-phosphate dehydrogenases (G6PDs) are important enzymes widely used in bioassay and biocatalysis. In this study, we reported the cloning, expression, and enzymatic characterization of G6PDs from the thermophilic bacterium Thermoanaerobacter tengcongensis MB4 (TtG6PD). SDS-PAGE showed that purified recombinant enzyme had an apparent subunit molecular weight of 60 kDa. Kinetics assay indicated that TtG6PD preferred NADP⁺ (k _cat/K _m = 2618 mM^?1 s^?1, k _cat = 249 s^?1, K _m = 0.10 ± 0.01 mM) as cofactor, although NAD⁺ (k _cat/K _m = 138 mM^?1 s^?1, k _cat = 604 s^?1, K _m = 4.37 ± 0.56 mM) could also be accepted. The K _m values of glucose-6-phosphate were 0.27 ± 0.07 mM and 5.08 ± 0.68 mM with NADP⁺ and NAD⁺ as cofactors, respectively. The enzyme displayed its optimum activity at pH 6.8–9.0 for NADP⁺ and at pH 7.0–8.6 for NAD⁺ while the optimal temperature was 80 °C for NADP⁺ and 70 °C for NAD⁺. This was the first observation that the NADP⁺-linked optimal temperature of a dual coenzyme-specific G6PD was higher than the NAD⁺-linked and growth (75 °C) optimal temperature, which suggested G6PD might contribute to the thermal resistance of a bacterium. The potential of TtG6PD to measure the activity of another thermophilic enzyme was demonstrated by the coupled assays for a thermophilic glucokinase.     

Cold stress promoting a psychrotolerant bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fragi</Emphasis> P121 producing trehaloase

A newly isolated Pseudomonas fragi P121 strain in a soil sample taken from the Arctic Circle is able to produce trehalose. The P121 strain was able to grow at temperatures ranging from 4 to 25 °C, had an optimum pH of 6.5, and an optimum salt concentration of 2 %. The P121 strain had a survival rate of 29.1 % after being repeatedly frozen and thawed five times, and a survival rate of 78.9 % when placed in physiological saline for 15 days at 20 °C after cold shock, which is far higher than the type strain Pseudomonas fragi ATCC 4973. The P121 strain could produce 2.89 g/L trehalose, which was 18.6 % of dry cell weight within 52 h in a 25 L fermention tank using the malt extract prepared from barley as medium at 15 °C, while only 11.8 % of dry cell weight at 20 °C. These results suggested that cold stress promoted the strain producing trehalose. It is the first reported cold-tolerant bacterium that produces trehalose, which may protect cells against the cold environment.     

Thermostable phycocyanin from the red microalga <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis>, a new natural blue food colorant

The demand for natural food colorants is growing as consumers question the use of artificial colorants more and more. The phycobiliprotein C-phycocyanin of Arthospira platensis is used as a natural blue colorant in certain food products. The thermoacidophilic red microalga Cyanidioschyzon merolae might provide an alternative source of phycocyanin. Cyanidioschyzon merolae belongs to the order Cyanidiophyceae of the phylum Rhodophyta. Its natural habitat are sulfuric hot springs and geysers found near volcanic areas in, e.g., Yellowstone National Park in the USA and in Java, Indonesia. It grows optimally at a pH between 0.5 and 3.0 and at temperatures up to 56 °C. The low pH at which C. merolae grows minimizes the risk of microbial contamination and could limit production loss. As C. merolae lacks a cell wall, phycocyanin with a high purity number of 9.9 could be extracted by an osmotic shock using a simple ultrapure water extraction followed by centrifugation. The denaturation midpoint at pH 5 was 83 °C, being considerably higher than the A. platensis phycocyanin (65 °C). The C. merolae phycocyanin was relatively stable at pH 4 and 5 up to 80 °C. The high thermostability at slightly acidic pH makes the C. merolae phycocyanin an interesting alternative to A. platensis phycocyanin as a natural blue food colorant.     

Identification of a New 1,4-beta-D-xylosidase <Emphasis Type="Italic">Pae</Emphasis>1263 from the Whole Genome Sequence of <Emphasis Type="Italic">Paenibacillus terrae</Emphasis> HPL-003

The gene of Pae1263 (2,196 bp, 732 aa) was found from the full-length sequence analysis of bacterium Paenibacillus terrae HPL-003 isolated from soil on Gara Mountain in Korea (CP003107, our previous study). Among the 20 open reading frames (ORFs) related with the xylose substrate, only the recombinant enzyme of ORF Pae1263 showed a 1,4-beta-D-xylosidase activity when all of the ORFs were transformed into E. coli. This gene is considered to be a new 1,4-beta-D-xylosidase because it has up to 93% similarity with other genes of ZP_10240221.1 from Lactococcus raffinolactis 4877 and ZP_11237858.1 from Paenibacillus peoriae in the GenBank blast search. The enzyme activity was confirmed by HPLC in which xylose was produced from xylobiose as a substrate by this recombinant enzyme. Mass production of the recombinant enzyme was done with the construction of the pET22(+)- Pae1263-6H expression vector system from E. coli. This new 1,4-beta-D-xylosidase was highly active at 50°C in a pH range between 6.0 and 8.0 and had thermo-stability for at least 24 h at 50°C and a K _m and V _max of 6.42 mg/mL and 75.76 U/mg on a xylobiose substrate, respectively.