Expression, purification, and characterization of the maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

The maltooligosyltrehalose trehalohydrolase (MTHase) mainly cleaves the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose to produce trehalose and the maltooligosaccharide with lower molecular mass. In this study, the treZ gene encoding MTHase was PCR-cloned from Sulfolobus solfataricus ATCC 35092 and then expressed in Escherichia coli. A high yield of the active wild-type MTHase, 13300 units/g of wet cells, was obtained in the absence of IPTG induction. Wild-type MTHase was purified sequentially using heat treatment, nucleic acid precipitation, and ion-exchange chromatography. The purified wild-type MTHase showed an apparent optimal pH of 5 and an optimal temperature at 85 degrees C. The enzyme was stable at pH values ranging from 3.5 to 11, and the activity was fully retained after a 2-h incubation at 45-85 degrees C. The k(cat) values of the enzyme for hydrolysis of maltooligosyltrehaloses with degree of polymerization (DP) 4-7 were 193, 1030, 1190, and 1230 s(-1), respectively, whereas the k(cat) values for glucose formation during hydrolysis of DP 4-7 maltooligosaccharides were 5.49, 17.7, 18.2, and 6.01 s(-1), respectively. The K(M) values of the enzyme for hydrolysis of DP 4-7 maltooligosyltrehaloses and those for maltooligosaccharides are similar at the same corresponding DPs. These results suggest that this MTHase could be used to produce trehalose at high temperatures.     

Identification of the essential catalytic residues and selectivity-related residues of maltooligosyltrehalose trehalohydrolase from the thermophilic archaeon Sulfolobus solfataricus ATCC 35092

Maltooligosyltrehalose trehalohydrolase (MTHase) catalyzes the release of trehalose by cleaving the alpha-1,4-glucosidic linkage next to the alpha-1,1-linked terminal disaccharide of maltooligosyltrehalose. Mutations at residues D255, E286, and D380 were constructed to identify the essential catalytic residues of MTHase, while mutations at residues W218, A259, Y328, F355, and R356 were constructed to identify selectivity-related residues of the enzyme. The specific activities of the purified D255A, E286A, and D380A MTHases were only 0.15, 0.09 and 0.01%, respectively, of that of wild-type MTHase, suggesting that these three residues are essential catalytic residues. Compared with wild-type MTHase, A259S, Y328F, F355Y, and R356K MTHases had increased selectivity ratios, which were defined as the ratios of the catalytic efficiencies for glucose formation to those for trehalose formation in the hydrolysis of maltooligosaccharides and maltooligosyltrehaloses, respectively, while W218A and W218F MTHases had decreased selectivity ratios. When starch digestion was carried out at 75 degrees C and wild-type and mutant MTHases were, respectively, used with isoamylase and maltooligosyltrehalose synthase (MTSase), the ratios of initial rates of glucose formation to those of trehalose formation were inversely correlated to the peak trehalose yields.     

Enzymatic Synthesis of Piceid Glucosides Using Maltosyltransferase from Caldicellulosiruptor bescii DSM 6725

Piceid is widely used in food, cosmetics, and pharmaceuticals because of its therapeutic benefits. However, the use of piceid as a drug is limited because of its low solubility. To increase solubility, we synthesized piceid glucosides using maltosyltransferase from Caldicellulosiruptor bescii . The MTase gene was cloned and expressed in Escherichia coli . The enzyme had a unique transfer specificity to the transfer of maltosyl units. Four piceid transglycosylation products were present and identified by thin-layer chromatography and recycling preparative high-performance liquid chromatography. The major product was purified by C(18) and gel filtration chromatography, and its molecular structure was determined using nuclear magnetic resonance spectroscopy to be α-d-maltosyl-(1→4)-piceid. The solubility of maltosyl piceid was 8.54 × 10(3) and 1.86 × 10(3) times those of natural resveratrol and piceid, respectively, suggesting that the transglycosylation greatly increased the water solubility. This suggests that dietary intake of this compound can enhance the bioavailability of resveratrol in the human body.     

Characterization of new soil thermophilic bacteria potentially involved in soil fertilization

Thermophilic bacteria were isolated from a soil of an olive grove in Alentejo (Portugal) and characterized morphologically, physiologically, and at the molecular level by partial sequencing of the 16S ribosomal RNA genes, followed by subsequent phylogenetic analysis. The isolates were shown to be gram‐positive rods, motile, and to belong to the Firmicutes Phylum. They were able to produce ammonium and sulfate during growth, the levels of which vary among the bacterial isolates. This ability suggests that these bacteria may have an important role in the mobilization of organic S and N in the soil and therefore in providing essential nutrients for plant growth. Tests using two isolates indicated a positive effect on initial seedling development suggesting their potential use in soil nutrient supplementation. The presence of these thermophiles in arable temperate soils might be increasingly important, particularly when the predicted global climate warming is considered, and their features are discussed in this context and considering actual strategies to improve soil fertilization.     

一株产高温蛋白酶耐热菌BY25的产酶条件与酶学性质研究

对菌株BY25的生长条件、产酶条件及其产生的蛋白酶的酶学性质进行了研究。结果发现,BY25的最高生长温度为55℃,最适生长温度为50℃,最佳产酶温度为30℃,最佳培养起始pH为8.0,最佳C源为葡萄糖,高通气量明显提高菌株产酶能力。在以上条件下培养52 h,上清液蛋白酶活力达4 170 U/ml。酶学性质研究表明：该蛋白酶为高温中性金属蛋白酶,最适反应pH为7.0,最适反应温度为55℃,具有良好的pH耐受性和较好的热稳定性;EDTA能强烈抑制酶活力,而Fe2+、Cu2+对酶活力也具有一定抑制作用。     

Characterization of a metalloproteinase component extracted from soil

Summary A metalloproteinase was found to be the main component of a protease in the extract from an Andosol collected from a tomato field. The protease has a pH optimum of 7 for benzyloxycarbonyl-L-phenylalanyl-L-tyrosyl-L-leucine with tyrosylleucine as the main reaction product. The K_m value for the substrate was 0.4 mM. Activity was inhibited by EDTA but not by pepstatin, p-chloromercuribenzoate and phenylmethanesulfonyl fluoride. After the removal of EDTA from the inactivated enzyme by dialysis and the addition of metal ions (Zn²⁺, Mn²⁺ and Fe³⁺), the enzyme activity could be recovered. The apparent isoelectric points of the metalloproteinase components were estimated to be 4.9, 4.5 and 4.1 by isoelectric focusing. A fraction with an apparent isoelectric point of 4.9 was the main component. The apparent molecular weight of the main protease component was estimated to be 4.7 × 10⁴ by gel filtration of Sephadex G-100. The enzyme hydrolyzed a natural polypeptide, angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). Main split sites in the peptide were -Tyr⁷-Ile⁵- and -Pro⁷-Phe⁸-. The former was the most sensitive site to the soil metalloproteinase concerned.     

Characterization of Ash-Basin Waters from a Risk-Based Perspective

The purpose of this investigation was to characterize waters in ash basins of coal-fired thermoelectric power plants and to identify constituents of concern from an environmental risk-based perspective. The investigation incorporated results from literature review and from laboratory analysis of ash-basin waters. General chemistry and concentrations of major and trace elements were investigated. Risk was assessed for aquatic life by a risk quotient approach using environmental exposure and toxic effects concentrations. The following constituents of concern in ash-basin waters were identified by the risk quotient method: aluminum, cadmium, chromium, copper, iron, lead, mercury, selenium, and zinc. In addition, pH was considered a constituent of concern because the observed pH range for ash-basin waters exceeded the range indicating reproductive impairment or mortality for sentinel species used for toxicity evaluation. The results of this investigation can be used to assess potential effects (reasonable potential analysis) for receiving system aquatic biota as a consequence of exposure to ash-basin effluent and applied to developing remediation strategies. The constituents of concern are readily treatable, and ash-basin waters can be potentially reused within a power plant or for other purposes (e.g., irrigation or stream flow augmentation).     

Characterization of mannanase from a novel mannanase-producing bacterium

Locust bean gum (LBG) was employed to screen mannanase-producing bacteria. The bacterium with highest mannanase ability was identified as Paenibacillus cookii. It revealed highest activity (6.67 U/mL) when cultivated in 0.1% LBG with 1.5% soytone and 0.5% tryptone after 4 days incubation at 27 °C. Its mannanase was purified to electrophoretical homogeneity after DEAE-Sepharose and Sephacryl S-100 separation. The purified mannanase, with an N-terminus of GLFGINAY, had pH and temperature optimum at 5.0 and 50 °C, respectively, and was stable at pH 5.0-7.0, ≤ 50 °C. It was strongly activated by β-mercaptoethanol, dithiothreitol, cysteine, and glutathione, but inhibited by Hg(2+), Cu(2+), Zn(2+), Fe(3+), PMSF, iodoacetic acid, and EDTA. According to substrate specificity study, the purified mannanase had high specificity to LBG and konjac.     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Isolation and characterization of novel thermophilic nitrifying Bacillus sp. from compost

A thermophilic nitrifying bacterium, strain T3, was isolated from compost made of animal waste by using a novel selective medium. Strain T3 was classified into the genus Bacillus, close to Bacillus halodurans, but identified as a novel species. To evaluate the effect of adding strain T3 on ammonia emission during the process of composting animal waste, laboratory scale composting was done. Ammonia emission was lower when strain T3 was added than in the control material to which strain T3 was not added. Thermophilic nitrifying bacteria in the strain T3-containing material increased from 6.24 (log value) to 7.55 (log value) on average during the tests. These results suggested the possibility of reducing ammonia emission from composting of animal waste by adding strain T3.     

Characterization of organic phosphorus in leachate from a grassland soil

The degree of eutrophication in fresh water ecosystems may be influenced by the forms of phosphorus (P) leached from agricultural systems. Physico-chemical fractionation of P in leachate from a grassland soil carried out over a two year period indicated that the majority of the P loss from the Lismore soil occurred in unreactive particulate (55-76%) P forms. ³¹P nuclear magnetic resonance analysis of a selected leachate sample indicated that unreactive P was mainly comprised of monoester and diester forms of organic P. The presence of phosphomonoesterase (20-200 μg p nitrophenol l⁻¹ h⁻¹) and phosphodiesterase (68 μg bis-p nitrophenol l⁻¹ h⁻¹) activity in leachate resulted in hydrolysis of 10-21% of total unreactive P (TUP), indicating that some of the monoesters and diesters can be eventually hydrolyzed into inorganic P forms during P transport. Enzyme hydrolysis showed that 23% of the TUP was present as labile monoester P (LMP), followed by 20% as inositol hexakisphosphate (IHP) and 14% as diesters (phospholipids and nucleic acids). The findings of this study suggest that LMP, IHP and diesters are an important component of organic P leaching from the grassland soil.     

Characterization of a salt-independent pectin methylesterase purified from valencia orange peel

The pectin methylesterase (PME; EC 3.1.1.11) present in a commercial orange peel enzyme preparation was characterized to establish its identity among the multiple PME isozymes present in Valencia orange (Citrus sinensis L.) peel. We show the commercial enzyme corresponds to the major peak 2 PME previously separated by heparin-Sepharose chromatography (Cameron et al., J. Food Sci. 1998, 63, 253). Both PMEs have comparable elution profiles on cation-exchange and hydrophobic-interaction perfusion chromatography columns, molecular weights (ca. 34 kDa) and pI (pH 9.2), and biochemical properties, including a broad pH activity range and activity in the absence of added cations. An identical partial amino terminal peptide sequence was also obtained for the PMEs, which further demonstrated a structural identity with other plant PMEs. The biochemical and structural properties readily distinguish this Valencia orange PME from salt-dependent isozymes and further suggest that it is an ortholog to the salt-independent fruit-specific isozyme of tomato. This work provides a well-defined, enzymatically homogeneous, salt-independent (type 1) plant PME isozyme that is suitable for studying details of the enzyme's mode of action and for use in modifying methylester patterns for studying the structure-functional property relationships in pectin.     

Characterization of a benzyl alcohol dehydrogenase from Lactobacillus plantarum WCFS1

Aroma is an important sensory parameter of food products. Lactic acid bacteria have enzymatic activities that could be important in the modification of food aroma. The complete genome sequence from Lactobacillus plantarum WCFS1 shows a gene (lp_3054) putatively encoding a protein with benzyl alcohol dehydrogenase activity. To confirm its enzymatic activity lp_3054 from this strain has been overexpressed and purified. Protein alignment indicated that lp_3054 is a member of the family of NAD(P)-dependent long-chain zinc-dependent alcohol dehydrogenases. In lp_3054 all of the residues involved in zinc and cofactor binding are conserved. It is also conserved the residue that determines the specificity of the dehydrogenase toward NAD (+) rather than NADP (+) and, therefore, L. plantarum benzyl alcohol dehydrogenase is less active in the presence of NADP (+) than in the presence of NAD (+). The purified enzyme exhibits optimal activity at pH 5.0 and 30 degrees C. The kinetic parameters K m and V max on benzyl alcohol as a substrate were, respectively, 0.23 mM and 204 mumol h (-1) mg (-1). Besides its activity toward benzyl alcohol, it showed activity against nerol, geraniol, phenethyl alcohol, cinnamyl alcohol, and coniferyl alcohol, all of which are volatile compounds involved in determining food aroma. The biochemical demonstration of a functional benzyl alcohol dehydrogenase activity in this lactic acid bacteria species should be considered when the influence of bacterial metabolism in the aroma of food products is determined.     

Characterization of the chemical composition of a byproduct from siam benzoin gum

The mixture of bark and gum obtained after size-grading of Siam benzoin gum was studied to establish its potential application as a valuable new grade of the balsamic resin. An analysis of its volatile constituents by means of static headspace and SPME led to the identification of 26 and 50 compounds, respectively. Significant differences were observed in both the headspace composition and olfactory properties of the byproduct as compared to those of Siam benzoin gum. This prompted the further analysis of its volatile extract and its resinoid by GC techniques, resulting in the identification of 60 (99.5%) and 16 (89.1%) components, respectively. To examine the influence of bark pieces, different extracts obtained from the raw material and from a sorted sample were analyzed by GC and HPLC techniques. The chemical compositions and the yields determined for the two resinoids lead to the conclusion that this harvesting byproduct is a new grade of Siam benzoin gum, providing interesting olfactory notes that differ from those of other grades.     

病死猪辅热好氧发酵尾气中的恶臭物质分析

为了明确病死猪辅热好氧发酵过程中产生的恶臭气体种类及其排放规律,为控制恶臭气体排放浓度、降低病死猪无害化处理过程对环境的污染提供基础依据,该研究以病死猪为发酵原料,以玉米秸秆为辅料,开展病死猪辅热好氧发酵试验,发酵过程中,采集处理槽排放的尾气,分析尾气中的有机恶臭物质组分并测定其排放浓度,同时测定其中的氨气浓度,并对不...     

Supercritical carbon dioxide micronization of zeaxanthin from moderately thermophilic bacteria Muricauda lutaonensis CC-HSB-11T

Moderately thermophilic bacterial strain CC-HSB-11(T) (Muricauda lutaonensis), which was described recently from a coastal hot spring of Green Island, Taiwan, has been identified to produce zeaxanthin as a predominant xanthophyll by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cell culture in bioreactor produced 3.12 ± 0.18 mg zeaxanthin L(-1) of culture. Micronization of zeaxanthin was achieved through supercritical carbon dioxide antisolvent precipitation method. Yield of zeaxanthin after the process was 53.4%. Dynamic light scattering assay determined the polydisperse existence of micronized particles of size 3 nm to 2 μm. Field emission scanning electron microscopy revealed distinct morphology and size distribution heterogeneity of particles. Integrity of zeaxanthin after the antisolvent process was assessed by LC-MS/MS. The technique capitalizes on the inherent ability of CC-HSB-11(T) to synthesize zeaxanthin and the work demonstrated feasibility of antisolvent precipitation method to produce microparticles exploiting a bacterial strain.     

Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 degrees C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were approximately 40 kDa, as estimated by both gel filtration and SDS-PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N",N' "-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68-88% deacetylated.     

Characterization of humus-protease complexes extracted from soil

Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A_I with nominal molecular weight (nmw) > 100 kD, A_II with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-l-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-l-phenylalanyl l-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A_II > A_I. The lowest amino acid N/organic C was found in the R fraction, whereas A_II fraction was rich in humic acids, carbohydrates and amino acid N and A_I fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A_I, A_II and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity.     

Characterization of alcohol acyltransferase from olive fruit

Alcohol acyltransferase catalyzes the esterification of volatile alcohols with acyl-CoA derivatives to produce volatile esters typically present in the aroma of some fruits. This enzyme was detected in extracts from the pericarp tissues of ripe olive fruits using hexanol and acetyl-CoA as the substrates. Alcohol acyltransferase showed a very low activity level in these fruits, with an optimum pH value at 7.5 and high K(m) values for hexanol and acetyl-CoA. The substrate specificity of this enzyme for various alcohols was also studied. The involvement of the studied enzyme in the biogenesis of the volatile esters present in the aroma of virgin olive oil was discussed.