Characterization of a bifunctional alginate lyase as a new member of the polysaccharide lyase family 17 from a marine strain BP-2.

OBJECTIVES: Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate. RESULTS: A new BP-2 strain that produces alginate lyase was screened and identified from rotted Sargassum. A new alginate lyase, Alg17B, belonging to the polysaccharide lyase family 17, was isolated and purified from BP-2 fermentation broth by freeze-drying, dialysis, and ion exchange chromatography. The enzymatic properties of the purified lyase were investigated. The molecular weight of Alg17B was approximately 77 kDa, its optimum reaction temperature was 40-45 °C, and its optimum reaction pH was 7.5-8.0. The enzyme was relatively stable at pH 7.0-8.0, with a temperature range of 25-35 °C, and the specific activity of the purified enzyme reached 4036 U/mg. A low Na+ concentration stimulated Alg17B enzyme activity, but Ca2+, Zn2+, and other metal ions inhibited it. Substrate specificity analysis, thin-layer chromatography, and mass spectrometry showed that Alg17B is an alginate lyase that catalyses the hydrolysis of sodium alginate, polymannuronic acid (polyM) and polyguluronic acid to produce monosaccharides and low molecular weight oligosaccharides. Alg17B is also bifunctional, exhibiting both endolytic and exolytic activities toward alginate, and has a wide substrate utilization range with a preference for polyM. CONCLUSIONS: Alg17B can be used to saccharify the main carbohydrate, alginate, in the ethanolic production of brown algae fuel as well as in preparing and researching oligosaccharides.

Cloning and expression of a thermostable keratinase gene from Thermoactinomyces sp. YT06 in Escherichia coli and characterization of purified recombinant enzymes.

The feather-degrading strain Thermoactinomyces sp. YT06 secretes an extracellular keratinolytic protease (KERTYT); however, the gene encoding this protease remains unknown. The kerT1 gene (1170 bp) encoding keratinase was cloned and expressed in Escherichia coli BL21(DE3). Purified recombinant keratinase (rKERTYT) was achieved at a yield of 39.16% and 65.27-fold purification with a specific activity of 1325 U/mg. It was shown that rKERTYT has many similarities to the native enzyme (KERTYT) by characterization of rKERTYT. The molecular weight of rKERTYT secreted by recombinant E. coli was approximately 28 kDa. The optimal temperature and the pH values of rKERTYT were 65 °C and 8.5, respectively, and the protein remained stable from 50 to 60 °C and pH 6-11. The keratinase was strongly inhibited by phenyl methane sulfonyl fluoride (PMSF), suggesting that it belongs to the serine protease family. It was significantly activated by Mn2+ and ß-mercaptoethanol (ß-Me). rKERTYT showed stability and retained over 80% activity with the existence of organic solvents such as acetone, methylbenzene and dimethyl sulfoxide. These findings indicated that rKERTYT will be a promising candidate for the enzymatic processing of keratinous wastes.

Biochemical characterization and mutational studies of the 8-oxoguanine DNA glycosylase from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans.

8-oxoguanine (GO) is a major lesion found in DNA that arises from guanine oxidation. The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes an archaeal GO DNA glycosylase (Tg-AGOG). Here, we characterized biochemically Tg-AGOG and probed its GO removal mechanism by mutational studies. Tg-AGOG can remove GO from DNA at high temperature through a ß-elimination reaction. The enzyme displays an optimal temperature, ca.85-95 °C, and an optimal pH, ca.7.0-8.5. In addition, Tg-AGOG activity is independent on a divalent metal ion. However, both Co2+ and Cu2+ inhibit its activity. The enzyme activity is also inhibited by NaCl. Furthermore, Tg-AGOG specifically cleaves GO-containing dsDNA in the order: GO:C, GO:T, GO:A, and GO:G. Moreover, the temperature dependence of cleavage rates of the enzyme was determined, and from this, the activation energy for GO removal from DNA was first estimated to be 16.9 ± 0.9 kcal/mol. In comparison with the wild-type Tg-AGOG, the R197A mutant has a reduced cleavage activity for GO-containing DNA, whereas both the P193A and F167A mutants exhibit similar cleavage activities for GO-containing DNA. While the mutations of P193 and F167 to Ala lead to increased binding, the mutation of R197 to Ala had no significant effect on binding. These observations suggest that residue R197 is involved in catalysis, and residues P193 and F167 are flexible for conformational change.

Purification, characterization, and functional groups of an extracellular aflatoxin M1 -detoxifizyme from Bacillus pumilus E-1-1-1.

The experiment was conducted to purify high activity extracellular enzymes, which were produced by a strain that we previously screened was able to degrade aflatoxin effectively, and speculate the functional groups of the enzyme associated with degradation. An extracellular aflatoxin-detoxifizyme (DAFE) from Bacillus pumilus E-1-1-1 was purified through a process including ammonium sulfate precipitation, ultrafiltration, Sephadex chromatography, and ion exchange chromatography. The molecular mass of the enzyme assessed by SDS-PAGE was found to be approximately 58 kDa. The optimum reaction temperature and pH for the purified enzyme were 45°C and pH 7, respectively. The enzyme showed temperature stability of up to 60°C. Ba2+ , Ca2+ Na+ , Mn2+ , EDTA, and ß-mercaptoethanol showed inhibitory effects on the enzyme activity. Mg2+ , Fe3+ , Zn2+ and K+ were the activators of enzymes. This enzyme was composed of at least 15 kinds of amino acids. Lysine, tryptophan, and histidine residues were necessary and major functional groups to maintain enzyme activity, disulfide bonds were observed, serine residues had little effect on the enzyme activity, so it was not the necessary group to reflect the enzyme activity, and arginine had no effect on enzyme activity.

Identification of potential AMPK activator by pharmacophore modeling, molecular docking and QSAR study.

AMP-activated protein kinase (AMPK) plays a major role in maintaining cellular energy homeostasis by sensing and responding to AMP/ADP concentrations relative to ATP. AMPK has attracted widespread attention as a potential therapeutic target for metabolic diseases such as cancer and cardiovascular diseases. The structure-based 3D pharmacophore model was developed based on the training set. The best pharmacophore model Hypo5 was proposed and validated using a decoy set, an external test set. Hypo5, with the correlation coefficient value of 0.936, cost difference value of 112.08 and low RMS value of 1.63, includes a ionizable positive, a hydrogen bond donor, a hydrogen bond acceptor and two hydrophobic features, which showed a high goodness of fit and enrichment factor. Thus it was used as a 3D query to find potential activator from the SPECS Database. Then the ADMET descriptors were used to filter all of 158 screening molecules. The 41 filtering compounds were subsequently subjected to molecular docking and Quantitative structure-activity relationship (QSAR) analysis. Finally, the compound H2 was picked out from those filtering compounds based on the receptor-ligand interaction analysis and the prediction of the QSAR models. And then it was submitted for molecular dynamics (MD) simulations to explore the stability of complex. The result indicates that the candidate could be considered a potential AMPK activator.

Transcriptional analysis of the laccase-like gene from Burkholderia cepacia BNS and expression in Escherichia coli.

Bacterial laccases have received considerable attention because of several advantages associated with the higher environmental stability of these enzymes compared with fungal laccases. In this study, a laccase-like gene from Burkholderia cepacia BNS was successfully cloned. This gene was found to encode a mature protein of 279 amino acids that exhibited laccase activity in dimer form. The mature protein was found to contain approximately 4 mol of copper per monomer, and the metal ion-binding sites were predicted. BC_lacL gene transcription levels were analyzed by qRT-PCR to study expression patterns in the presence of different putative inducers (copper ions, guaiacol, veratryl alcohol, vanillin, coniferaldehyde, p-coumaric acid, sinapic acid, and ferulic acid). Copper ions had a positive effect on both transcription levels and intracellular laccase activity. Interestingly, upon induction with sinapic acid, BC_lacL gene transcription was lower than in the presence of copper ions, but laccase activity was highest under these conditions. The BC_lacL protein expressed in Escherichia coli exhibited a specific activity of 7.81 U/mg with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate and 12.3 U/mg with 2,6-dimethoxyphenol (2,6-DMP) as the substrate after purification through Ni-affinity chromatography. The optimal activity and kinetic parameters of the recombinant BC_lacL protein were observed (kcat/Km = 3.96 s-1 µM-1) at a pH of 4.0 at 55 °C for ABTS oxidization and (kcat/Km = 11.6 s-1 µM-1) at a pH of 10.0 at 75 °C for 2,6-DMP oxidization. The protein exhibited high stability in an alkaline environment, with a half-life of more than 12 h. The same results were obtained via decolorization of eight dyes. Hence, this laccase-like enzyme may have potential industrial applications.

Extracellular lipase from Pseudomonas aeruginosa JCM5962(T): Isolation, identification, and characterization.

The study was done to isolate, identify, and characterize a good lipolytic strain from soil. Lipolytic strain isolation was done using tributyrin agar medium. The biochemical testing and 16S rRNA gene sequencing analysis was done for identification. The enzyme was purified using ammonium sulfate precipitation and column chromatography. Results have shown a novel high lipolytic strain of P. aeruginosa JCM5962(T), isolated from soil of sugarcane field. The 16S rRNA sequence analysis confirmed the strain as P. aeruginosa JCM5962(T); further, the sequence was submitted to Genbank (KX946966.1). The isolate produced an extracellular lipase which was purified as single band of 31 kDa. Maximum lipase activity was observed at 50 °C and pH 8.0. Activity was enhanced in the presence of cobalt and benzene solvent, whereas mercury, sodium dodecyl sulfate, and chloroform inhibited it. The enzyme's marked stability and activity at high temperature, alkaline pH and organic solvents suggest that this can be effectively used in a variety of applications in industries and as biotechnological tools.

Expression, purification and characterization of two leucine aminopeptidases of the blood fluke, Schistosoma mansoni.

Schistosomiasis is a major neglected tropical disease (NTD) and considered the most important of the human helminthiases in terms of morbidity and mortality. Whereas treatment with praziquantel has been effective since the 1980s, the potential for the emergence of drug resistance has propelled the search for new interventions. Studies have revealed key roles of proteases in parasitic helminths during establishment of infection, tissue invasion, immune evasion, parasite feeding and development throughout the different developmental stages, pinpointing them as possible candidates. The leucine aminopeptidases (LAPs), members of the M17 family of Zn-metalloproteases, preferentially cleave leucine (Leu) residues at the N-terminal end of proteins and short peptides. These enzymes display broad proteolytic activities beyond Leu hydrolysis and are involved in processing, maturation, activation and/or degradation of substrates. As a vaccine immunogen, LAP induces protection against infection with the liver fluke Fasciola hepatica. Herein, two LAPs, SmLAP1 (Smp_030000) and SmLAP2 (Smp_083870) of the human blood fluke Schistosoma mansoni were cloned, expressed, purified and biochemically characterized. The enzymes differed in activity against diagnostic substrates, including leucine, methionine and arginine, with an optimal pH of 8.0. The activity increased in the presence of Mg+2 and Mn+2, and was inhibited by bestatin, a specific inhibitor of aminopeptidase. In addition, 1,10-phenanthroline and EDTA inhibited the enzymatic activity of SmLAP2. Finally, immunolocalization using antibodies specific for SmLAP1 and SmLAP2 identified the expression of these proteases in the egg and adult developmental stages of S. mansoni, and in intestinal epithelia, vitelline cells and sub-tegumental regions of the parasite. Characterization of schistosome proteases not only enhances understanding of the biology of schistosomes and schistosomiasis, but may also provide novel intervention approaches.

Polyphenol oxidase from Pectobacterium atrosepticum: identification and cloning of gene and characteristics of the enzyme.

In the present study, we attempted to elucidate if the harmful phytopathogenic bacteria of Pectobacterium genus (P. atrosepticum) possess the enzymes for oxidation of phenolic compounds. Polyphenol oxidase (laccase) activity was revealed in P. atrosepticum cell lysates. Using bioinformatic analysis, an ORF encoding a putative copper-containing polyphenol oxidase of 241 amino acids with a predicted molecular mass of 25.9 kDa was found. This protein (named Pal1) shares significant level of identity with laccases of a new type described for several bacterial species. Cloning and expression of the pal1 gene and the analysis of corresponding recombinant protein confirmed that Pal1 possessed laccase activity. The recombinant Pal1 protein was characterized in terms of substrate specificity, kinetic parameters, pH and temperature optimum, sensitivity to inhibitors and metal content. Pal1 demonstrated alkali- and thermo-tolerance. The kinetic parameters Km and kcat for 2,6-dimethoxyphenol were 0.353 ± 0.062 mM and 98.79 ± 4.9 s-1 , respectively. The protein displayed high tolerance to sodium azide, sodium fluoride, NaCl, SDS and cinnamic acid. The transcript level of the pal1 gene in P. atrosepticum was shown to be induced by plant-derived phenolic compound (ferulic acid) and copper sulfate.

Compounds that enhance the tailing activity of Moloney murine leukemia virus reverse transcriptase.

In a previous study, we showed that MMLV-RT has a strong terminal transferase activity, and that the C-, G-, and T-tailing activities are enhanced by dGMP, dCMP, and dAMP, respectively. In this study, to achieve faster reaction and higher tailing efficiency, we screened other compounds for the ability to enhance the tailing activities of MMLV-RT, and determined the corresponding optimal concentrations. The C-, G-, and T-tailing activities were enhanced by guanine, cytosine, and adenine, respectively, and by derivatives thereof, suggesting a transient Watson-Click base pairing between an enhancer molecule and the nucleotide to be incorporated. In the presence of some additives (GMP and GDP for C-tailing and CMP for G-tailing), the tail length increased continuously, resulting in tail lengths of 7 to 15 (GMP and GDP) or 13 to 22 (CMP) nucleotides. Among the compounds that do not induce continuous addition, adenosine, deoxycytidine, and deoxyguanosine mostly enhanced T-, G-, and C-tailings, respectively. The enhancing chemicals described here will improve the feasibility of N-tailing by MMLV-RT in various biotechnological applications.

Chilo iridescent virus (CIV) ORF 012L encodes a protein with both exonuclease and endonuclease functions.

Chilo iridescent virus (CIV) is the type member of the genus Iridovirus within the family Iridoviridae. The virions of CIV contain a single linear dsDNA molecule that is circularly permuted and terminally redundant. The genome of CIV contains an open reading frame (ORF 012L) encoding a protein homologous to exonuclease II of Schizosaccharomyces pombe. In this study, we focused on the characterization of CIV ORF 012L. The target ORF was cloned into the pET28a vector, expressed in E. coli strain BL21 (DE3) pLysS with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified CIV 012L confirmed that this viral protein is a functional 5'-3' exonuclease that digests 3'-biotin-labelled oligonucleotides and linear double-stranded DNA (dsDNA) molecules from their 5' termini in a highly processive manner. CIV 012L also has a potent endonuclease activity on dsDNA in vitro. In addition, CIV 012L converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) and then open circular form into linear form (RFIII). Endonuclease activity of CIV 012L was optimal in the presence of 10 mM Mg(2+) or 30 mM Mn(2+) ions and at 150 mM NaCl or KCl salt concentrations. The highest endonuclease activity was obtained at pH 8, and it reached a maximum at 55 °C. The CIV 012L protein showed deficiencies for both double- and single-stranded RNAs.

Identification and characterization of a novel (+)-γ-lactamase from Microbacterium hydrocarbonoxydans.

2-Azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) is an important precursor of many carbocyclic nucleoside analogs and pharmaceuticals. (-)-γ-Lactam has attracted much attention because of its role as an intermediate of antiviral drugs such as abacavir and carbovir. (+)-γ-Lactamase can be used for the kinetic resolution of γ-lactam to obtain (-)-γ-lactam. In this study, a novel (+)-γ-lactamase (Mh33H4-5540) was discovered from the gene library of Microbacterium hydrocarbonoxydans based on a colorimetric high-throughput screening method and it could be used to enantioselectively catalyze the bioresolution of racemic γ-lactam with high enantiomeric excess (ee) (>99 %) and yield (>49 %). An unexpected finding was that Mh33H4-5540 was unrelated to other known γ-lactamases (5.7, 4.8, 7.2, and 5.4 % similarities in amino sequence with (+)-γ-lactamase from Comamonas acidovorans, Bradyrhizobium japonicum, Aeropyrum pernix, and Sulfolobus solfataricus, respectively) but rather related to isochorismatases. The homolog analysis of Mh33H4-5540 revealed that it was similar in structure with bacterial isochorismatases (an isochorismatase from Pseudomonas putida (PDB number 4H17) and a putative isochorismatase from Oleispira antarctica (PDB number 3LQY)). Thus, Mh33H4-5540 represented another type of (+)-γ-lactamase. Mh33H4-5540 was overexpressed in E. coli Rosetta (DE3), purified to homogeneity and functionally characterized. The enzyme displayed optimal activity at 25 °C and pH 8.0. The activity showed a 5.5-fold increase in the presence of 0.5 M Ni2+ or Co2+. Mh33H4-5540 displayed much higher (+)-γ-lactamase activity than any other biochemically characterized (+)-γ-lactamases. Overall, we discovered a novel (+)-γ-lactamase Mh33H4-5540 which displayed the highest activity. It could be a promising candidate of biocatalyst for industrial applications of highly valuable chiral pharmaceutical chemicals.

The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation.

Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2 (45 %), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2 mg mL-1) and V max of 17.4 µmol min-1 mg-1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.

Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10

Abstract Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30 °C for 96 h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3 kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50 °C, and substrate concentration of 1.5%. The enzyme was thermostable at 60 °C for 1 h, and the optimum enzyme–substrate reaction time was 30 min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30 °C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn2+, followed by Mg2+ and Fe2+. Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1 mM) and the reducing, β-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10.

Enzyme production by Aspergillus terreus NCFT 4269.10 was studied under liquid static surface and solid-state fermentation using mustard oil cake as a substrate. The maximum lipase biosynthesis was observed after incubation at 30°C for 96h. Among the domestic oils tested, the maximum lipase biosynthesis was achieved using palm oil. The crude lipase was purified 2.56-fold to electrophoretic homogeneity, with a yield of 8.44%, and the protein had a molecular weight of 46.3kDa as determined by SDS-PAGE. Enzyme characterization confirmed that the purified lipase was most active at pH 6.0, temperature of 50°C, and substrate concentration of 1.5%. The enzyme was thermostable at 60°C for 1h, and the optimum enzyme-substrate reaction time was 30min. Sodium dodecyl sulfate and commercial detergents did not significantly affect lipase activity during 30-min incubation at 30°C. Among the metal ions tested, the maximum lipase activity was attained in the presence of Zn(2+), followed by Mg(2+) and Fe(2+). Lipase activity was not significantly affected in the presence of ethylenediaminetetraacetic acid, sodium lauryl sulfate and Triton X-100. Phenylmethylsulfonyl fluoride (1mM) and the reducing, ß-mercaptoethanol significantly inhibited lipase activity. The remarkable stability in the presence of detergents, additives, inhibitors and metal ions makes this lipase unique and a potential candidate for significant biotechnological exploitation.

Characterizing LipR from Pseudomonas sp. R0-14 and Applying in Enrichment of Polyunsaturated Fatty Acids from Algal Oil.

In this study, Pseudomonas R0-14, which was isolated from Arctic soil samples, showed a clear halo when grown on M9 medium agarose plates containing olive oil-rhodamine B as substrate, suggesting that it expressed putative lipase(s). A putative lipase gene, lipR, was cloned from R0-14 by genome walking and Touchdown PCR. lipR encodes a 562-amino-acid polypeptide showing a typical α/ß hydrolase structure with a catalytic triad consisting of Ser153-Asp202-His260 and one α-helical lid (residues 103-113). A phylogenetic analysis revealed that LipR belongs to the lipase subfamily I.3. LipR was successfully expressed in Escherichia coli, purified, and biochemically characterized. Recombinant LipR exhibited its maximum activity towards p-nitrophenyl butyrate at pH 8.5 and 60°C with a Km of 0.37 mM and a kcat of 6.42 s(-1). It retained over 90% of its original activity after incubation at 50°C for 12 h. In addition, LipR was activated by Ca(2+), Mg(2+), Ba(2+), and Sr(2+), while strongly inhibited by Cu(2+), Zn(2+), Mn(2+), and ethylenediaminetetraacetic acid. Moreover, it showed a certain tolerance to organic solvents, including acetonitrile, isopropanol, acetone, methanol, and tert-butanol. When algal oil was hydrolyzed by LipR for 24 h, there was an enrichment of n-3 long-chain polyunsaturated fatty acids, including eicosapentaenoic acid (1.22%, 1.65-fold), docosapentaenoic acid (21.24%, 2.04-fold), and docosahexaenoic acid (36.98%, 1.33-fold), and even a certain amount of diacylglycerols was also produced. As a result, LipR has great prospect in industrial applications, especially in food and/or cosmetics applications.

Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30.

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of- flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80°C in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50 °C and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni(2+), Mn(2+), and Cu(2+) to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The Km and Vmax values were estimated to be 35.7 mg/ml, and 5 × 10(4) U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

Purification and characterization of a novel ß-carotene-9',10'-oxygenase from Saccharomyces cerevisiae ULI3.

OBJECTIVES: A novel ß-carotene-9,10'-oxygenase (ScBCO2) has been characterized from Saccharomyces cerevisiae ULI3 to convert ß-carotene to ß-apo-10'-carotenal, which is a precursor of the plant hormone strigolactone. RESULTS: The ScBCO2 enzyme was purified to homogeneity by ammonium sulfate precipitation, Q sepharose and Superdex-200 chromatography. The molecular mass of the enzyme was ~50 kDa by SDS-PAGE. The purified ScBCO2 enzyme displayed optimal activity at 45 °C and pH 8. Tween 20 (1%, w/v), Trition X-100 (1%, w/v), Mg(2+) (5 mM), Zn(2+) (5 mM), Cu(2+) (5 mM), Ca(2+) (5 mM) or DTT (5 mM) increased in the activity by 3, 7, 14, 17, 23, 26 and 27%, respectively. ScBCO2 only exhibited cleavage activity towards carotenoid substrates containing two ß-ionone rings and its catalytic efficiency (kcat/Km) followed the order ß-carotene > α-carotene > lutein. CONCLUSION: ScBCO2 could be used as a potential candidate for the enzymatic biotransformation of ß-carotene to ß-apo-10'-carotenal in biotechnological applications.

Molecular modeling and identification of novel glucokinase activators through stepwise virtual screening.

The glucose phosphorylating enzyme glucokinase (GK) is a 50kD monomeric protein having 465 amino acids. It maintains glucose homeostasis inside cells, acts as a glucose sensor in pancreatic ß-cells and as a rate controlling enzyme for hepatic glucose clearance and glycogen synthesis. It has two binding sites, one for binding d-glucose and the other for a putative allosteric activator named glucokinase activator (GKA). The GKAs interact with the same region of the GK enzyme that is commonly affected by naturally occurring mutations in humans. However, many GKAs do not bind to GK in the absence of glucose. Recently, it has been reported that GKAs are highly effective in patients with type 2 diabetes mellitus. In this milieu a molecular modeling study has been carried out on three natural variants of GK that lie in the GKA binding site and are known to cause maturity onset diabetes of young (MODY). Additionally, a 10ns molecular dynamics simulation was done on each of the modeled variant in order to explore the flexibility of this site. Subsequently, a systematic virtual screening study was done to identify compounds which can bind with high affinity at GKA binding site of mutant GK.

Cloning, Overexpression, and Characterization of a Metagenome-Derived Phytase with Optimal Activity at Low pH.

A phytase gene was identified in a publicly available metagenome derived from subsurface groundwater, which was deduced to encode for a protein of the histidine acid phosphatase (HAP) family. The nucleotide sequence of the phytase gene was chemically synthesized and cloned, in order to further overexpress the phytase in Escherichia coli. Purified protein of the recombinant phytase demonstrated an activity for phytic acid of 298 ± 17 µmol P/min/mg, at the pH optimum of 2.0 with the temperature of 37 °C. Interestingly, the pH optimum of this phytase is much lower in comparison with most HAP phytases known to date. It suggests that the phytase could possess improved adaptability to the low pH condition caused by the gastric acid in livestock and poultry stomachs.