Synthesis of salicylic acid phenylethyl ester (SAPE) and its implication in immunomodulatory and anticancer roles.

Salicylic acid phenylethyl ester (SAPE) was synthesized by Zn(OTf)2-catalyzed selective esterification of salicylic acid and phenylethyl alcohol and studied for its role as an immunomodulatory and anticancer agent. Low toxicity and favorable physical, Lipinski-type, and solubility properties were elucidated by ADME-tox studies. Molecular docking of SAPE against COX-2 revealed favorable MolDockscore, rerank score, interaction energy, internal pose energy, and hydrogen bonding as compared to ibuprofen and indomethacin. An average RMSD of ~ 0.13 nm for the docked complex with stable dynamic equilibrium condition was noted during the 20 ns MD simulation. A low band gap predicting a strong binding affinity at the enzyme's active site was further predicted by DFT analysis. The ester caused a reduction in the percentage of erythrocyte hemolysis and was shown to be non-cytotoxic against human lymphocytes, CaCo-2, and HepG-2 cells by the MTT assay. Moreover, it's in vitro efficacy in inhibiting COX-2 enzyme under both LPS stimulated intestinal cells and direct sequestration assays was found to be higher than salicylic acid and indomethacin. The anticancer activity of SAPE was tested on the breast cancer cell line MCF-7, and potential efficacy was exhibited in terms of decreased cell viability. Flow cytometry analysis exhibited the arrest of the cell cycle at G1/G0 and S phases, during which induction of autophagic vesicle formation and decrease in mitochondrial membrane potential was observed owing to increased ROS production. Furthermore, at these phases, the onset of apoptosis along with DNA damage was also observed. Pre-treatment with SAPE in colitis-induced Wistar rats displayed low disease activity index and reduction in the extent of intestinal tissue disruption and lipid peroxidation. A marked increase of anti-oxidative enzymes viz., catalase, GGT, and GST, and a decrease of pro-inflammatory cytokines IL-6 and TNF-α in the intestinal tissue extracts of the treated groups was noted. The results of this study have sufficient credence to support that the synthesised ester (SAPE) be considered as an anti-oxidative and anti-inflammatory compound with therapeutic potential for the effective management of cancer.

Growth and metabolic characterization of four lactic acid bacteria species isolated from rice beer prepared in Assam, India.

Isolation and identification of lactic acid bacteria (LAB) from rice beer prepared in Assam, India was performed and their growth associated and functional properties were studied. LAB strains were identified as Lactobacillus casei , Pediococcus pentosaceus , Lactobacillus pentosus and Lactobacillus plantarum based on 16 s rRNA sequencing. Their growth characteristics at different pH, NaCl concentration, temperature and presence of carbohydrates were profiled. High tolerance against acid and bile salts was shown by all the strains, particularly L. pentosus TEZU174 and P. pentosaceus TEZU199 up to a pH of 1.5, and L. pentosus TEZU174 up to 14  % bile concentration. They were susceptible towards the common antibiotics, wherein erythromycin, chloramphenicol and linezolid were the most effective. The strains displayed antibiosis activity against Escherichia coli and Staphylococcus aureus and antioxidant activity in terms of resistance to H2O2, scavenging of ·OH and DPPH free radicals was also displayed, wherein L. casei TEZU374 and P. pentosaceus TEZU482 were the most effective with above 70  % scavenging activity. The strains displayed cellular aggregation and L. casei TEZU262 and L. casei TEZU309 were highly aggregated, which attained 100  % autoaggregation within a period of 5 h. High cell surface hydrophobicity was shown by L. casei TEZU309 towards xylene and chloroform, and P. pentosaceus TEZU427 towards ethyl acetate. The strains evinced good gut tolerance capacity, antioxidant activity and adherence properties, which are characteristics of probiotic bacteria and thus are candidates for therapeutic uses and also to be used as starter cultures.

Bioflavonoids from Artocarpus heterophyllus lam. and Cyclosorus extensus (blume) H. Itô as preservatives for increased storage stability of rice beer.

The purified ethanolic extracts of Artocarpus heterophyllus Lam. contained myricetin and epigallocatechin gallate and that of Cyclosorus extensus (Blume) H. Itô contained kaempferol, luteoline and quercetin, in high amount, along with some other flavonoids. Rice beers were fortified with these extracts and kept at 32 °C for 8 weeks, and storage parameters were compared with synthetic antioxidant added and filtered rice beers. It was observed that the aerobic plate counts were significantly less (p ≤ 0.01) in the fortified beers, along with a reduction in the rate of pH fall and change of colour (ΔE). The antioxidant activity (DPPH scavenging) was highest in the fortified beers, along with a delay in lipid peroxidation (POV and TBARS values). The overall study indicated effectiveness of the flavonoid rich extracts in improving the shelf-life stability of rice beer under accelerated conditions.

Amylolytic fungi in starter cakes for rice beer production.

Two types of starter cakes, viz. amou and perok-kushi, used in the production of rice beer in Assam, India, by the Bodo and Deori communities, respectively, were used for the isolation of amylolytic fungi. Based on the sequencing of their internal transcribed spacer (ITS) regions the fungi were identified as Amylomyces rouxii and Rhizopus oryzae, and given the strain names TU460 and TU465, respectively. Both the strains showed the ability to degrade and saccharify starch. The glucoamylase activity was considerably high in A. rouxii TU460 (14.92 µmol/min) as compared to R. oryzae TU465 (1.41 µmol/min), whereas α-amylase activity was found to be closely related, i.e. 7.02 and 6.09 unit mL-1, respectively. SDS PAGE for the determination of the molecular size of the glucoamylase enzymes revealed the production of two distinct units of 59 kDa and 31 kDa by A. rouxii TU460, and one unit of 72 kDa by R. oryzae TU465. LC MS/MS analysis revealed that no mycotoxins were produced by either of the strains. The overall study indicated a good amylolytic property of both strains and a potential for application in the starch processing industries.

Optimization of the extraction of phenolic compounds from Cyclosorus extensa with solvents of varying polarities.

The leaves of Cyclosorus extensa are used in the preparation of rice beer in Assam, India. The optimal conditions of time and temperature of fermentation for extraction of bioactive compounds from the dried leaves were obtained using response surface methodology. The central composite rotatable design was used and 13 experimental runs based on two-factor-five-level design were generated and performed for each of the solvents. The independent variables were extraction time (12 and 48 h) and temperature (25 and 55°C). The responses studied were total polyphenol content, radical scavenging activity, antibacterial activity, and antifungal activity. The analysis of variance of the test data was performed and the sequential sum of squares, F-value, R2, and adjusted R2 were deduced. The predicted models for all the response variables were adequately fitted to the observed experimental data (p ≤ 0.001). The maximum extraction of bioactive compounds under the optimum conditions of extraction temperature and time for hexane, ethyl acetate, methanol, and distilled water were found to be 25°C for 29.43 h, 28.28°C for 41.27 h, 43.95°C for 29.61 h, and 55.00°C for 48.00 h, respectively. It was also observed that the solubility of the polyphenols was higher in methanol, followed by ethyl acetate, and the highest antibacterial activity against Escherichia coli was shown by the ethyl acetate extracts.

Genome Sequences of Three Strains of Lactobacillus paracasei of Different Origins and with Different Cholate Sensitivities.

We report here the draft genome sequences of three strains of Lactobacillus paracasei (NRIC 0644, NRIC 1781, and NRIC 1917) isolated from different sources. The three genomes range from 2.95 to 3.15 Mb with a G+C content of 46% and contain approximately 2,700 protein coding sequences.

Molecular characterization of two genes with high similarity to the dihydroxyacetone synthase gene in the methylotrophic yeast Pichia methanolica.

The methylotrophic yeast Pichia methanolica possesses two genes, PmDAS1 and PmDLP1, whose amino acid sequences show high similarity to dihydroxyacetone synthase (DAS), the formaldehyde-fixing enzyme for methanol metabolism within the peroxisome. The PmDAS1 and PmDLP1 genes encode 709 and 707 amino acid residues respectively, and PmDas1p contains a type-1 peroxisomal targeting signal (PTS1), while PmDlp1p does not. Upon phylogenetic analysis, PmDas1p fit into the DAS group with other DASs, while PmDlp1p was grouped with the DAS-like proteins (DLP) of non-methylotrophic yeasts and fungi, a branch of the phylogenetic tree independent of the DAS and transketolase (TK) groups. While expression of PmDAS1 restored the methylotrophic growth of the Candida boidinii das1Delta strain, the PmDLP1 and PmDAS1-DeltaPTS1 genes did not. Taken together, these results indicate that PmDAS1 encodes a functional DAS and has an indispensable role in methanol metabolism, and that PmDlp1p share a common, as yet uncharacterized function in P. methanolica as well as in non-methylotrophic yeasts and fungi.

Acetaldehyde tolerance in Saccharomyces cerevisiae involves the pentose phosphate pathway and oleic acid biosynthesis.

To identify genes responsible for acetaldehyde tolerance, genome-wide screening was performed using a collection of haploid Saccharomyces cerevisiae strains deleted in single genes. The screen identified 49 genes whose deletion conferred acetaldehyde sensitivity, and these were termed the genes required for acetaldehyde tolerance. We focused on six of these genes required for acetaldehyde tolerance, ZWF1, GND1, RPE1, TKL1 and TAL1, which encode enzymes in the pentose phosphate pathway (PPP), and OAR1, which encodes for NADPH-dependent 3-oxoacyl-(acyl-carrier-protein) reductase. These genes were not only responsible for acetaldehyde tolerance but also turned out to be induced by acetaldehyde. Moreover, the content of oleic acid was remarkably increased in yeast cells under acetaldehyde stress, and supplementation of oleic acid into the media partially alleviated acetaldehyde stress-induced growth inhibition of strains disrupted in the genes required for acetaldehyde tolerance and OLE1. Taken together, our data suggest that the supply of NADPH and the process of fatty acid biosynthesis are the key factors in acetaldehyde tolerance in the yeast, and that oleic acid plays an important role in acetaldehyde tolerance.

Assessment of the microbial contribution to the processing of salted salmon roe (Sujiko).

As the microbial contributions to the processing of salted foods have been little investigated, there remains a possibility that excess sterilization of raw materials for salted foods leads to deterioration in food quality and safety. At a salmon roe (sujiko) processing company, we investigated salted sujiko made identically to commercial products, but that had been processed with or without antibiotics. The antibiotics caused no significant difference in the content of free amino acids, lactic acid or acetic acid. These results show that general aerobic bacteria have no impact on the formation of these flavor compounds.

Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica.

In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA.

Peroxisomal metabolism is regulated by an oxygen-recognition system through organelle crosstalk between the mitochondria and peroxisomes.

In the present study using Pichia methanolica, it was found that expressions of methanol-metabolic enzymes were strictly regulated by the presence of oxygen, and that induction of alcohol oxidase (AOD) isozymes was completely dependent on oxygen concentrations. A proportion of AOD-isozyme species responded to oxygen conditions, e.g. in a low oxygen condition, Mod1p was dominant, but with an increase in the oxygen concentration, the ratio of Mod2p increased. The K(m) value of Mod1p for oxygen was ca. one-seventh lower than that of Mod2p (0.47 and 3.51 mM, respectively). This shows that Mod1p is suitable at low oxygen concentrations and Mod2p at high oxygen concentrations. Also, zymogram changes for AOD isozymes were observed by inhibition of respiratory chain activity. These indicated that P. methanolica has the ability to recognize oxygen conditions and the respiratory chain should participate in the sensor for available oxygen. These facts indicate that there is organelle crosstalk between mitochondria and peroxisomes through nucleus gene regulation in order to control the consumption balance of available oxygen between the mitochondrial respiratory chain and peroxisomal AODs.

Distribution, diversity and regulation of alcohol oxidase isozymes, and phylogenetic relationships of methylotrophic yeasts.

In this study, we attempted to classify the methylotrophic yeasts based on diversities of alcohol oxidase (AOD), i.e. zymogram patterns and partial amino acid sequences. According to zymogram patterns for AOD, members of the methylotrophic yeasts separate into two major lineages, one group involving strains having a single AOD and the other group, including Pichia methanolica, Candida pignaliae and C. sonorensis, showing nine AOD isozymes. Based on partial amino acid sequences of AOD, the methylotrophic yeasts could be divided into five groups, and this classification agrees mostly with grouping based on 26S domain D1/D2 rDNA nucleotide sequences, except for some strains. Moreover, the strains having AOD isozymes constitute one group with P. trehalophila, P. glucozyma and Pichia sp. strain BZ159, although these strains are divided into two types, based on amino acid sequences of second AODs. On the other hand, these AOD isozymes consist of two subunits; the first subunits are induced not only by methanol but also by glycerol and pectin, although the second subunits are mainly induced by methanol. These data indicate that AOD isozymes and second AOD genes distribute widely in several methylotrophic yeasts in the natural environment, and second AOD genes may have evolved as methylotrophic genes that can adapt to the environmental conditions of higher methanol concentrations.

Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2.

Cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2 was overexpressed in Escherichia coli using the Cold expression system and the recombinant enzyme, rBglAp, was characterized. The purified rBglAp exhibited similar enzymatic properties to the native enzyme, e.g., (i) it had high activity at 0 degrees C, (ii) its optimum temperature and pH were 10 degrees C and 8.0, respectively, and (iii) it was possible to rapidly inactivate the rBglAp at 50 degrees C in 5 min. Moreover, rBglAp was able to hydrolyze both ONPG and lactose with K(m) values of 2.7 and 42.1mM, respectively, at 10 degrees C. One U of rBglAp could hydrolyze about 70% of the lactose in 1 ml of milk in 24h, and the enzyme produced trisaccharide from lactose. We conclude that rBglAp is a cold-active enzyme that is extremely heat labile and has significant potential application to the food industry.

Degradation of dimethyl disulfide by pseudomonas fluorescens strain 76.

Strain 76, which was able to utilize dimethyl disulfide (DMDS) as a sole sulfur source, was screened from our microbial collection. It was identified as Pseudomonas fluorescens by taxonomical characterization and 16S rDNA sequence analysis. It does not belong to the methylotrophs, because it did not grow on DMDS or other C1 compounds as sole carbon source, and DMDS degradation was not repressed in the presence of glucose, Na(2)SO(4), or nutrient broth. Moreover, it showed high resistance to DMDS by growing in DMDS at concentrations up to 9.04 mM. Based on these findings, strain 76 metabolizes DMDS and has dual physiological roles: sulfur assimilation and degradation. Thus it has advantages as a biological scavenger of DMDS.

Molecular characterization of the Tim9 homologue from the methylotrophic yeast Pichia methanolica.

In this paper we describe molecular characterization of the TIM9 gene encoding the essential mitochondrial inner-membrane protein in the methylotrophic yeast Pichia methanolica. PmTIM9 contains two exons corresponding to a gene product of 89 amino acid residues and a 140 bp intron. The deduced amino acid sequence exhibited high identity to those of other yeast Tim9ps, and possessed two CX(3)C motifs that contained two cysteine residues conserved among small Tim family proteins. Moreover, PmTIM9 had the ability to partially suppress the temperature sensitivity of Saccharomyces cerevisiae strain tim9-3, suggesting that PmTIM9 is a functional homologue of the ScTIM9 gene.

Purification and molecular characterization of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2.

In this study, we purified and molecularly characterized a cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2. The purified beta-galactosidase from strain F2 exhibited high activity at 0 degrees C, and its optimum temperature and pH were 10 degrees C and 8.0, respectively. It was possible to inactivate the beta-galactosidase rapidly at 45 degrees C in 5 min. The enzyme was able to hydrolyze lactose as a substrate, as well as o-nitrophenyl-beta-D-galactopyranoside (ONPG), the Km values with ONPG and lactose being calculated to be 2.8 mM and 50 mM, respectively, at 10 degrees C. Moreover, the bglA gene encoding the beta-galactosidase of strain F2 was cloned and analyzed. The bglA gene consists of a 3,084-bp open reading frame corresponding to a protein of 1,028 amino acid residues. BglAp, the gene product derived from bglA, had several conserved regions for glycosyl hydrolase family 2, e.g., the glycosyl hydrolase 2 (GH2) sugar binding domain, GH2 acid-base catalyst, GH2 triosephosphate isomerase barrel domain, GH2 signature 1, and several other GH2 conserved regions. From these facts, we conclude that the beta-galactosidase from A. psychrolactophilus strain F2, which is a new member of glycosyl hydrolase family 2, is a cold-active enzyme that is extremely heat labile and could have advantageous applications in the food industry.

Regulation of two distinct alcohol oxidase promoters in the methylotrophic yeast Pichia methanolica.

In this study, two Pichia methanolica alcohol oxidase (AOD) promoters, P(MOD1) and P(MOD2), were evaluated in a promoter assay system utilizing the acid phosphatase (AP) gene from Saccharomyces cerevisiae (ScPHO5) as a reporter. Heterologous gene expression driven by the P(MOD1) and P(MOD2) promoters was found to be strong and tightly regulated by carbon source at the transcriptional level. P(MOD1) was induced not only by methanol but also by glycerol. P(MOD2) was induced only by methanol, although it was not repressed on the addition of glycerol to a methanol medium, suggesting that P(MOD2) is regulated in a manner distinct from that of other AOD-gene promoters. On the other hand, methanol and oxygen level-influenced gene expression mediated by P(MOD1) and P(MOD2). P(MOD1) expression was optimal at low methanol concentrations, whereas P(MOD2) was predominantly expressed at high methanol and high oxygen concentrations. Based on these results, both P(MOD2) and P(MOD1) should be useful tools for controlling heterologous gene expression in P. methanolica. In particular, it should be possible to differentially control the production phases of two heterologous proteins, using P(MOD1) and P(MOD2) in the same host cell and in the same flask.

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans.

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.

Pectin utilization by the methylotrophic yeast Pichia methanolica.

The methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Delta and mod1Deltamod2Delta strains showed a severe defect in the growth yield, although the mod2Delta strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.

A cold-active pectin lyase from the psychrophilic and basidiomycetous yeast Cystofilobasidium capitatum strain PPY-1.

In the present study we purified a cold-active PNL (pectin lyase) from the extracellular fraction of the PPY (pectinolytic and psychrophilic yeast) Cystofilobasidium capitatum strain PPY-1. The purified PNL has a molecular mass of approx. 42 kDa, and its N-terminal amino acid sequence is ATGVTGSAYGFATGTTGGGSATPAY, which exhibits 72% identity with that of PNL F from Aspergillus niger. The purified PNL exhibited high activity at 10 degrees C, although its optimum temperature was 40 degrees C. Moreover, Km and Vmax for pectin as a substrate were found to have values 36.6 mg/ml and 3000 units/mg respectively. These findings may indicate that this enzyme from strain PPY-1 is a cold-active PNL that is able to degrade pectin compounds at low temperature.