Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis

Alcaligenes sp. MTCC 10675 has been isolated from soil sample using enrichment method and has nitrilase catalytic system which is highly specific for the hydrolysis of arylaliphatic nitriles. Optimization of culture conditions using response surface methodology and inducer-mediated approach enhanced arylacetonitrilase production significantly (2.4-fold). Isobutyronitrile acted as an effective inducer for the induction of arylacetonitrilase, and it is highly specific for arylacetonitriles (phenyl acetonitrile and mandelonitrile). Arylacetonitrilase has no effect on its relative velocity (V _r) up to 20 mM substrate (mandelonitrile) concentration and at 30 mM mandelonitrile, 23.4 % degree of inhibition (I _d) was recorded. Half life of arylacetonitrilase of Alcaligenes sp. MTCC 10675 was 27.5 h at 25 °C. Hg²⁺, Ag⁺, Pb³⁺, and Co²⁺ were strong inhibitor of arylacetonitrilase activity which resulted into 100 %, 91 %, 84 %, and 83 % inhibition, respectively. Polar protic solvent (dichloromethane, dimethylsulphooxide, and n-butanol) reduce arylacetonitrilase activity up to 80–94 % at 10 % concentration. Alcaligenes sp. MTCC 10675 has higher biocatalytic activity, i.e., 3.9 gg^-1 dcw, which is highest in comparison to till reported organism. Arylacetonitrilase-mediated hydrolysis of racemic mandelonitrile resulted into R-(-) mandelic acid with 99.0 % enantiomeric excess (e.e.)     

Streptomyces amritsarensis sp. nov., exhibiting broad-spectrum antimicrobial activity

A new actinobacterium strain, designated 2A^T, was isolated from a soil sample collected from Guru Nanak Dev University, Punjab (India) and characterized using a polyphasic taxonomic approach. It showed antimicrobial activity against various Gram-positive and Gram-negative bacteria including drug resistant bacteria and fungi. The strain had chemotaxononomic and morphological properties typical of the genus Streptomyces. The 16S rRNA gene sequence of the strain showed 99.9, 99.5 and 99.5 % similarity with Streptomyces flavotricini DSM 40152^T, Streptomyces toxytricini DSM 40178^T and Streptomyces globosus DSM 40815^T, respectively. This strain formed a coherent cluster with them and shared DNA–DNA homology of 37.6 ± 0.6, 34.4 ± 0.5 and 33.1 ± 0.4 % with type strains, S. flavotricini DSM 40152^T, S. globosus DSM 40815^T and S. toxytricini DSM 40178^T, respectively. Further, the strain was readily distinguished from the phylogenetic close relatives in a variety of morphological, physiological and biochemical properties. Based on the genotypic and phenotypic characteristics, it is proposed that strain 2A^T represents a novel species in the genus Streptomyces, for which the name Streptomyces amritsarensis sp. nov. is proposed, with the type strain 2A^T (=MTCC 11845^T=JCM 19660^T).     

Paenibacillus ihbetae sp. nov., a cold-adapted antimicrobial producing bacterium isolated from high altitude Suraj Tal Lake in the Indian trans-Himalayas

The assessment of bacterial diversity and bioprospection of the high-altitude lake Suraj Tal microorganisms for potent antimicrobial activities revealed the presence of two Gram-stain-variable, endospore-forming, rod-shaped, aerobic bacteria, namely IHBB 9852^T and IHBB 9951. Phylogenetic analysis based on 16S rRNA gene sequence showed the affiliation of strains IHBB 9852^T and IHBB 9951 within the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus lactis DSM 15596^T (97.8% and 97.7%) and less than 95.9% similarity to other species of the genus Paenibacillus. DNA-DNA relatedness among strains IHBB 9852^T and IHBB 9951 was 90.2%, and with P. lactis DSM 15596^T, was 52.7% and 52.4%, respectively. The novel strains contain anteiso-C_15:0, iso-C_15:0, C_16:0 and iso-C_16:0 as major fatty acids, and phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol were predominant polar lipids. The DNA G+C content for IHBB 9852T and IHBB 9951 was 52.1 and 52.2 mol%. Based on the results of phenotypic and genomic characterisations, we concluded that strains IHBB 9852^T and IHBB 9951 belong to a novel Paenibacillus species, for which the name Paenibacillus ihbetae sp. nov. is proposed. The type strain is IHBB 9852^T (=MTCC 12459^T = MCC 2795^T = JCM 31131^T = KACC 19072^T; DPD TaxonNumber TA00046) and IHBB 9951 (=MTCC 12458 = MCC 2794 = JCM 31132 = KACC 19073) is a reference strain.     

Purification and characterization of an extracellular antifungal chitinase from Penicillium ochrochloron MTCC 517 and its application in protoplast formation

A highly chitinolytic strain Penicillium ochrochloron MTCC 517 was procured from MTCC, Chandigarh, India. Culture medium supplemented with 1% chitin was found to be suitable for maximum production of chitinase. Purification of extracellular chitinase was done from the culture medium by organic solvent precipitation and DEAE-cellulose column chromatography. The chitinase was purified 6.92-fold with 29.9% yield. Molecular mass of purified chitinase was found to be 64 kDa by SDS-PAGE. The chitinase showed optimum temperature 40 °C and pH 7.0. The enzyme activity was completely inhibited by Hg²⁺, Zn²⁺, K⁺ and NH₄⁺. The enzyme kinetic study of purified chitinase revealed the following characteristics, such as apparent K_m 1.3 mg ml^?1, V_max 5.523 × 10^?5 moles l^?1 min^?1 and K_cat 2.37 s^?1 and catalytic efficiency 1.82 s^?1 M^?1. The enzyme hydrolyzed colloidal chitin, glycol chitin, chitosan, glycol chitosan, N,N′-diacetylchitobiose, p-nitrophenyl N-acetyl-β-d-glucosaminide and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide. The chitinase of P. ochrochloron MTCC 517 is an exoenzyme, which gives N-acetylglucosamine as the main hydrolyzate after hydrolysis of colloidal chitin. Protoplasts with high regeneration capacity were obtained from Aspergillus niger using chitinase from P. ochrochloron MTCC 517. Since it also showed antifungal activity, P. ochrochloron MTCC 517 seems to be a promising biocontrol agent.     

High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus

NZ2114, a new variant of plectasin, was overexpressed in Pichia pastoris X-33 via pPICZαA for the first time. The total secreted protein of fermentation supernatant reached 2,390 mg/l (29 °C) and 2,310 mg/l (25 °C), and the recombinant NZ2114 (rNZ2114) reached 860 mg/l (29 °C) and 1,309 mg/l (25 °C) at 96 h induction in a 5-l fermentor, respectively.The rNZ2114 was purified by cation exchange chromatography, and its yield was 583 mg/l with 94.8 % purity. The minimal inhibitory concentration (MIC) of rNZ2114 to four ATCC strains of Staphyloccocus aureus was evaluated from 0.028 to 0.90 μM. Meanwhile, it showed potent activity (0.11–0.90 μM) to 20 clinical isolates of MRSA. The rNZ2114 killed over 99.9 % of tested S. aureus (ATCC 25923 and ATCC 43300) in Mueller-Hinton medium within 6 h when treated with 4?×?MIC. The postantibiotic effect of rNZ2114 to S. aureus ATCC 25923 and ATCC 43300 was 18.6–45.6 and 1.7–3.5 h under 1×, 2×, and 4× MIC, respectively. The fractional inhibitory concentration index (FICI) indicated a synergistic effect between rNZ2114 and kanamycin, streptomycin, and vancomycin against S. aureus ATCC 25923 (FICI?=?0.125), and additivity between rNZ2114 and ampicillin, spectinomycin (FICI?=?0.625), respectively. To S. aureus ATCC 43300 [methicillin-resistant S. aureus (MRSA)], rNZ2114 showed a synergistic effect (FICI?=?0.125–0.3125) with kanamycin, ampicillin, streptomycin, and vancomycin, and antagonism with spectinomycin (FICI?=?8.0625). The rNZ2114 caused only less than 0.1 % hemolytic activity in the concentration of 128 μg/ml, and showed a good thermostability from 20 to 80 °C. In addition, it exhibited the highest activity at pH 8.0. These results suggested that large-scale production of NZ2114 is feasible using the P. pastoris expression system, and it could be a new potential antimicrobial agent for the prevention and treatment of S. aureus especially for MRSA infections.     

Functional properties of a manganese-activated exo-polygalacturonase produced by a thermotolerant fungus Aspergillus niveus

A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0–6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn²⁺. The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K _m, V _max, K _cat, and K _cat/K _m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.     

Survival,prophage induction,and invasive properties of lysogenic Salmonella Typhimurium exposed to simulated gastrointestinal conditions

This study was designed to evaluate the viability, prophage induction, invasive ability, and relative gene expression in lysogenic Salmonella Typhimurium exposed to the simulated gastric juice (SGJ) at pH 2 (SGJ-2), 3 (SGJ-3), 4 (SGJ-4), and 5 (SGJ-5) for 30 min followed by 0.5 % bile salts for 2 h. The susceptibility of lysogenic S. Typhimurium increased with decreasing pH value and increasing bile salt concentration. The lysogenic S. Typhimurium cells were least susceptible to SGJ-4 and SGJ-5, showing <1 log reduction. The highest prophage induction was observed by 3.34 log PFU/ml in lysogenic S. Typhimurium at SGJ-3 in the presence of 0.5 % bile salts. The numbers of invading lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5 were 3.57, 3.73, and 4.15 log CFU/cm², respectively. Most genes (hilA, hilC, hilD, invA, invE, invF, and sirA) were down-regulated in lysogenic S. Typhimurium treated at SGJ-3, SGJ-4, and SGJ-5. This study provides useful information for understanding physiological changes of lysogenic S. Typhimurium in the simulated gastrointestinal conditions.     

Effect of subcritical water hydrolysate in the brown seaweed Saccharina japonica as a potential antibacterial agent on food-borne pathogens

Seaweeds are rich in bioactive compounds which have well-documented antioxidant properties. They also have antimicrobial activities against food pathogenic microorganisms. This study uses an extract of the brown seaweed, Saccharina (Laminaria) japonica, produced by subcritical water hydrolysis (SWH) for investigating its potential to inhibit bacteria. De-oiled S. japonica was obtained by supercritical carbon dioxide extraction. The reaction temperatures for hydrolysis of raw and de-oiled S. japonica were maintained from 200 to 280 °C. The experiment was done with condition 1.3–6.0 MPa for the reaction pressure and 1:10 (w/v) for the ratio of material to water. The antibacterial activities of raw and de-oiled S. japonica produced by SWH were determined by using the agar diffusion method. Antibacterial activity was tested against two Gram-negative (Escherichia coli and Salmonella typhimurium) and two Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus). The antibacterial activities of hydrolysate water with catalyst at 240 °C showed better bacterial inhibition than the others. Strong antibacterial activity was found using de-oiled material with acetic acid added, with a zone of inhibition of S. typhimurium (14.33?±?0.06 mm) and E. coli (13.00?±?0.09 mm). On the other hand, the weakest antibacterial inhibition was found for S. aureus (12.83?±?0.10 mm) and B. cereus (12.50?±?0.09 mm).     

Geodermatophilus saharensis sp. nov., isolated from sand of the Saharan desert in Chad

A novel Gram-positive, aerobic, actinobacterial strain, CF5/5, was isolated from soil in the Sahara desert, Chad. It grew best at 20–35 °C and at pH 6.0–8.0 and with 0–4 % (w/v) NaCl, forming black-colored colonies. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G + C content was 75.9 mol%. The peptidoglycan contained meso-diaminopimelic acid; galactose and xylose were detected as diagnostic sugars. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, and phosphatidylinositol; MK-9(H₄) was the dominant menaquinone. The major cellular fatty acids were: iso-C_16:0 and iso-C_15:0. The 16S rRNA gene showed 95.6–98.3 % sequence similarity with the other named members of the genus Geodermatophilus. Based on the polyphasic taxonomy data, the isolate is proposed to represent a novel species, Geodermatophilus saharensis with the type strain CF5/5^T = DSM 45423 = CCUG 62813 = MTCC 11416.     

Kinetics study of pyridine biodegradation by a novel bacterial strain,Rhizobium sp. NJUST18

Biodegradation of pyridine by a novel bacterial strain, Rhizobium sp. NJUST18, was studied in batch experiments over a wide concentration range (from 100 to 1,000 mg l^?1). Pyridine inhibited both growth of Rhizobium sp. NJUST18 and biodegradation of pyridine. The Haldane model could be fitted to the growth kinetics data well with the kinetic constants μ* = 0.1473 h^?1, K _s = 793.97 mg l^?1, K _i = 268.60 mg l^?1 and S _m = 461.80 mg l^?1. The true μ _max, calculated from μ*, was found to be 0.0332 h^?1. Yield coefficient Y _X/S depended on S _i and reached a maximum of 0.51 g g^?1 at S _i of 600 mg l^?1. V _max was calculated by fitting the pyridine consumption data with the Gompertz model. V _max increased with initial pyridine concentration up to 14.809 mg l^?1 h^?1. The q _S values, calculated from $V_{ \hbox{max} }$ , were fitted with the Haldane equation, yielding q _Smax = 0.1212 g g^?1 h^?1 and q* = 0.3874 g g^?1 h^?1 at S _m′ = 507.83 mg l^?1, K _s′ = 558.03 mg l^?1, and K _i′ = 462.15 mg l^?1. Inhibition constants for growth and degradation rate value were in the same range. Compared with other pyridine degraders, μ _max and S _m obtained for Rhizobium sp. NJUST18 were relatively high. High K _i and K _i′ values and extremely high K _s and K _s′ values indicated that NJUST18 was able to grow on pyridine within a wide concentration range, especially at relatively high concentrations.     

Impact of Proteolytic <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> MTCC5463 on Production of Bioactive Peptides Derived from Honey Based Fermented Milk

In this study, Lactobacillus helveticus MTCC5463 was evaluated for its proteolytic activity and production of bioactive peptides during fermentation of honey supplemented milk under specified growth conditions. Generally, lactic acid bacteria have a strong proteolytic system. However, L. helveticus MTCC5463 showed maximum proteolytic activity at 4 % level of honey supplementation compared to 6 % and control. Similarly, water soluble extract derived from fermented honey based milks exhibited different level of bioactive peptides productions during fermentation. L. helveticus MTCC5463 showed maximum peptides production at 4 % level of honey supplementation compared to control during HPLC analysis and LC–MS analysis.     

Identification of 1-chloro-2-formyl indenes and tetralenes as novel antistaphylococcal agents exhibiting sortase A inhibition

Tetralene and indene compounds have shown inhibitory activity against human pathogen, Mycobacterium tuberculosis. Their potential use as antistaphylococcal agent against drug-resistant Staphylococcus aureus has not been explored so far. We determined in vitro antistaphylococcal activity and mechanism of action of these compounds as sortase A inhibitors through in silico analysis followed by biological assays. Tetralene and indene series were tested against S. aureus strains MTCC96, MRSA, and VA30. Three compounds showed significant reduction in MIC in both wild-type and drug-resistant S. aureus strains. In silico absorption, distribution, metabolism, excretion, and toxicity analysis of identified leads and cytotoxicity testing with colorimetric method using Vero and WRL-68 cell lines showed no significant cytotoxic effects. Molecular docking of these molecules with sortase A (PDB: 2KID) showed H-bond interaction with functional site residue Arg197 of sortase A. Sortase A inhibition assay was developed by expressing SrtA_?N from S. aureus strain MTCC96. Tetralene and indene compounds were found to have sortase A inhibitory potential. S. aureus strain MTCC96 treated with these compounds showed surface-sorting inhibition of fibronectin-binding protein and reduction in adherence to host extracellular matrix protein, fibronectin. 1-Chloro, 2-formyl, 6-methoxy, 1-tetralene (Tet-5), 1,5-dichloro, 2-formyl, 1-indene (Tet-20) and 1-chloro, 2-formyl, 5,6-methylenedioxy, and 1-indene (Tet-21) exhibited antistaphylococcal activity along with sortase A inhibition. The results also indicate the possible role of these leads in other reactions essential for cell viability.     

Development and characterization of essential oil component-based polymer films: a potential approach to reduce bacterial biofilm

The development of new polymeric materials aimed to control the bacterial biofilm appears to be an important practical approach. The goal of the present study was to prepare and characterize poly(ethylene-co-vinyl acetate) copolymer (EVA) films containing citronellol, eugenol, and linalool and evaluate their efficiency on growth and biofilm formation of Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa in monospecies and dual species. The results showed that the addition of oil components influenced the elastic modulus (15 % decrease), the tensile stress (30 % decrease), the elongation at break (10 % increase), and the contact angle values (10–20° decrease) while leaving the homogeneity of the surface unaltered. Among the polymeric films, EVA + citronellol and EVA + eugenol at 7 wt% had the best inhibitory effect. After 24–48 h of incubation, EVA + citronellol was more effective against the growth (30–60 % reduction) than EVA + eugenol (15–30 % inhibition). However, this inhibition decreased after 240 h of incubation. On the contrary, the biofilm evaluation revealed a strong inhibition trend also after prolonged incubation time: the amount of biomass per square centimeter formed on copolymer with oil components was significantly less (40–70 % decrease) than that on pure copolymer control for L. monocytogenes, S. aureus, and E. coli. When polymeric materials were simultaneously inoculated with combinations of S. aureus and E. coli, the biomass accumulated was higher for EVA + citronellol and lower for EVA + eugenol than that in monoculture biofilm. The findings were similar to the results obtained by 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assay that measures the metabolic activity of viable cells.     

In vitro antifungal,probiotic and antioxidant properties of novel Lactobacillus plantarum K46 isolated from fermented sesame leaf

This study aimed to describe the diversity of antifungal lactic acid bacteria (LAB) in popular traditional Korean fermented food. A total of 22 LAB strains was selected and subjected to a monophasic identical approach using 16S rRNA gene sequence analysis. Antifungal LAB associated with fermented food was identified as Lactobacillus plantarum (9), Lactobacillus graminis (5), Lactobacillus pentosus (4), Lactobacillus sakei (2), Lactobacillus paraplantarum (1), and Leuconostoc mesenteroides subsp. mesenteroides (1). Novel Lactobacillus plantarum strain K46 exhibited comparatively better antifungal activity against several spoilage fungi, and was deposited in the Korean Collection for Type Cultures (KACC91758P). Antifungal substances from the spent medium in which K46 was cultivated were extracted with ethyl acetate. Antifungal activity was assessed using the broth micro dilution technique. Compounds were characterized based on infrared, ¹³C nuclear magnetic resonance (NMR), and ¹H NMR spectral data. The minimum inhibitory concentration (MIC) of the compounds against Aspergillus clavatus, Aspergillus oryzae, Penicillium chrysogenum and Penicillium roqueforti was 2.5 mg/mL and that against Aspergillus fumigatus, Aspergillus niger, Curvularia lunata and Gibberella moniliformis was 5.0 mg/mL. K46 was able to survive gastrointestinal conditions simulating the stomach and the duodenum passage with the highest percentage of hydrophobicity. In addition, its resistance to hydrogen peroxide and highest hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, with inhibition rates of 43.53 % and 56.88 %, respectively, were to its advantage. An antimicrobial susceptibility pattern was an intrinsic feature of this strain, thus consumption does not represent a health risk to humans. The results showed the potential of K46 strain as an antifungal, probiotic and antioxidant culture, and hence it was determined to be suitable for application in functional foods.     

Changes in soil microbial community structure with planting years and cultivars of tree peony (Paeonia suffruticosa)

To understand the effects of planting tree peony (Paeonia suffruticosa) on soil microbial community structure, soil samples were collected from the tree peony gardens with three peony cultivars and three planting years, and adjacent wasteland at Luoyang, Henan Province of China. Soil microbial communities were analyzed by the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) of partial 16S rDNA and sequencing methods. With the succeeding development of tree peony garden ecosystems, soil pH, organic C, total P, and available P increased. Soil total N, the cell numbers of bacteria, fungi, and actinomycetes, the Shannon’s diversity index (H), richness (S), and Evenness (E _H ) first showed an increasing trend after wasteland was reclaimed and then a decreasing trend became apparent after 5 years of planting. Principal component analysis based on DGGE banding patterns showed that the microbial community structures were influenced by tree peony cultivars and planting years, and the influences of planting years were greater than those of tree peony cultivars. Sequence analysis of the DGGE bands revealed that the dominant bacteria in tree peony garden soils belonged to Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Acidobacteria, Planctomycetes, Euryarchaeota, and Unclassified-bacteria. Moreover, some bacteria closely related to Bradyrhizobium, Sphingopyxis, Novosphingobium, and Sphingomonas, which have been associated with nitrogen fixation and recalcitrant compounds degradation, disappeared with the increasing planting years of tree peony. The bacteria had similarity of 100 % compared with Pseudomonas mandelii which was a denitrifying bacteria, and increased gradually with increasing planting years of tree peony.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

Influence of fungal elicitors on biosynthesis of natamycin by Streptomyces natalensis HW-2

To investigate the effect of fungal elicitors on biosynthesis of natamycin in the cultures of Streptomyces natalensis HW-2, the biomass and filtrate of the broth from Aspergillus niger AS 3.6472, Penicillium chrysogenum AS 3.5163, A. oryzae AS 3.2068, and Saccharomyces cerevisiae AS 2.2081 were used to induce natamycin production in S. natalensis HW-2. The results showed that the biomass of P. chrysogenum AS 3.5163 could enhance the yield of natamycin from 0.639 to 0.875 g?l^?1. The elicitor from the fermentation broth of P. chrysogenum AS 3.5163 showed the highest inducing efficiency with the yield of natamycin enhanced from 0.632 to 1.84 g?l^?1. The elicitor that was cultured for 2 days showed the strongest inducing activity during the fermentation of S. natalensis HW-2 for 24 h, and the yield of natamycin was enhanced from 0.637 to 2.12 g?l^?1. The biochemical parameters were examined at the end of fermentation and the results demonstrated that both the growth of cells and the concentration of residual sugar could be influenced. The residual sugar decreased from 5.03 to 4.27 g?l^?1, and the biomass decreased from 10.26 to 6.87 g?l^?1. Finally, the elicitor was identified as a low molecular weight substance with a similar polarity to that of butyl alcohol by primary qualitative analysis.     

Production of tannase through solid state fermentation using Indian Rosewood (Dalbergia Sissoo)sawdust—a timber industry waste

The tannase producing strain Aspergillus heteromorphus MTCC 8818 was used in the present study for the production of tannase under solid state fermentation using Rosewood (Dalbergia sissoo) sawdust—a timber industry waste—as substrate. Various physico-chemical parameters were optimized for extracellular yield of tannase. Maximum tannase (1.84 U/g dry substrate) and gallic acid (5.4 mg/g ds) was observed at 30 °C after 96 h of incubation. Czapek dox medium was found to be the best moistening agent, with pH and relative humidity of 5.5 and 70 %, respectively. The constituents of Czapek dox medium were varied to enhance enzyme production. The optimum concentration of modified Czapek dox constituents contained 0.2 % NaNO₃, 0.05 % K₂HPO₄ and MgSO₄, 0.15 % KCl. Among the additional salts supplemented to Czapek dox medium, ZnSO₄ and CuSO₄ were found to have a stimulating effect, with a relative tannase activity of 116 and 111 %, respectively. Glucose as an external carbon source was found to be a repressor of enzyme production.     

Nocardia bhagyanesis sp. nov., a novel actinomycete isolated from the rhizosphere of Callistemon citrinus (Curtis), India

A novel actinomycete strain, designated VRC07^T, was isolated from a Callistemon citrinus rhizosphere sample collected from Hyderabad, India. Its taxonomic status was determined by using polyphasic approach. It is a Gram-positive, aerobic, non-motile, weakly acid-fast strain. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain VRC07^T is a member of the genus Nocardia. The highest levels of 16S rRNA gene sequence similarity was found between the strains Nocardia niwae W9241^T (99.6 %), Nocardia amikacinitolerans W9988^T (99.3 %) and Nocardia arthritidis IFM 10035^T (98.9 %); similarity to other type strains of the genus Nocardia was below 98.7 %. The organism had chemical and morphological features consistent with its classification in the genus Nocardia such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan. Arabinose and galactose as the diagnostic sugars. Diagnostic polar lipids were phosphatidylinositol, diphosphatidylglycerol, and phosphatidylglycerol. The predominant menaquinone was MK-8(H_{4, ω-cycl}). The major fatty acids were C_16:0, C_18:0, C_{18:1 w9c}, C_{18:0 10-methyl TBSA} and sum in feature 3 (16:1 w7c/16:1 w6c). The G+C content of the genomic DNA was 68.5 mol%. The DNA–DNA relatedness data, together with phenotypic differences clearly distinguished the isolate from its closest relatives. On the basis of these phenotypic and genotypic data, the isolate represents a novel species, for which the name Nocardia bhagyanesis sp. nov., is proposed. The type strain is VRC07^T (=KCTC 29209^T = MTCC 11725^T = ATCC BAA-2548).     

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V _max and K _m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 μmol mg^?1 min^?1 and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K _i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml ^? resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.