Partial purification and characterization of exoinulinase produced from Bacillus sp.

Inulinase are industrial food enzymes which have gained much attention in recent scenario. In this study, Inulinase producing eight bacterial colonies were isolated and screened from three different plant root tubers soil sample. Among 8 inulinase producing colonies, the higher yielding colony was selected with 25.10?U/mL for further studies. The best inulinase producing colony was identified by partial 16S rRNA gene sequence as Bacillus sp. The crude inulinase was purified by using ammonium sulphate precipitation, dialysis and ion exchange chromatography on DEAE – sephacel and obtained 1.9 purification fold with total activity 293 U. The purified enzyme was subjected to characterization studies and it was found to be stable at 30–60?°C and optimum temperature was at 55?°C. The enzyme was stable at pH 3.0–7.0 and optimum pH was at 6.5. The K_m and V_max value for inulinase was found to be 0.117?mg/mL and 4.45?μmol?min?mg^?1 respectively, demonstrate its greater affinity. Hence, this enzyme can be widely used for the production of fructose, and fructooligosaccharides, which are important ingredients in food and pharmaceutical industry.     

Molecular diversity and hydrolytic enzymes production abilities of soil bacteria

Soil is an integral part of ecosystem which is niche for varieties of microflora. The present study was investigated to isolate varied strains of bacteria from soil samples of three different geographical regions of Tamil Nadu (India) and evaluate their hydrolytic enzymes (amylase, cellulase, and inulinase) producing potentialities. Among 72 bacterial cultures isolated from Ambattur Industrial Estate, Neyveli Lignite Corporation, and Arignar Anna Zoological Park regions, 41.66, 38.88, and 36.11% of isolates were observed amylase, cellulase, and inulinase producers, respectively. On the other hand, 20.83% of total bacteria isolated from all three regions exhibited concurrent production of amylase, cellulase, and inulinase. Potent isolates depicting maximum enzyme activities were identified as Bacillus anthracis strain ALA1, Bacillus cereus strain ALA3, Glutamicibacter arilaitensis strain ALA4, and Bacillus thuringiensis strain ALA5 based on molecular characterization tools. Further, the thermodynamics parameters, open reading frames (ORFs) regions, and guanine-cytosine (GC) content were determined by distinct bioinformatics tools using 16S rRNA sequences of strains. Minimum free energy values for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 were calculated as −480.73, −478.76, −496.63, and −479.03 kcal/mol, respectively. Mountain plot and entropy predicted the hierarchical representation of RNA secondary structure. The GC content of sequence for strain ALA1, strain ALA3, strain ALA4, and strain ALA5 was calculated as 53.06, 52.94, 56.78, and 53.06%, respectively. Nine ORFs were obtained for strain ALA1, strain ALA3, and strain ALA5 while 10 ORFs were observed for strain ALA4. Additionally, bootstrap tree demonstrated close resemblance of strains with existing bacteria of similar genus. Findings showed higher variability of bacterial diversity as hydrolytic enzymes producers in the investigated geographical regions.     

A <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain HPC248 from an effluent treatment plant with antimicrobial activity

This study reports the identification and demonstration of an organism with antimicrobial activity isolated from activated biomass of an effluent treatment plant (ETP) treating wastewater containing pesticides. While assessing the heterotrophic diversity of biomass collected from ETP, clear zones were observed on Luria Bertani plates. The bacterial isolate producing the zone as well as the bacterial cells surrounding the zone were isolated and purified by sub-culturing. Both isolates were identified by partial sequencing of the 16S rDNA clone. Presence of antimicrobial activity was demonstrated against various laboratory strains, isolated from different treatment plants and also against waterborne pathogens. The isolate that produced antimicrobial activity was identified as Bacillus subtilis strain HPC248 and the sensitive strain was identified as Bacillus sphaericus strain HPC249.     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

重楼中一株产纤维素酶内生真菌的分离及鉴定

从重楼根茎中分离、鉴定具有产纤维素酶活性的内生真菌。采用表面消毒法从重楼块茎中分离内生真菌;用纤维素酶活性CMC平板检测分离菌株的产纤维素酶活性;对高产菌株进行形态学观察和分子生物学测序鉴定;探究影响纤维素酶活力的因素;利用平板法检测该株菌产其他胞外水解酶的活性。从3个来源的重楼中分离出41株内生真菌,通过平板检测发现AS-5、AS-7、AS-9和AS-18菌株能产生纤维素酶,其中AS-9菌株活性最强;通过形态学观察和ITS、LSU序列分析将AS-9菌株鉴定为Setophoma terrestris;该菌在pH值为7.0和温度为28℃时表现出最大纤维素酶活性,紫外线照射对产纤维素酶活性无明显作用;检测发现AS-9菌株同时具有产酪蛋白酶、脂肪酶、天冬酰胺酶、谷氨酰胺酶和脲酶活性。首次在重楼中发现内生真菌Setophoma terrestris,且具有较好的产纤维素酶能力,值得深入研究。     

Penicillium subrubescens, a new species efficiently producing inulinase

Inulin is a reserve carbohydrate in about 15 % of the flowering plants and is accumulated in underground tubers of e.g. chicory, dahlia and Jerusalem artichoke. This carbohydrate consists of linear chains of β-(2,1)-linked fructose attached to a sucrose molecule. Inulinases hydrolyse inulin into fructose and glucose. To find efficient inulin degrading fungi, 126 fungal strains from the Fungal Biotechnology Culture Collection (FBCC) at University of Helsinki and 74 freshly isolated strains from soil around Jerusalem artichoke tubers were screened in liquid cultures with inulin as a sole source of carbon or ground Jerusalem artichoke tubers, which contains up to 19 % (fresh weight) inulin. Inulinase and invertase activities were assayed by the dinitrosalicylic acid (DNS) method and a freshly isolated Penicillium strain originating from agricultural soil (FBCC 1632) was the most efficient inulinase producer. When it was cultivated at pH 6 and 28 °C in 2 litre bioreactors using inulin and Jerusalem artichoke as a carbon source, inulinase and invertase activities were on day 4 7.7 and 3.1 U mL^?1, respectively. The released sugars analysed by TLC and HPLC showed that considerable amounts of fructose were released while the levels of oligofructans were low, indicating an exoinulinase type of activity. Taxonomic study of the inulinase producing strain showed that this isolate represents a new species belonging in Penicillium section Lanata-divaricata. This new species produces a unique combination of extrolites and is phenotypically and phylogenetically closely related to Penicillium pulvillorum. We propose the name Penicillium subrubescens sp. nov. (CBS 132785^T = FBCC 1632^T) for this new species.     

Screening and molecular characterization of Serratia marcescens VITSD2: A strain producing optimum serratiopeptidase

The current work was attempted to isolate and characterize the serratiopeptidase producing Serratia sp. Among the 10 bacterial isolates 7 strains were identified as Serratia sp. Out of 7 strains one showed potent proteolytic activity and selected for further studies. Based on the morphological, biochemical and molecular characterization, the potent isolate （RH03） was identified as Serratia marcescens （GenBank accession number： KC961637） and the strain was designated as Serratia marcescens VITSD2. The production of serratiopeptidase was carried out in trypticase soya broth and the enzyme was partially purified using ammonium sulfate precipitation and dialysis. The specific activity was determined by casein hydrolysis assay and was found to be 12.00, 21.33, and 25.40 units/rag for crude, precipitated and dialysed samples. The molecular weight of the protease was determined by SDS-PAGE and it was found to be 50 kDa. The antibacterial activity of the produced serratiopeptidase showed moderate activity against Pseudomonas aeruginosa MTCC No. 4676 （12 mm） and Escherichia coli MTCC No. 1588 （15 mm）.     

Subsurface Associations of Acaryochloris-Related Picocyanobacteria with Oil-Utilizing Bacteria in the Arabian Gulf Water Body: Promising Consortia in Oil Sediment Bioremediation

Two picocyanobacterial strains related to Acaryochloris were isolated from the Arabian Gulf, 3 m below the water surface, one from the north shore and the other from the south shore of Kuwait. Both strains were morphologically, ultrastructurally, and albeit to a less extend, phylogenetically similar to Acaryochloris. However, both isolates lacked chlorophyll d and produced instead chlorophyll a, as the major photosynthetic pigment. Both picocyanobacterial isolates were associated with oil-utilizing bacteria in the magnitude of 10⁵ cells g^?1. According to their 16S rRNA gene sequences, bacteria associated with the isolate from the north were affiliated to Paenibacillus sp., Bacillus pumilus, and Marinobacter aquaeolei, but those associated with the isolate from the south were affiliated to Bacillus asahii and Alcanivorax jadensis. These bacterial differences were probably due to environmental variations. In batch cultures, the bacterial consortia in the nonaxenic biomass as well as the pure bacterial isolates effectively consumed crude oil and pure aliphatic and aromatic hydrocarbons, including very high-molecular-weight compounds. Water and diethylether extracts from the phototrophic biomass enhanced growth of individual bacterial isolates and their hydrocarbon-consumption potential in batch cultures. It was concluded that these consortia could be promising in bioremediation of hydrocarbon pollutants, especially heavy sediments in the marine ecosystem.     

Isolation and molecular characterization of a novel strain of Bacillus with antifungal activity from the sorghum rhizosphere

We looked for bacterial strains with antifungal activity in the sorghum rhizosphere. A prescreening procedure to search for hemolytic activity among the isolated strains allowed us to detect good fungitoxic activity in a bacterial isolate that we named UM96. This bacterial isolate showed strong growth inhibition in bioassays against the pathogens Diaporthe phaseolorum, Colletotrichum acutatum, Rhizoctonia solani, and Fusarium oxysporum. The supernatant of isolate UM96 also showed strong hemolytic activity, which was not observed in the protease-treated supernatant. However, the supernatant that was treated with protease had similar antagonistic effects to those exhibited by the supernatant that was not treated with this enzyme. These results suggest that a bacteriocin-like compound is responsible for the hemolytic activity; whereas, as far as antifungal effect is concerned, an antibiotic of nonribosomal origin, such as a lipopeptide, might be acting. Further molecular characterization by partial 16S rDNA sequencing placed isolate UM96 in a cluster with Bacillus amyloliquefaciens; however, the highest identity match found in databases of Bacillus species was 91% identity. This suggests that Bacillus sp UM96 might be a novel species.     

Enhanced inulinase production in solid state fermentation by a mutant of the marine yeast <Emphasis Type="Italic">Pichia guilliermondii</Emphasis> using surface response methodology and inulin hydrolysis

In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl₂. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.     

Molecular characterization of a lipase-producing <Emphasis Type="Italic">Bacillus pumilus</Emphasis> strain (NMSN-1d) utilizing colloidal water-dispersible polyurethane

Bacillus pumilus strain NMSN-1d isolated from polyurethane-contaminated water was found to grow in high salt concentration (NaCl 10%, w/v) and degrade Impranil-DLN, water-dispersible polyurethane. The genetic relatedness of the isolate has been established by standard molecular biological techniques and the enzyme(s) involved in polyurethane degradation were also studied. A total of nine bacterial strains were isolated from polyurethane-polluted sites and characterized by conventional, microbiological and biochemical methods. These isolates were subjected to 16S ribosomal RNA gene amplification by PCR using specific primers. The genetic relatedness of the isolates was also ascertained by ribotyping and BLAST analysis of the 16S ribosomal RNA gene sequences. The bacterial isolates were grown in yeast extract-salts minimal broth medium supplemented with water-dispersible polyurethane (Impranil DLN) as a sole source of carbon. The promising isolate utilizing polyurethane and producing lipase was identified as Bacillus pumilus NMSN-1d. The polyurethane degradation has been studied in polyurethane-Rhodamine-B and Luria-Bertani-polyurethane plate assays. The activity of hydrolytic enzymes such as lipase and esterase was confirmed on 2xYT-olive oil and tributyrin-Tween 20 plate assay. The newly isolated Bacillus pumilus appears promising in the management of polyurethane waste and in production of industrially important enzymes.     

Characterization of marine bacterial carbonic anhydrase and their CO2 sequestration abilities based on a soil microcosm

Abstract

The novel technology of biological carbon sequestration using microbial enzymes have numerous advantages over conventional sequestration strategies. In the present study, extracellular carbonic anhydrase (CA) producing bacteria were isolated from water samples in the Arabian Sea, India. A potential isolate, Bacillus safensis isolate AS-75 was identified based on 16S rDNA sequence analysis. The culture conditions suitable for CA production were 32?°C incubation temperature with 4% NaCl and 10?mM Zn supplementation. Experimental optimization of culture conditions enhanced enzyme activity to 265?U mL^?1. CA specific gene was characterized and based on the analysis, the CA of B. safensis isolate AS-75 was a leucine (11.3%) with α-helices as the dominant component in its secondary structure. Based on soil microcosm studies, CA could sequester CO₂ by 95.4%?±?0.11% in sterilized soil with enzyme microcosm. Hence, the application of enzyme was found to be more effective in removing CO₂.     

Molecular characterization and estimation of cellulolytic potential of gut bacteria isolated from four white grub species native to Indian Himalayas

An investigation was carried out to isolate, identify and molecularly characterize the cellulose-degrading bacterial isolates from the guts of four white grub species (Anomala bengalensis, Brahmina coriacea, Holotrichia longipennis and Holotrichia setticollis) native to Uttarakhand, Himalayas through 16S rRNA sequencing. A total of 178 bacterial strains were isolated from different gut compartments of selected white grub species, of which 95 bacterial isolates showed cellulose metabolizing activities in the CMC assay. Maximum degraders i.e., 38 were isolated from A. bengalensis, of which 18 were isolated from the fermentation chamber. The value of cellulolytic index ranged between 0.05 and 16 showing a variable cellulolytic activity by degraders. A total of 25 potent strains of cellulose-degrading bacteria recording cellulolytic activity > 1 were isolated and sequenced for 16S rRNA gene. Bacillus stratosphericus strain CBG4MG1 (10.78 ± 4.18), Bacillus cereus strain CBG2FC1 (10.33 ± 3.53), Bacillus sp. strain CBG3MG2 (7.28 ± 0.16) and Paenibacillus ginsengagri strain CBG1FC2 (5.66 ± 2.67) were the most potent cellulose-degrading bacteria isolated from the gut of B. coriacea, H. longipennis, H. setticollis and A. bengalensis, respectively. Thus, the cellulolytic bacteria isolated from the gut of selected white grub species may be good sources for profiling novel isolates for industrial use besides identifying eco-friendly solutions for agro-waste management.     

The Determination of Assay for Laccase of Bacillus subtilis WPI with Two Classes of Chemical Compounds as Substrates

Ligninolytic enzyme complexes are involved in lignin degradation. Among them laccases are outstanding because they use molecular oxygen as a co-substrate instead of hydrogen peroxide as used by peroxidases. Bacterial laccase of Bacillus genus was first reported in Claus and Filip (Microbiol Res 152:209–216, 1997), since then more bacterial laccases have been found. In this research, laccase-producing bacteria were screened from pulp and paper industry wastewater, bagass and sugarcane rhizosphere. Nutrient agar medium containing 0.5 mM of guaiacol was used. It was observed that the laccase-producing strains developed brown colour from which 16 strains of Bacillus were identified. One of the isolated strains was identified as Bacillus subtilis WPI based on the results of biochemical tests and 16S rDNA sequence analysis. This strain showed laccase-like activity towards the oxidizing substrates ABTS and guaiacol. In this study guaiacol was used as the substrate of laccase activity assay. For determination of laccase activity of this isolate guaiacol was used as a substrate of assay for the first time in this study. SDS-PAGE and Native-PAGE confirmed the presence of laccase.     

Identification and characterization of thermophilic amylase producing bacterial isolates from the brick kiln soil

The present experiment was designed to isolate bacterial strains from the brick kiln soil and to check the activity and enzyme kinetics of amylase from these isolates. The bacterial colonies were isolated from soil samples through the serial dilution method. The bacterial isolates were identified through morphological, electron microscopic and molecular analysis. The 16S ribosomal RNA sequences of the isolates IR-1, IR-2, IR-3, IR-8, and IR-9 showed high similarities with Bacillus tequilensis, Bacillus paramycoides, Proteus alimentorum, Bacillus wiedmannii, and Pseudomonas aeruginosa, respectively. All of the bacterial isolates showed a positive catalase activity except IR-9. Furthermore, the isolates showed variable antagonistic effects against different bacterial pathogens. All of the strains produced indole acetic acid (IAA), and the concentrations increased in the presence of tryptophan application. The isolates showed the amylase enzyme activity and maximum activity of isolates was achieved in 4% starch concentration. The IR-9 isolate showed the highest amylase activity of 5.9 U/ml. The V_max values of the extracellular amylase from different bacterial isolates ranged between 12.90 and 50.00 IU ml⁻¹. The lowest K_m value of 6.33 mg starch was recorded for IR-8 and the maximum K_cat value of 2.50 min⁻¹ was observed for IR-3. The amylase activity of the isolates was significantly affected by a range of different incubation time, temperature, and pH values. Further tests are required before the potential utilization of these isolates for amylase production, and in the biopesticide and biofertilizer applications.     

Effect of Carbon Source on the Antimicrobial Activity of the Air Flora

Summary In an attempt to screen for air flora producing new potent antimicrobial substances, Bacillus megaterium NB-3, Bacillus cereus NB-4, Bacillus cereus NB-5, Bacillus subtilis NB-6 and Bacillus circulans NB-7, were isolated and were found to be antagonistic to bacteria and/or fungi. Production of antimicrobial substances by the bacterial strains was greatly influenced by variation of carbon sources. Glycerol strongly enhanced the antimicrobial activity of strains NB-3 and NB-6, whereas glucose increased the antimicrobial activity of strains NB-4 and NB-5. The maximum antibiotic yield of NB-7 was achieved with fructose as a carbon source. Starch (Bacillus megaterium NB-3), maltose (Bacillus cereus NB-5), glycerol (Bacillus circulans NB-7), arabinose, ribose (Bacillus cereus NB-4) and arabinose, fructose, glucose, ribose and sucrose (Bacillus subtilis NB-6) repressed the production of antimicrobial substances by the respective bacterial strains.     

Antibacterial activity of soil bacteria isolated from Kochi,India and their molecular identification

The present study, deal about the antibiosis activity of soil bacteria, isolated from 10 different locations of rhizosphere and diverse cultivation at Kochi, Kerala, India. The bacteria were isolated by standard serial dilution plate techniques. Morphological characterization of the isolate was done by Gram’s staining and found that all of them gram positive. Isolated bacteria were tested against 6 human pathogens viz., Escherichia coli, Enterococcus sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Acinetobacter sp. Primary screening was carried out by perpendicular streaking and seed overlay method. Based on the result of primary screening most potential isolates of S1A1 and S7A3 were selected for secondary screening. Both the isolates showed positive results against Enterococcus sp. and S.aureus. The maximum antagonistic activity of 20.98 and 27.08?mm zone of inhibition was recorded at S1A1 against Enterococcus sp. and S. aureus respectively, at 180?µl concentration. Molecular identification was carried out by 16S rRNA sequence. The 16S rRNA was amplified from the DNA samples by using PCR. The amplified 16S rRNA PCR products were purified and sequenced. The sequences were subjected to NCBI BLAST. The isolates S1A1 and S7A3 BLAST results showed 99% and 95% respectively, similarity with the available database sequence of Bacillus amyloliquefaciens. The sequences were deposited in GenBank and the accession numbers KY864390 (S1A1) and KY880975 (S7A3) were obtained.     

Effects of microbial elicitor on production of hypocrellin by Shiraia bambusicola

Some endophyte isolates were isolated in a bamboo pole sample parasitized the fungus Shiraia bambusicola from Zhejiang Province. After screening through hypocrellin bacteriostatic effect and fermentation test, we got the isolate TX4 of bacterial elicitor and GZUIFR-TT1 of fungal elicitor which had certain effect to promote S. bambusicola to produce hypocrellin. The Plackett–Burman design was introduced to evaluate the effects of nine factors based on single-factor test. Yeast extract, glucose, and isolate GZUIFR-TT1 elicitor were found to be the critical activity factors for increasing the total hypocrellin production. So we identified the isolate GZUIFR-TT1 as Trametes sp. Through response surface methodology, we obtained the optimum production conditions as follows: yeast extract, 2.99 g/L; glucose, 32.45 g/L; and Trametes sp. elicitor, 81.40 μg/mL. Under the above conditions, the experimental value of hypocrellin production was 102.60 mg/L, compared with the control it increased about 7.90 times.     

Production of novel alkalitolerant and thermostable inulinase from marine actinomycete Nocardiopsis sp. DN-K15 and inulin hydrolysis by the enzyme

An actinomycete strain Nocardiopsis sp. DN-K15 showing high inulinolytic activity was isolated from marine sediment of Jiaozhou Bay in China. Under optimal conditions, Nocardiopsis sp. DN-K15 produced 25.1 U/ml of inulinase within 60 h of fermentation at shake flask level, which was 2.7-fold higher than the level in the basal medium. The optimal pH and temperature of the inulinase from strain DN-K15 were determined to be 60 °C and pH 8.0, respectively. The inulinase was highly active over a wide pH range (5.0–11.0) and retained more than 81 % of residual activity after incubation at 60 °C for 1 h, indicating its alkali-tolerant and thermostable nature. Thin layer chromatography analysis revealed that fructose was the main product of inulin hydrolysis, indicating its exoinulinase activity. The high yield of extracellular inulinase combined with its novel enzymatic property made Nocardiopsis sp. DN-K15 a potential candidate in biotechnological and industrial applications.     

Inhibition of Bacterial Growth, Enterotoxin Production, and Spore Outgrowth in Strains of Bacillus cereus by Bacteriocin AS-48

Bacteriocin AS-48 showed high bactericidal activity for mesophilic and psychrotrophic strains of Bacillus cereus over a broad pH range. AS-48 inhibition of the enterotoxin-producing strain LWL1 was enhanced by sodium nitrite, sodium lactate, and sodium chloride. The latter also enhanced AS-48 activity against strain CECT 131. Bacterial growth and enterotoxin production by strain LWL1 were completely inhibited at bacteriocin concentrations of 7.5 μg/ml. At subinhibitory bacteriocin concentrations, enterotoxin production decreased markedly and sporulation was delayed. Intact spores were resistant to AS-48 but became gradually sensitive to AS-48 during the course of germination.