Cellulase Producing Bacteria from the Wood-Yards on Kallai River Bank

This study evaluates the influence of growth parameters such as pH, temperature, Carboxy Methyl Cellulose (CMC) concentration and agitation on cellulase production from three bacterial strains, viz., Achromobacter xylosoxidans BSS4, Bacillus sp. BSS3 and Pseudomonas sp. BSS2 isolated from the wood-yards on Kallai river bank in Kerala. Production of cellulase by these isolates was detected using basal salt medium (BSM) with 0.5% CMC as supplement, and CMCase activity was confirmed by iodine test. Dinitrosalicylic acid method was employed for assaying the cellulase production by measuring the amount of glucose liberated in μmol/mL/min. Maximum enzyme production from Pseudomonas sp. BSS2 was at pH 8, 37℃ with 1% CMC and 150 rpm, and cellulase production increased from initial 49.84 U/mL to 91.28 U/mL after optimization. The highest enzyme activity from Bacillus sp. BSS3 was at pH 9, 37℃ with 1% CMC, 150 rpm, and cellulase production increased from initial 26.05 U/mL to 104.68 U/mL after optimization. The maximum enzyme production from A. xylosoxidans BSS4 was at pH 7, 40℃ with 0.5% CMC and 150 rpm, and cellulase production increased from initial 55.28 U/mL to 68.37 U/mL after optimization. Thus among the three isolates, Bacillus sp. BSS3 showed maximum enzyme yield which can be explored for further scale up studies with an industrial perspective.

Rubber Seed Kernel as Potent Solid Substrate for the Production of Lipase by Pseudomonas aeruginosa Strain BUP2

This study explored the utility of flours of rubber seed, coconut and groundnut kernels, and de-oiled cakes of coconut and groundnut as solid substrate for the production of lipase by Pseudomonas aeruginosa strain BUP2 (MTCC No. 5924), a novel bacterium reported from the rumen of Malabari goat. Various proportions (10%, 20%, 30%, 40% or 50%) of flours or cakes were prepared (w/v) with BUP medium (pH 4, 5, 6, 7 or 8), and incubated at different temperature (25°C, 28°C, 30°C or 32°C) for 24 to 96 h. The samples were assayed for lipase activity at 24 h intervals. The rubber seed flour (20%)-BUP medium supported the maximum lipase production (871 U/gds) at 48h incubation (pH 6, 28°C), followed by ground nut flour (398 U/gds), while ground nut cake supported the least lipase production (244 U/gds). From this, it is evident that the cheaply available rubber seed is an efficient substrate for the production of lipase, irrespective of its known demerit that it contains the limarin, a toxin;in fact, we could not detect limarin in the fermented matter. Thus, the utility of rubber seed for the production of a costly enzyme is reported from a novel rumen bacterium, which would be advantageous for rubber farmers.

Optimization of Parameters for the Production of Lipase from Pseudomonas sp. BUP6 by Solid State Fermentation

Solid-state fermentation (SSF) holds tremendous potentials for the production of industrially significant enzymes. The present study describes the production of lipase by a novel rumen bacterium, Pseudomonas sp. strain BUP6 on agro-industrial residues. Pseudomonas sp. strain BUP6 showed higher lipase production when grown in Basal salt medium (BSM) supplemented with oil cakes. Initially, five different oil cakes (obtained after extracting oil from coconut, groundnut, cotton seed, gingelly or soybean) were screened to find out the most suitable substrate-cum-inducer for the production of lipase. Among them, groundnut cake supported the maximum production of lipase (107.44 U/gds). Box-Behnken Design (BBD), followed by response surface methodology (RSM) was employed to optimize the culture parameters for maximizing the production of lipase. Using the software Minitab 14, four different parameters like temperature, pH, moisture content and incubation time were selected for the statistical optimization, which resulted in 0.7 fold increase (i.e., 180.75 U/gds) in production of lipase under the optimum culture conditions (temperature 28&#176C, pH 5.9, moisture 33% and incubation 2 d). Thus, this study signifies the importance of SSF for the production of industrially-significant lipase using agro-industrial residues as solid support.

Biotic Stress Induced Biochemical and Isozyme Variations in Ginger and Tomato by <i>Ralstonia solanacearum</i>

This unique study evaluates the effects of Ralstonia solanacearum (Rs) induced biotic stress in two cultivars, Zingiber officinale (ginger) and Lycopersicon esculentum (tomato). They were grown in pots and hydroponic systems with controls;to induce biotic stress, about 8 × 104 colony forming units of Rs suspension was injected into the healthy test plants. Upon induction of Rs stress, highly significant (p 0.01) biochemical changes (%) were noticed in respect to controls: carbohydrate content was generally high in both plants;while they showed decreased starch and protein contents;phenolics showed a swing of decrease or increase between pot and hydroponic systems;and all plants in general showed higher (3-6 fold) proline content upon induction of biotic stress. Regarding oxidative stress isozymes (OSE), superoxide dismutase (EC 1.15.1.1) isozymes were normally 3, but treated hydroponics had 4 with comparable relative mobility values;peroxidase (EC 1.11.1.7) isozymes were generally 2, except for treated hydroponic tomato. Briefly, Rs induced biotic stress caused wilt symptoms in ginger, but did not affect tomato though its biochemical and OSE patterns especially in those grown as hydroponics were elicited to significantly higher levels.

Versatility of microbial proteases

Proteases or peptidases constitute the largest group of enzymes in bio-industry with a long array of uses. They play an invincible role in industrial biotechnology, especially in detergent, food and pharmaceutical arena. This focused review encompasses an overview on alkaline proteases, mainly of microbial sources in a handy module. Following an introduction and general classification with evolutionary insight, major sources of proteases (animal, plant and microbial including fungal, bacterial), their general properties with mechanism of action and molecular masses are discussed. Proteases fromBacillusspp. have been given special attention. In addition to this, an overview on the applications of proteases in detergent, tannery, food, metal recovery and waste treatment industries is also addressed briefly.

An Overview on the Crystal Toxins from <i>Bacillus thuringiensis</i>

Strains of Bacillus thuringiensis (Bt) are known to produce crystalline proteins (δ-endotoxins) concomitantly with sporulation during their stationary phase of growth, which are demonstrated as lethal to lepidopeterous, coleopeterous and dipterous insects in addition to mites, nematodes, protozoa and flukes. Upon ingestion, the δ-nascent endotoxin is an inactive protoxin complex of (Cry alone or Cry and Cyt toxins together) high molecular mass, which is cleaved upon ingestion into the active component proteins at the high alkaline environments in the digestive tract of these agricultural pests. Conventionally, Bt-crystals are being produced employing submerged or liquid fermentation techniques in commercial media, but recently many workers have used solid-state fermentation strategy for the enhanced production of Bt-toxin at low cost. Apart from δ-endotoxin, some isolates of Bt produce another class of insecticidal small molecules called β-exotoxin (thuringiensin), which may be harmful to humans. Moreover, resistance to Bt developed in various target pest is yet another concern for Bt-industry. Following a brief introduction, this review addresses various toxins produced by various strains of Bt, Bt production media and media formulations with emphasis to solid-state fermentation, general structure of Cry toxin, its mode of action, target pests, bioassay, resistance to Bt toxins and resistance management. Briefly, this review would provide the readers an overview on the general aspects of Bt toxin, its general structure and mechanism of action.

<i>Ralstonia solanacearum</i>Induction Causes Biochemical and Oxidative Stress Isozyme Variations in Mangroves without Wilt

We evaluated the effects of Ralstonia solanacearum (Rs) induced biotic stress in three mangroves, viz., Avicennia officinalis, Derris trifoliata and Excoecaria agallocha. These plants were grown in pots as well as hydroponic systems with sufficient controls, and about 8 × 104 colony forming units of Rs suspension was injected into the healthy test plants (saplings). The plants were subjected to biochemical and isozyme analyses. Upon induction of Rs stress, highly significant (p 0.01) biochemical changes (%) were noticed in respect to controls: carbohydrate content was generally high (24-36) in all plants;hydroponic mangroves showed higher starch content, mangroves under hydroponic system showed increased reducing sugars (29-46), almost all mangroves showed increased protein content;phenolics showed a swing of decrease or increase between plants grown in pot and hydroponic systems;and all plants in general showed higher proline content. Regarding oxidative stress isozymes (OSE) and superoxide dismutase (EC1.15.1.1), mangroves showed 1 or 2 additional isozymes with comparable relative mobility;similarly 1 or 2 additional peroxidase (EC1.11.1.7) isozymes were found in mangroves grown under hydroponic system. Briefly, Rs induced biotic stress did not cause any wilt symptom in all the 3 mangroves tested, but their normal biochemical and OSE patterns, especially of those grown as hydroponics were elicited to significantly higher levels.

A monograph on amylases from <i>Bacillus</i>spp.

Owing to the production of alpha, beta and gamma amylase subtypes;starch degrading microbes, especially bacteria have an invincible role in the food, fermentation, textile and paper industries. Of them, α-amylases from Bacillus spp. have contributed tremendous advancements in bio-industry, especially in starch, detergent and pharmaceutical arena. Though general reviews are seen in literature on amylases, no focused review is available yet solely on α-amylases produced by Bacillus spp. Hence, this focused review on α-amylases from the genus Bacillus is designed in such a way that it should give a vivid picture on most of the aspects on bacillial α-amylases in a handy module with an industrial perspective. With a short introduction on amylases in general, α-amylases from various species of Bacillus reviewed herein encompasses production of α-amylases by submerged and solid-state fermentations;nutrients and other factors required for maximizing production;immobilization strategies for whole cells or purified enzyme;an overview on the molecular weight of the enzyme;followed by distinct sections for purification, characterisation, stability and crystal structure;and concluded with a section on industrial applications of the α-amylases from Bacillus spp.

<i>Pseudomonas aeruginosa</i>BUP2—A Novel Strain Isolated from Malabari Goat Produces Type 2 Pyoverdine

This study focuses on the isolation and characterization of a novel strain of siderophore producing bacterium, i.e., Pseudomonas aeruginosa BUP2 (Pa BUP2) from the rumen of Malabari goat, coupled with qualitative and quantitative analyses of the siderophore produced by it. Pa BUP2—a facultative anaerobe was tuned to be an aerobe by repeatedly growing in Benjamin flask. The new isolate was grown in a specially designed semi-synthetic medium, designated as BUP medium, and the yellowish-green pigment produced was identified as a typical siderophore by spectrophotometry, Chromazurol-S assay, thin layer chromatography and isolectric focusing (IEF). The characteristic orange fluorescence upon UV irradiation on chromatogram and absorption maximum at λ404 confirmed that the characteristic siderophore produced by Pa BUP2 was a typical pyoverdine (PVD). This PVD was further categorized under type 2 by comparing its profile on the IEF gel with that of the representative strains of each PVD types, viz., Pa O1, Pa ATCC 27853 and Pa6. Moreover, the type 2 PVD was purified by XAD-4 Amberlite column chromatography and quantified;maximum yield (11.17 mg/ml) was observed on day 4 of incubation (37°C). Thus, it was confirmed that the bacterium isolated from the rumen content of Malabari goat is a novel strain of Pa capable of producing large quantity of PVD type 2 in specially designed BUP medium under aerobic condition, and that its clinical and industrial implications remain elusive.

Solid-State Fermentation for the Concomitant Production of δ-Endotoxin and Endospore from <i>Bacillus thuringiensis</i>subsp. <i>kurstaki</i>

Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt);and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h;while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores;malachite green-saffranin stained both δ-crystals and endospores;and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.

Lignocellulolytic activities of a novel strain of <i>Trichoderma harzianum</i>

This study describes a novel dark-green spore producing strain of Trichoderma harzianum exhibiting higher activities of cellulase, hemicellulase and ligninase on specific plate assays. To assess the cellulase production in detail, basal salt medium (BSM) was supplemented with synthetic [carboxymethyl cellulose (CMC), glucose, sucrose, dextrose, lactose or maltose] and natural (flours of banana, banana peel, jack seed, potato or tapioca) carbon as well as nitrogen (yeast extract, beef extract, peptone, NaNO3 or NH4NO3) sources. Temperature and pH optima were 28?C and 4, respectively for the growth of the fungus in CMC- BSM with 146 U/ml cellulase activity. Flours of potato and banana supported comparable yields of cellulase to that of CMC (147 U/ml and 168 U/ml, respectively), while sodium nitrate was the preferred nitrogen source (150 U/ml). The water soluble yellowish-green pigment (a probable siderophore) extracted from the spores showed an absorption maximum at 414 nm. To comprise, this fungus shows the complete lignocellulolytic potential which offers great industrial significance, especially for the ethanol production from the lignocellulosic waste coupled with the production of a new pigment.

A novel strain of <i>Trichoderma viride</i>shows complete lignocellulolytic activities

In this study we describe a novel dark-green strain of Trichoderma viride exhibiting complete ensemble of cellulase, hemicellulase and ligninase activities on specific plate assays. To assess the cellulase production in detail, basal salt medium (BSM) was fortified with synthetic (carboxymethyl cellulose (CMC), glucose, sucrose, dextrose, lactose or maltose) and natural (flours of banana, banana peel, jack seed, potato or tapioca) carbon as well as nitrogen (yeast extract, beef extract, peptone, NaNO3 or NH4NO3) sources. Temperature and pH optima were 28°C and 4, respectively for the growth of the fungus in CMC-BSM with 137 U/mL cellulase activity, which was enhanced to 173 U/mL at 1.25% CMC concentration. Flours of potato and banana peel supported comparable yields of cellulase to that of CMC, while sodium nitrate was the preferred nitrogen source. The water soluble bluish-green pigment (a probable siderophore) extracted from the spores showed an absorption maximum at 292 nm. To sum up, the complete lignocellulolytic potential of this fungus offers great industrial significance, coupled with the production of a new pigment.

Conjugated linoleic acid isomers and their precursor fatty acids regulate peroxisome proliferator-activated receptor subtypes and major peroxisome proliferator responsive element-bearing target genes in HepG2 cell model

The purpose of this study was to examine the induction profiles(as judged by quantitative reverse transcription polymerase chain reaction(qRT-PCR)) of peroxisome proliferator-activated receptor(PPAR) α,β,γ subtypes and major PPAR-target genes bearing a functional peroxisome proliferator responsive element(PPRE) in HepG2 cell model upon feeding with cis-9,trans-11-octadecadienoic acid(9-CLA) or trans-10,cis-12-octadecadienoic acid(10-CLA) or their precursor fatty acids(FAs).HepG2 cells were treated with 100 μmol/L 9-CLA or 10-CLA or their precursor FAs,viz.,oleic,linoleic,and trans-11-vaccenic acids against bezafibrate control to evaluate the induction/expression profiles of PPAR α,β,γ subtypes and major PPAR-target genes bearing a functional PPRE,i.e.,fatty acid transporter(FAT),glucose transporter-2(GLUT-2),liver-type FA binding protein(L-FABP),acyl CoA oxidase-1(ACOX-1),and peroxisomal bifunctional enzyme(PBE) with reference to β-actin as house keeping gene.Of the three housekeeping genes(glyceraldehyde 3-phosphate dehydrogenase(GAPDH),β-actin,and ubiquitin),β-actin was found to be stable.Dimethyl sulfoxide(DMSO),the common solubilizer of agonists,showed a significantly higher induction of genes analyzed.qRT-PCR profiles of CLAs and their precursor FAs clearly showed upregulation of FAT,GLUT-2,and L-FABP(～0.5-2.0-fold).Compared to 10-CLA,9-CLA decreased the induction of the FA metabolizing gene ACOX-1 less than did PBE,while 10-CLA decreased the induction of PBE less than did ACOX-1.Both CLAs and precursor FAs upregulated PPRE-bearing genes,but with comparatively less or marginal activation of PPAR subtypes.This indicates that the binding of CLAs and their precursor FAs to PPAR subtypes results in PPAR activation,thereby induction of the target transporter genes coupled with downstream lipid metabolising genes such as ACOX-1 and PBE.To sum up,the expression profiles of these candidate genes showed that CLAs and their precursor FAs are involved in lipid signalling by modulating the PPAR α,β,or γ subtype for the indirect activation of the PPAR-target genes,which may in turn be responsible for the supposed health effects of CLA,and that care should be taken while calculating the actual fold induction values of candidate genes with reference to housekeeping gene and DMSO as they may impart false positive results.