Isolation of <Emphasis Type="Italic">Serratia marcescens</Emphasis> SR1 as a Source of Chitinase Having Potentiality of Using as a Biocontrol Agent

Serratia marcescens, strain SR₁ was isolated from the local soil of a cultivated farm and it was screened as potent strain for chitinase production. Maximum chitinase production (77.3 u Mh⁻¹ 100⁻¹) was observed after 96 h of incubation period with pH 5.5 at 30°C under shake conditions (120 rpm). Compare to still flasks, shake culture with prawn fish colloidal chitin of 0.5% (w/v) concentration, showed a better enzyme yield. Crude enzyme showed antifungal activity against plant pathogens.     

Chitinase Production in Solid-State Fermentation from <Emphasis Type="Italic">Oerskovia xanthineolytica</Emphasis> NCIM 2839 and its Application in Fungal Protoplasts Formation

The present study reports the economic production of thermostable chitinase production from Oerskovia xanthineolytica NCIM 2839 by solid-state fermentation (SSF) technique and its application in fungal protoplasts formation. The Oerskovia xanthineolytica NCIM 2839 was found to produce thermostable chitinase 148 U g⁻¹ of solid substrate in SSF using wheat bran with colloidal chitin as base. Protoplasts of A. niger were formed by using crude chitinase produced in SSF and formed protoplasts were confirmed by using scanning electron microscopy. This is the simple and economical method for protoplast formation which makes it possible applications in strain improvement of various fungi by protoplasts fusion in Biotechnological industries.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Study of Biochemical Pathway and Enzyme Involved in Metsulfuron-Methyl Degradation by <Emphasis Type="Italic">Ancylobacter</Emphasis> sp. XJ-412-1 Isolated from Soil

Ancylobacter sp. XJ-412-1, capable of degrading metsulfuron-methyl, was isolated from sulfonylurea-contaminated soil. When metsulfuron-methyl was provided as the sole carbon source, more than 90.5% of metsulfuron-methyl at concentration of 50 mg l⁻¹ was degraded by strain XJ-412-1 after incubation at 30°C for 7 days. The initial degradation products of metsulfuron-methyl (MSM), thifensulfuron-methyl (TSM), and bensulfuron-methyl (BSM) by XJ-412-1 were identified as corresponding deesterified derivatives by liquid chromatography-mass spectrometry, which indicated a primary pathway of the deesterification of these three sulfonylurea herbicides. The carboxyesterase activity of the cell-free extracts was assayed and strongly inhibited by 4-chloromercuribenzoic acid (PCMB), diethyl pyrocarbonate (DEPC), phenylmethylsulfonyl fluoride (PMSF), and malathion.     

Establishment of functional primary cultures of heart cells from the clam <Emphasis Type="Italic">Ruditapes decussatus</Emphasis>

Heart cells from the clam Ruditapes decussatus were routinely cultured with a high level of reproducibility in sea water based medium. Three cell types attached to the plastic after 2 days and could be maintained in vitro for at least 1 month: epithelial-like cells, round cells and fibroblastic cells. Fibroblastic cells were identified as functional cardiomyocytes due to their spontaneous beating, their ultrastructural characteristics and their reactivity with antibodies against sarcomeric α-actinin, sarcomeric tropomyosin, myosin and troponin T-C. Patch clamp measurements allowed the identification of ionic currents characteristic of cardiomyocytes: a delayed potassium current (I _K slow) strongly suppressed (95%) by tetraethylammonium (1 mM), a fast inactivating potassium current (I _K fast) inhibited (50%) by 4 amino-pyridine at 1 mM and, at a lower level (34%) by TEA, a calcium dependent potassium current (I _KCa) activated by strong depolarization. Three inward voltage activated currents were also characterized in some cardiomyocytes: L-type calcium current (I _Ca) inhibited by verapamil at 5 × 10⁻⁴ M, T-type Ca²⁺ current, rapidly activated and inactivated, and sodium current (I _Na) observed in only a few cells after strong hyperpolarization. These two currents did not seem to be physiologically essential in the initiation of the beatings of cardiomyocytes. Potassium currents were partially inhibited by tributyltin (TBT) (1 μM) but not by okadaic acid (two marine pollutants). DNA synthesis was also demonstrated in few cultured cells using BrdU (bromo-2′-deoxyuridine). Observed effects of okadaic acid and TBT demonstrated that cultured heart cells from clam Ruditapes decussatus can be used as an experimental model in marine toxicology.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Adaptive evolution of nontransgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> KC01 for improved ethanol tolerance and homoethanol fermentation from xylose

Due to its excellent capability to ferment five-carbon sugars, Escherichia coli has been considered one of the platform organisms to be engineered for production of cellulosic ethanol. Nevertheless, genetically engineered ethanologenic E. coli lacks the essential trait of alcohol tolerance. Development of ethanol tolerance is required for cost-effective ethanol fermentation. In this study, we improved alcohol tolerance of a nontransgenic E. coli KC01 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) through adaptive evolution. During ~350 generations of adaptive evolution, a gradually increased concentration of ethanol was used as a selection pressure to enrich ethanol-tolerant mutants. The evolved mutant, E. coli SZ470, was able to grow anaerobically at 40 g l⁻¹ ethanol, a twofold improvement over parent KC01. When compared with KC01 for small-scale (500 ml) xylose (50 g l⁻¹) fermentation, SZ470 achieved 67% higher cell mass, 48% faster volumetric ethanol productivity, and 50% shorter time to complete fermentation with ethanol titer of 23.5 g l⁻¹ and yield of 94%. These results demonstrate that an industry-oriented nontransgenic E. coli strain could be developed through incremental improvements of desired traits by a combination of molecular biology and traditional microbiology techniques.     

An antifungal chitinase produced by Bacillus subtilis using chitin waste as a carbon source

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish-processing waste. In this study, shrimp and crab shell powder, prepared by treating shrimp- and crab-processing waste by boiling and crushing, was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus subtilis NPU 001, a strain isolated from soil samples, excreted a chitinase when cultured in a medium containing 2% (w/v) shrimp and crab shell powder as the major carbon source. The chitinase, which was purified by sequential chromatography, had a Mw of 31 kDa and a pI of 5.4. The purified chitinase (2 mg ml⁻¹) inhibited hyphal extension of the fungus Fusarium oxysporum. Compared with other known bacterial chitinases, the unique characteristics of NPU 001 chitinase include antifungal activity against plant-pathogenic fungi and the production of chitotriose as the major enzymatic hydrolysate from colloidal chitin.     

Proficient biodegradation of shrimp shell waste by Aeromonas hydrophila SBK1 for the concomitant production of antifungal chitinase and antioxidant chitosaccharides

This study aimed to optimize the biodegradation of shrimp shell waste by Aeromonas hydrophila SBK1 for the co-production of chitinase and chitosaccharides (CS) under submerged fermentation and evaluation of their bioactivities. Canonical analysis and parametric optimization wrought the peakest production of chitinase (21.48 U/ml) and CS (124 μg/ml) after 66.4 h of fermentation at 37.6 °C. The medium containing 2.64% (w/v) shrimp shell powder, 0.38% (w/v) NaCl, 6.86 × 10⁶ cfu/ml inoculum concentration and an agitation speed of 120 rpm were found best. These optimized parameters were also authenticated by scale up of fermentation in 5 L fermentor and a reproducible results obtained with specific yield of chitinase (Y_P/S^chi) of 958.82 U/g and CS (Y_P/S^CS) 5.5 mg/g. A 59 kD chitinase was purified from culture filtrate by sequential chromatography techniques. The enzyme exhibited high degree of antifungal activity particularly against pathogenic Aspergillus flavus and Fusarium oxysporum by dissolving their cell wall components. The IC₅₀ values for A. flavus and F. oxysporum were 3.7 and 4.5 U/ml of purified chitinase, respectively. Chitosaccharides were extracted from the culture filtrate, quantitatively identified as admixture of N-acetylglucosamine monomer (57.5%) and dimer (39.2%). These chitosaccharides have potential antioxidant activity as detected by in vitro free radical scavenging assay.     

Optimization of Process Variables for Lipase Biosynthesis from <Emphasis Type="Italic">Rhizopus oligosporus</Emphasis> NRRL 5905 Using Evolutionary Operation Factorial Design Technique

Extracellular lipase was produced from Rhizopus oligosporus NRRL 5905 through solid state fermentation (SSF). To provide an optimum fermentation conditions for maximum lipase yield, five process variables (temperature, liquid–solid ratio, pH, incubation period and spore concentration) were optimized using evolutionary operation (EVOP) factorial design technique taking into account the interaction between the process variables. Optimization through EVOP resulted in around 3 fold increase in lipase activity (77 U gds⁻¹) at a liquid–solid ratio of 1.5:1, fermentation temperature of 35°C, initial fermentation pH 6, incubation period 5 days and a spore concentration of 10⁸ spores ml⁻¹.     

Chitinolytic enzymes from the newly isolated <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> SBK1: study of the mosquitocidal activity

Aeromonas hydrophila SBK1 (GenBank accession no. HM802878.1), a potent chitinolytic bacterium, was isolated from a pool of 30 chitinolytic isolates. The isolate showed higher chitinolytic activity in respect to clear zone to colony size ratio of 2.15. Maximum production of chitinolytic enzymes, viz., β-N-acetyl-glucosaminidase and chitinase (specific activity 655.3 and 71.6 U mg⁻¹, respectively) by A. hydrophila SBK1 was observed in the synthetic media, containing (w/v)-colloidal chitin, 4.0%; peptone, 0.3%; phosphate, 0.3% (0.15% of each KH₂PO₄ and K₂HPO₄); NaCl, 0.25%; MgSO₄, 0.05%; KCl, 0.05%; pH 7.0 and at 35°C after 72 h of incubation. Both carbon-to-nitrogen (C/N) and carbon-to-phosphate (C/P) ratio of 13.33 were found optimum for chitinase production. Enzyme productivity increased about twofold in optimized culture condition in respect to its un-optimized state. The crude enzyme showed optimum activity against Culex quinquefasciatus larvae in native water at pH 7.0 and 35°C (LD₅₀ 0.60 U ml⁻¹ at 48 h). Therefore, the studied chitinases can be used as an effective mosquitocidal agent.     

Atrazine degradation by a simple consortium of <Emphasis Type="Italic">Klebsiella</Emphasis> sp. A1 and <Emphasis Type="Italic">Comamonas</Emphasis> sp. A2 in nitrogen enriched medium

A simple consortium consisted of two members of Klebsiella sp. A1 and Comamonas sp. A2 was isolated from the sewage of a pesticide mill in China. One member of Klebsiella sp. A1 is a novel strain that could use atrazine as the sole carbon and nitrogen source. The consortium showed high atrazine-mineralizing efficiency and about 83.3% of 5 g l⁻¹ atrazine could be mineralized after 24 h degradation. Contrary to many other reported microorganisms, the consortium was insensitive to some nitrogenous fertilizers commonly used, not only in presence of 200 mg l⁻¹ atrazine but also in 5 g l⁻¹ atrazine mediums. After 24 h incubation, 200 mg l⁻¹ atrazine was completely mineralized despite of the presence of urea, (NH₄)₂CO₃ and (NH₄)₂HPO₄ in the medium. Very minor influence was observed when NH₄Cl was added as additional nitrogen source. Advantages of the simple consortium, high mineralizing efficiency and insensitivity to most of exogenous nitrogen sources, all suggested application potential of the consortium for the bioremediation of atrazine-contaminated soils and waters.     

Response of Arbuscular Mycorrhizal Fungi on Wheat (Triticum aestivum L.) Grown Conventionally and on Beds in a Sandy Loam Soil

The present study was undertaken to assess the benefit and compare the functioning of AM fungi on wheat grown conventionally and on beds. Ten treatment combinations were used, treatments 1 and 2: no fertilizers with and without arbuscular mycorrhizal (AM) fungi (In vitro produced Glomus intraradices); 3:100% of recommended NPK: (120 kg ha⁻¹ N; 60 kg ha⁻¹ P; 50 kg ha⁻¹ K), and 4 and 5: 75% of recommended NPK dose with and without AM inoculation in a 5 × 2 split-plot design on wheat using conventional/flat system and elevated/raised bed system. The maximum grain yield (3.84 t ha⁻¹) was obtained in AM fungi inoculated plots of raised bed system applied with 75% NPK and was found higher (although non- significant) than the conventional (3.73 t ha⁻¹) system. The AM inoculation at 75% fertilizer application can save 8.47, 5.38 kg P and 16.95, 10.75 kg N ha⁻¹, respectively, in bed and conventional system. While comparing the yield response with 100% fertilizer application alone, AM inoculation was found to save 20.30, 15.79 kg P and 40.60, 31.59 kg N ha⁻¹, respectively, in beds and conventional system. Mycorrhizal inoculation at 75% NPK application particularly in raised bed system seems to be more efficient in saving fertilizer inputs and utilizing P for producing higher yield and growth unlike non-mycorrhizal plants of 100% P. Besides the yield, mycorrhizal plants grown on beds had higher AM root colonization, soil dehydrogenases activity, and P-uptake. The present study indicates that the inoculation of AM fungi to wheat under raised beds is better response (although non-significantly higher) to conventional system and could be adopted for achieving higher yield of wheat at reduced fertilizer inputs after field validation.     

Biotransformation of Acetamide to Acetohydroxamic Acid at Bench Scale Using Acyl Transferase Activity of Amidase of <Emphasis Type="Italic">Geobacillus pallidus</Emphasis> BTP-5x MTCC 9225

The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaH₂PO₄/Na₂HPO₄ buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaH₂PO₄/Na₂HPO₄ buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90–95% (mol mol⁻¹) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization.     

Decolorization of Azo Dye (Orange MR) by an Autochthonous Bacterium, <Emphasis Type="Italic">Micrococcus</Emphasis> sp. DBS 2

Soil and sediment samples obtained from Orange MR dye contaminated habitat were screened for heterotrophic bacterial population. The heterotrophic bacterial density of dye-contaminated soil was 2.14 × 10⁶ CFU/g. The generic composition of heterotrophic bacterial population was primarily composed of 10% of Proteus sp., 15% Aeromonas sp., 20% Bacillus sp., 25% Pseudomonas sp. and 30% Micrococcus sp. The bacterial strain that decolorized the azo dye Orange MR up to 900 ppm was identified as Micrococcus sp. The optimum inoculum load, pH and temperature were found to be 5%, 6 and 35°C, respectively. The rate of decolorization was assessed using spectrophotometer at 530 nm and the percentage of decolorization was ascertained. The autochthonous bacterial isolate was able to utilize the dye as both nitrogen and carbon source.     

Changes of Curdlan Biosynthesis and Nitrogenous Compounds Utilization Characterized in <Emphasis Type="Italic">ntrC</Emphasis> Mutant of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

The regulatory function of global regulator NtrC on curdlan biosynthesis and nitrogen consumption under nitrogen-limited condition in Agrobacterium sp. ATCC 31749 was investigated. The ntrC mutant of Agrobacterium sp. was constructed by homologous recombination. The ability to utilize NH₄Cl and KNO₃ was impaired in the mutant. Other nitrogenous compounds, such as glutamic acid and glutamine, were utilized normally. Curdlan production capability was impaired severely in the mutant. Curdlan production was 5-fold lower than the wild type strain in batch fermentation with NH₄Cl as the sole nitrogen source. However, up to 6.5 g l⁻¹ of a newly found alkali-insoluble biopolymer was produced by the ntrC mutant when glutamic acid was used as nitrogen source. The new biopolymer had glycosidic bond and hydroxyl group but no β-configuration absorption peak on IR spectrum was found as different from curdlan. In addition, the mutant exhibited a rapid morphological change from the dot to rod form. These results deduced that the global regulator NtrC was involved in curdlan and other biopolymer biosynthesis in Agrobacterium sp. ATCC 31749 in response to nitrogen-limited condition.     

Toxicity of Crude Extracellular Products of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> on Rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis> (Ham.)

In the present study the haemolytic and proteolytic activity of extracellular products (ECP) secreted from Aeromonas hydrophila (CAHH14 strain) were studied with respect to temperature and different time of incubation as well as its lethal toxicity on rohu, Labeo rohita. The strain was isolated from Catla catla (showing abdominal dropsy symptom) collected from the pond of Central Institute of Freshwater Aquaculture (CIFA), Bhubaneswar, India and was characterized on the basis of biochemical tests. The highest production of haemolysin was achieved when the bacteria was grown at 35°C for 30 h. The proteolytic activity was found to be highest when the bacterium was grown at 30°C for 36 h. The haemolytic and proteolytic toxin produced by Aeromonas hydrophila was found to be lethal to rohu (LD₅₀ 1.7 × 10⁴ cfu/ml). The lethality of ECP was decreased by heating and completely inactivated by boiling at 100°C for 10 min. This indicates that protease activity and haemolytic activity of A. hydrophila ECP was temperature dependant.     

Continuous butanol production with reduced byproducts formation from glycerol by a hyper producing mutant of <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Butanol, a four-carbon primary alcohol (C₄H₁₀O), is an important industrial chemical and has a good potential to be used as a superior biofuel. Bio-based production of butanol from renewable feedstock is a promising and sustainable alternative to substitute petroleum-based fuels. Here, we report the development of a process for butanol production from glycerol, which is abundantly available as a byproduct of biodiesel production. First, a hyper butanol producing strain of Clostridium pasteurianum was isolated by chemical mutagenesis. The best mutant strain, C. pasteurianum MBEL_GLY2, was able to produce 10.8 g l⁻¹ butanol from 80 g l⁻¹ glycerol as compared to 7.6 g l⁻¹ butanol produced by the parent strain. Next, the process parameters were optimized to maximize butanol production from glycerol. Under the optimized batch condition, the butanol concentration, yield, and productivity of 17.8 g l⁻¹, 0.30 g g⁻¹, and 0.43 g l⁻¹ h⁻¹ could be achieved. Finally, continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was carried out using glycerol as a major carbon source at several different dilution rates. The continuous fermentation was run for 710 h without strain degeneration. The acetone–butanol–ethanol productivity and the butanol productivity of 8.3 and 7.8 g l⁻¹ h⁻¹, respectively, could be achieved at the dilution rate of 0.9 h⁻¹. This study reports continuous production of butanol with reduced byproducts formation from glycerol using C. pasteurianum, and thus could help design a bioprocess for the improved production of butanol.     

Efficient constitutive expression of chitinase in the mother cell of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and its potential to enhance the toxicity of Cry1Ac protoxin

Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry⁻B. The recombinant plasmid was stably maintained over 240 generations in Cry⁻B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l⁻¹ Na₂CO₃–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.