Evaluation of Various Unstructured Kinetic Models for the Production of Protease by Bacillus sphaericus MTTC511

Bacillus sphaericus MTCC511 was used for the production of protease in submerged batch fermentation. Maximum protease activity of 1010 U/L was obtained during a fermentation period of 24 h under optimized conditions of 30 °C in a medium with an initial pH of 7 and at a shaking rate of 120 rpm. The maximum biomass obtained in the batch fermentation was 2.55 g/L after 16 h. Various unstructured models were analyzed to simulate the experimental values of microbial growth, protease activity and substrate concentration. The unstructured models, i.e. the Monod model for microbial growth, the Monod incorporated Luedeking‐Piret model for the production of protease and the Monod‐incorporated modified Luedeking‐Piret model for the utilization of substrate were capable of predicting the fermentation profile with high coefficient of determination (R²) values of 0.9967, 0.9402 and 0.9729, respectively. The results indicated that the unstructured models were able to describe the fermentation kinetics more effectively.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

Combined application of native Trichoderma isolates possessing multiple functions for the control of Fusarium wilt disease in banana cv. Grand Naine

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is considered as a lethal disease of bananas worldwide. To manage the disease effectively, 20 rhizospheric and 43 endophytic Trichoderma isolates obtained from 12 different Foc resistant banana accessions were evaluated against Foc in vitro and in vivo. In vitro screening among Trichoderma isolates for their multiple functions (mycelial and spore germination inhibition, hydrogen cyanide, chitinolytic enzymes, non-volatile and volatile metabolites production) in suppressing Foc and promoting plant growth (IAA production and phosphate solubilisation) indicated that the multiple biocontrol actions were significantly higher in 6 isolates of rhizospheric Trichoderma and 10 isolates of endophytic Trichoderma compared to other isolates. The greenhouse evaluation of individual application of these rhizospheric and endophytic Trichoderma isolates against Fusarium wilt pathogen in cv. Grand Naine (AAA) indicated significant suppression of Fusarium wilt disease and increased plant growth characters as compared to Foc pathogen inoculated plants. However, none of these individual Trichoderma isolates recorded complete suppression of Fusarium wilt disease. Therefore, the greenhouse evaluation involving combination of rhizospheric Trichoderma sp. NRCB3 + endophytic Trichoderma asperellum Prr2 recorded 100% reduction of Fusarium wilt disease and increased plant growth parameters up to 250% when compared to individual isolates application and Foc alone-inoculated plants. Further, the field evaluation of this combination of Trichoderma isolates applied for three times: (1) at 15 days before planting, (2) second month after planting and (3) fourth month after planting resulting in significant reduction of Fusarium wilt disease and also increase in bunch weight as compared to untreated control plants. Therefore, these Trichoderma isolates may be used in combination for the effective suppression of Fusarium wilt disease in banana.     

Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.     

An Antifungal Protein Purified from Pearl Millet Seeds Shows Sequence Homology to Lipid Transfer Proteins

In the course of a search for antifungal proteins from plant seeds, we observed inhibition of mycelial growth of Trichoderma viride with extracts of pearl millet. We have identified several proteins with antifungal properties in the seeds of pearl millet. One of these proteins has been purified to homogeneity and characterized. The purified protein has a molecular mass of 25 kDa. The N-terminal sequence of the protein (25 residues) shows homology to non-specific lipid transfer proteins (LTPs) of cotton, wheat and barley. The purified LTP inhibited mycelial growth of T. viride and the rice sheath blight fungus, Rhizoctonia solani in vitro.     

Comparative anatomy of Sirhookera (Orchidaceae) growing in Western Ghats of southern India

We compared the anatomical characteristics of vegetative organs, peduncle and mycorrhizal morphology of the two known species of Sirhookera (Epidendroideae, Orchidaceae) to identify anatomical markers for identification and the ecological adaptations of these species. The leaves are hypostomatic bearing tetracytic stomata and the walls of subsidiary cells are smooth in Sirhookera lanceolata and undulate in Sirhookera latifolia. On the adaxial and abaxial surfaces the leaves are covered by a thick cuticle. The hypodermis is dimorphic and present on both sides of the leaf; chlorenchyma is homogenous and the vascular bundles are collateral. The rhizome of Sirhookera possesses a single-layered epidermis, thick cuticle, thin-walled parenchymatous ground tissue containing starch grains and scattered collateral vascular bundles. A thick-walled sclerenchymatous band separates the cortex from the parenchymatous ground tissue comprising of banded cells in the peduncle. Starch grains are present in the ground tissue of the S. latifolia peduncle. The roots consist of the velamen, ∩-thickened exodermis, thin-walled cortex consisting of water-storage cells, O-thickened endodermis and a vascular cylinder with parenchymatous pith. Starch grains are present in the root cortical cells of S. lanceolata but absent in S. latifolia. Fungal pelotons that aids in nutrient acquisition were observed in the root cortical region of both species. The study revealed significant differences between the anatomical characteristics of the two species and that most of the anatomical features of Sirhookera relate to their ecological adaptations.     

Deletion of the SNP1 trypsin protease from Stagonospora nodorum reveals another major protease expressed during infection

The wheat fungal pathogen Stagonospora nodorum produces an extracellular trypsin-like protease, SNP1, during early stages of hyphal growth on the surface of host leaves and during penetration. Variation of SNP1 mRNA levels and enzyme activity during infection, were correlated with levels of aggressiveness of three wild-type isolates. SNP1 was deleted in two wild-type isolates using a gene replacement strategy. SNP1-deleted mutants completely lacked trypsin activity in vitro and on inoculated wheat leaves, but were not reduced in pathogenicity. SNP1-deleted mutants still have 50% of the total alkaline protease activity of wild-type. This residual activity comes from a previously undetected alkaline protease with subtilisin-like substrate and inhibitor specificities, which is produced in vitro and on host leaves. We hypothesize that this subtilisin protease may act in concert with SNP1 and may compensate for the loss of trypsin protease activity in the SNP1-deletion mutants.