Medium optimization of protease production by Brevibacterium linens DSM 20158, using statistical approach

Various cultivation parameters were optimized for the production of extra cellular protease by Brevibacterium linens DSM 20158 grown in solid state fermentation conditions using statistical approach. The cultivation variables were screened by the Plackett-Burman design and four significant variables (soybean meal, wheat bran, (NH4)2SO4 and inoculum size were further optimized via central composite design (CCD) using a response surface methodological approach. Using the optimal factors (soybean meal 12.0g, wheat bran 8.50g, (NH4)2SO4) 0.45g and inoculum size 3.50%), the rate of protease production was found to be twofold higher in the optimized medium as compared to the unoptimized reference medium.

Potential of antioxidant enzymes in depicting drought tolerance of wheat (Triticum aestivum L.).

In an effort to determine the biochemical markers for identifying genotypes before sowing for drought tolerance, changes in activities of antioxidant enzymes were determined in the seedlings of five drought-tolerant and five drought-sensitive wheat (Triticum aestivum L.) genotypes, each with different genetic background growing under normal and water deficit conditions induced by 6% mannitol. In comparison with non-stressed seedlings, the catalase (CAT) activity was upregulated by more than 50% in the roots of water-stressed seedlings in drought-tolerant genotypes. Water deficit stress also led to the upregulation of ascorbate peroxidase (APX) in the endosperms and glutathione reductase (GR), CAT and peroxidase (POD) in the shoots of stressed seedlings in drought-tolerant genotypes. Superoxide dismutase (SOD) activity was very low in roots and shoots and showed non-significant increase under water-stress in tolerant genotypes. Out of five specified enzyme activities (CAT in roots and shoots, APX in endosperms, GR and POD in shoots), if any three are upregulated in the specified tissues under water deficit conditions, the genotype is likely to be drought-tolerant. Wheat seedlings with low GR and APX activities and high POD activity in shoots with a low ratio of GR activity of shoot to root of non-stressed seedlings are likely to perform better under rainfed conditions. The observed data showed that status of antioxidant enzymes could provide a meaningful tool for depicting drought tolerance of a wheat genotype.

Alkali pretreated of wheat straw and its enzymatic hydrolysis

The efficiency of enzymatic hydrolysis of cellulose can be improved by various pretreatments of the substrate. In order to increase the efficiency of enzymatic saccharification of the wheat straw, we determined the effect of different pretreatments on the physical structure, chemical components and enzymatic saccharification of wheat straw. Our results showed that combination of grinding and sodium hydroxide (NaOH) treatment had high effect on the enzymatic hydrolysis of wheat straws. The optimal pretreatment condition was to grind the wheat straws into the sizes of 120 meshes followed by treatment with 1.0% NaOH for 1.5 h (121°C/15psi). Under this condition, the cellulose content of wheat straw was increased by 44.52%, while the content of hemicellulose and lignin was decreased by 44.15% and 42.52%, respectively. Scanning Electronic Microscopy and infrared spectrum analyses showed that significant changes occurred in the structure of wheat straws after pretreatment, which is favorable for the hydrolysis and saccharification. Cellulase produced by Penicillium waksmanii F10-2 was used to hydrolyze the pretreated wheat straw and the optimal condition was determined to be 30 h of enzymatic reaction under the temperature of 55°C, pH 5.5 and substrate concentration of 3%.

Kinetic properties of cell wall bound superoxide dismutase in leaves of wheat (Triticum aestivum L.) following stripe rust (Puccinia striiformis) infection.

Stripe rust (Puccinia striiformis f.sp. tritici) is the most devastating disease of wheat (Triticum aestivum L.) accounting huge economical losses to the industry worldwide. HD 2329 was a widely grown wheat cultivar which had become highly susceptible to stripe rust and was used to understand the biochemical aspects of the host pathogen interaction through characterization of superoxide dismutase (SOD). In the present study, two types of SOD, ionically or covalently bound to the particulate fraction were found in the stripe rust infected and uninfected wheat leaves of susceptible cultivar HD 2329. Cell walls of leaves contained a high level of SOD, of which 41-44% was extractable by 2 M NaCl and 10-13% by 0.5% EDTA in infected and uninfected leaves. The NaCl-released SOD constituted the predominant fraction. It exhibited maximum activity at pH 9.0, had a Km value of 1.82-2.51 for uninfected and 1.77-2.37 mM for infected, respectively with pyrogallol as the substrate, and a Vmax of 9.55-21.4 and 12.4-24.1 A min-1g-1FW. A temperature optimum of 20oC was observed for SOD of both uninfected and infected leaves. SOD showed differential response to metal ions, suggesting their distinctive nature. Inhibition of wall bound SOD by iodine and its partial regeneration of activity by mercaptoethanol suggested the involvement of cysteine in active site of the enzyme. These two forms showed greater differences with respect to thermodynamic properties like energy of activation (Ea) and enthalpy change (H), while entropy change (S) and free energy change (G) were similar. The results further showed that pathogen infection of the leaves of susceptible wheat cultivar induced a decrease in the SOD activity and kinetics which might be critical during the response of plant cells to the infection.

Characterization of cellulolytic activities of Bjerkandera adusta and Pycnoporus sanguineus on solid wheat straw medium / Caracterización de las actividades celulolíticas de Bjerkandera adusta y Pycnoporus sanguineus en un medio sólido de paja de trigo

Cellulolytic properties of two white rot fungi, Bjerkandera adusta and Pycnoporus sanguineus, cultivated on wheat straw agar medium, were characterized and compared. Optimal growing parameters for maximum enzyme production for both fungi were wheat straw medium pH 5 and 28ºC. B. adusta showed, on the 6th day of culture, carboxymethylcellulose (CMC)ase activity levels 1.6 times higher than maximal P. sanguineus activity, achieved on the 8th day. B. adusta supernatants also displayed higher activity levels towards xylan (3.6-fold) compared to those of P. sanguineus. However, enzymes from P. sanguineus were more robust resisting one hour incubation at high temperatures (up to 80ºC), and exhibiting activity and stability in pH range from 2 to 8. Cellulolytic activities, with molecular masses ranging from 25 to 90 kDa, from the two species were detected in zymograms.

Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars.

The effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism (alpha and beta amylases, sucrose phosphate synthase, sucrose synthase, acid and alkaline invertases) in wheat (Triticum aestivum L.) was investigated in the seedlings of drought-sensitive (PBW 343) and drought-tolerant (C 306) cultivars. The water deficit was induced by adding 6% mannitol (water potential -0.815 Mpa) in the growth medium. The water deficit reduced starch content in the shoots of tolerant seedlings as compared to the sensitive ones, but increased sucrose content in the shoots and roots of tolerant seedlings, indicating their protective role during stress conditions. It also decreased the alpha-amylase activity in the endosperm of seedlings of both the cultivars, but increased alpha and beta amylase activities in the shoots of tolerant ones. Sucrose phosphate synthase (SPS) activity showed a significant increase at 6 days of seedling growth (DSG) in the shoots of stressed seedlings of tolerant cultivar. However, SPS activity in the roots of stressed seedlings of sensitive cultivar was very low at 4 DSG and appeared significantly only at day 6. Sucrose synthase (SS) activity was lower in the shoots and roots of stressed seedlings of tolerant cultivar than sensitive ones at early stage of seedling growth. Higher acid invertase activity in the shoots of seedlings of tolerant cultivar appeared to be a unique characteristic of this cultivar for stress tolerance. Alkaline invertase activity, although affected under water deficit conditions, but was too low as compared to acid invertase activity to cause any significant affect on sucrose hydrolysis. In conclusion, higher sucrose content with high SPS and low acid invertase and SS activities in the roots under water deficit conditions could be responsible for drought tolerance of C 306.

Photosynthetic acclimation to elevated CO2 in relation to Rubisco gene expression in three C3 species.

Wheat (Triticum aestivum L. var. DL 1266-5), sunflower (Helianthus annuus L. var. MSFH 17) and mungbean [Vigna radiata (L.) Wilczek var. P 9072] were grown in field under atmospheric (360 +/- 10 cm3 m(-3), AC) and elevated (650 +/- 50 cm3 m(-3), EC) CO2 concentrations in open top chambers for entire period of growth and development. Photosynthetic acclimation to elevated CO2 was examined by comparing photosynthesis rate (Pn), Pn/Ci curves, leaf contents of RuBP carboxylase/oxygenase (Rubisco), change in the transcripts of Rubisco small subunit (SSU) gene and leaf carbohydrate constituents in AC and EC grown plants. The study indicated that photosynthetic acclimation to elevated CO2 concentration in wheat occurred because of down regulation of Rubisco, through limitation imposed on Rubisco SSU gene expression, as a consequence of sugar accumulation in the leaves. Leaf starch accumulators, sunflower and mungbean, showed no down regulation of Pn under EC. The Rubisco contents (%) in leaf soluble protein and rbcS transcript levels were not significantly affected in EC plants compared to AC plants of sunflower and mungbean. The study indicated that accumulation of excess assimilates in the leaves as starch was less inhibitory to Pn and would, therefore, be an important trait for sustenance of Pn not only under EC, but also under AC, where Pn inhibited by end products.

Enhanced proteolysis leads to pre-mature cell death under the influence of elicitor like mycelial components from karnal bunt (tilletia indica) pathogen in wheat callus cultures.

Calli raised from mature embryos of susceptible wheat cultivar WH 542 were used in the present study as in vitro bioassay system to study the influence of disease determinant(s) of Karnal bunt (Tilletia indica), a semi-biotrophic fungal pathogen of wheat. Influence of elicitor and conditioned medium (CM) prepared from fungal cultures of T. indica was investigated on induction of programmed cell death (PCD). Induction of PCD was observed as hypersensitive response (HR) in terms of browning at localized regions of callus cultures and induction of proteolytic enzyme(s). Elicitor treated calli showed higher induction of protease activity than untreated and CM-treated cultures, which showed not much change in the activity. It was further substantiated by gel protease assay and activation of caspase-3 like protein(s) in callus cultures that clearly suggested the presence of signaling molecule(s) in the fungal elicitor preparation rather than in conditioned medium. This study further demonstrated that only elicitor preparation possesses such molecule(s), which might be cell wall bound components, rather than secretory in nature as CM was unable to induce PCD in wheat callus cultivars.

Effect of different temperature on starch synthase activity in excised grains of wheat cultivars.

Excised grains of wheat (Triticum aestivum) varieties HD 2285 (relatively tolerant) and HD 2329 (susceptible type) were incubated for 1 hr at 15 degrees, 25 degrees, 35 degrees and 45 degrees C. In an another treatment, excised grains were incubated for 1 hr at increasing temperature (15 degrees, 25 degrees, 35 degrees and 45 degrees C) continuously, thus exposing the grains to gradual rise in temperature. The above treated grains were then analysed for the activity of soluble starch synthase (SSS) and granule bound starch synthase (GBSS) assayed at 20 degrees C. SSS activity decreased as the pre-exposure temperature was higher, though the tolerant variety showed lesser decrease. Decrease in SSS activity was lesser when excised grains were exposed to gradual rise in temperature from 15 degrees to 45 degrees C as compared to direct exposure to 45 degrees C. Pre-exposure of excised grains to different temperatures however, had no significant effect on GBSS activity.

Interaction of guanidine hydrochloride and guanidine thiocyanate with wheat germ lipase.

Effect of two classical and potent denaturants, guanidine hydrochloride (GuHCl) and guanidine thiocyanate (GuHSCN) on purified wheat germ lipase has been studied. Lipase was found to be active only up to 5 M GuHCl and 1.5 M GuHSCN. The extent of interaction was determined by the measurement of apparent partial specific volume of the enzyme in presence of these two denaturants. While the preferential interaction parameter (zeta 3) has values of 0.08 +/- 0.02 and 0.14 +/- 0.03 g/g, the interaction parameter (delta m3/delta m2)T,mu 1, mu3 has values of 35 +/- 9 and 50 +/- 10 mole/mole for GuHCl and GuHSCN, respectively. The number of denaturant molecules bound to the enzyme, A3, obtained experimentally were 0.486 +/- 0.020 and 0.348 +/- 0.020 g/g and the calculated values were 0.459 +/- 0.023 and 0.567 +/- 0.030 g/g for 6 M GuHCl and 3 M GuHSCN, respectively. The volume change occurring upon denaturation results in -420 +/- 42 and -462 +/- 84 ml/mole in 6 M GuHCl and 3 M GuHSCN, respectively. The denaturation is accompanied by exposure of hydrophobic groups to the bulk solvent as confirmed by fluorescence emission measurements of the enzyme. The Tm measurements indicated a control value of 56 +/- 1 degree C. In presence of 6 M GuHCl/3 M GuHSCN, the value was 42 +/- 1 degree C. These results explain the retention of lipase activity even at 5 M GuHCl from a mechanistic point of view.

Characterization of amyloplastic phosphohexose isomerase from immature wheat (Triticum aestivum L.) endosperm.

Phosphohexose isomerase from amyloplasts of immature wheat endosperm was purified 133-fold. The enzyme had a molecular weight of 130 kDa and maximum activity at pH 8.6. It showed normal hyperbolic kinetics for both fructose-6-P and glucose-6-P with Km of 0.12 mM and 0.44 mM, respectively. pH had a great influence on Km for fructose-6-P. Using glucose-6-P as the substrate, the equilibrium was reached at 23% fructose-6-P and 77% glucose-6-P and an equilibrium constant of about 3.0. The delta F calculated from the apparent equilibrium constant was +742 cal.mol-1. The activation energy calculated from the Arrhenius plot was 7450 cal.mol-1. None of the sulphydryl reagents at 2.5 mM concentration inactivated the enzyme. The enzyme was competitively inhibited by 6-phosphogluconate, ribose-5-P and ribulose-5-P with Ki values of 0.18, 0.14, and 0.13 mM, respectively. The probable role of the enzyme in starch biosynthesis in amyloplasts is discussed.