Improved antifungal activity of barley derived chitinase I gene that overexpress a 32 kDa recombinant chitinase in Escherichia coli host

Abstract Agricultural crops suffer many diseases, including fungal and bacterial infections, causing significant yield losses. The identification and characterisation of pathogenesis-related protein genes, such as chitinases, can lead to reduction in pathogen growth, thereby increasing tolerance against fungal pathogens. In the present study, the chitinase I gene was isolated from the genomic DNA of Barley (Hordeum vulgare L.) cultivar, Haider-93. The isolated DNA was used as template for the amplification of the ∼935 bp full-length chitinase I gene. Based on the sequence of the amplified gene fragment, class I barley chitinase shares 93% amino acid sequence homology with class II wheat chitinase. Interestingly, barley class I chitinase and class II chitinase do not share sequence homology. Furthermore, the amplified fragment was expressed in Escherichia coli Rosetta strain under the control of T7 promoter in pET 30a vector. Recombinant chitinase protein of 35 kDa exhibited highest expression at 0.5 mM concentration of IPTG. Expressed recombinant protein of 35 kDa was purified to homogeneity with affinity chromatography. Following purification, a Western blot assay for recombinant chitinase protein measuring 35 kDa was developed with His-tag specific antibodies. The purified recombinant chitinase protein was demonstrated to inhibit significantly the important phytopathogenic fungi Alternaria solani, Fusarium spp, Rhizoctonia solani and Verticillium dahliae compared to the control at concentrations of 80 µg and 200 µg.

Introduction of a synthetic Thermococcus-derived α-amlyase gene into barley genome for increased enzyme thermostability in grains

Background: The enzymes utilized in the process of beer production are generally sensitive to higher temperatures. About 60% of them are deactivated in drying the malt that limits the utilization of starting material in the fermentation process. Gene transfer from thermophilic bacteria is a promising tool for producing barley grains harboring thermotolerant enzymes. Results: Gene for α-amylase from hydrothermal Thermococcus, optimally active at 75­85°C and pH between 5.0 and 5.5, was adapted in silico to barley codon usage. The corresponding sequence was put under control of the endosperm-specific promoter 1Dx5 and after synthesis and cloning transferred into barley by biolistics. In addition to model cultivar Golden Promise we transformed three Slovak barley cultivars Pribina, Levan and Nitran, and transgenic plants were obtained. Expression of the ~50 kDa active recombinant enzyme in grains of cvs. Pribina and Nitran resulted in retaining up to 9.39% of enzyme activity upon heating to 75°C, which is more than 4 times higher compared to non-transgenic controls. In the model cv. Golden Promise the grain α-amylase activity upon heating was above 9% either, however, the effects of the introduced enzyme were less pronounced (only 1.22 fold difference compared with non-transgenic barley). Conclusions: Expression of the synthetic gene in barley enhanced the residual α-amylase activity in grains at high temperatures.

Optimization of extracellular xylanase production by Sclerotinia sclerotiorum S2 using factorial design.

The improvement of xylanase production by Sclerotinia sclerotiorum S2 using a liquid fermentation culture was investigated. The optimized process was divided into three basic steps: (i) evaluating xylanase inducers using different agricultural residues such as wheat bran, oat bran, orange peel and barley bran at 1% final concentration, and also filter paper. Among these, wheat bran showed the maximum activity (2.5 U/ml) at 12 days post-inoculation; (ii) for optimization, we determined the optimal concentration of inducer, the effect of phosphate anion (K2HPO4/KH2PO4) and culture aeration using a rotary shaker at 100 and 180 rpm. The optimal conditions for these three factors were determined in an experimental panel using factorial data, in which a mathematical model (Minitab software) was fitted; (iii) The optimized culture medium containing a high level of wheat bran (3%) without KH2PO4-K2HPO4 and submitted to a high agitation (180 rpm/min) increased the xylanase production from 2.5 U/ml to 4 U/ml (1.6-fold).

Enzyme electrode based on oxalate oxidase immobilized in gelatin for specific determination of oxalate.

A biosensor for the specific determination of oxalate was developed using oxalate oxidase (EC 1.2.3.4) from barley (Hordeum vulgare) seedling roots in combination with a dissolved oxygen probe. Oxalate oxidase immobilized with gelatin using glutaraldehyde and fixed on pretreated teflon membrane served as an enzyme electrode. The electrode response was maximum when 50 mM succinate buffer was used at pH 3.2 and 35 degrees C. The biosensor response depends linearly on oxalate concentration between 5 x 10(-6)-2 x 10(-4) M with response time 30 sec and substrate specificity of the oxalate oxidase electrode of 100%. The sensor is stable for more than 3 months during which time more than 400 assays can be performed.

Barley oxalate oxidase immobilized on zirconia-coated alkylamine glass using glutaraldehyde.

A method for immobilizing barley oxalate oxidase to zirconia coated alkylamine glass through the process of glutaraldehyde coupling has been described. The immobilized enzyme retained 97.2% of the specific activity, with a conjugation yield of 6.63 mg/g support and showed an increase in optimum pH. The Km value of immobilized enzyme was unaltered but Vmax was decreased compared to free enzyme. The conjugated enzyme was stable at 4 degrees C for 2 years. A number of inorganic ions and metabolic substances did not denature the immobilized enzyme. The clinical importance of this work is demonstrated.