Biochemical characterization of recombinant thermostable cohnella sp. A01 Beta -Glucanase

Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of Beta-1,6- glucanase [bg16M] from native thermophilic bacteria, Cohnella A01

Methods: bg16M gene was cloned and expressed in E. coli BL21 [DE3]. The enzyme was purified utilizing Ni-NTA nikcle sepharose column. Pustulan and laminarin were selected as substrates in enzyme assay. The purified bg16M enzyme was treated with different pH, temperature, metal ions, and detergents

Results: The expressed protein, including 639 amino acids, showed a high similarity with the hydrolytic glycosylated family 30. The molecular weight of enzyme was 64 kDa, and purification yield was 46%. The bg16M demonstrated activity as 4.83 U/ml on laminarin and 2.88 U/ml on pustulan. The optimum pH and temperature of the enzyme were 8 and 50 degree C, respectively. The enzyme had an appropriate stability at high temperatures and in the pH range of 7 to 9, showing acceptable stability, while it did not lose enzymatic activity completely at acidic or basic pH. None of the studied metal ions and chemical compounds was the activator of bg16M, and urea, SDS, and copper acted as enzyme inhibitors

Conclusion: Biochemical characterization of this enzyme revealed that bg16M can be applied in beverage industries and medical sectors because of its high activity, as well as thermal and alkaline stability

nested-splicing by overlap extension PCR improves specificity of this standard method

Background: splicing by overlap extension [SOE] PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function

Objectives: we introduced a nested-SOE-PCR [N -SOE-PCR] in order to increase the specificity and generating mutations in a gene by SOE-PCR

Materials and Methods: genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application

Results: in comparison to the conventional SOE-PCR, the improved method [i.e. N-SOE-PCR] increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products

Conclusions: by applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE

Bacterial secretome analysis in hunt for novel bacteriocins with ability to control Xanthomonas citri subsp. Citri

Background: Xanthomonas citri subsp. citri [Xcc], the causative agent of bacterial citrus canker, has affected citriculture worldwide. Varieties of means have been used to minimize its devastating effects, but no attention has been given to bacteriocins

Objectives: here and for the first time, we report the isolation and characterization of two novel bacteriocins

Materials and Methods: secretome containing bacteriocins of isolated bacteria was separated via SDS-PAGE. Each isolated protein band was characterized and checked for its efficacy in controlling two pathogenic isolates of Xcc via disk diffusion assay. The effects of varieties of carbon, nitrogen and phosphate sources were evaluated on both bacterial growth and bacteriocin production via Taguchi orthogonal method

Results: the two bacteriocins showed an activity up to 55[degree]C that were sensitive to proteases suggesting being protein in nature. Analysis of SDS-PAGE purified protein bands of bacterial secretomes with demonstrated potency against Xcc revealed the presence of peptides with relative molecular masses of 16.9 and 17 kDa for Cronobacter and Enterobacter, respectively. Sequence analysis of peptides revealed an HCP1 family VI secretion system homologue for Cronobacter [YP_001439956] and pilin FimA homologue for Enterobacter [CBK85798.1]. A Taguchi orthogonal array was also implemented to determine the effect of temperature and eight other chemical factors on bacteriocin production for each bacterium

Conclusions: two peptides with novel antibacterial activities effective against Xcc were isolated, characterized and conditions were optimized for their higher production

effects of non-starch polysaccharides content of wheat and xylanase supplementation on the intestinal amylase, aminopeptidase and lipase activities, ileal viscosity and fat digestibility in layer diet

The effects of non-starch polysaccharide [NSP] content of wheat and xylanase supplementation [XS] of the diet, on intestinal enzyme activity [amylase, aminopeptidase and lipase], fat digestibility and ileal viscosity of laying hens has been studied. Two hundred forty Hy-Line W-36 layer from 20 to 25 week of age were studied under a factorial experiment [4x2] in completely randomized design with 8 treatments including four levels of wheat [0, 23, 46 and 69%] corresponding to a dietary xylose content of 1.9, 2.1, 2.3 and 2.5% and two levels of xylanase [none or added at the dosage recommended by the supplier]. Each treatment was replicated five times each with six hens. Wheat inclusion in the diet increased amylase and lipase activity in the duodenum and jejunum, respectively [P<0.001]. Wheat inclusion, increased ileal viscosity while adding xylanase to diet, reduced it [P<0.001]. Fat digestibility was decreased by wheat increment levels [P<0.001]. The pH of the digesta content in different segments of gastro-intestinal tract [GIT] was not affected by wheat levels or XS, except for cecum that decreased with increasing the level of wheat. Relative weight and length of duodenum, jejunum and ileum were not affected by dietary treatment. Results suggested that wheat inclusion at high levels increased endogenous enzyme activity but could not alleviate the adverse effect of NSP content of diet on fat digestibility

[Serratia marcescens B4A chitinase product optimization using Taguchi approach]

Chitinase production by newly isolated Serratia marcescens B4A was optimized following Taguchi's array methods. Twenty-three bacterial isolates were screened from shrimp culture ponds in the South of Iran. A chitinase-producing bacterium was isolated based on it's ability to utilize chitin as the sole carbon source. The isolate designated as B4A, was identified as Serratia marcescens based on its 16S rRNA sequence and key morphological, physiological and biochemical characteristics. The cultivation of Serratia marcescens B4A in the appropriate liquid medium resulted in production of high levels of chitinase. The malt extract and colloidal chitin represented the best nitrogen and carbon sources, respectively. Chitinase production by Serratia marcescens B4A was optimized following the Taguchi orthogonal array [OA] for the design of experiments [DOE]. Statistical experimental design via the Taguchi method was applied to determine the optimal levels of physical parameters and key media components in the medium, such as temperature, pH, NaCl and chitin concentrations. The results of this study showed that temperature of 30§C, pH 7.9, NaCl 0.1% [w/v] and chitin 1% [w/v] are optimal conditions for this protocol