Overexpression of a Bacillus subilis amylase in E.coli and application in bread making

Bacterial alpha-amylases (EC3.2.1.1) have potential use in wide number of industrial applications such as textile, paper, detergent, food, fermentation and pharmaceutical industries. Recombinant DNA technology for amylase production involves the selection of an amylase gene, its insertion into an appropriate vector system, transformation in an efficient bacterial system to produce high amount of recombinant protein. In this context, the aim of this work is the overexpression of an α-amylase gene from Bacillus subtilis US572 in E.coli strain, the characterization of the recombinant enzyme and test the effect of different quantities added of amylase on wheat flours and bread characterization

Structure/function/properties relationships and application of a GH11 xylanase

Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of the heteroxylans constituting the lignocellulosic plant cell wall. Due to their variety, xylanases have been classified in glycoside hydrolase families GH5, GH8, GH10, GH11, GH30 and GH43 in the CAZy database. In this work, we focus on GH11 family, which is one of the best characterized GH families with bacterial and fungal members. GH11 xylanases have for a long time been used as biotechnological tools in various industrial applications and represent in addition promising candidates for future other uses

Overexpression. of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato

[EN] One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K+ is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na+ accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K+ and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na+ accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term. (C) 2011 Elsevier GmbH. All rights reserved.This work was supported by the Spanish Ministry of Science and Innovation through grant AGL2009-13388-C03 and by the Council of Science and Technology from the Region of Murcia (Spain) (Fundacion SENECA) through grant 04553/GERM/06.Muñoz Mayor, A.; Pineda Chaza, BJ.; García Abellán, JO.; Antón Martínez, MT.; García Sogo, B.; Sánchez Bel, P.; Flores, FB.... (2012). Overexpression. of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato. Journal of Plant Physiology. 169(5):459-468. https://doi.org/10.1016/j.jplph.2011.11.018S459468169

Biotechnological Perspective of Reactive Oxygen Species (ROS)-Mediated Stress Tolerance in Plants

All environmental cues lead to develop secondary stress conditions like osmotic and oxidative stress conditions that reduces average crop yields by more than 50% every year. The univalent reduction of molecular oxygen (O2) in metabolic reactions consequently produces superoxide anions (O2•−) and other reactive oxygen species (ROS) ubiquitously in all compartments of the cell that disturbs redox potential and causes threat to cellular organelles. The production of ROS further increases under stress conditions and especially in combination with high light intensity. Plants have evolved different strategies to minimize the accumulation of excess ROS like avoidance mechanisms such as physiological adaptation, efficient photosystems such as C4 or CAM metabolism and scavenging mechanisms through production of antioxidants and antioxidative enzymes. Ascorbate-glutathione pathway plays an important role in detoxifying excess ROS in plant cells, which includes superoxide dismutase (SOD) and ascorbate peroxidase (APX) in detoxifying O2•−radical and hydrogen peroxide (H2O2) respectively, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR) involved in recycling of reduced substrates such as ascorbate and glutathione. Efficient ROS management is one of the strategies used by tolerant plants to survive and perform cellular activities under stress conditions. The present chapter describes different sites of ROS generation and and their consequences under abiotic stress conditions and also described the approaches to overcome oxidative stress through genomics and genetic engineering

An enigma in the genetic responses of plants to salt stresses

Soil salinity is one of the main factors restricting crop production throughout the world. Various salt tolerance traits and the genes controlling these traits are responsible for coping with salinity stress in plants. These coping mechanisms include osmotic tolerance, ion exclusion, and tissue tolerance. Plants exposed to salinity stress sense the stress conditions, convey specific stimuli signals, and initiate responses against stress through the activation of tolerance mechanisms that include multiple genes and pathways. Advances in our understanding of the genetic responses of plants to salinity and their connections with yield improvement are essential for attaining sustainable agriculture. Although a wide range of studies have been conducted that demonstrate genetic variations in response to salinity stress, numerous questions need to be answered to fully understand plant tolerance to salt stress. This chapter provides an overview of previous studies on the genetic control of salinity stress in plants, including signaling, tolerance mechanisms, and the genes, pathways, and epigenetic regulators necessary for plant salinity tolerance

Biochemical and molecular characterization of a recombinant \u3b1-amylase from Bacillus subtilis

Alpha amylase (EC3.2.1.1), one of more widespread enzymes in the industrial world, is a glycoside hydrolase. It hydrolyzes the \u3b1- (1,4) glucoside linkage between the glucose units of a polysaccharide. Microbial amylases, particularly those from Bacillus genus are more in demand than those from other sources. Alpha-amylases have potential application in wide number of industrial applications such as textile, paper, detergent, food, fermentation and pharmaceutical industries. Recombinant DNA technology for amylase production involves the selection of an efficient amylase gene, its insertion into an appropriate vector system, transformation in an efficient bacterial system to produce high amount of recombinant protein. In this context, the aim of this work is the overexpression of an\u3b1amylase gene from Bacillus subtilisUS572in E. coli strain and the characterization of the recombinant amylase which is an interesting candidate for biotechnological applications

His-tag effect on biochemical properties of B. subtilis US572 a-amylase produced in E. coli: application of the recombinant enzyme in breadmaking

The gene encoding Bacillus subtilis α-amylase was cloned into pET-21a (+). The expression level of the recombinant enzyme is 10.7-fold higher than the expression level of the native one (0.13 mg mL-1). The recombinant enzyme (His6-rAmyKS) was purified in one step using Ni-NTA column affinity with a specific activity of 664.28 U.mg-1. The biochemical properties of the His6-rAmyKS were determined and compared to those of the non-tagged enzyme. Interestingly, differences were found between the two enzymes mainly for the optimal temperature and pH. Experiment tests and molecular modeling confirmed that the extra residues (C-terminal His-tag fusion peptide and cleavage thrombin site) could be responsible for the slight increase in total activity and the improvement of biochemical properties of the His-tagged enzyme compared to the native one. The His6-rAmyKS was used as an additive in breadmaking. It showed a significant effect in improving the dough texture and the bread quality

Identification of salt-stress induced transcripts in potato leaves by cDNA-AFLP

Potato (Solanum tuberosum L.) is highly sensitive to salt stress, which is one of the most important factors limiting plant cultivation. The investigation of plant response to high salinity was envisaged in this report using cDNA–amplified fragment length polymorphism (AFLP). This technique was applied to salt- stressed and control potato plants (cv. Nicola). The expression profiles showed approx 5000 bands. Of these, 154 were upregulated and 120 were repressed by salt stress. In this study we have only considered cDNA fragments that seem to be originated from salt-induced mRNA. Eighteen fragments were then reamplified, cloned, and sequenced. Sequence comparison of these cDNA, identified in response to salt stress in potato, revealed that some of them present homologies with proteins in other species that are involved in cell wall structure and turnover such as proline-rich proteins and β-galactosidase. A number of identified clones encoded putative stress response proteins such as NADP-dependant glyceraldehyde dehy- drogenase and wound-induced protein. In addition, some of them encode proteins related to hypersensitive response against pathogens such as putative late blight and nematode as well as putative pathogenesis- related proteins. These cDNA seem to be differentially expressed in the presence of salt stress as shown by Northern blot or reverse Northern hybridization experiments

Enzyme Storage and Recycling: Nanoassemblies of \u3b1-Amylase and Xylanase Immobilized on Biomimetic Magnetic Nanoparticles

Immobilization of enzymes has been extensively required in a wide variety of industrial applications, as a way to ensure functionality and the potential of enzyme recycling after used. In particular, enzyme immobilization on magnetic nanoparticles (MNPs) could offer reusability by means of magnetic recovery and concentration, along with increased stability and robust activity of enzyme at different physicochemical conditions. In the present work, microbial \u3b1-amylase (AmyKS) and xylanase (XAn11) were both immobilized on different types of magnetic nanoparticles [MamC mediated biomimetic magnetic nanoparticles (BMNPs) and inorganic magnetic nanoparticles (MNPs)] by using two different strategies (electrostatic interaction and covalent bond). AmyKS immobilization was successful using electrostatic interaction with BMNPs. Instead the best strategy to immobilize XAn11 was using MNPs through the hetero-crosslinker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The immobilization protocols were optimized by varying glutaraldehyde (GA) concentration, enzyme quantity and reaction time. Under optimal conditions, 92% of AmyKS and 87% of XAn11 were immobilized on BMNPs and MNPs-E/N respectively (here referred as AmyKS-BMNPs and XAn11-MNPs nanoassemblies). The results show that the immobilization of the enzymes did not extensively alter their functionality and that increased enzyme stability compared to that of the free enzyme following upon storage at 4 \ub0C and 20 \ub0C. Interestingly, the immobilized amylase and xylanase were reused for 15 and 8 cycles respectively without signi\ufb01cant loss of activity upon magnetic recovering of the nanoassemblies. Results suggest the great potential of these nanoassemblies in bio-industry applications