Cloning and characterization of a putative β-glucosidase (NfBGL595) from Neosartorya fischeri

Whole genome sequence of Neosartorya fischeri NRRL181 revealed four putative GH1 β-glucosidases (BGLs). One BGL, NfBGL595 was successfully expressed and characterized. DNA sequence analysis revealed an open reading frame of 1590 bp, encoding a polypeptide of 529 amino acid residues. The gene was cloned in pET28a and overexpressed in Escherichia coli. The purified recombinant BGL showed high levels of catalytic activity, with V_max of 1693 U mg-protein⁻¹ and a K_m of 2.8 mM for p-nitrophenyl-β-d-glucopyranoside (pNPG). The optimal temperature and pH for enzyme activity were 40 °C and 6.0, respectively. The enzyme exhibited broad substrate specificity towards aryl glycosides including pNP-mannose, pNP-galactose, pNP-xylose, and pNP-cellobioside. A homology model of NfBGL595 was constructed based on the X-ray crystal structure of Trichoderma reesei BGL2. Molecular dynamics simulation studies of the enzyme with the pNPG and cellobiose, shed light on the substrate specificity of N. fischeri BGL595 only towards aryl glycoside.     

Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin-binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. Several engineering studies on CBCRs were attempted, especially for designing near-infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far-red light-emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches.     

Cell wall damage-induced lignin biosynthesis is regulated by a reactive oxygen species- and jasmonic acid-dependent process in Arabidopsis

The plant cell wall is a dynamic and complex structure whose functional integrity is constantly being monitored and maintained during development and interactions with the environment. In response to cell wall damage (CWD), putatively compensatory responses, such as lignin production, are initiated. In this context, lignin deposition could reinforce the cell wall to maintain functional integrity. Lignin is important for the plant's response to environmental stress, for reinforcement during secondary cell wall formation, and for long-distance water transport. Here, we identify two stages and several components of a genetic network that regulate CWD-induced lignin production in Arabidopsis (Arabidopsis thaliana). During the early stage, calcium and diphenyleneiodonium-sensitive reactive oxygen species (ROS) production are required to induce a secondary ROS burst and jasmonic acid (JA) accumulation. During the second stage, ROS derived from the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D and JA-isoleucine generated by JASMONIC ACID RESISTANT1, form a negative feedback loop that can repress each other's production. This feedback loop in turn seems to influence lignin accumulation. Our results characterize a genetic network enabling plants to regulate lignin biosynthesis in response to CWD through dynamic interactions between JA and ROS.     

Characterization of a β-1,4-mannanase from a newly isolated strain of Pholiota adiposa and its application for biomass pretreatment

A highly efficient β-1,4-mannanase-secreting strain, Pholiota adiposa SKU0714, was isolated and identified on the basis of its morphological features and sequence analysis of internal transcribed spacer rDNA. P. adiposa β-1,4-mannanase was purified to homogeneity from P. adiposa culture supernatants by one-step chromatography on a Sephacryl gel filtration column. P. adiposa β-1,4-mannanase showed the highest activity toward locust bean gum (V _max = 1,990 U/mg protein, K _m = 0.12 mg/mL) ever reported. Its internal amino acid sequence showed homology with hydrolases from the glycoside hydrolase family 5 (GH5), indicating that the enzyme is a member of the GH5 family. The saccharification of commercial mannanase and P. adiposa β-1,4-mannanase-pretreated rice straw by Celluclast 1.5L (Novozymes) was compared. In comparison with the commercial Novo Mannaway^® (113 mg/g-substrate), P. adiposa β-1,4-mannanase-pretreated rice straw released more reducing sugars (141 mg/g-substrate). These properties make P. adiposa β-1,4-mannanase a good candidate as a new commercial β-1,4-mannanase to improve biomass pretreatment.     

Meta-topolin and liquid medium enhanced in vitro regeneration in Scaevola taccada (Gaertn.) Roxb

In Vitro Cellular & Developmental Biology - Plant - Scaevola taccada (Gaertn.) Roxb. is a hemi-sclerophyllous littoral shrub of the family Goodeniaceae. It is a salt-tolerant plant and used in...     

Mitigating in vitro induced macro- and micro-morphological anomalies using seismic stress in Vitex negundo L.

Plant Cell, Tissue and Organ Culture (PCTOC) - Present study explores the potential of seismo-mechanical stress in alleviating the macro- and micro-morphological aberrations observed in the in...     

Meta-topolin induced morphometric and structurally stable bulblets in Malabar River Lily (Amaryllidaceae)

The current investigation revealed a highly proficient and reproducible method for comprehensive in vitro regeneration and structural differentiation of bulblets in Crinum malabaricum Lekhak & Yadav using meta-topolin [mT; 6-(3-hydroxybenzylamino) purine] [6-benzylaminopurine] for the first time. Cultures were established from twin scales explants using optimized sterilization method and growth regulators for regeneration of in vitro plantlets. It was observed that mT at 2.0 mg L^?1 was the main factor involved in direct bulblets formation from the basal plate, and the highest mean number of bulblets per twin scale explant was 26 within 8 weeks. On maturation, the bulblets were rooted simultaneously on the mT containing medium. Regenerated plantlets were acclimatized with a 100% survival rate after 8 weeks. Light microscopic examination of bulblet scales/cataphylls was done to characterize the in vitro bulblets developed from mT and BAP containing media. The bulblet scales differed in quantitative traits of tissue systems, and the meta-topolin derived bulblets represented well differentiated dermal, ground and vascular tissues. The results obtained may provide a new tool to improve the production of pharmaceutically important compounds (viz. galanthamine) in bulblets and give the guarantee of survival of plants under natural conditions which can reaffirm the conservation measure of this critically endangered medicinal plant.

     

Repeated Random Mutagenesis of alpha-Amylase from Bacillus licheniformis for Improved pH Performance

The alpha-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis alpha-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.