Multi-point enzyme immobilization,surface chemistry,and novel platforms: a paradigm shift in biocatalyst design

Engineering enzymes with improved catalytic properties in non-natural environments have been concerned with their diverse industrial and biotechnological applications. Immobilization represents a promising but straightforward route, and immobilized biocatalysts often display higher activities and stabilities compared to free enzymes. Owing to their unique physicochemical characteristics, including the high-specific surface area, exceptional chemical, electrical, and mechanical properties, efficient enzyme loading, and multivalent functionalization, nano-based materials are postulated as suitable carriers for biomolecules or enzyme immobilization. Enzymes immobilized on nanomaterial-based supports are more robust, stable, and recoverable than their pristine counterparts, and are even used for continuous catalytic processes. Furthermore, the unique intrinsic properties of nanomaterials, particularly nanoparticles, also confer the immobilized enzymes to be used for their broader applications. Herein, an effort has been made to present novel potentialities of multi-point enzyme immobilization in the current biotechnological sector. Various nano-based platforms for enzyme/biomolecule immobilization are discussed in the second part of the review. In summary, recent developments in the use of nanomaterials as new carriers to construct robust nano-biocatalytic systems are reviewed, and future trends are pointed out in this article.     

Enhanced decolorization of Solar brilliant red 80 textile dye by an indigenous white rot fungus Schizophyllum commune IBL-06

An indigenously isolated white rot fungus, Schizophyllum commune IBL-06 was used to decolorize Solar brilliant red 80 direct dye in Kirk’s basal salts medium. In initial screening study, the maximum decolorization (84.8%) of Solar brilliant red 80 was achieved in 7 days shaking incubation period at pH 4.5 and 30 °C. Different physical and nutritional factors including pH, temperature and fungal inoculum density were statistically optimized through Completely Randomized Design (CRD), to enhance the efficiency of S. commune IBL-06 for maximum decolorization of Solar brilliant red 80 dye. The effects of inexpensive carbon and nitrogen sources were also investigated. Percent dye decolorization was determined by a reduction in optical density at the wavelength of maximum absorbance (λ_max, 590 nm). Under optimum conditions, the S. commune IBL-06 completely decolorized (100%) the Solar brilliant red 80 dye using maltose and ammonium sulfate as inexpensive carbon and nitrogen sources, respectively in 3 days. S. commune IBL-06 produced the three major ligninolytic enzymes lignin peroxidase (LiP), manganase peroxidase (MnP) and lacaase (Lac) during the decolorization of Solar brilliant red 80. LiP was the major enzyme (944 U/mL) secreted by S. commune IBL-06 along with comparatively lower activities of MnP and Laccase.     

Enhanced lignin peroxidase synthesis by Phanerochaete Chrysosporium in solid state bioprocessing of a lignocellulosic substrate

Summary A solid state fermentation (SSF) process for the production of lignin peroxidase was optimized to enhance enzyme production by Phanerochaete chrysosporium. Optimization of the corncob SSF medium caused a significant reduction in fermentation time to give maximum lignin peroxidase yield. Supplementation of the SSF medium by low concentrations of peptone, yeast extract and Tween-80 enhanced lignin peroxidase production. Maximum yield of lignin peroxidase was 13.7 U/gds (units per gram dry substrate) noted after 5 days of SSF with 70% moisture and 20% (v/w) inoculum.     

Decolorization of Some Reactive Textile Dyes by White Rot Fungi Isolated in Pakistan

Summary Four white-rot fungi isolated in Pakistan were used for decolorization of widely used reactive textile dyestuffs. Phanerochaete chrysosporium, Coriolus versicolor, Ganoderma lucidum and Pleurotus ostreatus were grown in defined nutrient media for decolorization of Drimarene Orange K-GL, Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR for 10 days in shake flasks. Samples were removed every day, centrifuged and the absorbances of the supernatants were read to determine percentage decolorization. It was observed that P. chrysosporium and C. versicolor could effectively decolorize Remazol Brilliant Yellow 3GL, Procion BluePX-5R and Cibacron Blue P-3RGR. Drimarene Orange K-GL was completely decolorized (0.2 g/l after 8 days) only by P.chrysosporium, followed by P. ostreatus (0.17 g/l after 10 days). P. ostreatus also showed good decolorization efficiencies (0.19–0.2 g/l) on all dyes except Remazol Brilliant Yellow (0.07 g/l after 10 days). G. lucidum did not decolorize any of the dyestuffs to an appreciable extent except Remazol Brilliant Yellow (0.2 g/l after 8 days).     

Decolorization of practical textile industry effluents by white rot fungus Coriolus versicolor IBL-04

Textile industry discharges a vast amount of unused synthetic dyes in effluents. The discharge of these effluents into rivers and lakes leads to a reduction in sunlight penetration in natural water bodies, which, in turn, decreases both photosynthetic activity and dissolved oxygen concentration rendering it toxic to living beings. This paper describes the decolorization potential of a local white rot fungus, Coriolus versicolor IBL-04 for practical industrial effluents collected from five different textile industries of Faisalabad, Pakistan. Screening of C. versicolor IBL-04 on five effluents showed best decolorization results (36.3%) for Arzoo Textile Industry (ART) effluent in 6 days followed by Crescent Textile Industry (CRT), Itmad Textile Industry (ITT), Megna Textile Industry (MGT) and Ayesha Textile Industry (AST) effluents. Optimization of different process parameters for ART effluent decolorization by C. versicolor IBL-04 showed that manganese peroxidase (MnP) (486 U/mL) was the lignolytic enzyme present in the culture filtrates with undetectable lignin peroxidase (LiP) and laccase. The MnP synthesis and effluent decolorization could be enhanced to 725 U/mL and 84.4%, respectively, with a significant time reduction to 3 days by optimizing pH and temperature and using 1% starch as a supplementary carbon source.     

Cross-talk between sulfur assimilation and ethylene signaling in plants

Sulfur (S) deficiency is prevailing all over the world and becoming an important issue for crop improvement through maximising its utilization efficiency by plants for sustainable agriculture. Its interaction with other regulatory molecules in plants is necessary to improve our understanding on its role under changing environment. Our knowledge on the influence of S on ethylene signaling is meagre although it is a constituent of cysteine (Cys) required for the synthesis of reduced glutathione (GSH) and S-adenosyl methionine (SAM), a precursor of ethylene biosynthesis. Thus, there may be an interaction between S assimilation, ethylene signaling and plant responses under optimal and stressful environmental conditions. The present review emphasizes that responses of plants to S involve ethylene action. This evaluation will provide an insight into the details of interactive role of S and ethylene signaling in regulating plant processes and prove profitable for developing sustainability under changing environmental conditions.     

Focus on Ethylene: Role of Ethylene and Its Cross Talk with Other Signaling Molecules in Plant Responses to Heavy Metal Stress

Excessive heavy metals (HMs) in agricultural lands cause toxicities to plants, resulting in declines in crop productivity. Recent advances in ethylene biology research have established that ethylene is not only responsible for many important physiological activities in plants but also plays a pivotal role in HM stress tolerance. The manipulation of ethylene in plants to cope with HM stress through various approaches targeting either ethylene biosynthesis or the ethylene signaling pathway has brought promising outcomes. This review covers ethylene production and signal transduction in plant responses to HM stress, cross talk between ethylene and other signaling molecules under adverse HM stress conditions, and approaches to modify ethylene action to improve HM tolerance. From our current understanding about ethylene and its regulatory activities, it is believed that the optimization of endogenous ethylene levels in plants under HM stress would pave the way for developing transgenic crops with improved HM tolerance.In addition to common abiotic stresses seen in agricultural production, such as drought, submerging, and extreme temperatures (Thao and Tran, 2012; Xia et al., 2015), heavy metal (HM) stress has arisen as a new pervasive threat (Srivastava et al., 2014; Ahmad et al., 2015). This is mainly due to the unrestricted industrialization and urbanization carried out during the past few decades, which have led to the increase of HMs in soils. Plants naturally require more than 15 different types of HM as nutrients serving for biological activities in cells (Sharma and Chakraverty, 2013). However, when the nutritional/nonnutritional HMs are present in excess, plants have to either suffer or take these up from the soil in an unwilling manner (Nies, 1999; Sharma and Chakraverty, 2013). Upon HM stress exposure, plants induce oxidative stress due to the excessive production of reactive oxygen species (ROS) and methylglyoxal (Sharma and Chakraverty, 2013). High levels of these compounds have been shown to negatively affect cellular structure maintenance (e.g. induction of lipid peroxidation in the membrane, biological macromolecule deterioration, ion leakage, and DNA strand cleavage; Gill and Tuteja, 2010; Nagajyoti et al., 2010) as well as many other biochemical and physiological processes (Dugardeyn and Van Der Straeten, 2008). As a result, plant growth is retarded and, ultimately, economic yield is decreased (Yadav, 2010; Anjum et al., 2012; Hossain et al., 2012; Asgher et al., 2015). Moreover, the accumulation of metal residues in the major food chain has been shown to cause serious ecological and health problems (Malik, 2004; Verstraeten et al., 2008).Plants employ different strategies to detoxify the unwanted HMs. Among the common responses of plants to HM stress are increases in ethylene production due to the enhanced expression of ethylene-related biosynthetic genes (Asgher et al., 2014; Khan and Khan, 2014; Khan et al., 2015b) and/or changes in the expression of ethylene-responsive genes (Maksymiec, 2007). Conventionally, this hormone has been established to modulate a number of important plant physiological activities, including seed germination, root hair and root nodule formation, and maturation (fruit ripening in particular; Dugardeyn and Van Der Straeten, 2008). On the other hand, although ethylene has also been suggested to be a stress-related hormone responding to a number of biotic and abiotic triggers, little is known about the exact role of elevated HM stress-related ethylene in plants (Zapata et al., 2003). Enhanced production of ethylene in plants subjected to toxic levels of cadmium (Cd), copper (Cu), iron (Fe), nickel (Ni), and zinc (Zn) has been shown (Maksymiec, 2007). As an example, Cd- and Cu-mediated stimulation of ethylene synthesis has been reported as a result of the increase of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) activity, one of the enzymes involved in the ethylene synthesis pathway (Schlagnhaufer and Arteca, 1997; Khan et al., 2015b).Plants tend to adjust or induce adaptation or tolerance mechanisms to overcome stress conditions. To develop stress tolerance, plants trigger a network of hormonal cross talk and signaling, among which ethylene production and signaling are prominently involved in stress-induced symptoms in acclimation processes (Gazzarrini and McCourt, 2003). Therefore, the necessity of controlling ethylene homeostasis and signal transduction using biochemical and molecular tools remains open to combat stress situations. Stress-induced ethylene acts to trigger stress-related effects on plants because of the autocatalytic ethylene synthesis. Autocatalytic stress-related ethylene production is controlled by mitogen-activated protein kinase (MAPK) phosphorylation cascades (Takahashi et al., 2007) and through stabilizing ACS2/6 (Li et al., 2012). Strong lines of evidence have shown the multiple facets of ethylene in plant responses to different abiotic stresses, including excessive HM, depending upon endogenous ethylene concentration and ethylene sensitivities that differ in developmental stage, plant species, and culture systems (Pierik et al., 2006; Kim et al., 2008; Khan and Khan, 2014). Under HM stress conditions, plants show a rapid increase in ethylene production and reduced plant growth and development, suggesting a negative regulatory role of ethylene in plant responses to HM stress (Schellingen et al., 2014; Khan et al., 2015b). On the other hand, a potential involvement of ETHYLENE INSENSITIVE2 (EIN2), a central component of the ethylene signaling pathway, as a positive regulator in lead (Pb) resistance in Arabidopsis (Arabidopsis thaliana) has also been demonstrated (Cao et al., 2009). More recently, Khan and Khan (2014) showed that ethylene-regulated antioxidant metabolism maintained a higher level of reduced glutathione (GSH) and alleviated photosynthetic inhibition in mustard (Brassica juncea) plants exposed to Ni, Zn, or Cd through the optimization of ethylene homeostasis (Masood et al., 2012). Taken together, the purpose of this review is to update the research community with our current understanding of the roles of ethylene and its signaling in plant responses to HM stress. Moreover, the cross talk of ethylene with other phytohormones and signaling molecules upon HM stress will also be discussed.     

Photosynthetic and growth responses of two mustard cultivars differing in phytocystatin activity under cadmium stress

Cadmium inhibits photosynthetic capacity of plants by disturbing protein conformations, whereas phytocystatins prevent degradation of target proteins and are involved in abiotic stress tolerance. Two mustard (Brassica juncea L.) cultivars, Ro Agro 4001 and Amruta, were grown with Cd (50 µM) in order to study physiological and biochemical basis of differences in Cd tolerance. Amruta accumulated higher Cd and H₂O₂ concentrations in leaves than that of Ro Agro 4001. Cd significantly decreased photosynthesis and growth of plants in both cultivars by reducing a chlorophyll content, gas exchange parameters, and activity of Rubisco; the effects were more prominent in Amruta than those in Ro Agro 4001. The greater photosynthesis and growth of Ro Agro 4001 under Cd stress might be attributed to its higher phytocystatin activity together with greater ascorbate peroxidase activity, photosynthetic nitrogen-use efficiency, sulphur assimilation (ATP-sulphurylase activity and S content), and contents of cysteine and reduced glutathione compared to Amruta. In contrast, the activity of superoxide dismutase (SOD) was higher in Amruta than that of Ro Agro 4001 under control conditions, whereas the Cd treatment increased significantly the SOD activity in both cultivars with the greater increase in Ro Agro 4001. The fluorescence spectra of phytocystatin showed a lesser change in Ro Agro 4001 under Cd stress than that in Amruta suggesting higher resistance of Ro Agro 4001 to Cd. The higher phytocystatin activity under Cd stress in Ro Agro 4001 compared to Amruta enabled the plants to protect their proteins more efficiently. This resulted in a greater increase of photosynthetic capacity in Ro Agro 4001 than that of Amruta. Thus, the phytocystatin activity may be considered as a physiological parameter for augmenting photosynthesis and growth of mustard under Cd stress.     

Ethylene Supplementation Increases PSII Efficiency and Alleviates Chromium-Inhibited Photosynthesis Through Increased Nitrogen and Sulfur Assimilation in Mustard

Journal of Plant Growth Regulation - The effect of chromium (Cr) toxicity on four cultivars of mustard (Brassica juncea), Varuna, Pusa Bold, Rohini and SS2, was studied to select the cultivar with...     

Purification and Characterization of a Xylanase Produced by Chaetomium thermophile NIBGE

Summary Xylanase was produced by growing Chaetomium thermophile NIBGE in a submerged liquid culture using wheat straw and urea as carbon and nitrogen sources respectively. The xylanase was purified to electrophoretic homogeneity after ammonium sulphate precipitation, anion exchange chromatography by FPLC and gel filtration. The molecular mass of this xylanase BII was 50 kDa. The pH and temperature optima were 6.5 and 70 °C respectively. The xylanase BII showed reasonable stability at high pH and 65 °C temperature. Some metal ions and EDTA caused little inhibition at low concentrations but complete inhibition was observed at concentrations higher than 2 mM. The K_m and V_max values with oat spelt xylan as the substrate were found to be 12.5 mg/ml and 83.3 IU/mg protein, respectively. Liberation of reducing sugars from commercial paper pulp samples suggest the feasibility of a biopulping process using this xylanase.