Centre for Chemistry and Biotechnology

The efficient immobilization of enzymes using nanostructured materials has recently been demonstrated. The materials used for this purpose, such as nanoparticles, nanofibers, nanotubes, nanoporous media, nanocomposites, and graphene all possess large surface areas that improve biocatalytic efficiency for industrial applications by increasing enzyme loading and facilitating reaction kinetics . In this report, we present the research opportunities for nanoscale materials in enzyme biotechnology and highlight recent developments in biofuel production using more advanced material supports for enzyme immobilization and stabilization.

Hybrid lethality is an important problem for cross-breeding; however, its molecular mechanism is not clear. The purpose of the present study was to identify the genes expressed during hybrid lethality in the hybrid cells (Nicotiana suaveolens · N. tabacum). In order to identify these genes, we employed suppression subtractive hybridization (SSH) between RNA isolated from cells expressing lethality (lethal hybrid line; LH line) and cells overcoming lethality fortuitously (a surviving hybrid line; SH line). Four populations of cDNA were created from the time points corresponding to before and during induction, and at and after the point of no return (PNR) during the process of programmed cell death (PCD) that occurs during hybrid lethality. By SSH and following dot-blot macroarray analysis, 99 genes out of 138 isolated clones were identified as hybrid lethality-related (HLR) genes. Quantitative real-time PCR analysis data indicated that ten clones were expressed specifically in LH line cells. The HLR genes in these clones show homology to genes involved in disease resistance, ethylene-induced reactions, phosphorylation, ubiquitination, jasmonic acid-related reactions, calcium signaling and self-incompatibility. These data suggested that at least some parts of the mechanism of hybrid lethality are shared with those of the putative functions of the HLR gene-related pathways.

Metagenome represent an unlimited resource for discovery of novel genes. Here we report, sequence analysis of a salt tolerant metagenomic clone (6B4) from a pond water metagenomic library. Clone 6B4 had an insert of 2254 bp with G+C composition of 64.06%. DNA sequence from 6B4 showed homology to DNA sequences from proteobacteria indicating origin of 6B4 metagenomic insert from a yet uncharacterized proteobacteria. Two encoded proteins from clone 6B4 showed match with ATP-dependent Clp protease adaptor protein (ClpS) and phasin, while two truncated encoded proteins showed match with poly-3-hydroxybutyrate synthase and permease. Clp complex is known to play a role in stress tolerance. Expression of ClpS from metagenomic clone is proposed to be responsible for salt tolerance of the metagenomic clone 6B4.

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity. virus Ribosome inactivating proteins (RIPs) are a group of plant enzymes that inhibit polypeptide chain elongation by inactivating the ribosome [24] . All plant RIPs share the common property of inactivating ribosomes and inhibiting protein synthesis. RIPs damage ribosomes in an irreversible manner by removing adenine residue from 28S rRNA by N-glycosidase activity . The removal of one adenine base renders the 60S subunit of eukaryotic ribosomes unable to bind the elongation factor 2 (EF-2), with consequent arrest of protein synthesis .

A newly isolated strain of Kluyveromyces marxianus YS-1 was used for the production of extra cellular inulinase in a medium containing inulin, meat extract, CaCl 2 and sodium dodecyl sulphate (SDS). Fermentation medium pH 6.5, cultivation temperature 30°C and 5% (v/v) inoculum of 12 h-old culture were optimal for enzyme production (30.8 IU/ml) with a fermentation time of 72 h at shake flask level. Raw inulin (2%, w/v) extracted from dahlia tubers by processing at 15 kg/cm 2 for 10 min was optimum for bioreactor studies. Maximum enzyme production (55.4 IU/ml) was obtained at an agitation rate of 200 rpm and aeration of 0.75 vvm in a stirred tank reactor with a fermentation time of 60 h.

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40°C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca +2 caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5l reactor in 34 h. An increase in sugar concentration (15 gl −1 ) in the fermentation medium further increased naringinase production (8.9 IUml −1 ) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.

Naringinase (EC 3.2.1.40) from Penicillium sp was immobilized by covalent binding to woodchips to improve its catalytic activity. The immobilization of naringinase on glutaraldehyde-coated woodchips (600 mg woodchips, 10 U naringinase, 45 • C, pH 4.0 and 12h) through 1% glutaraldehyde cross-linking was optimized. The pH-activity curve of the immobilized enzyme shifted toward a lower pH compared with that of the soluble enzyme. The immobilization caused a marked increase in thermal stability of the enzyme. The immobilized naringinase was stable during storage at 4 • C. No loss of activity was observed when the immobilized enzyme was used for seven consecutive cycles of operations. The efficiency of immobilization was 120%, while soluble naringinase afforded 82% efficacy for the hydrolysis of standard naringin under optimal conditions. Its applicability for debittering kinnow mandarin juice afforded 76% debittering efficiency.

Cellulase Biofuel Lignocellulose Pretreatment a b s t r a c t Lignocellulosic biomass is a highly rigid and recalcitrant structure which requires pretreatment to loosen chemical bonds to make accessible monomeric sugars for biofuel production. In this study, locally available biomass, that is hemp (Cannabis sativa), a low cost feedstock for ethanol production, has been used for the production of fermentable sugars. Hemp hurd biomass (HHB) was exposed to five different pretreatments which included dilute acid (H 2 SO 4 ), alkaline (NaOH), alkaline peroxide, hot water and one stage dilute acid (H 2 SO 4 ). Different pretreatments resulted in loosening and degradation of HHB structure thus facilitating enzymatic saccharification at optimized parameters (pHe4.8 and 50 C). The changes in the reactive groups (hydroxyl or acetyl) of the HHB were confirmed by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Scanning electron microscopy (SEM) was employed to characterize the surface morphology of untreated and treated HHB. Finally, enzymatic saccharification demonstrated maximum yield of total sugars (743 mg g À1 ) that are suitable for biofuel production. ª b i o m a s s a n d b i o e n e r g y 5 8 ( 2 0 1 3 ) 1 8 0 e1 8 7 0961-9534/$ e see front matter ª

Momordica balsamina, African pumpkin (Cucurbitaceae), is a tendril-bearing, wild climber containing wide spectrum of medicinal and nutritional values and has been used as a traditional folk medicine in many countries. The leaves, fruits, seeds, and bark of the plant contain resins, alkaloids, flavonoids, glycosides, steroids, terpenes, cardiac glycoside, saponins having various medicinal importance viz. anti-HIV, anti-plasmodial, shigellocidal, anti-diarrheal, antiseptic, anti-bacterial, anti-viral, anti-inflammatory, anti-microbial, hypoglycemic, antioxidant, analgesic and hepatoprotective properties. The therapeutic agent 'Momordin' is capable of inhibiting the growth of HIV and other viruses. The leaves are also important source of nutrients having 17 amino acids with adequate mineral composition like potassium, magnesium, phosphorus, calcium, sodium, zinc, manganese and iron. It also helps to combat the problem of micronutrient deficiencies in soil and high value of protein and fat with low fibre content. High potassium content is a good source for the management of hypertension and other cardiovascular conditions. This plant is being promoted as a protein supplement for cereal-based diets in poor rural communities. The commercial exploitation of this plant for biopharmaceuticals and neutraceuticals are some of the prospective future potential of this wild herb. This review discusses the potential of medicinal and nutritional importance of this wild herb for health care management.

Screening of matrices for the immobilization of naringinase demonstrated that 2% sodium alginate was an optimal matrix. Upon immobilization, 30 U of naringinase gave 82% naringin hydrolysis in 3 h, broadening of pH optima has attributed desirable flexibility for debittering kinnow juice of varying pHs, and temperature profiles indicated an improved thernwstability which could be handy during the reduction in cost of debittering. Alginate permitted attainment of equilibrium readily with no hindrance in the inj7ow of naringin and the outflow of naringenidprunin, and adequate mechanical stability, cumulatively indicating the feasibility of its commercial exploitation. The application of kinetic parameters optimized with pure naringin to kinnow juice resulted in 60% debittering. It suggested the desirability of ultrafiltration to minimize product inhibition in order to maximize naringin hydrolysis, and subsequent fortification by the permeate at the end of debittering to formulate sweetened kinnow juice with nourishing and natural characteristics. Efforts to change over from the batch process to ~7 continuous column process are underway.

Marine microbes are competent organisms, some of which can accumulate large amounts of lipids. A yeast strain, Rhodotorula mucilaginosa AMCQ8A was isolated from the marine water of the Queenscliff region, Victoria, Australia. The yeast isolate was identified by sequencing 18s rDNA genes. Scanning electron microscopy images revealed scars on the surface of the yeast cells. The Fourier transform infrared spectroscopy microspectroscopy studies demonstrated the presence of unsaturated fatty acids by differential microscopic analysis. The sharp band at 1745 cm L1 was represented by n(C]O) stretches of ester functional groups from lipids and fats, and therefore indicated the presence of total lipids produced by the cells. Over 65% of the fatty acids from the yeast strain were analyzed as C 16 and C 18:1 with omega-3 content from about 6% to 7%. Thus, this marine-derived yeast could be a potential source of lipids, including omega-3 fatty acids.

Marine heterotrophic microbes are capable of accumulating large amounts of lipids, omega-3 fatty acids, carotenoids, and have potential for biodiesel production. Pollen baiting using Pinus radiata pollen grain along with direct plating techniques were used in this study as techniques for the isolation of oil-producing marine thraustochytrid species from Queenscliff, Victoria, Australia. Thirteen isolates were obtained using either direct plating or using pine pollen, with pine pollen acting as a specific substrate for the surface attachment of thraustochytrids. The isolates obtained from the pollen baiting technique showed a wide range of docosahexaenoic acid (DHA) accumulation, from 11 to 41 % of total fatty acid content (TFA). Direct plating isolates showed a moderate range of DHA accumulation, from 19 to 25 % of TFA. Seven isolates were identified on the basis of 18S rRNA sequencing technique as Thraustochytrium species, Schizochytrium species, and Ulkenia species. Although both methods appear to result in the isolation of similar strains, pollen baiting proved to be a simpler method for the isolation of these relatively slow-growing organisms.

Ribosome-inactivating proteins (RIPs) are endowed with several medicinal properties, including antiviral activity. We demonstrate here that the recently identified type I RIP from Momordica balsamina also possesses antiviral activity, as determined by viral growth curve assays and single-round infection experiments. Importantly, this activity is at play even as doses where the RIP has no cytotoxic effect. In addition, balsamin inhibits HIV-1 replication not only in T cell lines but also in human primary CD4 + T cells. This antiviral compound exerts its activity at a viral replicative step occurring later than reversetranscription, most likely on viral protein translation, prior to viral budding and release. Finally, we demonstrate that balsamin antiviral activity is broad since it also impedes influenza virus replication. Altogether our results demonstrate that type I RIP can exert a potent anti-HIV-1 activity which paves the way for new therapeutic avenues for the treatment of viral infections. Citation: Kaur I, Puri M, Ahmed Z, Blanchet FP, Mangeat B, et al. (2013) Inhibition of HIV-1 Replication by Balsamin, a Ribosome Inactivating Protein of Momordica balsamina. PLoS ONE 8(9): e73780.

Background: The aim of this work is to investigate the structure and function of enzymes immobilised on nanomaterials. This work will allow better understanding of enzyme-nanomaterial interactions, as well as designing functional proteinnanomaterial conjugates.

A bacterial strain, MAK-2, was isolated as a producer of a-L-rhamnosidase from a soil sample of Dehradoon, India. The strain was identified based on morphology, physiological tests and 16S rDNA analysis. The phylogenetic analysis based on the 16S rDNA sequence, identified the isolate as Staphylococcus xylosus, a nonpathogenic member of CNS (coagulase-negative staphylococci) family. The strain was capable of producing a-L-rhamnosidase by hydrolysing flavonoids thus confirming potential application in the citrus-processing industry.