V.S. Bisaria

Microbial β-glucosidases: Cloning, properties, and applications

ABSTRACT:  β-Glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and amino-glucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial β-glucosidases, yeast β-glucosidases, thermostable fungal β-glucosidase, and the physiological functions, characteristics, and catalytic action of native β-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant β-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.

Plant cell reactors—A perspective

Abstract The potential of plant cell culture for the production of commercially valuable secondary metabolites is reviewed. The techniques employed for large-scale plant cell suspension culture are largely those developed for microbial culture, so the basic characteristics of the two types of culture are compared. Technological problems associated with plant cell culture—mixing, oxygen transfer, and cell aggregation and adhesion—are then considered in detail. Various bioreactors currently used for plant cell cultivation are analyzed according to the dynamics of growth and product formation in microbial systems exhibiting no cellular differentiation. Criteria for choosing a bioreactor type for optimal growth and metabolite production are also discussed. Finally a brief economic outlook and future prospects for plant cell culture processes are presented.

Effect of formulated root endophytic fungus Piriformospora indica and plant growth promoting rhizobacteria fluorescent pseudomonads R62 and R81 on Vigna mungo

In the present investigation, the effect of three beneficial organisms (root endophytic fungus Piriformospora indica (Pi) and pseudomonads strains R62 and R81) and their four different consortia (Pi+R62, Pi+R81, R62+R81, Pi+R62+R81) was investigated on the plant Vigna mungo through their inorganic carrier-based (talcum powder and vermiculite) formulations. All the treatments resulted in significant increase in growth parameters under glasshouse as well as field conditions and showed a consistency in their performance on moving from glasshouse to field conditions. In glasshouse conditions, a maximum increase of 4.5-fold in dry root weight and 3.9-fold in dry shoot weight compared to control was obtained with vermiculite-based consortium formulation of Pi+R81. In field studies using vermiculite as carrier, a maximum enhancement of 3.2-fold in dry root weight, 3.0-fold in dry shoot weight, 8.4-fold in number of nodules and 4.0-fold in number of pods in comparison to control was obtained with the bio-inoculant formulation containing consortium of Pi+R81. The same treatment also caused the highest improvement of 1.9-fold in nitrogen content and 1.7-fold in phosphorus content, while the highest increase of 1.4-fold in potassium content was obtained with Pi alone.

Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium

Multi-analogue resistant mutants of Corynebacterium lilium were developed using UV and N -methyl-N ?-nitro-nitrosoguanidine (NTG) mutagenesis in order to overproduce methionine. Although the methionine yield at each stage of mutation by UV and NTG mutagenesis were comparable, it was observed that the NTG mutants had higher cell growth and glucose utilization rates. From the parent NTG mutant C. lilium E 4 resistant to ethionine, the highest methionine producing strain C. lilium M-128 resistant to 3.5 mg ml � 1 of ethionine, 3.0 mg ml � 1 of norleucine, 3.8 mg ml � 1 of methionine sulphoxide and 3.8 mg ml � 1 of methionine methyl sulphoniumchloride was derived. When C. lilium M-128 was grown under optimised conditions, it produced 2.3 g l � 1 of methionine in a 15 l batch reactor. The maximum cell mass concentration obtained was 17 g l � 1 dry cell weight resulting in a cell yield coefficient of 0.34 g g � 1 . The dissolved oxygen concentration was controlled at 40% saturation by cascade control of the airflow rate and agitation speed. # 2002 Elsevier Science Ltd. All rights reserved.

Influence of L-Sorbose on Growth and Enzyme Synthesis of Trichoderma reesei C-5

SUMMARY: The effects of L-sorbose on the growth and biosynthesis of cellulases and other polysaccharide-degrading enzymes of Trichoderma reesei C-5 were studied. The specific growth rate and yield of this strain in batch culture were reduced by 23% and 46% respectively on addition of 1% (w/v) sorbose to Vogels medium containing 1% (w/v) glucose. The specific consumption rate of both sorbose and glucose decreased in the presence of the other sugar at 1% (w/v) concentration. The addition of sorbose (1-5%) to cultures grown in 1% glucose resulted in enhanced activities of all cellulase enzymes, and particularly endoglucanase activity, which increased sevenfold in the presence of 5% sorbose. There was no significant effect on the activities of β-glucosidase, acid phosphatase and amylase. While the increased enzyme activities seemed to be correlated with a decreased rate of glucose consumption, a direct effect on some extracellular enzymes could not be ruled out.

Cell culture of Holarrhena antidysenterica : growth and alkaloid production

Abstract Callus and suspension culture of Holarrhena antidysenterica were established for production of steroidal alkaloids, especially conessine. The doubling time and specific growth rate of cells in suspension culture were computed to be 47.5 hr and 0.35 per day respectively. A maximum of 300 mg alkaloids per 100 g dry cell wt in 40 days and 130 mg per 100 g dry cell wt in eight days were obtained in the callus and suspension cultures respectively. Alkaloid production in suspension culture was a combined growth and non-growth associated phenomena. About 90% of the total alkaloids produced in the cell culture was conessine, which was confirmed by spectral data.

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25μg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.

Effect of Piriformospora indica on Enhanced Biosynthesis of Anticancer Drug, Podophyllotoxin, in Plant Cell Cultures of Linum album

Plant cell technology provides an alternative to the whole plant cultivation for the production of valuable plant-based secondary metabolites. The treatment of plant cells with elicitors is one of the most effective strategies for improving secondary metabolite production by plant tissue and cell cultures. These elicitors stimulate secondary metabolite production and thereby decrease the process time to attain high product concentrations. A variety of fungal products (cell wall polysaccharides, culture filtrate, mycelia, spores, polypeptides, glycoproteins, lipid molecules, and metabolites) have been used as elicitors to trigger secondary metabolite biosynthesis by plant cell cultures. This chapter provides information about effects of culture filtrate, cell extract, and live cells of root endophytic fungus Piriformospora indica as elicitors, on growth and lignan production by Linum album hairy root cultures.

Enzymatic synthesis of oligosaccharides, alkyl and terpenyl glucosides, by recombinant Escherichia coli-expressed Pichia etchellsii β-glucosidase II

The biosynthetic activity of yeast Pichia etchellsii β-glucosidase II (BglII) expressed in recombinant Escherichia coli was utilized for synthesis of cellooligosaccharides, alkyl and terpene glucosides. Cellooligosaccharides with a degree of polymerization of 3 and greater were resolved by thin-layer chromatography (TLC) using an ethyl acetate: 1-propanol:2-propanol:water (8:5:1:1) solvent system followed by visualization with 0.2% naphthoresorcinol reagent. Using 2M cellobiose and 15 IU of partially purified BglII, 57 mmol/L of oligosaccharides (comprising mostly cellotriose and cellopentaose) was synthesized in 16 h. Similarly, alkyl glucosides with chain lengths from 6 to 10 carbons were synthesized and products extracted to near purity by ethylacetate extraction. The same extraction method was employed to separate, to near purity, various monoterpenyl (nerol, geraniol, citronellol) glucosides. A reliable and simple method for separation of cellooligosaccharides using a combination of Bio-Gel P-2 gel filtration and charcoal celite adsorption chromatography was developed. The cellooligosaccharides were separated to purity as confirmed by TLC. The enzyme was among the very few that could synthesize a wide variety of glycoconjugates.

Xylanases from Thermophilic Fungi: Classification, Structure, and Case Study of Melanocarpus albomyces

Xylanases are an important category under the glycoside hydrolase families and together with cellulases constitute nearly 25% market in enzyme sector. Some of the major applications of this enzyme are in bleaching of pulp and paper, food and feed sector, etc. For several of these applications, enzymes from thermophilic sources are preferred. In this chapter, we present information on classification of family 11 xylanases, used in pulp and paper industry. Factors underlying thermostability, such as the length and composition of the N-terminus, Ser/Thr ratio, presence of Arg on enzyme surface, core packing, and hydrophobic interactions, have been described. Based on these principles, protein engineering approaches to achieve thermostability of fungal xylanases are reviewed. Our own work on development of hyper-xylanase-producing mutant and process strategies adopted to enhance production of this enzyme from a thermophilic fungus Melanocarpus albomyces is summarized. Role of nitrogen source, pH, temperature, aeration, and agitation is emphasized through this case study whereby productivities of 22,000 IU/L/h have been achieved. Additives, currently in use, to make stable xylanase preparation are also described. Special emphasis is laid on downstream processing, which includes role of carriers and binders in producing the product of desired quality.