Saroj Mishra

Adhesives and plastics based on soy protein products

Significance of eco-friendly materials based on easily renewable natural resources, and the finite nature of petrochemical resources, has necessitated the development of polymers from agricultural processing by products such as soy proteins from oil processing. Although, considerable work was done in the early part of last century on polymers based on soy protein, there was almost no activity in this field for the last fifty years. There is a need to critically analyse the available literature on soy protein based polymeric materials. Therefore, an attempt is made to review the state-of-the-art of the polymeric materials with emphasis on adhesives and plastics derived from soy protein, a renewable resource abundantly available in nature.

Enzymatically-modified soy protein part 2: adhesion behaviour

Adhesive properties of soy protein isolated from soy protein concentrate (SPC) and its modification by proteolytic enzymes (such as papain, trypsin, chymotrypsin and pepsin) and urease were investigated on different types of woods (such as rubberwood, Bhutan pine, teakwood and plywood). Adhesive strength and viscosity of native and enzyme modified soy protein were compared with commercially available adhesives using the same types of woods. Papain and urease modified soy protein isolate (SPI) showed better adhesive strength compared to unmodified SPI adhesive on rubberwood, while chymotrypsin modified SPI showed zero adhesive strength. All enzymatically-modified adhesives showed lower viscosity in comparison to unmodified SPI adhesive. Hydrophobicity of the native and modified SPIs was determined by the sodium dodecyl sulfate (SDS) binding method. Chymotrypsin-modified SPI (CSPI) showed maximum value of hydrophobicity followed by papain and trypsin modified SPI. Urease modified SPI showed only slight increase in hydrophobicity with respect to unmodified SPI. Structural changes after hydrolysis of SPI by different enzymes were investigated by FT-IR spectroscopy. The ratio of the intensity of νc=o of carboxyl and amide I or ratio of νc=o of carboxyl and amide II showed maximum value in case of pepsin modified SPI followed by chymotrypsin modified SPI. Papain, trypsin and urease modified SPIs showed only slight increase in the ratio of carboxyl/amide I or amide II.

Bibliometric study of bioinformatics literature

Bioinformatics is a multidisciplinary and comparatively new area of science that has made a significant impact within a short period. A systematic analysis of the rise in bioinformatics literature is, however, not available. This study analyses the growth of the scientific literature in this area as available from NCBI PubMed using standard bibliometric techniques. Bradford’s law of scattering was used to identify core journals and Lotka’s law employed to analyze author’s productivity pattern. Study also explored publication type, language and the country of publication. Twenty core journals were identified and the primary mode of dissemination of information was through journal articles. Authors with single publication were more predominant (73.58%) contrary to that predicted by Lotka’s law. The study provides useful information to scientists wishing to undertake work in this area.

The 69 kDa Escherichia coli maltodextrin glucosidase does not get encapsulated underneath GroES and folds through trans mechanism during GroEL/GroES-assisted folding

Escherichia coli chaperonin GroEL and GroES assist in folding of a wide variety of substrate proteins in the molecular mass range of ~50 kDa, using cis mechanism, but limited information is available on how they assist in folding of larger proteins. Considering that the central cavity of GroEL can accommodate a non‐native protein of ~60 kDa, it is important to study the GroEL‐GroES‐assisted folding of substrate proteins that are large enough for cis encapsulation. In this study, we have reported the mechanism of GroEL/ GroES‐assisted in vivo and in vitro folding of a 69 kDa monomelic E. coli protein maltodextrin glucosidase (MalZ). Coexpression of GroEL and GroES in E. coli causes a 2‐fold enhancement of exogenous MalZ activity in vivo. In vitro, GroEL and GroES in the presence of ATP give rise to a 7‐fold enhancement in MalZ refolding. Neither GroEL nor single ring GroEL (SR1) in the presence or absence of ATP could enhance the in vitro folding of MalZ. GroES could not encapsulate GroEL‐bound MalZ. All these experimental findings suggested that GroEL/GroES‐assisted folding of MalZ followed trans mechanism, whereas denatured MalZ and GroES bound to the opposite rings of a GroEL molecule.—Paul, S., Singh, C., Mishra, S., Chaudhuri, T. K. The 69 kDa Escherichia coli maltodextrin glucosidase does not get encapsulated underneath GroES and folds through trans mechanism during GroEL/GroES‐assisted folding. FASEB J. 21, 2874–2885 (2007)

Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase

BackgroundLaccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase.ResultsIn this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg-1 protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac.ConclusionLaccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L-1 in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation.

Fibril formation from cellulose by a novel protein from Trichoderma reesei: A non-hydrolytic cellulolytic component?

A low molecular weight protein, named fibril-forming protein (FFP), was isolated from the culture supernatant of Avicel-grown Trichoderma reesei. The protein was purified to homogeneity and it exhibited a molecular weight of 11,400Da. Low amounts of this protein caused apparently non-hydrolytic disruption of filter paper, releasing fibrils without any detectable release of reducing sugars. It displayed no hydrolytic activity on carboxymethylcellulose (CMC), p-nitrophenyl-β-d-glucoside (pNPG) or 4-methylumbelliferyl cellobioside. The pH optimum of the protein was between 4 and 5. The temperature optimum was 40°C and the computed activation energy (Ea) for the filter paper disruption process was 4.18kcal/mol, suggesting disruption of non-covalent bonds. It had no immunological cross reactivity with reported cellulase components of T. reesei.

Bioprocess strategies for enhanced production of xylanase by Melanocarpus albomyces IITD3A on agro-residual extract

The production of high titer xylanase without cellulase is required for prebleaching of pulps in pulp and paper industry. The mutant IITD3A of Melanocarpus albomyces developed from the spores of the wild type organism was used in this study. The statistical optimization of the process parameters by response surface methodology revealed that the production of xylanase was most affected by changes in the pH of the production medium which contained a soluble extract of wheat straw as the sole carbon source. When the pH of the production medium in a 14 L bioreactor was controlled on-line at 7.8, xylanase activity of 415 IU mL⁻¹ was obtained after 36 h fermentation. On cycling the pH between 7.8 and 8.2, the same activity could be attained in 24 h with an overall productivity of 16,670 IU L⁻¹ h⁻¹. The production of xylanase was also influenced by the fungus morphology; the activity being maximum when it exhibited pellet form at an agitation speed of 600 rpm. On optimization of aeration rate to 0.25 vvm, the xylanase activity further increased to 550 IU mL⁻¹ with a very high overall volumetric productivity of 22,000 IU L⁻¹ h⁻¹. Thus, a 5.2-fold enhancement in overall volumetric productivity of xylanase could be obtained by the mutant in comparison to that obtained on insoluble wheat straw.

Enzymatically modified Soy Protein

Optimum temperature and pH for the isolation of soy protein isolate (SPI) from soy protein concentrate (SPC) were established. Enzymatic hydrolysis of SPI with enzymes of different specificities such as trypsin, chymotrypsin, papain and urease was carried out and the products of hydrolysis were characterized by molecular mass determination [sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] and thermal techniques [differential scanning calorimetry (DSC) and thermogravimetric analysis (TG)]. Enzymatic hydrolysis resulted in a significant reduction in molecular masses. However the thermal stability of hydrolysed SPI was similar to native SPI indicating that it is independent of molecular mass. DSC studies indicated an increase in temperatures of endothermic transition associated with SPI denaturation and loss of absorbed moisture in samples of lower molecular masses.

Enhancement of over expression and chaperone assisted yield of folded recombinant aconitase in Escherichia coli in bioreactor cultures.

A major portion of the over expressed yeast mitochondrial aconitase, a large 82 kDa monomeric TCA cycle enzyme, in Escherichia coli led to the formation of inclusion bodies. Bacterial chaperonin GroEL mediated the correct folding of aconitase with the assistance of its co-chaperonin GroES in an ATP dependent manner. Till date the chaperonin assisted folding of aconitase was limited to the shake flask studies with relatively low yields of folded aconitase. No attempt had yet been made to enhance the yield of chaperone mediated folding of aconitase using a bioreactor. The current report deals with the effect of co-expression of GroEL/GroES in the production of soluble, biologically active recombinant aconitase in E. coli by cultivation in a bioreactor at different temperatures under optimized conditions. It revealed that the yield of functional aconitase was enhanced, either in presence of co-expressed GroEL/ES or at low temperature cultivation. However, the outcome from the chaperone assisted folding of aconitase was more pronounced at lower temperature. A 3-fold enhancement in the yield of functional aconitase from the bioreactor based chaperone assisted folding was obtained as compared to the shake flask study. Hence, the present study provides optimized conditions for increasing the yield of functional aconitase by batch cultivation in a bioreactor.

Cloning, characterization ofPichia etchellsii β-glucosidase II and effect of media composition and feeding strategy on its production in a bioreactor

The cloning and expression of β-glucosidase II, encoded by the geneßglu2, from thermotolerant yeastPichia etchellsii intoEscherichia coli is described. Cloning of the 7.3 kbBamHI/SalI yeast insert containingßglu2 in pUC18, which allowed for reverse orientation of the insert, resulted in better enzyme expression. Transformation of this plasmid intoE. coli JM109 resulted in accumulation of the enzyme in periplasmic space. At 50°C, the highest hydrolytic activity of 1686 IU/g protein was obtained on sophorose. Batch and fed-batch techniques were employed for enzyme production in a 14 L bioreactor. Exponential feeding rates were determined from mass balance equations and these were employed to control specific growth rate and in turn maximize cell growth and enzyme production. Media optimization coupled with this strategy resulted in increased enzyme units of 1.2 kU/L at a stabilized growth rate of 0.14 h−1. Increased enzyme production in bioreactor was accompanied by formation of inclusion bodies.