Arvind Lali

Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates

ABSTRACT Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G4S)3-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.

Enhanced performance of expanded bed chromatography on rigid superporous adsorbent matrix.

Rigid spherical macroporous adsorbent beads with surface hydroxyl groups were prepared by cross-linking of cellulose. These beads had diameter in the range 100-200 microm and a mean pore size of about 3 microm with about 60% pore volume. The matrix (bulk density approximately 1600 kg m(-3)) could be expanded into a stable bed and used for protein chromatography. Chromatographic runs were performed on a 10 mm diameter column under non-retaining and retaining conditions on the prepared matrix (called Celbeads) and performance of the runs was measured in terms of the height equivalent to a theoretical plate (HETP). The HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents, i.e. increasing and levelling with velocity. Unimpaired performance of the matrix at increasing flow-rates permitted expanded bed elution of adsorbed solutes without loss of efficiency in terms of purification factor and product concentration. As a model system, Celbeads was used to purify lactate dehydrogenase from porcine muscle homogenate by dye-affinity chromatography. The prepared matrix provided about 100 theoretical plates per meter for the enzyme system at a linear flow velocity of 1.27 cm x min(-1) in an expanded bed elution mode, and gave enzyme yields of 100% with a purification factor of 31 using an optimized procedure. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.

Comparison of batch, packed bed and expanded bed purification of A. niger cellulase using cellulose beads

Rigid macroporous cross-linked cellulose beads were prepared and used as a useful affinity medium for purification of A. niger cellulase from commercial preparation, in batch; packed bed and expanded bed modes. The beads bound 99% activity in both packed bed and expanded bed modes and upto 91% activity could be recovered by washing the adsorbent with 1 M phosphate buffer, pH 7.0. While batch adsorption and elution gave only 4-fold purification, packed bed operation gave 14-fold purification and expanded bed, the highest, 36-fold purification.

Expanded bed affinity purification of bacterial α-amylase and cellulase on composite substrate analogue–cellulose matrices

Abstract Expanded bed purification of α-amylase and cellulase directly from unclarified fermentation broth was carried out on specially prepared composite affinity matrices. The concept used was incorporation of polymeric substrates/substrate analogue during cross-linking of cellulose to prepare rigid, porous, cross-linked composite affinity matrices for target enzymes. Of the several polymeric substrates/substrate-analogue used, alginic acid (AA) and microcrystalline cellulose (MCC) when used to prepare cross-linked composite matrices with cellulose, resulted in best affinity purification matrices for α-amylase and cellulase, respectively. These matrices were suitable for purification of the enzymes by batch, packed bed as well as expanded bed purification protocols. The optimized expanded bed protocol for α-amylase from Bacillus spp. B3 gave 51-fold purification on AA-CELBEADS with 69% recovery, whereas, cellulase from Bacillus spp. B21 was purified on MCC-CELBEADS to 18-fold purification with 97% recovery. The SDS-PAGE of both purified preparations showed single bands indicating significant purification on composite affinity adsorbents in a single step strategy.

AbSep--an amino acid based pseudobioaffinity adsorbent for the purification of immunoglobulin G.

The present work deals with the development and characterization of a tryptophan based pseudobioaffinity adsorbent for the purification of monoclonal and polyclonal antibodies. Tryptophan as a ligand was selected based on molecular docking and experimental screening studies of the amino acids involved in IgG-Protein A interaction. The ligand was coupled to a polymethacrylate based rigid, porous SEPABEADS beaded matrix to obtain the desired affinity adsorbent, which was named AbSep. Characterization studies with regards to the effect of matrix properties (pore size, particle size, nature of matrix, spacer arm) and the medium properties (pH, conductivity, additives) were performed to elucidate the nature of IgG-AbSep interactions and to determine the optimal conditions for obtaining high binding and purity of IgG. The equilibrium binding capacity of AbSep and dissociation constant was found to be 78 mg/ml and 5.31×10(-6)M respectively. AbSep was able to successfully purify polyclonal human IgG from plasma and monoclonal antibody (chimeric IgG1) from CHO cell culture supernatant. Both binding and elution steps were performed at near neutral pH resulting in a purity and recovery of more than 90% and 85% respectively. Additionally, AbSep was shown to be stable to 0.5M NaOH solutions, the preferred agent for cleaning and sanitization of chromatographic media.

An integrated process for the extraction of fuel and chemicals from marine macroalgal biomass.

We describe an integrated process that can be applied to biomass of the green seaweed, Ulva fasciata, to allow the sequential recovery of four economically important fractions; mineral rich liquid extract (MRLE), lipid, ulvan, and cellulose. The main benefits of our process are: a) its simplicity and b) the consistent yields obtained from the residual biomass after each successive extraction step. For example, dry Ulva biomass yields ~26% of its starting mass as MRLE, ~3% as lipid, ~25% as ulvan, and ~11% as cellulose, with the enzymatic hydrolysis and fermentation of the final cellulose fraction under optimized conditions producing ethanol at a competitive 0.45 g/g reducing sugar. These yields are comparable to those obtained by direct processing of the individual components from primary biomass. We propose that this integration of ethanol production and chemical feedstock recovery from macroalgal biomass could substantially enhance the sustainability of marine biomass use.

Exacting predictions by cybernetic model confirmed experimentally: Steady state multiplicity in the chemostat

We demonstrate strong experimental support for the cybernetic model based on maximizing carbon uptake rate in describing the microorganisms regulatory behavior by verifying exacting predictions of steady state multiplicity in a chemostat. Experiments with a feed mixture of glucose and pyruvate show multiple steady state behavior as predicted by the cybernetic model. When multiplicity occurs at a dilution (growth) rate, it results in hysteretic behavior following switches in dilution rate from above and below. This phenomenon is caused by transient paths leading to different steady states through dynamic maximization of the carbon uptake rate. Thus steady state multiplicity is a manifestation of the nonlinearity arising from cybernetic mechanisms rather than of the nonlinear kinetics. The predicted metabolic multiplicity would extend to intracellular states such as enzyme levels and fluxes to be verified in future experiments.

Expanded bed adsorption on supermacroporous cross-linked cellulose matrix

Rigid spherical macroporous adsorbent beads (CELBEADS) prepared by cross-linking of cellulose were characterised and found eminently suitable for use as expanded bed affinity chromatography matrix. Chromatographic runs were performed on a 10 mm diameter column with three solutes tyrosine, papain and bovine serum albumin under non-retaining conditions on CELBEADS and StreamlineTM DEAE, a commercial agarose based expanded bed matrix. Performance of the runs was measured in terms of height equivalent to theoretical plate, HETP. Variation in HETP with velocity on StreamlineTM DEAE gave flat profiles in packed bed and increasing trend in expanded bed. On CELBEADS, the HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents i.e. increasing and levelling with velocity. HETP values obtained for papain and bovine serum albumin on CELBEADS were lower than those obtained on StreamlineTM DEAE at all velocities. Lactate dehydrogenase was purified from porcine muscle homogenate using Cibacron blue conjugated to CELBEADS using a protocol reported for supports with surface hydroxyl groups. Elution of the enzyme was investigated both in packed mode as well as in expanded mode at a flow rate of 1 ml min-1. The purification procedure took about 60 minutes and a purification fold of about 14 was achieved in both cases. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.

Reversible precipitation of proteins on carboxymethyl cellulose

Abstract Precipitation, one of the steps used most routinely in protein purifications, suffers from a general lack of selectivity. In an attempt to impart controllable selectivity of a definite nature, carboxymethyl cellulose was developed as a soluble ion-exchange precipitant for isolation and purification of proteins. Carboxymethyl cellulose was used as a reversibly soluble/insoluble ion exchange matrix, which after binding proteins from ‘crude’ could be precipitated quantitatively with a combination of calcium and polyethylene glycol. The precipitated proteins were recovered by solubilising in a dissociating buffer devoid of any one or both of the precipitants i.e. calcium and polyethylene glycol; followed by reprecipitating of carboxymethyl cellulose. Lactoperoxidase was used as a model enzyme and purified from milk whey using the proposed system. A purification factor of about four was achieved in a single step of precipitation with a suitable choice of binding and elution conditions.

Carboxymethyl cellulose as a new heterobifunctional ligand carrier for affinity precipitation of proteins

Affinity precipitation is a technique that imparts selectivity to the widely used primary purification step of precipitation of proteins from crude extracts. Hetero-bifunctional affinity precipitation involves use of reversibly soluble/insoluble polymers that can be used as backbones to conjugate affinity ligands for specific separations. A variety of such polymers have been reported in literature. In this work we report development of carboxymethyl cellulose (CM cellulose) as a cheap, readily available and versatile reversibly soluble polymer system. Available CM cellulose as sodium salt could be quantitatively precipitated from its aqueous solution in presence of about 50 mM calcium and 7.2% w/v polyethylene glycol-4000, and could be resolubilised in the working buffer in absence of calcium, polyethylene glycol or both. Effectiveness of the CM cellulose-calcium-polyethylene glycol system was demonstrated by purifying lactate dehydrogenase from porcine muscle extractusing covalently conjugated Cibacron blue dye-ligand. By careful choice of conditions that suppressed non-specific interactions, the system was shown to be an effective affinity precipitation polymer system inspite of the polyelectrolytic nature of CM cellulose. Up to 23 fold purification of the enzyme from crude extarct was obtained in one single precipitation sequence.