Search

Globally, the reserves of heavy crude oil are seven times more abundant than that of light crude, and yet, they are underutilized because of their high viscosity and density, which is largely due to the presence of large amounts of asphaltenes. Biotransformation of heavy oil asphaltenes into smaller metabolites can be used for reducing their viscosity. Several microorganisms capable of asphaltene biodegradation have been reported but only few have been characterized for its biotransformation. In the present study, a 9-membered microbial consortium was isolated from an oil contaminated soil. About 72% and 75% asphaltene biotransformation was achieved by growing cells at shake flask level and in a 1.5 l bioreactor, respectively. A representative structure of asphaltene was constructed based on LC–MS, 1H-NMR, 13C-NMR, FT-IR, ICPMS and elemental analysis (CHNS) of n-heptane purified asphaltene from Maya crude oil. Biotransformation of asphaltene, as analyzed by performing 1H-NMR, FT-IR and elemental analysis, resulted in 80% decrease in S and N when compared to the control along with incorporation of oxygen in the structure of asphaltene. About 91% decrease in the viscosity of the Maya crude oil was observed after two weeks when oil: aqueous phase ratio was 1:9. The results suggest that the isolated microbial consortium can be used for biological upgradation of heavy crude oil. To our knowledge, this is the first report where a microbial consortium resulted in such high asphaltene biotransformation.

Termitomyces sp. OE 147 is one of the active cellulose degraders in the ecosphere and produces large amount of cellobiose dehydrogenase (CDH) and β-glucosidases when cultivated on cellulose. In ord...

Background The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. Results Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57–70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30–43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47–49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30–43 in the WT-GCSF was partially restored (by 60%) when the Δ57–70 was added. The effect of Δ47–49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57–70 indicating the importance of the 47–49 region. A stimulatory effect of Δ57–70 was confirmed in the double deletion (Matα:Δ57–70;47–49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57–70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. Conclusions Among the different truncations (Matα:Δ57–70, Matα:Δ47–49, Matα:Δ30–43, Matα:Δ57–70;30–43, Matα:Δ57–70;47–49) in pro-peptide of α-MAT secretion signal, Matα:Δ57–70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30–43 and Matα:Δ47–49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

Here, we report the whole-genome sequence of Bacillus sp. strain IITD106. The bacterium has the unique ability to produce saponins. The complete nucleotide sequence will provide insights into the various genes and regulators involved in the biosynthesis of saponin.

The human granulocyte colony-stimulating factor (G-CSF) is one of the hematopoietic growth factors administered for chemotherapy induced neutropenia and is currently produced through recombinant route in Escherichia coli. The methylotrophic unicellular yeast Pichia pastoris (syn. Komagataella phaffii) makes a good host for production of human therapeutics as the proteins are low-mannose glycosylated, disulfide bonded and correctly folded on their way to the cell exterior. Given the low level of production of G-CSF in P. pastoris, the present study examined modification of the Saccharomyces cerevisiae derived α-mating type secretory signal sequence to enhance its production. The substitution of Glu, at the P1' position of the Kex2 cleavage site, by Val/Ala led to extracellular production of ~ 60 mg/L of G-CSF in the extracellular medium. Production was further increased to ~ 100 mg/L by putting these mutations against rarely occurring methanol slow utilization P. pastoris X-33 host. Analysis of the modelled structure of the signal peptide indicated exposed loop structures, created by presence of Val/Ala, that favour cleavage by the Kex2 peptidase thereby leading to enhanced production of G-CSF. The conformational changes, induced on account of binding between the signal sequence and the cargo protein (G-CSF), also appear to play an important role in the final yield of the extracellular protein.

Laccases (EC 1.10.3.2) are multi-copper oxidases that can degrade several xenobiotics, including textile dyes. Present study investigated the nature of laccase isoforms induced by 2,6-dimethylaniline in Cyathus bulleri cultivated on basal salt medium. Two isoforms, LacI and LacII were identified and purified by a combination of ultrafiltration and ion-exchange chromatography. The MS spectrum of the two proteins displayed a number of non-identical and identical molecular peaks (m/z), and, the latter were mapped to protein originating from the previously reported Laccase (Lcc) 1 gene. The LacI isoform exhibited higher catalytic efficiency (Kcat/Km) towards 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), 2,6-dimethoxyphenol, guaiacol and pyrogallol and was tolerant to high levels of chloride ions and resistant to EDTA. Higher decolorization of several dyes such as Direct Scarlet B (67%), Reactive Brilliant blue-R (96%), Direct Orange 34 (50%) and Reactive Red198 (95%) by the LacI isoform makes it a good candidate for degradation of synthetic dyes. The decolorization of Direct Orange 34 by laccases is being reported for the first time. Many of the properties exhibited by this isoform make it a good candidate for large scale production and applications for use in the dyeing industry.

Protein folding is an extremely complex and fast, yet perfectly defined process, involving interplay of many intra and inter-molecular forces. In vitro, these molecular interactions are reversible for many proteins e.g., smaller and monomeric, organized into single domains. However, refolding of larger multi-domain/multimeric proteins is much more complicated, proceeds in a hierarchal way and is often irreversible. In a comparative study on two large, multi-domain and multimeric isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from Pichia etchellsii, we studied spontaneous and assisted refolding under three denaturing conditions viz. GdnHCl, alkaline pH and heat. During refolding, higher refolding yields were obtained for BGLII in case of pH induced unfolding (13.89% ± 0.25) than BGLI (6% ± 0.85) while for GdnHCl induced unfolding, refolding was marginal (BGLI = 5% ± 0.5; BGLII = 6% ± 0.69). Thermal unfolding was irreversible while assisted refolding also showed little structural gain for both proteins. When the apparent free energies of unfolding (ΔGUapp) were calculated from GdnHCl unfolding data, their values were strikingly found to be lower (BGLI ΔGUapp = 3.02 kcal/mol; BGLII ΔGUapp = 2.99 kcal/mol) than reported for globular (ΔGU = 5–15 kcal/mol)/multimeric proteins (ΔGU = 23–29 kcal/mol) indicating marginal stability results in low refolding.

This study reports extraction and characterization of carotenoid pigments from Microbacterium paraoxydans, a non-photosynthetic bacterium, cultivated in Luria–Bertani (LB) medium. The isolate was identified to be moderately halo- and osmo-tolerant capable of withstanding high (~ 6%) salt and sugar (30% w/v sucrose, 20% w/v glucose) concentrations after a brief period of adaptation. The pigments were characterized using a combination of UV–Vis spectral analysis with the λmax at 407, 436 and 466 nm and ESI–MS with an m/z value at 536.44. The absorption profile of the pigments and their nature was influenced by carbon, nitrogen source and presence of salt in the growth medium. Highest level of pigment (~ 16 g kg dry wt cells−1) was produced in NH4Cl supplemented LB medium. The pigment displayed free radical scavenging, anticancer activity, characteristic of the plant carotenoids. Based on the accumulation of pigments under different conditions, a biochemical pathway for synthesis of neurosporene was proposed.

In this study, the white-rot fungus Cyathus bulleri was cultivated on low-cost agro-residues, namely wheat bran (WB), wheat straw (WS), and domestic waste orange peel (OP) for production of ligninolytic enzymes. Of the three substrates, WB and OP served as good materials for the production of laccase with no requirement of additional carbon or nitrogen source. Specific laccase activity of 94.4 U mg−1 extracellular protein and 21.01 U mg−1 protein was obtained on WB and OP, respectively. Maximum decolorization rate of 13.6 μmol h−1 U−1 laccase for reactive black 5 and 22.68 μmol h−1 U−1 laccase for reactive orange 16 (RO) was obtained with the WB culture filtrate, and 11.7 μmol h−1 U−1 laccase for reactive violet 5 was observed with OP culture filtrate. Importantly, Kiton blue A (KB), reported not to be amenable to enzymatic degradation, was degraded by culture filtrate borne activities. Products of degradation of KB and RO were identified by mass spectrometry, and a pathway of degradation proposed. WB-grown culture filtrate decolorized and detoxified real and simulated textile effluents by about 40%. The study highlights the use of inexpensive materials for the production of enzymes effective on dyes and effluents.

Highlights • Use of culture filtrates of Termitomyces sp. OE147. • Decolourization and degradation of Reactive blue 21. • Cellobiose dehydrogenase and laccase as effective redox couple., This study evaluates culture filtrate, rich in cellobiose dehydrogenase and laccases, of Termitomyces sp. OE 147, in decolouration and degradation of Reactive blue (RB) 21. About 35% decolouration was achieved at low volumes of the culture supernatant without addition of external redox mediators. An optimized dye to culture fluid ratio (75 ppm: 0.1 ml) at a pH of 4–5 resulted in removal of colour by 60%. The degradation products of RB21 were analysed by Electron Spray Ionization-Mass Spectrometry and several small molecules (of m/z 106–199) were detected. These were concluded to be o-Xylene, 2,3-Dihydro-1H-isoindole, Isoindole-1,3-dione, 2,Benzenesulfonyl-ethanol, (4-Hydroxy-phenyl)-sulfamic acid, 2,3-Dihydro-1H-isoindole-5-sulfonic acid and proposed to result from joint action of cellobiose dehydrogenase, laccase, peroxidases and unidentified oxidoreductases present in the culture fluids. Based on the products formed and the known reactions of these enzymes, a degradation pathway was proposed for RB21. The culture fluid was also effective in decolouration (by about 50%) and detoxification (by ∼25%) of the combined effluent collected from a local mill indicating a treatment process that bypasses use of H2O2 and toxic mediators.

The synthesis of fructo-oligosaccharides was carried out using whole cells of Microbacterium paraoxydans. Reactions were carried out using un-induced, inulin-induced and sucrose-induced cells displaying different amounts of invertase and inulinase activities out of which the best transfructosylation occurred using sucrose-induced cells displaying 12 I.U. invertase/0.75 I.U. inulinase activities. Using 40% w/v sucrose and in the sucrose-induced cells, a maximum fructo-oligosaccharide yield of 155 g/l (corresponding to product yield of 0.38 g/g initial substrate) was obtained. The major products synthesized were the tri-saccharide, 1-kestose [1F (1-β- d -fructofuranosyl)sucrose] and the tetrasaccharide, nystose [1F (1-β- d -fructofuranosyl)kestose]. A Box–Behnken design was used to optimize the factors affecting the fructo-oligosaccharide synthesis and these were at 31.5% sucrose, 10.96 I.U. invertase/0.69 I.U. inulinase and 14.32 h incubation time leading to an overall yield of 0.44 g/g initial substrate. The synthesized 1-kestose and nystose were purified to homogeneity by preparative TLC and structurally characterized by ESI-MS and 2D NMR.

A number of limitations exist for production of human granulocyte colony-stimulating factor (G-CSF) in Pichia pastoris. In this study, two different specific growth rates (0.015 h−1, 0.01 h−1) were used sequentially in the mixed substrate feeding period during methanol induction phase to enhance the G-CSF titer in the culture broth. Necessary parameters required for implementing the feeding strategy, such as specific product yield on biomass (YP/X) and maintenance coefficient (m) on glycerol, methanol, and sorbitol were estimated using continuous culture technique. Using this strategy, for the same volumetric productivity, about 20% increase in protein titer was achieved over that obtained from the run carried out at a single pre-set value of 0.015 h−1 alone. Thus, implementation of higher specific growth rate (0.015 h−1) set during initial stages of the methanol induction phase followed by a lower specific growth rate (0.01 h−1) helped in achieving increased protein titers.

Human serum albumin (HSA), sourced from human serum, has been an important therapeutic protein for several decades. Pichia pastoris is strongly considered as an expression platform, but proteolytic degradation of recombinant HSA in the culture filtrate remains a major bottleneck for use of this system. In this study, we have reported the development of a medium that minimized proteolytic degradation across different copy number constructs. A synthetic codon-optimized copy of HSA was cloned downstream of &alpha, &ndash, factor secretory signal sequence and expressed in P. pastoris under the control of Alcohol oxidase 1 promoter. A two-copy expression cassette was also prepared. Culture conditions and medium components were identified and optimized using statistical tools to develop a medium that supported stable production of HSA. Comparative analysis of transcriptome data obtained by cultivation on optimized and unoptimized medium indicated upregulation of genes involved in methanol metabolism, alternate nitrogen assimilation, and DNA transcription, whereas enzymes of translation and secretion were downregulated. Several new genes were identified that could serve as possible targets for strain engineering of this yeast.

The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major β-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.

Sodium alginate immobilized cells of Kluyveromyces marxianus NCIM 3551 cells were used for synthesis of galacto-oligosaccharides (GOS) using lactose as a substrate through transgalactosylation approach. The immobilized cells retained 84.4% of the original β-galactosidase activity displayed by free cells. An increase in thermo-stability and a slight shift in pH optimum was observed for β-galactosidase in immobilized cells. Maximum GOS concentration of 72.0 g/L and specific GOS productivity 60.0 g/L/U was obtained using free cells as opposed to 42.6 g/L and productivity of 35.6 g/L/U for immobilized cells. A repeated batch reaction was carried out using free and immobilized cells for 10 cycles, of 3 h each, using 20% lactose (w/v) with a nearly similar productivity of 24.0 g/L/h and 21.3 g/L/h respectively. Based on the stability of the immobilized cells, continuous synthesis of GOS was carried out for 10 days in packed bed bioreactor using immobilized cells at a loading of 28.8 units/reactor volume. A yield of 64.0 g/L of GOS and a productivity of 21.3 g/L/h was achieved at an optimized flow rate of 1.0 mL/min. The system here demonstrated the feasibility of using whole cells for production of GOS in a packed bed reactor.

A process was developed for enrichment of galacto-oligosaccharides (GOS), synthesized from a whole cell driven system, from a sugar reaction mixture (SRM) containing non prebiotic sugars (monosaccharides and disaccharides) as impurities. SRM containing 38% (w/w of total carbohydrates) of GOS was enriched by 7 and 27%, attaining a purity of 45 and 65% respectively using Saccharomyces cerevisiae followed by Kluyveromyces lactis var. lactis treatment. The two cell types could be recycled for consecutive 12 and 10 cycles respectively. The microbial purified GOS (MPG) was characterized by mass spectrometry and quantitated by HPLC. MPG was further evaluated for its prebiotic potential on Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus casei Shirota and Saccharomyces boulardii. The growth profile and colony forming units were determined and compared with the profiles obtained on glucose, used as a control. MPG was efficiently utilized by L. acidophilus and L. plantarum which showed antimicrobial activity with zone of lysis (12 and 10 mm) against Escherichia coli and Citrobacter (14 and 9 mm) respectively and performed better than Vivinal (commercial GOS), fructo-oligosaccharides and inulin. The synergistic effect of the MPG with L. acidophilus and L. plantarum was found to be most effective against pathogens as compared to other tested commercial oligosaccharides.

To determine the transglycosylation activity of cell-bound enzymes from Microbacterium paraoxydans to catalyze the synthesis of hexyl-α-d-glucoside (HG) and -polyglucosides using sucrose as a glycosyl donor. Maximum HG yield (14.8 %) was achieved at 0.96 water activity in 12 h with sucrose at 0.5 M with lyophilized cells (equivalent to 8 IU α-glucosidase activity). The synthesized alkyl-glucosides and-polyglucosides were characterized by ESI-MS. Structural elucidation of the main product (purified by solid phase chromatography) was done by HSQC (2D NMR) which was confirmed as 1-hexyl-α-d-glucopyranoside. The synthesis was scaled up in a fed-batch reactor, with continuous feeding of whole cells every 6 h and a total yield of ~44 % was obtained for hexyl-glucoside and -polyglucosides under the optimized conditions. Synthesis of HG, hexyl di- and tri-glucosides has been achieved using a novel method.

Isomalto-oligosaccharides (IMOs), one of the emerging prebiotics, were synthesized by transglycosylation activity of the cell bound α-glucosidase of a novel strain of Microbacterium sp. using maltose as a glycosyl donor. Parameters affecting yield were optimized and a yield of 85 g/l was obtained at 40% (w/v) initial substrate concentration after 24 h using whole cells (∼25 IU α-glucosidase activity). A mixture of isomaltotriose, isomaltotetraose, isomaltopentaose and isomaltohexaose were obtained as determined by electrospray ionization mass spectrometry. Addition of 0.5 M LiCl to the reaction mixture lead to a further 15% increase in the yield. The synthesized IMOs were purified for structure analysis by preparative thin layer chromatography and structures elucidated by 1H and 13C NMR. The chemical shift values of 13C NMR clearly confirmed that the products formed were isomaltotriose and isomaltotetraose (α-1,6 linked). The reactions were scaled up in a column to 100 ml level using immobilized cells. An increase in productivity by 4.2 times was achieved in this system with a conversion efficiency of 24%.

Textile effluent is characterized by high colour, chemical oxygen demand and conductivity due to presence of a large number of recalcitrant compounds. The effluent is often discharged without an effective treatment leading to contamination of water bodies. In the present study, a combination of mediator assisted laccase and chemical treatment was used for decolorization of effluent from a local textile mill in a continuous enzyme membrane reactor (EMR). Treatment of the effluent first with laccase and ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) lead to about 60% decolorization but was accompanied by membrane fouling. Addition of alum further coagulated 90% of the residual colour but the process was associated with dye sludge formation and ABTS could not be recovered from the treated effluent. Reversal of the treatment sequence was effective in that 85% decolorization was achieved in the EMR and the process could be operated for over a period of 15 days. No sludge formation was noticed and membrane fouling was negligible. Most importantly, about 60% ABTS could be recovered from the treated effluent. Analysis of the treated effluent by mass spectrometery indicated extensive breakdown of the dye molecules by laccase and ABTS and the breakdown products were neither toxic nor mutagenic as assessed by measurement of the oxygen consumption rate and the standard Ames test.

Class I cellobiose dehydrogenases (CDHs) are extracellular hemoflavo enzymes produced at low levels by the Basidiomycetes (white rot fungi). In presence of suitable electron acceptors, e.g., cytochrome c, 2,6-dichlorophenol-indophenol, or metal ions, it oxidizes cellobiose to cellobionolactone. A stringent requirement for disaccharides makes CDH also useful for conversion of lactose to lactobionic acid, an important ingredient in pharma and detergent industry. In this work, class I CDH was produced using a newly identified white rot fungus Termitomyces sp. OE147. Four media were evaluated for CDH production, and maximum enzyme activity of 0.92 international unit (IU)/ml was obtained on Ludwig medium under submerged conditions. Statistical optimization of N source, which had significant effect on CDH production, using Box-Behnken design followed by optimization of inoculum size and age resulted in an increase in activity to 2.9 IU/ml and a productivity of ~25 IU/l/h. The nearly purified CDH exhibited high activity of 26.4 IU/mg protein on lactose indicating this enzyme to be useful for lactobionic acid synthesis. Some of the internal peptide sequences bore 100 % homology to the CDH produced in Myceliophthora thermophila. The fungal isolate was amenable to scale up, and an overall productivity of ~18 IU/l/h was obtained at 14-l level.

Enzymatic synthesis of oligosaccharides with absolute stereo-selectivity and regio-selectivity provides an economical alternative to classical chemical methods. Here we demonstrate, for the first time, that whole cells of P. etchellsii are highly efficient biocatalysts and can be used for oligosaccharide synthesis using p-nitrophenyl-β-D-glucopyranoside, o-nitrophenyl-β-D-glucopyranoside and p-nitrophenyl-β-D-xylopyranoside as both donors and acceptors. Auto-condensation of p-nitrophenyl-β-D-glucopyranoside and o-nitrophenyl-β-D-glucopyranoside resulted in formation of β-(1→6) linked disaccharide as major products in 4 and 12% yield respectively. By contrast, auto condensation of p-nitrophenyl-β-D-xylopyranoside exclusively lead to formation of β-(1→4) linked disaccharide in 24% yield.

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.

Octyl-β- d -glucopyranoside was synthesized by transglucosylation between p -nitrophenyl β- d -glucopyranoside ( p NPG) and octanol as an acceptor using whole cells of thermo tolerant yeast Pichia etchellsii displaying cell wall bound β-glucosidase. Effect of several parameters such as glucosyl donor concentration, enzyme units and initial water activity was studied to optimize product yield. An initial water activity interval of 0.33–0.64 was favorable and increase in total enzyme units had marginal effect on conversion yield. An empirical model was developed to describe the relationship between various parameters and octyl glucoside yield. These factors were combined in a batch replacement strategy whereby octyl-β- d -glucopyranoside was synthesized in 4 h to a concentration of 30 mM (9.25 mg/ml) with a conversion yield of nearly 70% with p NPG as a glucosyl donor. Quantitative analysis was done by a highly reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method and detection was achieved using refractive index detector. The structure of the product was confirmed by 13 C and 1 H NMR spectroscopy. Additional products like octyl diglucoside were also formed, the structure of which was confirmed by mass spectrometry.

β-Glucosidases (EC 3.2.1.21) are industrially important glycosyl hydrolases used for cellulose saccharification as well as for synthesis of glyco-conjugates. Crystal structure of only one β-glucosidase of family 3 of the glycosyl hydrolase families is available due to difficulty in purification of these closely related enzymes from a given source. Multiple steps used during purification result in low yield, making it difficult to study their properties. Conditions for purification of two closely related β-glucosidases (BGL I and BGL II) of family 3 from Pichia etchellsii were investigated in this study. Two weak anion exchange columns convective interaction media-diethyl amino ethyl (CIM-DEAE) and CIM-ethylenediamine (CIM-EDA) were used for this purpose. The results obtained at 0.34 ml disk (CIM-DEAE) level were scaled up to 8 ml CIM-DEAE tube column wherein BGL I and BGL II were separated from the major contaminants in the cell-free extract. The recovered enzymes were completely resolved in the second step using CIM-EDA. A final specific activity of 9,180 IU/mg and 2,345.3 IU/mg was achieved for BGL I and BGL II respectively with an overall yield of 33%. The system should be applicable to resolution of other closely related enzymes from this family.

Apo-aconitase, the Fe(4)S(4) cluster free form of TCA cycle enzyme aconitase, binds with GroEL and dissociates itself upon maturation through insertion of the cluster. It is not clearly established as to why apo-protein binds with GroEL. In order to explore the possibility that stability is a factor responsible for the aggregation of apo-form at low ionic strengths and hence it associates with GroEL to avoid the unfavorable event, we carried out the unfolding studies with holo- and apo-aconitase. By probing the unfolding process through the changes in secondary structural element, exposed surface hydrophobicity, and the microenvironment around tryptophan residues, we were able to establish the relevant changes associated with the event. Apparent guanidine hydrochloride concentration required for unfolding of 50% of aconitase indicates that aconitase is destabilized in the absence of the Fe(4)S(4) cluster. The destabilization of the apo-aconitase was further reflected through its three times higher rate of unfolding as compared to the holo-protein. It was also observed that the apo-form has higher surface hydrophobicity than the holo-form. Hence, the lower ground state stability and higher solvent exposed hydrophobic surface of the apo-form makes it aggregation prone. Based on the present observation and earlier findings, we propose that binding of apo-aconitase to GroEL not only rescues it from the aggregation, but also assists in the final stage of maturation by orienting the cluster insertion site of GroEL bound apo-protein. This information sheds new light on the potential role of GroEL in the biosynthetic pathway of the metallo proteins.

Banana fiber, a waste product of banana cultivation, has been used to prepare banana fiber reinforced soy protein composites. Alkali modified banana fibers were characterized in terms of density, denier and crystallinity index. Fourier transformed infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and thermogravimetric analysis (TGA) were also performed on the fibers. Soy protein composites were prepared by incorporating different volume fractions of alkali-treated and untreated fibers into soy protein isolate (SPI) with different amounts of glycerol (25%–50%) as plasticizer. Composites thus prepared were characterized in terms of mechanical properties, SEM and water resistance. The results indicate that at 0.3 volume fraction, tensile strength and modulus of alkali treated fiber reinforced soy protein composites increased to 82% and 963%, respectively, compared to soy protein film without fibers. Water resistance of the composites increased significantly with the addition of glutaraldehyde which acts as cross-linking agent. Biodegradability of the composites has also been tested in the contaminated environment and the composites were found to be 100% biodegradable.

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% alpha-helical, 26% beta-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher beta-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.

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. Consider- ing 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 monomeric E. coli protein maltodextrin glucosidase (MalZ). Coexpression of GroEL and GroES in E. coli causes a 2-fold enhancement of exogenous MalZ activ- ity in vivo. In vitro, GroEL and GroES in the presence of ATP give rise to a 7-fold enhancement in MalZ refold- ing. 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, 000-000 (2007)

Granulocyte colony-stimulating factor (GCSF) has therapeutic applications due to its proven efficacy in different forms of neutropenia and chemotherapy-induced leucopenia. The original 564-bp nucleotide sequence from NCBI was codon optimized and assembled by overlapping PCR method comprising of 16 oligos of 50-nt length with 15 base overhang. The synthetic gene (CO-GCSF) was cloned under glucose utilizing glyceraldehyde 3-phosphate dehydrogenase (GAP) and methanol-utilizing alcohol oxidase (AOX1) promoters and expressed in Pichia pastoris SMD1168 strain. Constitutive expression under GAP resulted in cellular toxicity while AOX1 promoter controlled expression was stable. Variation in the levels of expression was observed among the transformant colonies with transformant #2 secreting up to ∼4 mg/L of GCSF. The molecular mass of the expressed GCSF in P. pastoris was ∼19.0 kDa. Quatitation of the expressed protein was carried out by a highly reproducible gel densitometric method. Effect of several operational and nutritional conditions was studied on GCSF production and the results suggest a general approach for increasing the yield of GCSF several folds (2- to 5-fold) over the standard conditions employed currently. Cultivation of the single-copy integrant in the chemically defined medium in a 5-L fermenter resulted in a volumetric productivity of ∼0.7 mg/L/h at the end of the induction phase, which was about 4-fold higher than attained in the shake flask.

Genomic DNA fragment encoding a novel β-glucosidase-like activity of the yeast Pichia etchellsii was cloned and expressed in Escherichia coli. An open-reading frame of 1515 bp, termed mugA, coding for a protein of predicted molecular mass of approximately 54 kDa was confirmed for this activity. The sequence of the deduced protein did not show homology with the generic β-glucosidases but a high degree of identity was seen with several Ser-Asp (SD)-rich cell-surface-associated proteins. The secondary structure prediction program 3D-PSSM indicated the protein to be composed of largely helical and coiled structures, which was confirmed by circular dichroism spectroscopy. The encoded protein, MUGA, was purified by about 53-fold and characterized as a monomer of 52.1 kDa by SDS–PAGE and MALDI-TOF. The protein displayed high hydrolytic activity on methylumbelliferyl β- d -glucoside but relatively very little hydrolysis of p-nitrophenyl β- d -glucoside and gentiobiose, characteristic substrates for β-glucosidases. The binding experiments performed between P. etchellsii cells and the purified E. coli expressed MUGA indicated binding with the cell surface, which was monitored by fluorescence microscopy. In competition experiments with the SD dipeptide, less protein was shown to bind to the cell surface, in a concentration-dependent manner.

Beta-glucosidase II (Bgl II), encoded by the betaglu2 gene of the thermo-tolerant yeast Pichia etchellsii, was purified from recombinant Escherichia coli pBG22:JM109. The enzyme had a molecular mass of 176 kDa and was a dimer with an apparent subunit mass of 83 kDa. It exhibited broad substrate specificity and hydrolyzed beta-linked gluco-disaccharides and oligosaccharides, salicin, and cyanogenic glucoside amygladin. The unusually high hydrolytic activity of 7,680 units min(-1) g(-1) protein was obtained on sophorose. Competition experiments performed using differently linked beta-disaccharides indicated these to be hydrolyzed at the same active site. Transglycosylation activity leading to the biosynthesis of several disaccharides and oligosaccharides was observed. The enzyme was placed in glycosyl hydrolase family 3, based on a statistical approach using amino acid composition data. The involvement of His as a catalytically important residue was confirmed by diethylpyrocarbonate modification. Pre-incubation of the purified enzyme with 5 mM p-nitrophenyl-beta-D-glucoside offered 2.5-fold higher residual activity compared with unbound enzyme, indicating protection at the active site. The feasibility of this enzyme as a biocatalyst of choice for the synthesis of glyco-conjugates is discussed.

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.

The stability of almond ?-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30�C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl ?-D-glucopyranoside as donor and ?-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond ?-glucosidase and cloned Pichia etchellsii ?-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1 ? 3)- as well as (1 ? 6)-regioisomeric disaccharides, the former being the major product in cloned ?-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of ?-1-N-acetamido-D-mannopyranose and ?-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond ?-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme. ? 2004 Taylor & Francis Ltd.

The biosynthetic activity of yeast Pichia etchellsii beta-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 ethyl acetate 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.

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 incr...

The thermo-tolerant yeast Pichia etchellsii produced two cell-wall-bound inducible beta-glucosidases, BGLI (molecular mass 186 kDa) and BGLII (molecular mass 340 kDa), which were purified by a simple, three-step method, comprising ammonium sulfate precipitation, ion-exchange and hydroxyapatite chromatography. The two enzymes exhibited a similar pH and temperature optima, inhibitory effect by glucose and gluconolactone, and stability in the pH range of 3.0-9.0. Placed in family 3 of glycosylhydrolase families, BGLI was more active on salicin, p-nitrophenyl beta-D-glucopyranoside and alkyl beta-D-glucosides whereas BGLII was most active on cellobiose. k(cat) and K(M) values were determined for a number of substrates and, for BGLI, it was established that the deglycosylation step was equally effective on aryl- and alkyl-glucosides while the glycosylation step varied depending on the substrate used. This information was used to synthesize alkyl-glucosides (up to a chain length of C(10)) using dimethyl sulfoxide stabilized single-phase reaction microenvironment. About 12% molar yield of octyl-glucoside was calculated based on a simple spectrophotometric method developed for its estimation. Further, detailed comparison of properties of the enzymes indicated these to be different from the previously cloned beta-glucosidases from this yeast.

The amino acid composition and adsorption properties of the fibril forming protein (FFP), identified previously as a non-hydrolytic component of Trichoderma reseei cellulase enzymes, are described. The amino acid composition was markedly different from the reported endoglucanases (EG) and cellobiohydrolases (CBH) of T. reesei . FFP was bound to Avicel cellulose and acid swollen cellulose rapidly and the equilibrium was reached within 20–30 min. Adsorption on Avicel cellulose was highly dependent on temperature: at lower temperature the rate of adsorption was low although higher amount of FFP bound to cellulose. The energy of activation for binding to cellulose was 6.4 kcal mol −1 . The adsorption equilibrium constant ( K A ) and theoretical maximum amount of protein bound ( A max ) were calculated using modified form of the Langmuir equation, and the values were found to be 3.42×10 5 l mol −1 and 6.0 mg g −1 of cellulose, respectively. Binding of FFP was also studied on laboratory prepared cellulose of different crystallinity index (CrI), prepared by ball milling of Whatman filter paper. Cellulose with higher CrI bound higher amount of FFP (3.6 mg g −1 of cellulose) than cellulose with lower CrI, which was 2.38 mg g −1 of cellulose. Prolonged incubation of filter paper with FFP resulted in reduction of CrI. Scanning electron micrograph of treated filter paper indicated loosening of the structure of cellulose microfibrils which is hypothesized to result in reduction of CrI.

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.

The cloning and expression of β-glucosidase II, encoded by the genesglu2, from thermotolerant yeastPichia etchellsii intoEscherichia coli is described. Cloning of the 7.3 kbBamHI/SalI yeast insert containingsglu2 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.

The hydrolysis and biosynthetic reactions of partially purified Pichia etchellsii beta-glucosidase II from recombinant Escherichia coli pBG22:JM109 are described. With 167 mmol/L of initial glucose, the products of synthetic reactions, glucobiose and glucotriose, accumulated to 18 and 6 mmol/L, respectively. In transglycosylation reactions with 79 mmol/L of initial cellobiose, glucotriose and glucopentaose were obtained at 4.5 and 2 mmol/L, respectively. The effects of incubation time and substrate concentration were studied on the yield of synthesized oligosaccharides. In a reaction time of 24 h with 468 mmol/L of initial cellobiose, glucotriose and glucopentaose levels of 21.6 and 6.6 mmol/L, respectively, were obtained. The addition of dimethyl sulfoxide (DMSO) further increased the yields of the products by 10%. Detailed kinetic analysis indicated a significant (about twofold) increase in Vmax/KM of synthetic reactions in the presence of DMSO. A study of other disaccharides in transglycosylation reactions indicated biosynthetic activity in the order of sophorose > gentiobiose > cellobiose.

Among a number of yeast strains screened for whole cell transgalactosylating activity, Kluyveromyces marxianus NCIM 3551 was found to be most suitable biocatalyst for production of galacto-oligosaccharides (GOS). Cell permeabilization lead to an efficient bioconversion by β-galactosidase resulting in synthesis of GOS. A maximum GOS yield of 36% (w/w) of total sugars was achieved and the products consisted of tri- and tetra-galacto-oligosaccharides. A lactose conversion rate of 80% and productivity of 24g/L/h was obtained under the optimum conditions at lactose concentration of 20% (w/v), temperature 40°C, pH 6.5 and enzyme units after 3h of reaction time. Tetrasaccharides were the main component of the reaction mixture. The products were quantitated by HPLC and structurally characterized by mass spectrometry.

Addition of L-sorbose, a non-metabolizable non-inducing ketohexose, toTrichoderma reesei cultures growing on cellobiose or Avicel-cellulose lead to increased cellulase activities. Addition of sorbose resulted in a 6-fold increase in cellodextrins (cellotriose, cellotetraose, cellopentaose) concentration on day 3 in cellobiose cultures and 1.3-fold increase in cellodextrins concentrations on day 4 in Avicel cellulose cultures. This increase in intracellular cellodextrins concentration matched closely with the increase in endoglucanase activity at these time points. Treatment of the cell-free extracts with cellulase preparation led to disappearance of the cellodextrins and increase of glucose. These observations suggested a more direct involvement of cellodextrins in cellulase induction process. The cellulases produced in sorbose-supplemented cellobiose medium hydrolyzed microcrystalline cellulose as effectively as the ones produced on Avicel cellulose medium.

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.

The beta-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme beta-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl beta,1-4 linkage, and beta (1-2), beta(1-4), beta(1-6) and beta(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.

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.

Alkyl-glucosides and alkyl-polyglucosides are the new-generation biodegradable surfactants with good emulsifying and wetting properties. The α-forms of these glucosides occur in antibiotics and also stimulate nasal absorption of many drugs. In this paper, we report the synthesis of hexyl α-glucoside and α-polyglucosides using cell-bound α-glucosidase activity of a novel strain of Microbacterium paraoxydans. A number of cell-bound glycosyl hydrolase activities were detected in the isolate with the maximum hydrolytic activity of 180 IU g(-1) dry wt cells on p-nitrophenyl-α-D-glucopyranoside. In a micro-aqueous system, at a water activity of 0.69, 1.8 g l(-1) of hexyl α-glucoside (corresponding to about 25 % yield) was synthesized by whole cells with maltose and hexanol as substrates. The concentration was enhanced to 11 g l(-1) (~60 % yield) in a biphasic system at a water content of 60 %. (1)H and (13)C NMR spectra of the purified compound confirmed the synthesized product to be hexyl-α-D-glucopyranoside, while the presence of hexyl di- and tri-glucosides was confirmed by electrospray ionization mass spectrometry. The cell-driven synthesis makes this an extremely attractive alternative for synthesis of such compounds.

A 4.8-kilobase pairs DNA fragment from thermophilic yeast Pichia etchellsii was cloned into the vector plasmid pUC19 to form plasmid pBG55 and the encoded β-glucosidase expressed in Escherichia coli . The effect of different carbon sources on growth and enzyme synthesis was studied in the pBG55 transformant and 0.2% (w/v) cellobiose found to be the most suitable carbon source for enzyme biosynthesis. The level of intracellularly produced β-glucosidase was slightly reduced on 0.2% (w/v) glucose and 0.2% (w/v) maltose. The partially purified enzyme from the β- glu transformant was active against a wide range of aryl β-glucosides and β-linked disaccharides and the preferred substrates were p -nitrophenyl-β- d -glucoside ( p NPG), cellobiose, gentiobiose, sophorose and sucrose. While maximum enzyme activity of 62 U/ l was against p NPG at 50°C, the activities in the range of 120–170 U/ l were against various β-linked disaccharides at 37°C. The enzyme displayed glucose tolerance and a temperature optima prolile slightly different from that exhibited by the native yeast glycosylated enzyme. The β-glucosidase in the crude extract of pBG55 transformant was identified as a stably produced protein of 200 kDa by PAGE-Zymogram analysis.

Background Laccases 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. Results In 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. Conclusion Laccase 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.

The folding of proteins in the milieu of the cellular environment involves various interactions among the residues of the polypeptide chain and the microenvironment where it resides. These interactions are responsible for stabilizing the protein molecule, and disruption of the same provides information about the stability of the molecule. β-Glucosidase isozymes, despite having high homology in their primary and tertiary designs, show deviations in their properties such as unfolding, refolding, and stability. In a comparative study on two large cell-wall-bound isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from a thermo-tolerant yeast, Pichia etchellsii, we have investigated guanidine hydrochloride (GdnHCl)-induced, alkali-induced, and thermal-unfolding transitions using CD and fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Using spectral parameters (MRE 222 nm) to monitor the conformational transitions of the GdnHCl-induced unfolding phenomenon, it was observed that the midpoints of unfolding, apparent C (m), occurred at 1.2 M ± 0.05 and 0.8 M ± 0.03 GdnHCl, respectively, for BGLI and BGLII. The alkali-induced unfolding process indicated that BGLI showed a mid-transition point at pH 11 ± 0.17, while for BGLII it was at pH 10 ± 0.40, further indicating BGLI to be more stable to alkali denaturation than BGLII. In the case of thermal unfolding, the midpoint of transition was observed at 63 ± 0.12°C for BGLI and at 58 ± 0.55°C for BGLII. Analysis by high sensitivity differential scanning calorimeter supported the unfolding data in which BGLI showed higher melting temperature, T (m), (56.07°C ± 0.34) than BGLII (54.02°C ± 0.36). Our results clearly indicate that BGLI is structurally more rigid and stable than BGLII.