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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...

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.

Protein fusion technology has emerged as one of the important strategies to increase the level of expression and half-life of therapeutic proteins in heterologous expression systems. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor and is clinically used against neutropenia. Enhanced expression and stability of G-CSF were achieved in Pichia pastoris by the way of constructing a fusion protein with human serum albumin (HSA). The strategy involved polymerase chain reaction (PCR) amplification of fragments corresponding to codon-optimized G-CSF and domain 3 of HSA. Overlapping PCR was used to obtain the full-length fused gene (1,184 bp) with a 15-bp linker sequence comprising of 4 Gly and 1 Ser residues. Extracellular expression was carried out downstream of α-factor secretion signal sequence under the control of alcohol oxidase 1 promoter using pPICZαB. Excreted protein in the range of 110–380 mg L−1 was observed among the transformants. Effect of aeration and temperature was investigated in one of the transformants (35) overexpressing fusion protein and levels of G-CSF enhanced by 1.8-fold and 2.3-fold, respectively. Assay of biological activity indicated the fusion protein to retain similar cell proliferation activity as the commercial G-CSF preparation.

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 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.

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.

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.

Random mutagenesis was applied for development of organic solvent tolerant β-glucosidase I (BGLI) of Pichia etchellsii to extend its use for glyco-conjugate synthesis in non-aqueous medium. Previously cloned BGL1 encoding β-glucosidase I (BGLI) was subjected to error prone PCR and the mutant library was cloned in pPIC9 followed by transformation into Pichia pastoris. Transformants were first selected for functional β-glucosidase phenotype followed by screening for organic solvent tolerance in acetonitrile, dimethylformamide, dimethyl sulfoxide, methanol, 2-propanol. One of the enzyme variants, named, OT-BGLI exhibited 1.7- to 10-fold higher stability in 20–30% (v/v) methanol, 2-propanol, dimethylformamide compared to the wild type enzyme. This property was also accompanied by higher thermostability of the enzyme in aqueous buffer solution. Three of the six mutations (other 3 being silent) were mapped in the non-conserved regions of the gene. Modeled structure of the enzyme pin pointed increased hydrogen bonds, intra-ionic interactions (contributed by G414D and N789D replacements), facilitated proton exchange in organic solvents (contributed by Y722H) to be the causative agents for enhanced organic solvent stability and thermostability. Secondary structure of the mutant enzyme was similar to the wild type enzyme. Simultaneous enhanced organic solvent and thermal stability makes this enzyme an ideal candidate for glycoconjugate synthesis in aqueous-organic solvent systems.

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.

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.

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.

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.

Over last two decades many researchers have demonstrated the mechanisms of how the Escherichia coli chaperonin GroEL and GroES work in the binding and folding of different aggregation prone substrate proteins both in vivo and in vitro. However, preliminary aspects, such as influence of co-expressing GroEL and GroES on the over expression of other recombinant proteins in E. coli cells and subsequent growth aspects, as well as the conditions for optimum production of recombinant proteins in presence of recombinant chaperones have not been properly investigated. In the present study we have demonstrated the temperature dependent growth characteristics of E. coli cells, which are over expressing recombinant aconitase and how the co-expression of E. coli chaperonin GroEL and GroES influence the growth rate of the cells and in vivo folding of recombinant aconitase. Presence of co-expressed GroEL reduces the aconitase over-expression drastically; however, exogenous GroELGroES together compensate this reduction. For the aconitase over-expressing cells the growth rate decreases by 30% at 25 degrees C when compared with the M15 E. coli cells, however, there is an increase of 20% at 37 degrees C indicating the participation of endogenous chaperonin in the folding of a fraction of over expressed aconitase. However, in presence of co-expressed GroEL and GroES the growth rate of aconitase producing cells was enhanced by 30% at 37 degrees C confirming the assistance of exogenous chaperone system for the folding of recombinant aconitase. Optimum in vivo folding of aconitase requires co-production of complete E. coli chaperonin machinery GroEL and GroES together.

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.

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.

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.

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.

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.

Purification of biotechnologically important proteins is of vital interest to the biotech industry. b-Glucosidases, belonging to Family 1 and Family 3 of the glycosylhydrolases, have varied applications as carbohydrate hydrolyzing and synthesizing enzymes. Obtaining high quantities of these enzymes is important for exploring their biosynthetic potential, structural information and catalytic activities. Classical methods for their preparation fail to deliver high yields because of adoption of several/ hydroxyapatite chromatography steps. We report here a preparative method for purification of large quantities of two closely related cell bound b-glucosidases (BGL I and BGL II) from Pichia etchellsii that belong to Family 3 glycosylhydrolases. A combination of ion-exchange and gel filtration chromatography was used to process milligram quantities of protein with recoveries of up to 53%. A simple affinity based separation resulted in resolution of BGL I and BGL II with high recovery and high specific activities of 74 IU/mg and 32 IU/mg protein respectively. Peptide sequences of BGL II indicated it to be a novel member of Family 3. Methods reported here present a successful strategy for obtaining large quantities of these enzymes.

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.

Laccase from basidiomycete fungus Cyathus bulleri was evaluated for its ability to decolorize a number of reactive and acidic dyes in the presence of natural and synthetic mediators. The extent of decolorization was monitored at different mediator/dye concentrations and incubation time. Among the synthetic mediators, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was effective at low mediator/dye ratios and resulted in 80-95% decolorization at rates that varied from 226 +/- 4 nmol min(-1) mg(-1) for Reactive Orange 1 to 1,333 +/- 15 nmol min(-1) mg(-1) for Reactive Red 198. Other synthetic mediators like 1-hydroxybenzotriazole and violuric acid showed both concentration- and time-dependent increases in percent decolorization. Natural mediators like vanillin, on the other hand, were found to be less effective on all the dyes except Reactive Orange 1. Computed rates of decolorization were about twofold lower than that with ABTS. The laccase-ABTS system also led to nearly 80% decolorization for the simulated dye mixture. No clear correlation between laccase activity on the mediator and its ability to decolorize dyes was found, but pH had a significant effect: Optimum pH for decolorization coincided with the optimum pH for mediator oxidation. The treated samples were also evaluated for toxicity in model microbial systems. The laccase-mediator system appears promising for treatment of textile wastewaters.

Beta-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 aminoglucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial beta-glucosidases, yeast beta-glucosidases, thermostable fungal beta-glucosidase, and the physiological functions, characteristics, and catalytic action of native beta-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 beta-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.

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.

The fungus Fusarium oxysporum was cultivated on glucose and lactose medium in a 2 l bioreactor to evaluate its carbon conversion efficiency. The initial optimization of culture conditions indicated NH 4 NO 3 or NH 4 Cl as the best N source at a C/N ratio between 1:4.5 to 1:11.5 at 1% glucose concentration. The pattern of allocation of the substrate carbon and energy into the production of biomass, carbon dioxide, and heat during growth was identified by applying mass and energy balance equations. It is indicated that glucose-containing substrates are better for SCP production from the organism than substrates with lactose.

Summary: A mutant (D6-13) of Trichoderma reesei that produced low levels of extracellular β-glucosidase was physiologically and biochemically characterized. The mutant was unable to grow and produced very little cellulase activity on Solka-Floc, Avicel, micro-crystalline cellulose and acid-swollen cellulose. Whereas growth was nearly normal on cellobiose, lactose and glucose, cellulase production was slightly decreased on cellobiose and lactose. The mutant produced nearly normal levels of FPA (filter-paper activity) and endoglucanase on sophorose. A distinct lag and a lowered specific sugar consumption rate was observed for the mutant on 0·3% cellobiose; this appeared to be due to lowered wall-bound β-glucosidase activity. The multiplicity and role of different β-glucosidases in T. reesei is discussed.

The cellulase enzyme system consists of cellobiohydrolase, endoglucanase, and beta-glucosidase and has been extensively studied with respect to its biosynthesis, properties, mode of action, application, and, most recently, secretion mechanisms. A knowledge of the factors governing the biosynthesis and secretion of these enzymes at the molecular level will be useful in maximizing enzyme productivity in extracellular fluid. Among other topics, the regulatory effects of sorbose (a noninducing sugar which is not a product of cellulose hydrolysis) on cellulase synthesis and release are described. Cellulase genes have recently been cloned into a number of microorganisms with a view to understanding the gene structure and expression and to obtaining the enzyme components in pure form. The factors governing biosynthesis and secretion of cellulases in recombinant cells are also discussed. Cellulases are known to be glycoproteins, therefore, the role of O- and N-linked glycosylation on enzyme stability and secretion is also detailed.

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 Vogel's 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.

Techniques for measuring methanol accumulated by resting cells of Methylosinus trichosporium in the narrow range of 70-80mM phosphate concentration have been standardized. The maximum amount of methanol, 6μmol•mg-1•hr-1, was accumulated at a cell density of 2mg•ml-1 (w/v) of cells in the early stationary phase at pH 6.5, temperature 35°C. The phosphate-dependent inhibition of methanol dehydrogenase was found to be uncompetitive and reversible. Further, 80mM phosphate inhibited methane monooxygenase and formate dehydrogenase by 16% and 20% respectively. A possible biochemical explanation of the role of phosphate inhibition is offered.

The single major factor limiting the economy of enzymatic hydrolysis of cellulose is the cost of the cellulase enzymes. Most of the work to date in reducing this cost has centered around selection of hyperproducing strains using the standard cellulose medium. Cellulose is an insoluble substrate with obvious design disadvantages in bioreactors. It is often indicated that another reasonable approach to the problem would be the isolation of constitutive mutants which require no inducer to form cellulase. In fact, the goal of several laboratories around the world working on the hydrolysis of cellulosic substances has been to isolate a constitutive mutant. Reported here is the isolation of a constitutively cellulase producing mutant in T. reesei that produces high levels of all of the component enzymes in the cellulase complex in the presence of glucose or sucrose. With this constitutive mutant, it is hoped that a major hurdle of working with an insoluble substrate will be overcome. The mutant will allow easier operation and control of cellulose bioreactors and can be used for continuous enzyme production, as opposed to the present batch production system. Also, a number of other unusable susbtrates like whey and molasses can be used for commercial preparationsmore » of cellulases. (Refs. 2).« less