Improvement of Laccase Production by Thielavia terrestris Co3Bag1. Enhancing the Bio-Catalytic Performance of the Native Thermophilic TtLacA via Immobilization in Copper Alginate Gel Beads.

A 32-fold increase in laccase activity production by the thermophilic biomass-degrading fungus T. terrestris Co3Bag1 was achieved when the microorganism was grown on a modified medium containing fructose, sodium nitrate, and copper. A 70 kDa laccase (TtLacA), produced under the above conditions, was purified, immobilized in copper alginate gel beads, and characterized. TtLacA, both free and immobilized enzymes, exhibited optimal activity at pH 3.0, at a temperature of 65 and 70 °C, respectively, although both displayed 70% of activity from 40 to 70 °C. Free and immobilized enzymes retained at least 80% of relative activity in the pH range from 3 to 4.6. Immobilized TtLacA manifested a 2.3-fold higher thermal stability than the free form of the enzyme at 60 and 70 °C. Immobilized TtLacA retained 95% initial activity for six consecutive reuse cycles at 60 °C, and also retained 86% of initial activity after 12 days of storage at 4 °C. Based on the biochemical features, thermophilic TtLacA may be an efficient enzyme for dye decolorization and other industrial applications at high temperatures or acidic conditions. This work represents the first report about the immobilization and biochemical characterization of a thermophilic laccase from a member of the genus Thielavia.

TtCel7A: A Native Thermophilic Bifunctional Cellulose/Xylanase Exogluclanase from the Thermophilic Biomass-Degrading Fungus Thielavia terrestris Co3Bag1, and Its Application in Enzymatic Hydrolysis of Agroindustrial Derivatives.

The biomass-degrading thermophilic ascomycete fungus Thielavia terrestris Co3Bag1 produces TtCel7A, a native bifunctional cellulase/xylanase GH7 family. The purified TtCel7A, with an estimated molecular weight of 71 kDa, was biochemically characterized. TtCel7A displayed an optimal pH of 5.5 for both activities and an optimal temperature of 60 and 50 °C for cellulolytic and xylanolytic activities, respectively. The half-lives determined for cellulase activity were 140, 106, and 41 min at 50, 60, and 70 °C, respectively, whereas the half-lives observed for xylanase activity were 24, 10, and 1.4 h at 50, 60, and 70 °C, respectively. The KM and Vmax values were 3.12 mg/mL and 50 U/mg for cellulase activity and 0.17 mg/mL and 42.75 U/mg for xylanase activity. Circular dichroism analysis suggests changes in the secondary structure of TtCel7A in the presence of CMC as the substrate, whereas no modifications were observed with beechwood xylan. TtCel7A displayed the excellent capability to hydrolyze CMC, beechwood xylan, and complex substrates such as oat bran, wheat bran, and sugarcane bagasse, with glucose and cellobiose being the main products released; also, slightly less endo cellulase and xylanase activities were observed. Thus, suggesting TtCel7A has an exo- and endomode of action. Based on the characteristics of the enzyme, it might be considered a good candidate for industrial applications.

Rahnella sp., a Dominant Symbiont of the Core Gut Bacteriome of Dendroctonus Species, Has Metabolic Capacity to Degrade Xylan by Bifunctional Xylanase-Ferulic Acid Esterase.

Rahnella sp. ChDrAdgB13 is a dominant member of the gut bacterial core of species of the genus Dendroctonus, which is one of the most destructive pine forest bark beetles. The objectives of this study were identified in Rahnella sp. ChDrAdgB13 genome the glycosyl hydrolase families involved in carbohydrate metabolism and specifically, the genes that participate in xylan hydrolysis, to determine the functionality of a putative endo-1,4-ß-D-xylanase, which results to be bifunctional xylanase-ferulic acid esterase called R13 Fae and characterize it biochemically. The carbohydrate-active enzyme prediction revealed 25 glycoside hydrolases, 20 glycosyl transferases, carbohydrate esterases, two auxiliary activities, one polysaccharide lyase, and one carbohydrate-binding module (CBM). The R13 Fae predicted showed high identity to the putative esterases and glycosyl hydrolases from Rahnella species and some members of the Yersiniaceae family. The r13 fae gene encodes 393 amino acids (43.5 kDa), containing a signal peptide, esterase catalytic domain, and CBM48. The R13 Fae modeling showed a higher binding affinity to ferulic acid, α-naphthyl acetate, and arabinoxylan, and a low affinity to starch. The R13 Fae recombinant protein showed activity on α-naphthyl acetate and xylan, but not on starch. This enzyme showed mesophilic characteristics, displaying its optimal activity at pH 6.0 and 25°C. The enzyme was stable at pH from 4.5 to 9.0, retaining nearly 66-71% of its original activity. The half-life of the enzyme was 23 days at 25°C. The enzyme was stable in the presence of metallic ions, except for Hg2+. The products of R13 Fae mediated hydrolysis of beechwood xylan were xylobiose and xylose, manifesting an exo-activity. The results suggest that Rahnella sp. ChDrAdgB13 hydrolyze xylan and its products could be assimilated by its host and other gut microbes as a nutritional source, demonstrating their functional role in the bacterial-insect interaction contributing to their fitness, development, and survival.

Bioremediation of an agricultural saline soil contaminated with endosulfan and Escherichia coli by an active surface agent induced in a Penicillium crustosum culture.

This study evaluates the production of a biological active surface agent (BASA) through its surface tension (ST) and emulsifying activity (E24) for endosulfan degradation (ED) and Escherichia coli growth inhibition (EcGI) in an agricultural saline soil. The fungus, identified as Penicillium crustosum was isolated from the Citrus sinensis peel (CsP), then the surface properties were evaluated in 9 culture media through a Taguchi L9 experimental design. The culture conditions included: stirring speed, pH, carbon (C) and nitrogen (N) sources; being glucose, NH4N03, 120 rpm and pH of 5, the most significant parameters in the BASA production. The BASA identified as a lipopeptide type, showed a ST = 38 mN m-1 and E24=71%. Both properties were stable at 80 °C, while ST presented stability in the pH range of 2 - 12, and a saline concentration of 200 g L-1; E24 was also stable at a pH between 8-12. Further application of BASA and fungal inoculum to a contaminated agricultural saline soil presented an EcGI of 99.8% on the 8th day, and ED of 92.9 ± 4.7% in 30 days, respectively; being the first report that uses this fungus for pesticide and bacteria elimination from an agricultural saline soil.

Effect of the single mutation N9Y on the catalytical properties of xylanase Xyn11A from Cellulomonas uda: a biochemical and molecular dynamic simulation analysis.

Cellulomonas uda produces Xyn11A, moderately thermostable xylanase, with optimal activity at 50 °C and pH 6.5. An improvement in the biochemical properties of Xyn11A was achieved by site-directed mutagenesis approach. Wild-type xylanase, Xyn11A-WT, and its mutant Xyn11A-N9Y were expressed in Escherichia coli, and then both enzymes were purified and characterized. Xyn11A-N9Y displayed optimal activity at 60 °C and pH 7.5, an upward shift of 10 °C in the optimum temperature and an upward shift of 1 unit in optimum pH; also, it manifested an 11-fold increase in thermal stability at 60 °C, compared to that displayed by Xyn11A-WT. Molecular dynamics simulations of Xyn11A-WT and Xyn11A-N9Y suggest that the substitution N9Y leads to an array of secondary structure changes at the N-terminal end and an increase in the number of hydrogen bonds in Xyn11A-N9Y. Based on the significant improvements, Xyn11A-N9Y may be considered as a candidate for several biotechnological applications.

An Overview of Genes From Cyberlindnera americana, a Symbiont Yeast Isolated From the Gut of the Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae), Involved in the Detoxification Process Using Genome and Transcriptome Data.

Bark beetles from Dendroctonus genus promote ecological succession and nutrient cycling in coniferous forests. However, they can trigger outbreaks leading to important economic losses in the forest industry. Conifers have evolved resistance mechanisms that can be toxic to insects but at the same time, bark beetles are capable of overcoming tree barriers and colonize these habitats. In this sense, symbiont yeasts present in the gut of bark beetles have been suggested to play a role in the detoxification process of tree defensive chemicals. In the present study, genes related to this process were identified and their response to a terpene highly toxic to bark beetles and their symbionts was analyzed in the Cyberlindnera americana yeast. The genome and transcriptome of C. americana (ChDrAdgY46) isolated from the gut of Dendroctonus rhizophagus were presented. Genome analysis identified 5752 protein-coding genes and diverse gene families associated with the detoxification process. The most abundant belonged to the Aldo-Keto Reductase Superfamily, ATP-binding cassette Superfamily, and the Major Facilitator Superfamily transporters. The transcriptome analysis of non-α-pinene stimulated and α-pinene stimulated yeasts showed a significant expression of genes belonging to these families. The activities demonstrated by the genes identified as Aryl-alcohol dehydrogenase and ABC transporter under (+)-α-pinene suggest that they are responsible, that C. americana is a dominant symbiont that resists high amounts of monoterpenes inside the gut of bark beetles.

Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Taxonomic identification of the thermotolerant and fast-growing fungus Lichtheimia ramosa H71D and biochemical characterization of the thermophilic xylanase LrXynA.

The zygomycete fungus Lichtheimia ramosa H71D, isolated from sugarcane bagasse compost, was identified by applying phylogenetic analysis based on the DNA sequence of the Internal Transcribed Spacer (ITS), and subsequent secondary structure analysis of ITS2. L. ramosa H71D was able to grow over a wide range of temperatures (25-45 °C), manifesting optimal growth at 37 °C. A 64 kDa xylanase (named LrXynA) was purified from the culture supernatant of L. ramosa H71D grown on 2% carboxymethylcellulose (CMC), as the only carbon source. LrXynA displayed optimal activity at pH 6 and temperature of 65 °C. The enzyme retained more than 50% of its maximal activity over a broad range of pH values (4.5-7.5). Enzyme half-life (t½) times at 55, 65 and 75 °C were 80, 25, and 8 min, respectively. LrXynA showed higher affinity (k M of 2.87 mg/mL) and catalytic efficiency (k cat /k M of 0.651 mg s/mL) towards Beechwood xylan in comparison to other substrates such as Birchwood xylan, Oat-spelt xylan, CMC, Avicel and Solka floc. The predominant final products from LrXynA-mediated hydrolysis of Beechwood xylan were xylobiose and xylotriose, suggesting that the enzyme is an endo-ß-1,4 xylanase. Scanning electron microscopy (SEM) imaging of sugar cane bagasse (SCB) treated with LrXynA, alone or in combination with commercial cellulases, showed a positive effect on the hydrolysis of SCB. To our knowledge, this is the first report focusing on the biochemical and functional characterization of an endo-ß-1,4 xylanase from the thermotolerant and fast-growing fungus Lichtheimia ramosa.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable ß-1,4-xylanase with exo- and endo-activity.

A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production.

Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae).

Serratia proteamaculans CDBB-1961, a gut symbiont from the roundheaded pine beetle Dendroctonus adjunctus, displayed strong cellulolytic activity on agar-plates with carboxymethyl cellulose (CMC) as carbon source. Automatic genome annotation of S. proteamaculans made possible the identification of a single endoglucanase encoding gene, designated spr cel8A. The predicted protein, named Spr Cel8A shows high similarity (59-94 %) to endo-1,4-ß-D-glucanases (EC 3.2.1.4) from the glycoside hydrolase family 8 (GH8). The gene spr cel8A has an ORF of 1113 bp, encoding a 371 amino acid residue protein (41.2 kDa) with a signal peptide of 23 amino acid residues. Expression of the gene spr cel8A in Escherichia coli yields a mature recombinant endoglucanase 39 kDa. Cel8A displayed optimal activity at pH 7.0 and 40 °C, with a specific activity of 0.85 U/mg. The enzyme was stable at pH from 4 to 8.5, retaining nearly 40-80 % of its original activity, and exhibited a half-life of 8 days at 40 °C. The K m and V max values for Spr Cel8A were 6.87 mg/ml and 3.5 µmol/min/mg of protein, respectively, using CMC as substrate. The final principle products of Spr Cel8A-mediated hydrolysis of CMC were cellobiose, cello oligosaccharides and a small amount of glucose, suggesting that Spr Cel8A is an endo-ß-1,4-glucanase manifesting exo-activity. This is the first report regarding the functional biochemical and molecular characterization of an endoglucanase from S. proteamaculans, found in the gut-associated bacteria community of Dendroctonus bark beetles. These results contribute to improved understanding of the functional role played by this bacterium as a symbiont of bark beetles.

Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties.

Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses.

Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma.

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.

Behavior of transition state regulator AbrB in batch cultures of Bacillus thuringiensis.

The transition state regulator AbrB is involved in the regulation of various cellular functions such as exponential growth, transition state and sporulation onset, due to its ability to activate, suppress or prevent the inappropriate expression of various genes in Bacillus subtilis. In order to understand combined behavior in batch cultures of AbrB in Bacillus thuringiensis, we cloned and expressed the abrB gene of B. thuringiensis in Escherichia coli. The deduced sequence of abrB gene coded for a protein consisting of 94 amino acids with ~10.5 kDa protein that shares 100 and 85 % identity with those from Bacillus cereus and Bacillus subtilis. The recombinant AbrB protein was used as antigen for the production of rabbit polyclonal antibodies anti-AbrB. Two media cultures with carbon: nitrogen ratios of 7.0, but varying access to nutrients were tested in batch cultures. In the case of both media, AbrB accumulation occurred from the beginning of the process and was maximal during early exponential growth. Thereafter, the level of AbrB decreased when there were no nutrient limitations and coincided with a decreased value in specific growth rate, although growth continued exponentially. Nonetheless, sporulation onset was determined 3 h and 4 h later, in media with highly metabolizable nutrients clean medium and Farrera medium, respectively. Hence, the maximal level of AbrB accumulation in batch cultures of B. thuringiensis is not influenced by limiting nutrients; however, nutrient availability affects the required time lapse for transition state regulator accumulation.

The family II carbohydrate-binding module of xylanase CflXyn11A from Cellulomonas flavigena increases the synergy with cellulase TrCel7B from Trichoderma reesei during the hydrolysis of sugar cane bagasse.

Synergy between Cellulomonas flavigena xylanase CflXyn11A and Trichoderma reesei endoglucanase TrCel7B was assessed during hydrolysis of alkaline pretreated sugar cane bagasse (SCB) after 12-48 h, applying the individual enzymes and mixtures of the enzymes. A high degree of synergy (6.3) between CflXyn11A and TrCel7B in hydrolysis of SCB was observed after 12h in the equimolar mixture. A threefold decrease in the degree of synergy was observed with TrCel7B and the catalytic module of CflXyn11A; suggesting an important role played by the carbohydrate-binding module of CflXyn11A (CflXyn11A-CBM) in the observed synergy. Affinity electrophoresis and binding assays showed that CflXyn11A-CBM binds to xylans and to a lesser extent to cellulose. Our results suggest that synergy is more pronounced at early stages of hydrolysis. Furthermore, for the first time it is described that a CBM carried by a xylanase significantly enhances the synergy with a cellulase (threefold increase in synergy).

Cloning and expression of a novel, moderately thermostable xylanase-encoding gene (Cflxyn11A) from Cellulomonas flavigena.

The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production.

Expression, purification and immobilization of the intracellular invertase INVA, from Zymomonas mobilis on crystalline cellulose and Nylon-6.

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBDCex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 degrees C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. Km and Vmax were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 degrees C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to Vmax increased up to 5.7-fold, following immobilization, whereas Km increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.

Immobilization of the recombinant invertase INVB from Zymomonas mobilis on Nylon-6.

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 degrees C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 degrees C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 degrees C. The values for kinetic parameters were determined as: 984 and 98 mM for Kappm of immobilized and free re-INVB, respectively. Kappcat values for immobilized and free enzymes were 6.1x10(2) and 1.2x10(4) s(-1), respectively, and immobilized re-INVB showed Vappmax of 158.73 micromol h min(-1) mg(-1). Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 degrees C and 70% at 40 degrees C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.

Immobilization of recombinant invertase (re-INVB) from Zymomonas mobilis on D-sorbitol cinnamic ester for production of invert sugar.

The recombinant invertase (re-INVB) from Zymomonas mobilis was immobilized by adsorption onto the totally cinnamoylated derivative of D-sorbitol. The polymerization and cross-linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of re-INVB on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. Enzyme concentration, immobilization time, and irradiation time were important parameters affecting the immobilization efficiency. The optimum reaction pH of immobilized enzyme was 5, and the optimal reaction temperature was 40 degrees C. The apparent Michaelis constant and the apparent catalytic constant of re-INVB immobilized on the SOTCN derivative acting on sucrose was 78+/-5 mM and 5x10(4)+/-3x10(2) s(-1), respectively, while for the free enzyme, it was 98.0+/-4 mM and 1.2x10(4)+/-2.5x10(2) s(-1), respectively, suggesting a better apparent affinity of the enzyme for the substrate and a better hydrolysis rate when immobilized than when in solution. Immobilized re-INVB also showed good thermal stability and good operational stability (40% of the initial activity remaining after 45 cyles of 1 min duration and 90.6 mg of sucrose being hydrolyzed in 45 min per 2.5 mg of immobilized protein). The results showed that cinnamic carbohydrate esters of D-sorbitol are an appropriate support for re-INVB immobilization and the production of invert sugar.

Purification, characterization and modular organization of a cellulose-binding protein, CBP105, a processive beta-1,4-endoglucanase from Cellulomonas flavigena.

A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose (CMC) at pH 7.5 and 60 degrees C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM, two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive CMC degradation.