Microenzymes: Is There Anybody Out There?

Biological macromolecules are found in different shapes and sizes. Among these, enzymes catalyze biochemical reactions and are essential in all organisms, but is there a limit size for them to function properly? Large enzymes such as catalases have hundreds of kDa and are formed by multiple subunits, whereas most enzymes are smaller, with molecular weights of 20-60 kDa. Enzymes smaller than 10 kDa could be called microenzymes and the present literature review brings together evidence of their occurrence in nature.

Mapping secondary substrate-binding sites on the GH11 xylanase from Bacillus subtilis.

Xylanases are of significant interest for biomass conversion technologies. Here, we investigated the allosteric regulation of xylan hydrolysis by the Bacillus subtilis GH11 endoxylanase. Molecular dynamics simulations (MDS) in the presence of xylobiose identified binding to the active site and two potential secondary binding sites (SBS) around surface residues Asn54 and Asn151.

Biocatalytic potential of CAZymes (Sciaroidea, Diptera) in degrading plant and fungal cell wall polysaccharides.

Soil biota has a crucial impact on soil ecology, global climate changes, and effective crop management and studying the diverse ecological roles of dipteran larvae deepens the understanding of soil food webs. A multi-omics study of comb. nov.

Xylose isomerase from Piromyces sp. E2 is a promiscuous enzyme with epimerase activity.

Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose with promiscuous activity for other saccharides including D-glucose, D-allose, and L-arabinose. The xylose isomerase from the fungus Piromyces sp. E2 (PirE2_XI) is used to engineer xylose usage by the fermenting yeast Saccharomyces cerevisiae, but its biochemical characterization is poorly understood with divergent catalytic parameters reported.

Glycomic profiling identifies key-structural differences in three arabinoxylan fractions from sugarcane culms.

Sugarcane is an important food and bioenergy crop, and although the residual biomass is potentially available for biorefinery and biofuels production the complex plant cell wall matrix requires pretreatment prior to enzymatic hydrolysis. Arabinoxylans require multiple enzymes for xylose backbone and saccharide side-branch hydrolysis to release xylooligosaccharides and pentoses. The effect of arabinoxylan structure on xylooligosaccharide release by combinations of up to five xylanolytic enzymes was studied using three arabinoxylan fractions extracted from sugarcane culms by sodium chlorite, DMSO and alkaline treatments.

The Glycoside Hydrolase Family 35 β-galactosidase from Trichoderma reesei debranches xyloglucan oligosaccharides from tamarind and jatobá.

Trichoderma reesei (anamorph Hypocrea jecorina) produces an extracellular beta-galactosidase from Glycoside Hydrolase Family 35 (TrBga1). Hydrolysis of xyloglucan oligosaccharides (XGOs) by TrBga1 has been studied by hydrolysis profile analysis of both tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seed storage xyloglucans using PACE and MALDI-ToF-MS for separation, quantification and identification of the hydrolysis products. The TrBga1 substrate preference for galactosylated oligosaccharides from both the XXXG- and XXXXG-series of jatobá xyloglucan showed that the doubly galactosylated oligosaccharides were the first to be hydrolyzed.

Selective xyloglucan oligosaccharide hydrolysis by a GH31 α-xylosidase from Escherichia coli.

Xyloglucan is ubiquitous in the cell walls of land plants and is also an essential storage polymer in seeds of many species. We studied the hydrolysis of the non-reducing end xylosyl residue of xyloglucan oligosaccharides (XGOs) by the Escherichia coli α-xylosidase (YicI). Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS) and ion fragmentation analysis together with high performance anion exchange chromatography with pulsed amperometric detection revealed that YicI preferentially removes the xylosyl residue from the glycosyl residue of non-galactosylated oligosaccharides.

Covalent Immobilization of Chondrostereum purpureum Endopolygalacturonase on Ferromagnetic Nanoparticles: Catalytic Properties and Biotechnological Application.

Pectinases are widely used in a variety of industrial processes. However, their application is limited by low catalytic processivity, reduced stability, high cost, and poor re-use compatibility. These drawbacks may be overcome by enzyme immobilization with ferromagnetic nanoparticles, which are easily recovered by a magnetic field.

Enhanced hydrolytic efficiency of an engineered CBM11-glucanase enzyme chimera against barley β-d-glucan extracts.

The β-d-glucans are abundant cell wall polysaccharides in many cereals and contain both (1,3)- and (1,4)-bonds. The β-1,3-1,4-glucanases (EC 3.2.

A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) β-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source.

A Halotolerant Endo-1,4-β-Xylanase from Aspergillus clavatus with Potential Application for Agroindustrial Residues Saccharification.

The use of non-potable water (such as seawater) is an attractive alternative for water intensive processes such as biomass pretreatment and saccharification steps in the production of biochemicals and biofuels. Identification and application of halotolerant enzymes compatible with high-salt conditions may reduce the energy needed for non-potable water treatment and decrease waste treatment costs. Here we present the biochemical properties of a halotolerant endo-1,4-β-xylanase produced by Aspergillus clavatus in submerged fermentation, using paper sludge (XPS) and sugarcane bagasse (XSCB), and its potential application in the hydrolysis of agroindustrial residues.

A novel Trichoderma reesei mutant RP698 with enhanced cellulase production.

A new strain of Trichoderma reesei (teleomorph Hypocrea jecorina) with high cellulase production was obtained by exposing the spores from T. reesei QM9414 to an ultraviolet light followed by selecting fast-growing colonies on plates containing CMC (1% w/v) as the carbon source. The mutant T.

Biochemical and kinetic characterization of the recombinant GH28 Stereum purpureum endopolygalacturonase and its biotechnological application.

Endopolygalacturonase (EndoPG) from Stereum purpureum was expressed as a soluble protein in Pichia pastoris GS115, where after 3 days methanol induction the enzyme activity in the culture supernatant was 40 U mL. After purification by IMAC, SDS-PAGE analysis showed that the molecular weight of EndoPG was approximately 60.0 kDa.

Immobilization of a β-glucosidase and an endoglucanase in ferromagnetic nanoparticles: A study of synergistic effects.

Nanoparticles can act as support materials for enzymatic immobilization, introducing a balance of characteristics that modulate the efficiency of biocatalysts, such as specific surface area, resistance to mass transfer and effective enzymatic loading. Magnetic nanoparticles can be easily separated using an external magnetic field, and in this work two recombinant enzymes, the β-glucosidase from Humicola insolens (Bglhi) and the endoglucanase from Scytalidium thermophilum (Egst) were immobilized on synthetized FeO nanoparticles derivatized with chitosan/glutaraldehyde/N-(5-amino-1-carboxy-pentyl) iminodiacetic acid and functionalized with NiCl. The immobilization yields were about 20% for Bglhi and Egst with efficiencies of 132% and 115%, respectively.

Glucose tolerant and glucose stimulated β-glucosidases - A review.

The β-glucosidases (β-D-glucoside glucohydrolase, EC 3.2.1.

Overexpression of a Cellobiose-Glucose-Halotolerant Endoglucanase from Scytalidium thermophilum.

Enzyme reaction products and by-products from pretreatment steps can inhibit endoglucanases and are major factors limiting the efficiency of enzymatic lignocellulosic biomass hydrolysis. The gene encoding the endoglucanase from Scytalidium thermophilum (egst) was cloned and expressed as a soluble protein in Pichia pastoris GS115. The recombinant enzyme (Egst) was monomeric (66 kDa) and showed an estimated carbohydrate content of 53.

Engineering the GH1 β-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose.

The activity of the GH1 β-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-β-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites.

Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A.

Background: Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A (Lys49-PLA) from the venom of which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLAs is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA-induced nociception and inflammation.

The role of local residue environmental changes in thermostable mutants of the GH11 xylanase from Bacillus subtilis.

A thermostable variant of the mesophilic xylanase A from Bacillus subtilis (BsXynA-G3_4x) contains the four mutations Gln7His, Gly13Arg, Ser22Pro, and Ser179Cys. The crystal structure of the BsXynA-G3_4x has been solved, and the local environments around each of these positions investigated by molecular dynamics (MD) simulations at 328K and 348K. The structural and MD simulation results were correlated with thermodynamic data of the wild-type enzyme, the 4 single mutants and the BsXynA-G3_4x.

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

Background: Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose.

Results: The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E.

GH53 Endo-Beta-1,4-Galactanase from a Newly Isolated Bacillus licheniformis CBMAI 1609 as an Enzymatic Cocktail Supplement for Biomass Saccharification.

Galactanases (endo-β-1,4-galactanases-EC 3.2.1.

Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase.

Background: Product inhibition can reduce catalytic performance of enzymes used for biofuel production. Different mechanisms can cause this inhibition and, in most cases, the use of classical enzymology approach is not sufficient to overcome this problem. Here we have used a semi-rational protein fusion strategy to create a product-stimulated enzyme.

Increased biomass saccharification by supplementation of a commercial enzyme cocktail with endo-arabinanase from Bacillus licheniformis.

Objectives: The use of endo-arabinanase from Bacillus licheniformis (ABNase) for sugarcane saccharification has been evaluated by enzyme immobilization and commercial cocktail supplement with the immobilized heterologous protein.

Results: Biochemical characterization of the purified ABNase showed that the catalytic activity was strongly inhibited by 5 mM Cu(2+), Zn(2+) or Fe(3+). The optimum pH and temperature for activity were 5.

Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening.

There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification.

Concommitant adaptation of a GH11 xylanase by directed evolution to create an alkali-tolerant/thermophilic enzyme.

As part of an ongoing directed evolution program, the catalytic performance of the Xylanase A from Bacillus subtilis (XynA), which presents temperature and pH optima of 50°C and 6.0, respectively, has been enhanced to create a highly thermostable and alkali-tolerant enzyme. A library of random XynA mutants generated by error-prone polymerase chain reaction was screened by halo formation on agar containing xylan at pH 8.