Characterization of temperature dependent and substrate-binding cleft movements in Bacillus circulans family 11 xylanase: A molecular dynamics investigation

Background: Xylanases (EC 3.2.1.8) hydrolyze xylan, one of the most abundant plant polysaccharides found in nature, and have many potential applications in biotechnology. Methods: Molecular dynamics simulations were used to investigate the effects of temperature between 298 to 338 K and xylobiose binding on residues located in the substrate-binding cleft of the family 11 xylanase from Bacillus circulans (BcX). Results: In the absence of xylobiose the BcX exhibits temperature dependent movement of the thumb region which adopts an open conformation exposing the active site at the optimum catalytic temperature (328 K). In the presence of substrate, the thumb region restricts access to the active site at all temperatures, and this conformation is maintained by substrate/protein hydrogen bonds involving active site residues, including hydrogen bonds between Tyr69 and the 2` hydroxyl group of the substrate. Substrate access to the active site is regulated by temperature dependent motions that are restricted to the thumb region, and the BcX/substrate complex is stabilized by extensive intermolecular hydrogen bonding with residues in the active site. General significance: These results call for a revision of both the ""hinge-bending"" model for the activity of group 11 xylanases, and the role of Tyr69 in the catalytic mechanism. (C) 2009 Elsevier B.V. All rights reserved.

Effect of Bacillus circulans D1 thermostable xylanase on biobleaching of eucalyptus kraft pulp

The alkalophilic Bacillus circulans D1 was isolated from decayed wood. It produced high levels of extracellular cellulase-free xylanase. The enzyme was thermally stable up to 60°C, with an optimal hydrolysis temperature of 70°C. It was stable over a wide pH range (5.5-10.5), with an optimum pH at 5.5 and 80% of its activity at pH 9.0. This cellulase-free xylanase preparation was used to biobleach kraft pulp. Enzymatic treatment of kraft pulp decreased chlorine dioxide use by 23 and 37% to obtain the same kappa number (κ number) and brightness, respectively. Separation on Sephadex G-50 isolated three fractions with xylanase activity with distinct molecular weights.

An insight into the thermostability of a pair of xylanases: the role of hydrogen bonds

Xylanases are enzymes that are very tolerant to temperature. Their potential use in several biotechnological applications, such as animal food manufacture and pulp bleaching, is due to their intrinsic thermostability. The present report deals with two xylanases, the mesophilic xylanase from Bacillus circulans, BCX, and the thermophilic xylanase from Thermomyces lanuginosus,TLX. These enzymes belong to family 11, and they are the most structurally similar mesophilic-thermophilic pair. Molecular dynamics simulations were employed to investigate the factors responsible for the different thermostabilities exhibited by these structurally similar enzymes. Their active site is their most rigid region, and it is equally rigid at all temperatures. Inter and intramolecular interactions, hydrogen bonds in particular, are the key to the main differences between BCX and TLX. The intramolecular hydrogen bonds and salt bridges are important for maintenance of the backbone rigidity even at high temperature, and the highly solvated surface is a clear optimization in TLX compared with BCX. The main differences between these two enzymes can be found on the fingers domain, which indicates that this domain must be the target for the site-directed mutagenesis responsible for improving the temperature tolerance of this family of enzymes.

Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Biochemical properties of a beta-mannanase and a beta-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan, Its activity was optimal at pH 4.5. The K(m) value with galactoglucomannan as substrate was 0.50 mg ml (1). One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 degrees C and pH 8.0. Its K(m) value with birchwood xylan as substrate was 1.65 mg ml (1). Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn(+2), Fe(+3), and Cu(+2) were strong inhibitors for the mannanase. (C) 2008 Elsevier Ltd. All rights reserved.

Thermostable variants of the recombinant xylanase A from Bacillus subtilis produced by directed evolution show reduced heat capacity changes

Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P, and S179C that showed an increase in melting temperature of 20 degrees C. The thermodynamic properties Of a representative subset of nine XylA variants showing a range of thermostabilities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (Delta C(p)) with an increase in the midpoint of the transition temperature (T(m)) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in Delta C(p) on should include effects arising from the prot inlsolvent interface.

Purification and characterization of two xylanases from alkalophilic and thermophilic Bacillus licheniformis 77-2

The alkalophilic bacteria Bacillus licheniformis 77-2 produces significant quantities of thermostable cellulase-free xylanases. The crude xylanase was purified to apparent homogeneity by gel filtration (G-75) and ionic exchange chromatography (carboxymethyl sephadex, Q sepharose, and Mono Q), resulting in the isolation of two xylanases. The molecular masses of the enzymes were estimated to be 17 kDa (X-I) and 40 kDa (X-II), as determined by SDS-PAGE. The K(m) and V(max) values were 1.8 mg/mL and 7.05 U/mg protein (X-I), and 1.05 mg/mL and 9.1 U/mg protein (X-II). The xylanases demonstrated optimum activity at pH 7.0 and 8.0-10.0 for xylanase X-I and X-II, respectively, and, retained more than 75% of hydrolytic activity up to pH 11.0. The purified enzymes were most active at 70 and 75 degrees C for X-I and X-II, respectively, and, retained more than 90% of hydrolytic activity after 1 h of heating at 50 degrees C and 60 degrees C for X-I and X-II, respectively. The predominant products of xylan hydrolysates indicated that these enzymes were endoxylanases.

Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Optimization of xylanase production by Bacillus circulans D1 in submerged fermentation using response surface methodology

In this work, a 3(3) factorial design was performed with the aim of optimizing the culture conditions for xylanase production by an alkalophilic thermophilic strain of Bacillus circulans, using response surface methodology. The variables involved in this study were xylan concentration (X-1), pH (X-2) and cultivation time (X-3). The optimal response region was approached without using paths of steepest ascent. Statistical analysis of results showed that, in the range studied, only pH did not have a significant effect on xylanase production. A second-order model was proposed to represent the enzymic activity as a function of xylan concentration (X-1) and cultivation time (X-3). The optimum xylan concentration and cultivation time were 5 g/l and 48 h, respectively. Under these conditions, the model predicted a xylanase activity of 19.1 U/ml. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Thermophilic fungi as new sources for production of cellulases and xylanases with potential use in sugarcane bagasse saccharification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Xylooligosaccharides Production from Alkali-Pretreated Sugarcane Bagasse Using Xylanases from Thermoascus aurantiacus

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of L-arabinose, 4-O-methyl-D-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50 degrees C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5-3.5% (w/v), enzyme load 40-80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 22 factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.

Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts

In the present work, a thermophilic esterase from Thermus thermophilus HB27 was cloned into Kluyveromyces marxianus and into Kluyveromyces lactis using two different expression systems, yielding four recombinant strains. K. lactis showed the highest esterase expression levels (294 units per gram dry cell weight, with 65% of cell-bound enzyme) using an episomal system with the PGK promoter and terminator from Saccharomyces cerevisiae combined with the K. lactis k1 secretion signal. K. marxianus showed higher secretion efficiency of the heterologous esterase (56.9 units per gram dry cell weight, with 34% of cell-bound enzyme) than K. lactis. Hydrolytic activities for the heterologous esterases were maximum at pH values between 8.0 and 9.0 for both yeast species and at temperatures of 50 A degrees C and 45 A degrees C for K. marxianus and K. lactis, respectively. When compared to previously published data on this same esterase produced in the original host or in S. cerevisiae, our results indicate that Kluyveromyces yeasts can be considered good hosts for the heterologous secretion of thermophilic esterases, which have a potential application in biodiesel production or in resolving racemates.

Sensory evaluation of probiotic Minas fresh cheese with Lactobacillus acidophilus added solely or in co-culture with a thermophilic starter culture

Sensory acceptance of formulations of probiotic Minas fresh cheese was investigated. Cheeses were prepared and supplemented with Lactobacillus acidophilus (T1 - probiotic), Lactobacillus acidophilus + Streptococcus thermophilus (T2 - probiotic + starter) or produced with no addition of cultures (T3 - control). Sensory acceptance tests were performed after 7 and 14 days of storage at 5 degrees C, using a 9-point hedonic scale (1 = dislike extremely; 9 = like extremely). After 7 days, no significant difference was detected among cheeses T1, T2 and T3 (P > 0.05). After 14 days, cheeses T1 and T2 presented higher acceptance and differed significantly from cheeses T3. Cheeses T3 presented significant difference between 7 and 14 days of storage (P < 0.05), whereas probiotic cheeses T1 and T2 were stable in the same period (P > 0.05). The addition of L. acidophilus, either solely or in co-culture with a thermophilic starter culture, resulted in good acceptance of Minas fresh cheese, improving sensory performance of the product during storage.

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).