Huoqing Huang

Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge

In order to increase the stability of thermophilic Thermomyces lanuginosus GH11 xylanase, TLX, a disulfide bridge Q1C-Q24C was introduced into the N-terminal region of the enzyme. The apparent temperature optimum shifted upwards at pH 6.5 by about 10°C to 75°C. The resistance to thermal inactivation also increased by about 10°C. The melting temperature measured by CD spectroscopy increased from 66 to 74°C. Therefore the N-terminal disulfide bridge increased both kinetic and thermodynamic stability almost equally. At pH 8 and 70°C, the disulfide bridge increased the enzyme half-life 20-fold in the presence of substrate. In contrast to the situation in acidic-neutral pH, the substrate decreased the thermostability of xylanases in alkaline pH. The upper limit for the performance of the disulfide bridge mutant at pH 9 was 75°C. This study showed that N-terminal disulfide bridges can stabilize even thermostable family GH11 xylanases.

Diversity of Beta-Propeller Phytase Genes in the Intestinal Contents of Grass Carp Provides Insight into the Release of Major Phosphorus from Phytate in Nature

ABSTRACT Phytate is the most abundant organic phosphorus compound in nature, and microbial mineralization of phytate by phytase is a key process for phosphorus recycling in the biosphere. In the present study, beta-propeller phytase (BPP) gene fragments were readily amplified from the intestinal contents of grass carp (Ctenopharyngodon idellus) directly or from phytate-degrading isolates from the same source, confirming the widespread occurrence of BPP in aquatic communities. The amounts of sequences collected using these two methods differed (88 distinct genes versus 10 isolates), but the sequences showed the same general topology based on phylogenetic analysis. All of the sequences fell in five clusters and were distinct from those of Anabaena, Gloeobacter, Streptomyces, Flavobacterium, Prosthecochloris, and Desulfuromonas, which have never been found in the grass carp intestine. Analysis of the microbial diversity by denaturing gradient gel electrophoresis demonstrated that unculturable bacteria were dominant bacteria in the grass carp intestine and thus the predominant phytate-degrading organisms. The predominant cultured species corresponding to the phytate-degrading isolates, Pseudomonas, Bacillus and Shewanella species, might be the main source of known BPPs. A phytase from Brevundimonas was first obtained from cultured species. Combining our results with Lim et al.s inference that phytate-mineralizing bacteria are widely distributed and highly diverse in nature (B. L. Lim, P. Yeung, C. Cheng, and J. E. Hill, ISME J. 1:321-330, 2007), we concluded that BPP is the major phytate-degrading enzyme in nature, that most of this enzyme might originate from unculturable bacteria, and that the distribution of BPP may be related to the type of niche. To our knowledge, this is the first study to experimentally estimate BPP diversity in situ.

Two xylose-tolerant GH43 bifunctional β-xylosidase/α-arabinosidases and one GH11 xylanase from Humicola insolens and their synergy in the degradation of xylan

Two β-xylosidases of family 43 (Xyl43A and Xyl43B) and one xylanase of family 11 (Xyn11A) were identified from the genome sequence of Humicola insolens Y1, and their gene products were successfully expressed in heterologous hosts. The optimal activities of the purified Xyl43A, Xyl43B, and Xyn11A were found at pH 6.5-7.0 and 50-60 °C. They were stable over a pH range of 5.0-10.0 and temperatures of 50 °C and below. Xyl43A and Xyl43B had the activities of β-xylosidase, α-arabinosidase and xylanase, and showed xylose tolerance up to 79 and 292 mM, respectively. Xyn11A and Xyl43A or Xyl43B showed significant synergistic effects on the degradation of various xylans, releasing more reduced sugars (up to 1.29 folds) by simultaneous or sequential addition. This study provides several enzymes for synergistic degradation of xylan and contributes to the formulation of optimised enzyme mixtures for the efficient hydrolysis of plant biomass.

A novel cold-active xylanase gene from the environmental DNA of goat rumen contents: Direct cloning, expression and enzyme characterization

A xylanase-coding gene, xynGR40, was cloned directly from the environmental DNA of goat rumen contents and expressed in Escherichia coli BL21 (DE3). The 1446-bp full-length gene encodes a 481-residue polypeptide (XynGR40) containing a catalytic domain belonging to glycosyl hydrolase (GH) family 10. Phylogenetic analysis indicated that XynGR40 was closely related with microbial xylanases of gastrointestinal source. Purified recombinant XynGR40 exhibited high activity at low temperatures, and remained active (∼10% of the activity) even at 0°C. The optimal temperature of XynGR40 was 30°C, much lower than other xylanases from rumen. Compared with mesophilic and thermophilic counterparts, XynGR40 had fewer hydrogen bonds and salt bridges, and lengthened loops in the catalytic domain. The enzyme also had relatively better stability at mesophilic temperatures and a higher catalytic efficiency than other known GH 10 cold active xylanases. These properties suggest that XynGR40 is a novel cold active xylanase and has great potential for basic research and industrial applications.

An α-galactosidase from an acidophilic Bispora sp. MEY-1 strain acts synergistically with β-mannanase

An alpha-galactosidase gene (AgalB) was cloned from the acidophilic fungus Bispora sp. MEY-1 and expressed in Pichia pastoris. The deduced amino acid sequence showed highest identity (35%) to the alpha-galactosidase from Penicillium simplicissimum, belonging to the glycosyl hydrolase family 27. The purified recombinant alpha-galactosidase (r-AgalB) exhibited optimal activity at pH 3.5 and 55 degrees C, was stable at pH 2.2-8.0, and showed higher hydrolytic activity towards galactomannan polysaccharides (guar gum and locust bean gum) than toward small galacto-oligosaccharides (melibiose, raffinose and stachyose). A synergistic (3-fold) increase in guar gum hydrolysis was observed when beta-mannanase Man5A from Bispora sp. MEY-1 and r-AgalB were combined. Further, an increase in the reaction time from 5h to 12h or increase of the temperature from 37 degrees C to 55 degrees C enhanced guar gum degradation by the enzyme combination. These properties make r-AgalB a good candidate for extensive application in the pulp/paper, food, and feed industries.

Paenibacillus sp. Strain E18 Bifunctional Xylanase-Glucanase with a Single Catalytic Domain

ABSTRACT Xylanases are utilized in a variety of industries for the breakdown of plant materials. Most native and engineered bifunctional/multifunctional xylanases have separate catalytic domains within the same polypeptide chain. Here we report a new bifunctional xylanase (XynBE18) produced by Paenibacillus sp. E18 with xylanase and β-1,3-1,4-glucanase activities derived from the same active center by substrate competition assays and site-directed mutagenesis of xylanase catalytic Glu residues (E129A and E236A). The gene consists of 981 bp, encodes 327 amino acids, and comprises only one catalytic domain that is highly homologous to the glycoside hydrolase family 10 xylanase catalytic domain. Recombinant XynBE18 purified from Escherichia coli BL21(DE3) showed specificity toward oat spelt xylan and birchwood xylan and β-1,3-1,4-glucan (barley β-glucan and lichenin). Homology modeling and molecular dynamic simulation were used to explore structure differences between XynBE18 and the monofunctional xylanase XynE2, which has enzymatic properties similar to those of XynBE18 but does not hydrolyze β-1,3-1,4-glucan. The cleft containing the active site of XynBE18 is larger than that of XynE2, suggesting that XynBE18 is able to bind larger substrates such as barley β-glucan and lichenin. Further molecular docking studies revealed that XynBE18 can accommodate xylan and β-1,3-1,4-glucan, but XynE2 is only accessible to xylan. These results indicate a previously unidentified structure-function relationship for substrate specificities among family 10 xylanases.

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

ABSTRACT Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.

Characterization of three novel thermophilic xylanases from Humicola insolens Y1 with application potentials in the brewing industry.

Three xylanase genes (xynA, xynB, xynC) of glycosyl hydrolase family 10 were identified in Humicola insolens Y1. The deduced protein sequences showed the highest identity of ⩽83% to known fungal xylanases and of ⩽38% with each other. Recombinant XynA-C produced in Pichia pastoris showed optimal activities at pH 6.0-7.0 and at high temperature (70-80°C), and exhibited good stability over a broad pH range and temperatures at 60°C. The gene xynC produced by H. insolens Y1 (named XynW) was similar in enzyme properties with XynC expressed by Pichia. XynA exhibited better alkaline adaptation and thermostability, and had higher catalytic efficiency and wider substrate specificity. Under simulated mashing conditions, addition of XynA-C showed better performance on filtration acceleration (37.4%) and viscosity reduction (13.5%) than Ultraflo from Novozyme. Thus the three xylanases represent good candidates for application in the brewing industry.

An Acidophilic and Acid-Stable β-Mannanase from Phialophora sp. P13 with High Mannan Hydrolysis Activity under Simulated Gastric Conditions

A beta-mannanase gene, man5AP13, was cloned from Phialophora sp. P13 and expressed in Pichia pastoris. The deduced amino acid sequence of the mature enzyme, MAN5AP13, had highest identity (53%) with the glycoside hydrolase family 5 beta-mannanase from Bispora sp. MEY-1. The purified recombinant beta-mannanase was acidophilic and acid stable, exhibiting maximal activity at pH 1.5 and retaining >60% of the initial activity over the pH range 1.5-7.0. The optimum temperature was 60 degrees C. The specific activity, K(m) and V(max) for locust bean gum substrate were 851 U/mg, 2.5 mg/mL, and 1667.7 U/min.mg, respectively. The enzyme had excellent activity and stability under simulated gastric conditions, and the released reducing sugar of locust bean gum was significantly enhanced by one-fold in simulated gastric fluid containing pepsin in contrast to that without pepsin. All these properties make MAN5AP13 a potential additive for use in the food and feed industries.

Molecular detection and diversity of xylanase genes in alpine tundra soil

Xylan is a major polysaccharide in plant cell walls, and its degradation is mainly conducted by microbial xylanases in nature. To explore the xylanase diversity in the environment, two sets of degenerate primers were designed based on the microbial xylanase sequences in Pfam database of glycosyl hydrolase (GH) family 10 and 11 and were used to amplify objective gene fragments directly from the alpine tundra soil DNA of the Tianshan Mountains, China. Ninety-six distinct GH 10 and 31 GH 11 xylanase gene fragments were retrieved, and most of them have low identities with known sequences in GenBank. Based on phylogenetic analysis, all of the GH 10 xylanase sequences fell into six clusters and were related to xylanases from Actinobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Firmicutes, and Acidobacteria. Three clusters of GH 11 xylanase sequences were established, and two of them were related with enzymes from fungi. These results indicated the diversity of xylanase genes in this cold environment. Four xylanolytic strains were isolated from the soil, and GH 10 xylanase gene fragments were cloned using the same primers. A full-length gene was obtained and expressed in Escherichia coli, and the recombinant enzyme showed some cold-related characteristics. Our study provides an efficient molecular approach to study xylanase in complex environments and casts an insight into the diversity and distribution of xylanases in a cold environment, which is very meaningful to understand their roles in xylan degradation in nature.