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A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases.

ABSTRACT: Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Escherichia coli. The C-terminal oligopeptide had significant effects on the function and structure of XynA. Compared with XynA-Tr, XynA exhibited improved specific activity (12-fold) and catalytic efficiency (14-fold), a higher temperature optimum (50°C versus 45°C), and broader ranges of temperature and pH optima (pH 5.0 to 7.5 and 40 to 60°C versus pH 5.5 to 6.5 and 40 to 50°C). Moreover, XynA released more xylose than XynA-Tr when using beech wood xylan and wheat arabinoxylan as the substrate. The underlying mechanisms responsible for these changes were analyzed by substrate binding assay, circular dichroism (CD) spectroscopy, isothermal titration calorimetry (ITC), and xylooligosaccharide hydrolysis. XynA had no ability to bind to any of the tested soluble and insoluble polysaccharides. However, it contained more ? helices and had a greater affinity and catalytic efficiency toward xylooligosaccharides, which benefited complete substrate degradation. Similar results were obtained when the C-terminal sequence was fused to another GH10 xylanase from sheep rumen. This study reveals an engineering strategy to improve the catalytic performance of enzymes.

SUBMITTER: Li Z

PROVIDER: S-EPMC4018843 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases.

Li Zhongyuan Z Xue Xianli X Zhao Heng H Yang Peilong P Luo Huiying H Zhao Junqi J Huang Huoqing H Yao Bin B

Applied and environmental microbiology 20140321 11

Efficient degradation of plant polysaccharides in rumen requires xylanolytic enzymes with a high catalytic capacity. In this study, a full-length xylanase gene (xynA) was retrieved from the sheep rumen. The deduced XynA sequence contains a putative signal peptide, a catalytic motif of glycoside hydrolase family 10 (GH10), and an extra C-terminal proline-rich sequence without a homolog. To determine its function, both mature XynA and its C terminus-truncated mutant, XynA-Tr, were expressed in Esc ...[more]

PMID: 24657866

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Project description:BackgroundThe rumen harbors a complex microbial ecosystem for efficient hydrolysis of plant polysaccharides which are the main constituent of the diet. Xylanase is crucial for hemicellulose hydrolysis and plays an important role in the plant cell wall degradation. Xylanases of ruminal strains were widely studied, but few studies have focused on their diversity in rumen microenvironment.Methodology/principal findingsWe explored the genetic diversity of xylanases belonging to two major glycosyl hydrolase families (GH 10 and 11) in goat rumen contents by analyzing the amplicons generated with two degenerate primer sets. Fifty-two distinct GH 10 and 35 GH 11 xylanase gene fragments (similarity <95%) were retrieved, and most had low identities with known sequences. Based on phylogenetic analysis, all GH 10 xylanase sequences fell into seven clusters, and 88.5% of them were related to xylanases from Bacteroidetes. Five clusters of GH 11 xylanase sequences were identified. Of these, 85.7% were related to xylanases from Firmicutes, and 14.3% were related to those of rumen fungi. Two full-length xylanase genes (one for each family) were directly cloned and expressed in Escherichia coli. Both the recombinant enzymes showed substantial xylanase activity, and were purified and characterized. Combined with the results of sheep rumen, Bacteroidetes and Firmicutes are the two major phyla of xylan-degrading microorganisms in rumen, which is distinct from the representatives of other environments such as soil and termite hindgut, suggesting that xylan-degrading microorganisms are environment specific.Conclusion/significanceThe numerous new xylanase genes suggested the functional diversity of xylanase in the rumen microenvironment which may have great potential applications in industry and agriculture. The phylogenetic diversity and different distributions of xylanase genes will help us understand their roles in plant cell wall degradation in the rumen microenvironment.

Project description:The challenge of wheat straw as a ruminant feed is its low ruminal digestibility. This study investigated the impact of a xylanase called RuXyn, derived from the rumen metagenome of beef cattle, on the in vitro ruminal fermentation of wheat straw. RuXyn encoded 505 amino acids and was categorized within subfamily 8 of the glycosyl hydrolase 30 family. RuXyn was heterologously expressed in Escherichia coli and displayed its highest level of activity at pH 6.0 and 40 °C. RuXyn primarily hydrolyzed xylan, while it did not show any noticeable activity towards other substrates, including carboxymethylcellulose and Avicel. At concentrations of 5 mM, Mn2+ and dithiothreitol significantly enhanced RuXyn's activity by 73% and 20%, respectively. RuXyn's activity was almost or completely inactivated in the presence of Cu2+, even at low concentrations. The main hydrolysis products of corncob xylan by RuXyn were xylopentose, xylotriose, and xylotetraose. RuXyn hydrolyzed wheat straw and rice straw more effectively than it did other agricultural by-products. A remarkable synergistic effect was observed between RuXyn and a cellulase cocktail on wheat straw hydrolysis. Supplementation with RuXyn increased dry matter digestibility; acetate, propionate, valerate, and total volatile fatty acid yields; NH3-N concentration, and total bacterial number during in vitro fermentation of wheat straw relative to the control. RuXyn's inactivity at 60 °C and 70 °C was remedied by mutating proline 151 to phenylalanine and aspartic acid 204 to leucine, boosting activity to 20.3% and 21.8% of the maximum activity at the respective temperatures. As an exogenous enzyme preparation, RuXyn exhibits considerable potential to improve ruminal digestion and the utilization of wheat straw in ruminants. As far as we know, this is the first study on a GH30 xylanase promoting the ruminal fermentation of agricultural straws. The findings demonstrate that the utilization of RuXyn can significantly enhance the ruminal digestibility of wheat straw by approximately 10 percentage points. This outcome signifies the emergence of a novel and highly efficient enzyme preparation that holds promise for the effective utilization of wheat straw, a by-product of crop production, in ruminants.

Project description:Xylanases play a crucial role in the degradation of xylan in both terrestrial and marine environments. The endoxylanase XynB from the marine bacterium Glaciecola mesophila KMM 241 is a modular enzyme comprising a long N-terminal domain (NTD) (E44 to T562) with xylan-binding ability and a catalytic domain (CD) (T563 to E912) of glycoside hydrolase family 8 (GH8). In this study, the long NTD is confirmed to contain three different functional regions, which are NTD1 (E44 to D136), NTD2 (Y137 to A193), and NTD3 (L194 to T562). NTD1, mainly composed of eight ?-strands, functions as a new type of carbohydrate-binding module (CBM), which has xylan-binding ability but no sequence similarity to any known CBM. NTD2, mainly forming two ?-helices, contains one of the ?-helices of the catalytic domain's (?/?)6 barrel and therefore is essential for the activity of XynB, although it is far away from the catalytic domain in sequence. NTD3, next to the catalytic domain in sequence, is shown to be helpful in maintaining the thermostability of XynB. Thus, XynB represents a kind of xylanase with a new domain architecture. There are four other predicted glycoside hydrolase sequences with the same domain architecture and high sequence identity (?80%) with XynB, all of which are from marine bacteria. Phylogenetic analysis shows that XynB and these homologs form a new group in GH8, representing a new class of marine bacterial xylanases. Our results shed light on xylanases, especially marine xylanases.IMPORTANCE Xylanases play a crucial role in natural xylan degradation and have been extensively used in industries such as food processing, animal feed, and kraft pulp biobleaching. Some marine bacteria have been found to secrete xylanases. Characterization of novel xylanases from marine bacteria has significance for both the clarification of xylan degradation mechanisms in the sea and the development of new enzymes for industrial application. With G. mesophila XynB as a representative, this study reveals a new group of the GH8 xylanases from marine bacteria, which have a distinct domain architecture and contain a novel carbohydrate-binding module. Thus, this study offers new knowledge on marine xylanases.

Dataset Information

A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases.

Publications

A C-terminal proline-rich sequence simultaneously broadens the optimal temperature and pH ranges and improves the catalytic efficiency of glycosyl hydrolase family 10 ruminal xylanases.

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