The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan.

BACKGROUND: Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. RESULTS: In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. CONCLUSION: This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.

Functional Analysis of a Highly Active ß-Glucanase from Bispora sp. MEY-1 Using Its C-terminally Truncated Mutant.

A ß-1,3-1,4-glucanase-encoding gene, Bisglu16B, was identified in Bispora sp. MEY-1. The deduced BisGlu16B consists of an N-terminal signal peptide, a catalytic module of glycoside hydrolase family 16 (GH16), and a C-terminal serine/proline-rich module. After expression in Pichia pastoris GS115, the purified recombinant BisGlu16B showed maximal activity at pH 4.0 and 55 °C and had broad substrate specificity (ß-1,3-/ß-1,4-mixed, ß-1,3-, ß-1,4-, and ß-1,6-linked glucan, and ß-1,4-mannan). The enzyme possessed high specific activities toward barley ß-glucan (34 700 U·mg-1), lichenan (23 900 U·mg-1), and laminarin (9 000 U·mg-1). After removing the C-terminal module, the truncated mutant, BisGlu16B-ΔC, retained similar enzymatic properties to the wild type but displayed significantly enhanced activities (up to 2.5-fold). Functional and structural analyses indicated that the C-terminal module plays a key role in the substrate binding of BisGlu16B. This study provided an excellent candidate glucanase for industrial purposes and revealed the functions of a C-terminal serine/proline-rich region.

[Use of rpoB and 16S rDNA genes to analyze rumen bacterial diversity of goat using PCR and DGGE].

DNA extraction from the rumen of three species of goat (boer goat, Nanjiang yellow goat, Inner Mongolia cashmere goat) was followed by Polymerase Chain Reaction (PCR) amplification of the beta subunit of the RNA polymerase (rpoB) and 16S rDNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare the predominant bacterial community structure. The results showed the rpoB DGGE profiles comprised fewer bands than those of 16S rDNA profiles and were easier to analyze. The gene for rpo B is a single copy gene unlike 16S rDNA. So using the rpoB gene offeres a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE. The bacteria community structure of different goats were similar to each other. The similarities within species were noticeably higher than that between species. Goat species were found to influence the rumen microbe community. Phylogenetic and sequence similarity analyses of the resultant 14 clone sequences in16S rDNA DGGE libraries revealed that 4 clone show similarity over 97% with that of database sequences, while the rest present similarity in a range of 89%-96%, and 13 clone of all were similar to those unidentified rumen bacteria. These results suggest that DGGE followed by clone technique is a practicable protocol to research the complex community of rumen microbe.

[Purification and characterization of a novel alpha-galactosidase from penicillium sp. F63 CGMCC1669].

An a-galactosidase-producing fungus was screened out of 26 filamentous fungi isolated from soil by us. Phylogenetic analysis based on the alignment of 18S rDNA sequences, combined with the morphological identification, indicated that the strain F63 was a member of the genus Penicillium. The a-galactosidase from Penicillium sp. F63 was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange and gel filtration chromatography. The molecular size of the purified enzyme is approximately 82kDa estimated by SDS-PAGE. The a-galactosidase has an optimum pH of 5.0 and an optimum temperature of 45 degrees C. The enzyme is stable between pH5.0 and 6.0 below 40 degrees C. The a-galactosidase activity is slightly inhibited by Ag+ , which is dissimilar to other a-galactosidases. Kinetic studies of the a-galactosidase showed that the Km and the Vmax for pNPG are 1.4mmol/L and 1.556mmol/L. min(-1) x mg- 1, respectively. The enzyme is able to degrade natural substrates such as melibiose, raffinose and stachyose but not galactose-containing polysaccharides. The alpha-galactosidase was identified by MALDI-TOF-MS and its inner peptides were sequenced by ESI-MS/MS. The results show that the a-galactosidase is a novel one.

[Improving phytase expression by increasing the gene copy number of appA-m in Pichia pastoris].

In order to improve the fermentation potency of phytase in recombinant host and decrease the production cost, the pichia expression vector pGAPZalpha-A was modified by introduction of an AOX1 promoter from vector pPIC9 and the resulted vector pAOXZalpha is an methanol induced vector. After that, a phytase gene appA-m was cloned into pAOXZalpha to construct the recombinant vector pAOXZalpha-appA-m. The recombinant Pichia pastoris 74#, which already contains one copy of appA-m and its fermentation potency exceeded 7.5 x 10(6) IU/mL, was used as the host strain for the transformation of pAOXZalpha-appA-m. The Pichia pastoris transformants were gained by electroporation. PCR results indicated that the appA-m expression box has integrated into the genome of Pichia pastoris and the original construction of phytase gene has not changed. SDS-PAGE analysis revealed that phytase was overexpressed and secreted into the medium supernatant. Recombinants with high expression level were screened and used for fermentation. In 5L fermentor, the expression level of phytase protein achieved 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2 x 10(7) IU/mL, which was about 1.6-fold compared with that of the host strain 74#. Moreover, the improved recombinant Pichia pastoris is excellent at expression stability and heredity stability.

[Improvement of the thermostability of xylanase by N-terminus replacement].

The hybrid xylanase TB was constructed by the substitution of the N-terminus segment of the Streptomyces olivaceoviridis xylanase XYNB with corresponding region of Thermomonosporafusca xylanase TfxA. The hybrid gene tb, encoding the TB, was correctly expressed in Escherichia coli BL21 and Pichia pastoris GS115. TB was purified and its enzymatic properties were determined. The results revealed that the optimal temperature and optimal pH of TB were at 70 degrees C and 6.0, which have been obviously improved compared with those of XYNB. The thermostability of TB were all about six-fold of XYNB's after incubating the properly diluted enzyme solutions at 80 degrees C and 90 degrees C for 3min, respectively. The pH stability of TB was 5 to approximately 9, which was narrower than that of XYNB. Still, TB remains a high specific activity as XYNB does. Analysis of a homology modeling and sequence similarity were used to reveal the factors influencing the enzymatic properties of TB and the discussion for the relationship between structure and function of xylanase was given.

[Overexpression of Citrobacter braakii phytase with high specific activity in Pichia pastoris].

Utilization of the phytase with high specific activity is an effective way to improve the fermentation potency of phytase in recombinant host and decrease the production cost. Up to now, the phytase APPA from Citrobacter braakii exhibits the highest specific activity in the all phytases recorded previously. The gene AppA encoding phytase was modified according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence and artificially synthesized. The modified gene, AppA ( m) , was inserted into the Pichia pastoris expression vector pPIC9 under the control of AOX1 promoter, and the resulted expression vector pPIC9-AppA ( m) was introduced into the host Pichia pastoris by electroporation. PCR analysis of the recombinant yeast indicated that AppA (m) gene was integrated into the chromosome of Pichia pastoris. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The recombinant phytase APPA was purified by simple methods, such as dialysis, ultrafiltration and chromatography. After the simple purification, the purity of the recombinant phytase reached to electrophoresis purity, and the recombinant phytase was shown to be glycosylated by Endo-H treatment. The specific activity of the purified recombinant APPA was 3.5 x 10(6) IU/mg of protein. Recombinant phytase APPA showed activity at pH values from 2.0 through 7.0 with the optimum at 4.5. The temperature optimum was 55 degrees C at pH 4.5.The Km value for sodium phytate was 0.165mmol/L with a Vmax of 3.3 x 10(6)IU/mg min. In 5-liter fermentor in fed-batch fermentation, the expression level of phytase in recombinant Pichia pastoris was 3.2mg/mL and the fermentation potency exceeded 1.4 x 10(7) IU/mL, which is the highest level among all of the reported phytase recombinant strains at present.

[Hydrophobic interaction between beta-sheet B1 and B2 in xylanase XYNB influencing the enzyme thermostability].

A homology modeling of xylanase XYNB from Streptomyces olivaceoviridis A1 was made by Swiss-Model. The hydrophobic Interaction between beta-sheet B1 and B2 in the tertiary structure model of XYNB was compared with other thermophilic xylanase. A T11Y mutation was introduced in XYNB by site-dirrected mutagenesis to improve the thermostability of the enzyme. The XYNB and mutant xylanase (XYNB') expressed in Pichia pastoris were purified and their enzymatic properties were determined. The result revealed that the thermostability of XYNB' was obviously higher than that of XYNB. The optimal temperature of XYNB' for its activity was 60 degrees C, similar to XYNB. But, compare to XYNB, the optimal pH value, the Km value and the specific activity of XYNB' had also been changed. The research results suggested that the aromatic interaction between beta-sheet B1 and B2 in xylanase should increase enzyme thermostability. The mutant xylanase XYNB' is a good material for further research in the relationship between structure and function of xylanase.