Biochemical characterization, molecular cloning, and structural modeling of an interesting ß-1,4-glucanase from Sclerotinia sclerotiorum.

The filamentous fungus Sclerotinia sclerotiorum produces a complete set of cellulolytic enzymes needed for efficient solubilization of native cellulose, the major component of plants. In this work, we reported the molecular characterization of an important glycosyl-hydrolase enzyme classified as endo-ß-1,4-glucanase. The importance of this enzyme was revealed with the in-gel activity staining, showing a high degradation capacity of cellulose. When purified from native gel and ran in denaturing polyacrylamide gel, the polypeptide has an apparent molecular mass of about 34 kDa called Endo2. For further characterization of this protein, a mass spectrometry approach was carried out. The LC-MS/MS analysis revealed two peptides belonging to this enzyme. The genomic DNA and cDNA sequences were resolved by PCR amplification and sequencing, revealing a gene with two intron sequences. The open reading frame of 987 bp encoded a putative polypeptide of 328 amino acids having a calculated molecular mass of 33,297 Da. Yet, the molecular modeling and comparative investigation of different 3D cellulase structures showed that this endoglucanase isoform has probably two domains. A core domain having a high similarity with endoglucanases family 5 and a cellulose-binding domain having similarities with those of exo-type cellulases of family 1, linked together by a serine-threonine-rich region. These results are with great interests and show new characteristics of S. sclerotiorum glucanase.

Expression of an extremely acidic beta-1,4-glucanase from thermoacidophilic Alicyclobacillus sp. A4 in Pichia pastoris is improved by truncating the gene sequence.

BACKGROUND: Alicyclobacillus sp. A4 is thermoacidophilic and produces many glycoside hydrolases. An extremely acidic beta-1,4-glucanase (CelA4) has been isolated from Alicyclobacillus sp. A4 and purified. This glucanase with a molecular mass of 48.6 kDa decreases the viscosity of barley-soybean feed under simulated gastric conditions. Therefore, it has the potential to improve the nutrient bioavailability of pig feed. For the study reported herein, the full-length gene, CelA4, of this glucanase (CelA4) was identified using the sequences of six peptides and cloned from strain A4. The gene fragment (CelA4F) encoding the mature protein was expressed in Pichia pastoris. Sequence truncation and glycosylation were found for recombinant CelA4F, both of which affected the expression efficiency. The physical properties of various forms of CelA4 as they affected enzymatic activity were characterized. RESULTS: We located the full-length 2,148-bp gene for CelA4 (CelA4) in the genome of Alicyclobacillus sp. A4. CelA4 encodes a 715-residue polypeptide with a calculated molecular mass of 71.64 kDa, including an N-terminal signal peptide (residues 1-39), a catalytic domain (residues 39-497), and a C-terminal threonine-rich region (residues 498-715). Its deduced amino acid sequence and that of an Alicyclobacillus acidocaldarius endo-beta-1,4-glucanase were identical at 44% of the residue positions. When the experimental molecular mass of CelA4F--a recombinant protein designed to mimic the CelA4 sequence lacking the N-terminal signal peptide that had been expressed in Pichia pastoris--was compared with its hypothetical molecular mass, it was apparent that CelA4F was truncated, possibly at residue 497. An artificially truncated gene fragment (CelA4T) without C-terminal threonine-rich region was expressed in P. pastoris, and the expression efficiency of CelA4T was substantially greater than that of CelA4F. Purified CelA4F and CelA4T had similar molecular masses (~60 kDa) and enzymatic properties (optimum pH, 3.4; optimum temperature, 60 degrees C); they were relatively stable between pH 1.2 and 8.2 at 70 degrees C and resistant to acidic and neutral proteases. However, their molecular masses and thermostabilities differed from those of CelA4 isolated from Alicyclobacillus sp. A4. A deglycosylated form of CelA4 (CelA4D) had properties similar to that of CelA4 except that it was thermoliable at 60 degrees C. CONCLUSIONS: Truncation during expression of CelA4F or artificial truncation of its gene--both of which produced a form of CelA4 lacking a threonine-rich region that includes a putative linker--increased the level of enzyme produced in comparison with that produced by cultivation of Alicyclobacillus sp. A4. Glycosylation increased the thermostability of CelA4. Of the four forms of CelA4 studied, CelA4T was produced in highest yield and had the most favorable physical properties; therefore, it has potential for use in the feed industry.

[Screening, identifying of cellulose-decomposing strain L-06 and its enzyme-producing conditions].

Cellulases are relatively costly enzymes that are sold in large volumes for use in different industrial applications, and a significant reduction in cost will be important for their commercial use in biorefineries. The production of cellulase is a major factor in the hydrolysis of cellulosic materials. Hence it is essential to make the process economically viable. A strain (L-06) with high cellulase activity was screened from rice straw compost and classified as Penicillium decumbens by the analysis of its morphology and 18S rRNA gene sequences. Different conditions of liquid fermentation medium including nitrogen source, carbon source, surfactant, temperature, initial pH, inoculation quantity for the production of cellulase had been studied. The maximal beta-1, 4-glucosidase(BGL) activity was 1662 u/mL which is 1.49 times of the previous and the maximal exo-beta-1, 4-glucanases(CBH) activity was 2770 u/mL which is 1.36 times of the previous, cultured in the optimal condition for three days. And the maximal endo-beta-1, 4-glucanases (EG) activity was 18064 u/mL which is 1.87 times of the previous and the maximal filter paper enzyme(FPase) activity was 4035 u/mL which is 1.47 times of the previous, cultured in the optimal condition for four days. In the optimization experiments, the EG and CBH in the culture condition (pH10) maintained 70% and 43% activity. In the culture condition (50 degrees C) EG and CBH maintained 59% and 68% activity, which showed heat and alkali resistant characteristics.

Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus--production of extracellular enzymes and characterization of the major cellulases.

Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65% loss of dry mass within 98 days, and it produced endo-1,4-beta-glucanase (EG), endo-1,4-beta-xylanase, endo-1,4-beta-mannanase, 1,4-beta-glucosidase (BG), 1,4-beta-xylosidase, 1,4-beta-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and beta-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2.6-2.8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed beta-xylosidase and endoxylanase activities. The K(m) for carboxymethylcellulose was 3.5 g l(-1), with the highest activity at pH 3.5 and 70 degrees C. BG1 was a protein of 36 kDa with a pI around 2.6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K(m) for p-nitrophenyl-1,4-beta-glucoside was 1.8 mM, with the highest activity at pH 4 and 60 degrees C, and the enzyme was competitively inhibited by glucose (K(i)=5.8 mM). The fungus produced mainly beta-glucosidase and beta-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and beta-xylosidase were found in fungus-colonized wood.