Accelerated degradation of cellulose in silkworm excrement by the interaction of housefly larvae and cellulose-degrading bacteria.

The environmental pollution caused by silkworm (Bombyx mori) excrement is prominent, and rich in refractory cellulose is the bottleneck restricting the efficient recycling of silkworm excrement. This study was performed to investigate the effects of housefly larvae vermicomposting on the biodegradation of cellulose in silkworm excrement. After six days, a 58.90% reduction of cellulose content in treatment groups was observed, which was significantly higher than 11.5% of the control groups without housefly larvae. Three cellulose-degrading bacterial strains were isolated from silkworm excrement, which were identified as Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis based on 16S rRNA gene sequence analysis. These three bacterial stains had a high cellulose degradation index (HC value ranged to between 1.86 and 5.97 and FPase ranged from 5.07 U/mL to 7.31 U/mL). It was found that housefly larvae increased the abundance of cellulose-degrading bacterial genus (Bacillus and Pseudomonas) by regulating the external environmental conditions (temperature and pH). Carbohydrate metabolism was the bacterial communities' primary function during vermicomposting based on the PICRUSt. The results of Tax4Fun indicated that the abundance of endo-ß-1,4-glucanase and exo-ß-1,4-glucanase increased rapidly and maintained at a higher level in silkworm excrement due to the addition of housefly larvae, which contributed to the accelerated degradation of cellulose in silkworm excrement. The finding of this investigation showed that housefly larvae can significantly accelerate the degradation of cellulose in silkworm excrement by increasing the abundance of cellulose-degrading bacterial genera and cellulase.

Effects of exogenous ß-glucanase on ileal digesta soluble ß-glucan molecular weight, digestive tract characteristics, and performance of coccidiosis vaccinated broiler chickens fed hulless barley-based diets with and without medication.

INTRODUCTION: Limited use of medication in poultry feed led to the investigation of exogenous enzymes as antibiotic alternatives for controlling enteric disease. The objective of this study was to evaluate the effects of diet ß-glucanase (BGase) and medication on ß-glucan depolymerization, digestive tract characteristics, and growth performance of broilers. MATERIALS AND METHODS: Broilers were fed hulless barley (HB) based diets with BGase (Econase GT 200P from AB Vista; 0 and 0.1%) and medication (Bacitracin and Salinomycin Na; with and without) arranged as a 2 × 2 factorial. In Experiment 1, 160 broilers were housed in cages from d 0 to 28. Each treatment was assigned to 10 cages. In Experiment 2, broilers (2376) were housed in floor pens and vaccinated for coccidiosis on d 5. Each treatment was assigned to one floor pen in each of nine rooms. RESULTS: In Experiment 1, the soluble ß-glucan weighted average molecular weight (Mw) in the ileal digesta was lower with medication in the 0% BGase treatments. Peak molecular weight (Mp) and Mw were lower with BGase regardless of medication. The maximum molecular weight for the smallest 10% ß-glucan (MW-10%) was lower with BGase addition. In Experiment 2, Mp was lower with medication in 0% BGase treatments. Beta-glucanase resulted in lower Mp regardless of medication, and the degree of response was lower with medication. The MW-10% was lower with BGase despite antibiotic addition. Body weight gain and feed efficiency were higher with medication regardless of BGase use through-out the trial (except d 11-22 feed efficiency). Beta-glucanase resulted in higher body weight gain after d 11 and worsened and improved feed efficiency before and after d 11, respectively, in unmedicated treatments. CONCLUSION: BGase and medication caused the depolymerization of soluble ileal ß-glucan. Beta-glucanase acted as a partial replacement for diet medication by increasing growth performance in coccidiosis vaccinated broilers.

Identification of a cold-adapted and metal-stimulated ß-1,4-glucanase with potential use in the extraction of bioactive compounds from plants.

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

Identification of a unique 1,4-ß-D-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-ß-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl ß-D-cellobioside (ρ-NPC) and ρ-nitrophenyl ß-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth. KEY POINTS: • CHU_0961 and its homologues formed a novel group (group C) of GH9 enzymes. • CHU_0961 was identified as a 1,4-ß-d-glucan glucohydrolase with unique properties. • CHU_0961 may play an important role in the early stage of C. hutchinsonii growth.

Enzymatic Characteristics of a Highly Thermostable ß-(1-4)-Glucanase from Fervidobacterium islandicum AW-1 (KCTC 4680).

A highly thermostable ß-(1-4)-glucanase (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward ß-D-glucan from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4-nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at 90°C and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at 85°C. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.

The family 22 carbohydrate-binding module of bifunctional xylanase/ß-glucanase Xyn10E from Paenibacillus curdlanolyticus B-6 has an important role in lignocellulose degradation.

A newly isolated endo-ß-1,4-xylanase (Xyn10E) from Paenibacillus curdlanolyticus B-6 has a modular structure consisting of a family 22 carbohydrate-binding module (CBM), a glycoside hydrolase (GH) family 10 catalytic domain, two fibronectin type III (Fn3) domains, and a family 3 CBM at the C-terminus. Intact Xyn10E (rXyn10E), CBM22-deleted Xyn10E (X-CBM3), CBM3-deleted Xyn10E (X-CBM22), and GH10 catalytic domain only (X-GH10) were expressed in Escherichia coli. rXyn10E showed bifunctional degradation activity toward xylan and ß-glucan and also degraded microcrystalline cellulose. Although X-CBM3 and X-GH10 had drastically reduced xylanase and ß-glucanase activities, X-CBM22 mostly retained these activities. Similar Km values were obtained for rXyn10E and X-CBM3, but kcat and kcat/Km values for X-CBM3 and X-GH10 were lower than those for rXyn10E, suggesting that CBM22 of Xyn10E may contribute to catalytic efficiency. In binding assays, X-CBM3 was still able to bind to ß-glucan, soluble xylan, insoluble xylan, and cellulose through GH10 and CBM3. These results indicate that CBM22 has an important role not only in binding to xylan and ß-glucan but also in feeding both polysaccharides into the neighboring GH10 catalytic domain. rXyn10E showed remarkable synergism with rXyn11A, a major xylanase subunit of P. curdlanolyticus B-6, in the degradation of untreated corn stover and sugarcane bagasse; however, the combination of X-CBM3 and rXyn11A was not synergistic. These results indicate that Xyn10E and Xyn11A act synergistically on lignocellulosic biomass, and CBM22 is essential for efficient degradation of lignocellulosic materials.

Synergistic growth in bacteria depends on substrate complexity.

Both positive and negative interactions among bacteria take place in the environment. We hypothesize that the complexity of the substrate affects the way bacteria interact with greater cooperation in the presence of recalcitrant substrate. We isolated lignocellulolytic bacteria from salt marsh detritus and compared the growth, metabolic activity and enzyme production of pure cultures to those of three-species mixed cultures in lignocellulose and glucose media. Synergistic growth was common in lignocellulose medium containing carboxyl methyl cellulose, xylan and lignin but absent in glucose medium. Bacterial synergism promoted metabolic activity in synergistic mixed cultures but not the maximal growth rate (µ). Bacterial synergism also promoted the production of ß-1,4-glucosidase but not the production of cellobiohydrolase or ß-1,4-xylosidase. Our results suggest that the chemical complexity of the substrate affects the way bacteria interact. While a complex substrate such as lignocellulose promotes positive interactions and synergistic growth, a labile substrate such as glucose promotes negative interactions and competition. Synergistic interactions among indigenous bacteria are suggested to be important in promoting lignocellulose degradation in the environment.

Induced mutations in tomato SlExp1 alter cell wall metabolism and delay fruit softening.

Fruit ripening and softening are key traits for many fleshy fruit. Since cell walls play a key role in the softening process, expansins have been investigated to control fruit over ripening and deterioration. In tomato, expression of Expansin 1 gene, SlExp1, during fruit ripening was associated with fruit softening. To engineer tomato plants with long shelf life, we screened for mutant plants impaired in SlExp1 function. Characterization of two induced mutations, Slexp1-6_W211S, and Slexp1-7_Q213Stop, showed that SlExp1 loss of function leads to enhanced fruit firmness and delayed fruit ripening. Analysis of cell wall polysaccharide composition of Slexp1-7_Q213Stop mutant pointed out significant differences for uronic acid, neutral sugar and total sugar contents. Hemicelluloses chemistry analysis by endo-ß-1,4-d-glucanase hydrolysis and MALDI-TOF spectrometry revealed that xyloglucan structures were affected in the fruit pericarp of Slexp1-7_Q213Stop mutant. Altogether, these results demonstrated that SlExp1 loss of function mutants yield firmer and late ripening fruits through modification of hemicellulose structure. These SlExp1 mutants represent good tools for breeding long shelf life tomato lines with contrasted fruit texture as well as for the understanding of the cell wall polysaccharide assembly dynamics in fleshy fruits.

Production of cellulolytic enzymes by Pleurotus species on lignocellulosic wastes using novel pretreatments.

In the present investigation three species of Pleurotus i.e. P. sajor—caju (P1), P. florida (P2) and P. flabellatus (P3) along with two lignocellulosic substrates namely paddy straw and wheat straw were selected for evaluation of production of extracellular cellulolytic enzymes. During the cultivation of three species of Pleurotus under in vivo condition, the two lignocellulosic substrates were treated with plants extracts (aqueous extracts of ashoka leaves (A) and neem oil (B)), hot water (H) and chemicals (C).Among all treatments, neem oil treated substrates supported better enzyme production followed by aqueous extract of ashoka leaves, hot water and chemical treatment. Between the two substrates paddy straw supported better enzyme production than wheat straw. P. flabellatus showed maximum activity of exoglucanase, endoglucanase and β—glucosidase followed by P. florida and P. sajor—caju.

Comparison of seasonal soil microbial process in snow-covered temperate ecosystems of northern China.

More than half of the earth's terrestrial surface currently experiences seasonal snow cover and soil frost. Winter compositional and functional investigations in soil microbial community are frequently conducted in alpine tundra and boreal forest ecosystems. However, little information on winter microbial biogeochemistry is known from seasonally snow-covered temperate ecosystems. As decomposer microbes may differ in their ability/strategy to efficiently use soil organic carbon (SOC) within different phases of the year, understanding seasonal microbial process will increase our knowledge of biogeochemical cycling from the aspect of decomposition rates and corresponding nutrient dynamics. In this study, we measured soil microbial biomass, community composition and potential SOC mineralization rates in winter and summer, from six temperate ecosystems in northern China. Our results showed a clear pattern of increased microbial biomass C to nitrogen (N) ratio in most winter soils. Concurrently, a shift in soil microbial community composition occurred with higher fungal to bacterial biomass ratio and gram negative (G-) to gram positive (G+) bacterial biomass ratio in winter than in summer. Furthermore, potential SOC mineralization rate was higher in winter than in summer. Our study demonstrated a distinct transition of microbial community structure and function from winter to summer in temperate snow-covered ecosystems. Microbial N immobilization in winter may not be the major contributor for plant growth in the following spring.

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.

Specific fusion of ß-1,4-endoglucanase and ß-1,4-glucosidase enhances cellulolytic activity and helps in channeling of intermediates.

Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel ß-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a ß-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G(4)S)(3)-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for ß-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.

Cloning, characterization and molecular docking of a highly thermostable ß-1,4-glucosidase from Thermotoga petrophila.

A genomic DNA fragment, encoding a thermotolerant ß-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5 kDa protein (by SDS-PAGE) encoded by 1.341 kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-ß-D-glucopyranoside were 2.8 mM and 42.7 mmol min(-1) mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.

Structure and activity of exo-1,3/1,4-ß-glucanase from marine bacterium Pseudoalteromonas sp. BB1 showing a novel C-terminal domain.

UNLABELLED: Following the discovery of an exo-1,3/1,4-ß-glucanase (glycoside hydrolase family 3) from a seaweed-associated bacterium Pseudoalteromonas sp. BB1, the recombinant three-domain protein (ExoP) was crystallized and its structure solved to 2.3 Å resolution. The first two domains of ExoP, both of which contribute to the architecture of the active site, are similar to those of the two-domain barley homologue ß-d-glucan exohydrolase (ExoI) with a distinctive Trp-Trp clamp at the +1 subsite, although ExoI displays broader specificity towards ß-glycosidic linkages. Notably, excision of the third domain of ExoP results in an inactive enzyme. Domain 3 has a ß-sandwich structure and was shown by CD to be more temperature stable than the native enzyme. It makes relatively few contacts to domain 1 and none at all to domain 2. Two of the domain 3 residues involved at the interface, Q683 (forming one hydrogen bond) and Q676 (forming two hydrogen bonds) were mutated to alanine. Variant Q676A retained about half the activity of native ExoP, but the Q683A variant was severely attenuated. The crystal structure of Q683A-ExoP indicated that domain 3 was highly mobile and that Q683 is critical to the stabilization of ExoP by domain 3. Small-angle X-ray scattering data lent support to this proposal. Domain 3 does not appear to be an obvious carbohydrate-binding domain and is related neither in sequence nor structure to the additional domains characterized in other glycoside hydrolase 3 subgroups. Its major role appears to be for protein stability but it may also help orient substrate. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 3UT0, 3USZ, 3F95 and 3RRX.

Cloning and characterization of a novel cold-active endoglucanase establishing a new subfamily of glycosyl hydrolase family 5 from a psychrophilic deep-sea bacterium.

The gene of a novel endo-ß-1,4-glucanase (named Cel5M) was isolated from the psychrophilic deep-sea bacteria Pseudomonas sp. MM15. The deduced protein sequence lacked the typical cellulase domain structures of the carbohydrate-binding module and the linker region. Cel5M showed relatively higher activity toward carboxymethyl cellulose, but much lower activity toward p-nitrophenyl-ß-D-galactopyranoside and no activity toward avicel. Cel5M was identified as a cold-active cellulase with an optimal temperature of 30 °C and it was active within a narrow pH range with an optimum at pH 4.5. Phylogenetic analysis showed that Cel5M represented a new subfamily of the glycosyl hydrolase family 5, representing an opportunity for research into and applications of novel cold-active cellulases.

Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference.

Cellulases are pathogenic substances suspected to be responsible for the development of the early symptoms of nematode disease. The pine wood nematode, Bursaphelenchus xylophilus (Parasitaphelenchidae), is the causal agent of pine wilt disease, which kills millions of pine trees. We used RNA interference (RNAi), a reverse genetic tool, to analyze the function of the endo-ß-1,4-glucanase gene of B. xylophilus, which causes the most serious forest tree disease in China and the rest of eastern Asia. Silencing of this gene was detected through real-time PCR and cellulase activity assays after soaking for 24 h in dsRNA. The cellulase gene silencing effects differed among various siRNAs. The propagation and dispersal ability of these nematodes decreased when the endo-ß-1,4-glucanase gene was silenced. It is important to select an effective siRNA before performing an RNAi test.

Thermal denaturation of ß-glucosidase B from Paenibacillus polymyxa proceeds through a Lumry-Eyring mechanism.

ß-glucosidase B (BglB), 1,4-ß-D: -glucanohydrolase, is an enzyme with various technological applications for which some thermostable mutants have been obtained. Because BglB denatures irreversibly with heating, the stabilities of these mutants are assessed kinetically. It, therefore, becomes relevant to determine whether the measured rate constants reflect one or several elementary kinetic steps. We have analyzed the kinetics of heat denaturation of BglB from Paenibacillus polymyxa under various conditions by following the loss of secondary structure and enzymatic activity. The denaturation is accompanied by aggregation and an initial reversible step at low temperatures. At T ≥ T ( m ), the process follows a two-state irreversible mechanism for which the kinetics does not depend on the enzyme concentration. This behavior can be explained by a Lumry-Eyring model in which the difference between the rates of the irreversible and the renaturation steps increases with temperature. Accordingly, at high scan rates (≥1 °C min(-1)) or temperatures (T ≥ T ( m )), the measurable activation energy involves only the elementary step of denaturation.

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.

Sugar treatment inhibits IAA-induced expression of endo-1,3:1,4-beta-glucanase EI transcripts in barley coleoptile segments.

The degradation of 1,3:1,4-beta-glucan by glucanases is believed to be critical for auxin-induced elongation in Gramineae coleoptile. In the present study, we reinvestigated the relationship between auxin-induced elongation and gene expression of glucanases upon treatment of coleoptile segments with sugars. Gene expression of exo-beta-1,3:1,4-glucanase ExoII was not affected by treatment with IAA and/or sucrose. In contrast, levels of endo-beta-1,3:1,4-glucanase EI transcripts increased in response to IAA treatment, which was negated by the addition of glucose or sucrose, although the addition of sucrose or glucose did not suppress IAA-induced elongation. Sugar composition analysis of the hemicellulosic fraction revealed that the addition of glucose suppressed the IAA-induced reduction of beta-glucan. In the coleoptile segments that were starved by pre-incubation in water, the IAA-induced accumulation of EI mRNA was accelerated, as compared with the non-starved segments, which suggests that the level of carbon source in the cytoplasm regulates EI expression. Moreover, in the basal region of coleoptiles, where IAA treatment does not induce elongation growth, high levels of EI transcripts were observed in the presence and absence of IAA treatment. These results strongly demonstrated that the expressions of exo- and endo-beta-glucanase genes are not directly involved in the IAA-induced loosening of cell walls associated with elongation and also suggests that cell walls may degrade 1,3:1,4-beta-glucan to provide glucose as an energy source for cell elongation.

Fluorescence study on interactions of alpha-crystallin with the molten globule state of 1, 4-beta-D-glucan glucanohydrolase from Thermomonospora sp. induced by guanidine hydrochloride.

In this paper, the interaction between alpha- crystallin and molten globule structure of 1,4-beta-D-Glucan Glucohydrolase (TSC) from an alkalothermophilic Thermomonospora sp. was investigated mainly by fluorescence quenching spectra, circular dichroism and three dimensional fluorescence spectra under simulative physiological conditions. Denaturation studies using GdnCl indicated that TSC folds through a partially folded state that resembles molten globule at 1.8 M GdnCl. The chaperone activity of alpha- crystallin was employed to study refolding of TSC. Here we studied the refolding of GdnCl denatured TSC from its molten globule state (TSC-m complex) in the presence and absence of alpha-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and ANS binding studies, suggest that alpha-crystallin formed a complex with a putative intermediate molten globule-like intermediate in the refolding pathway of TSC. Reconstitution of the active TSC was observed on cooling the alpha-crystallin * TSC-m complex to 4 degrees C. Addition of alpha-crystallin to the molten globule-like intermediate of TSC (TSC-m complex) complex initiated the refolding of TSC with 69% recovery of the biological activity of the enzyme.