Expression and biochemical characterization of a novel thermostable alkaline ß-1,3-1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1.

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

Transcriptomic analysis provides an insight into the function of CmGH9B3, a key gene of ß-1, 4-glucanase, during the graft union healing of oriental melon scion grafted onto squash rootstock.

The melon (Cucumis melo L.) is a globally cherished and economically significant crop. The grafting technique has been widely used in the vegetative propagation of melon to promote environmental tolerance and disease resistance. However, mechanisms governing graft healing and potential incompatibilities in melons following the grafting process remain unknown. To uncover the molecular mechanism of healing of grafted melon seedlings, melon wild type (Control) and TRV-CmGH9B3 lines were obtained and grafted onto the squash rootstocks (C. moschata). Anatomical differences indicated that the healing process of the TRV-CmGH9B3 plants was slower than that of the control. A total of 335 significantly differentially expressed genes (DEGs) were detected between two transcriptomes. Most of these DEGs were down-regulated in TRV-CmGH9B3 grafted seedlings. GO and KEGG analysis showed that many metabolic, physiological, and hormonal responses were involved in graft healing, including metabolic processes, plant hormone signaling, plant MAPK pathway, and sucrose starch pathway. During the healing process of TRV-CmGH9B3 grafted seedlings, gene synthesis related to hormone signal transduction (auxin, cytokinin, gibberellin, brassinolide) was delayed. At the same time, it was found that most of the DEGs related to the sucrose pathway were down-regulated in TRV-CmGH9B3 grafted seedlings. The results showed that sugar was also involved in the healing process of melon grafted onto squash. These results deepened our understanding of the molecular mechanism of GH9B3, a key gene of ß-1, 4-glucanase. It also provided a reference for elucidating the gene mechanism and function analysis of CmGH9B3 in the process of graft union healing.

Expression and characterization of ß-1,3-1,4-glucanase of Aspergillus usamii in Escherichia coli and its application in sourdough bread making.

A ß-1,3-1,4-glucanase gene (Auglu12A) from Aspergillus usamii was successfully expressed in Escherichia coli BL21(DE3). The recombinant enzyme, reAuglu12A was efficiently purified using the one-step nickel-nitrilotriacetic acid affinity chromatography. The specific activity of reAuglu12A was 694.8 U/mg, with an optimal temperature of 55°C and pH of 5.0. The reAuglu12A exhibited stability at temperatures up to 60°C and within the pH range of 4.0-5.5. The reAuglu12A hydrolytic activity was increased in the presence of metal ions, especially K+ and Na+ , whereas it exhibited a Km and Vmax of 8.35 mg/mL and 1254.02 µmol/min/mg, respectively, toward barley ß-glucan at pH 5.0 and 55°C. The addition of reAuglu12A significantly increased the specific volume (p < 0.05) and reduced crumb firmness and chewiness (p < 0.05) of wheat-barley sourdough bread during a 7-day storage period compared to the control. Overall, the quality of wheat-barley sourdough bread was improved after incorporation of reAuglu12A (especially at 3000 U/300 g). These changes were attributed to the synergistic effect of acidification by sourdough and its metabolites which provided a conducive environment for the optimal action of reAuglu12A in the degradation of ß-glucans of barley flour in sourdough. This stabilized the dough structure, thereby enhancing the quality, texture, and shelf life of the bread. These findings suggest that reAuglu12A holds promise as a candidate for ß-glucanase application in the baking industry.

Potential of insect endogenous cellulases for lignocellulosic break down deciphered using molecular docking studies.

Insects possess cellulolytic system capable of producing variegate enzymes with multifarious specificities to break down complex lignocellulosic products. Astonishingly, endoglucanases, exoglucanases and ß-glycosidases act sequentially in a synergistic system to facilitate the breakdown of cellulose to utilisable energy source glucose. In silico docking studies of endo-ß-1,4-glucanase from 19 different insects belonging to six different orders identified that it possesses high affinity for all the six substrates, including CMC, cellulose, cellotriose, cellotetraose, cellopentose and cellohexaose. Additionally, ß-glucosidase from nearly all the reported insect sources also showed considerable affinity towards cellobiose. Van der Waals, conventional hydrogen bonds and carbon-hydrogen bonds stabilise the interaction between the enzyme and different substrates. Molecular dynamics simulations also held up the stability of various complexes. Efficient breakdown of lignocelluloses-based substrates becoming a major focus of industrial and academic communities worldwide, this study can perhaps complement the propensity of insect cellulases for prospected applications.

Safety evaluation of the food enzyme cellulase from the non-genetically modified Aspergillus niger strain 294.

The food enzyme cellulase (4-(1,3;1,4)-ß-d-glucan-4-glucanohydrolase; EC 3.1.2.4) is produced with the non-genetically modified Aspergillus niger strain 294 by Kerry Ingredients & Flavours Ltd. The food enzyme is considered free from viable cells of the production organism. The enzyme is intended to be used in eight food manufacturing processes: baking processes, cereal-based processes, brewing processes, grain treatment for the production of starch and gluten fractions, fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juices, distilled alcohol production and wine and wine vinegar production. Since residual amounts of total organic solids (TOS) are removed during distilled alcohol production and grain treatment for the production of starch and gluten fractions, dietary exposure was only calculated for the remaining six food manufacturing processes. It was estimated to be up to 5.706 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 794 mg TOS/kg bw per day, the highest dose tested. The calculated margin of exposure for each age group was 184 (infants), 146 (toddlers), 139 (children), 219 (adolescents), 305 (adults) and 441 (the elderly). A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and four matches were found. The Panel considered that, under the intended conditions of use (other than distilled alcohol production), the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use for adolescents, adults and the elderly.

Biochemical Characterization of Novel GH6 Endoglucanase from Myxococcus sp. B6-1 and Its Effects on Agricultural Straws Saccharification.

Cellulase has been widely used in many industrial fields, such as feed and food industry, because it can hydrolyze cellulose to oligosaccharides with a lower degree of polymerization. Endo-ß-1,4-glucanase is a critical speed-limiting cellulase in the saccharification process. In this study, endo-ß-1,4-glucanase gene (CelA257) from Myxococcus sp. B6-1 was cloned and expressed in Escherichia coli. CelA257 contained carbohydrate-binding module (CBM) 4-9 and glycosyl hydrolase (GH) family 6 domain that shares 54.7% identity with endoglucanase from Streptomyces halstedii. The recombinant enzyme exhibited optimal activity at pH 6.5 and 50 °C and was stable over a broad pH (6-9.5) range and temperature < 50 °C. CelA257 exhibited broad substrate specificity to barley ß-glucan, lichenin, CMC, chitosan, laminarin, avicel, and phosphoric acid swollen cellulose (PASC). CelA257 degraded both cellotetrose (G4) and cellppentaose (G5) to cellobiose (G2) and cellotriose (G3). Adding CelA257 increased the release of reducing sugars in crop straw powers, including wheat straw (0.18 mg/mL), rape straw (0.42 mg/mL), rice straw (0.16 mg/mL), peanut straw (0.16 mg/mL), and corn straw (0.61 mg/mL). This study provides a potential additive in biomass saccharification applications.

Evolution of a Cockroach Allergen into the Major Protein of Termite Royal Jelly.

Termites live in colonies, and their members belong to different castes that each have their specific role within the termite society. In well-established colonies of higher termites, the only food the founding female, the queen, receives is saliva from workers; such queens can live for many years and produce up to 10,000 eggs per day. In higher termites, worker saliva must thus constitute a complete diet and therein resembles royal jelly produced by the hypopharyngeal glands of honeybee workers that serves as food for their queens; indeed, it might as well be called termite royal jelly. However, whereas the composition of honeybee royal jelly is well established, that of worker termite saliva in higher termites remains largely unknown. In lower termites, cellulose-digesting enzymes constitute the major proteins in worker saliva, but these enzymes are absent in higher termites. Others identified a partial protein sequence of the major saliva protein of a higher termite and identified it as a homolog of a cockroach allergen. Publicly available genome and transcriptome sequences from termites make it possible to study this protein in more detail. The gene coding the termite ortholog was duplicated, and the new paralog was preferentially expressed in the salivary gland. The amino acid sequence of the original allergen lacks the essential amino acids methionine, cysteine and tryptophan, but the salivary paralog incorporated these amino acids, thus allowing it to become more nutritionally balanced. The gene is found in both lower and higher termites, but it is in the latter that the salivary paralog gene got reamplified, facilitating an even higher expression of the allergen. This protein is not expressed in soldiers, and, like the major royal jelly proteins in honeybees, it is expressed in young but not old workers.

Genome-Wide Characteristics of GH9B Family Members in Melon and Their Expression Profiles under Exogenous Hormone and Far-Red Light Treatment during the Grafting Healing Process.

ß-1,4-glucanase can not only promote the wound healing of grafted seedlings but can also have a positive effect on a plant's cell wall construction. As a critical gene of ß-1,4-glucanase, GH9B is involved in cell wall remodeling and intercellular adhesion and plays a vital role in grafting healing. However, the GH9B family members have not yet been characterized for melons. In this study, 18 CmGH9Bs were identified from the melon genome, and these CmGH9Bs were located on 15 chromosomes. Our phylogenetic analysis of these CmGH9B genes and GH9B genes from other species divided them into three clusters. The gene structure and conserved functional domains of CmGH9Bs in different populations differed significantly. However, CmGH9Bs responded to cis elements such as low temperature, exogenous hormones, drought, and injury induction. The expression profiles of CmGH9Bs were different. During the graft healing process of the melon scion grafted onto the squash rootstock, both exogenous naphthyl acetic acid (NAA) and far-red light treatment significantly induced the upregulated expression of CmGH9B14 related to the graft healing process. The results provided a technical possibility for managing the graft healing of melon grafted onto squash by regulating CmGH9B14 expression.

Asn57 N-glycosylation promotes the degradation of hemicellulose by ß-1,3-1,4-glucanase from Rhizopus homothallicus.

N-glycosylation alters the properties of different enzymes in different ways. Rhizopus homothallicus was first described as an environmental isolate from desert soil in Guatemala. A new gene encoding glucanase RhGlu16B was identified in R. homothallicus. It had high specific activity (9673 U/mg) when barley glucan was used as a substrate, and ß-glucan is hemicellulose that is abundant in nature. RhGlu16B has only one N-glycosylation site in its Ala55-Gly64 loop. It was found that N-glycosylation increased its Tm value and catalytic efficiency by 5.1 °C and 59%, respectively. Adding N-glycosylation to the same region of GH16 family glucanases TlGlu16A (from Talaromyces leycettanus) increased its thermostability and catalytic efficiency by 6.4 °C and 38%, respectively. In a verification experiment using GH16 family glucanases BisGlu16B (from Bisporus) in which N-glycosylation was removed, N-glycosylation also appeared to promote thermostability and catalytic efficiency. N-glycosylation reduced the overall root mean square deviation of the enzyme structure, creating rigidity and increasing overall thermostability. This study provided a reference for the molecular modification of GH16 family glucanases and guided the utilization of ß-glucan in hemicellulose.

More efficient barley malting under catalyst: Thermostability improvement of a ß-1,3-1,4-glucanase through surface charge engineering with higher activity.

ß-1,3-1,4-Glucanase is an indispensable biocatalyst in barley brewing industry for its crucial effect in reducing the viscosity of mash. However, the unsatisfactory thermostability greatly limited its application performance. In this study, structure-based surface charge engineering was conducted aiming at thermostability improvement of BisGlu16B, a highly active ß-1,3-1,4-glucanase from Bispora sp. MEY-1. By applying the enzyme thermal stability system (ETSS), residues D47, D213, and D253 were inferred to be critical sites for thermal properties. Single (D47A, D213A, and D253A) and combination (D47A/D213A/D253A) mutants were generated and compared with BisGlu16B. Among all improved mutants, D47A/D213A/D253A outstanded in thermostability. In comparison with BisGlu16B, its T50 and Tm were respectively increased by 7.0 °C and 4.3 °C, while the t1/2 at 70 °C was 8.1 times that of the wild type. Furthermore, the catalytic activity of D47A/D213A/D253A also increased by 42.5%, compared with BisGlu16B (42,900 ± 300 U/mg vs. 30,100 ± 800 U/mg). Comparing with BisGlu16B and commercial enzyme treatment groups, under simulated malting conditions, efficiency improvement was observed in decreasement of viscosity (35.5% and 90.7%) and filtration time (30.9% and 34.6%) for D47A/D213A/D253A treatment group. Molecular dynamics simulation showed that mutation sites A47, A213, and A253 increased the protein rigidity by lowering the overall root mean square deviation (RMSD). This study may bring optimization of technology and improvement of producing efficiency to the present brewing industry.

Novel, acidic, and cold-adapted glycoside hydrolase family 8 endo-ß-1,4-glucanase from an Antarctic lichen-associated bacterium, Lichenicola cladoniae PAMC 26568.

Endo-ß-1,4-glucanase is a crucial glycoside hydrolase (GH) involved in the decomposition of cellulosic materials. In this study, to discover a novel cold-adapted ß-1,4-D-glucan-degrading enzyme, the gene coding for an extracellular endo-ß-1,4-glucanase (GluL) from Lichenicola cladoniae PAMC 26568, an Antarctic lichen (Cladonia borealis)-associated bacterium, was identified and recombinantly expressed in Escherichia coli BL21. The GluL gene (1044-bp) encoded a non-modular polypeptide consisting of a single catalytic GH8 domain, which shared the highest sequence identity of 55% with that of an uncharacterized protein from Gluconacetobacter takamatsuzukensis (WP_182950054). The recombinant endo-ß-1,4-glucanase (rGluL: 38.0 kDa) most efficiently degraded sodium carboxymethylcellulose (CMC) at pH 4.0 and 45°C, and showed approximately 23% of its maximum degradation activity even at 3°C. The biocatalytic activity of rGluL was noticeably enhanced by >1.3-fold in the presence of 1 mM Mn2+ or NaCl at concentrations between 0.1 and 0.5 M, whereas the enzyme was considerably downregulated by 1 mM Hg2+ and Fe2+ together with 5 mM N-bromosuccinimide and 0.5% sodium dodecyl sulfate. rGluL is a true endo-ß-1,4-glucanase, which could preferentially decompose D-cellooligosaccharides consisting of 3 to 6 D-glucose, CMC, and barley ß-glucan, without other additional glycoside hydrolase activities. The specific activity (15.1 U mg-1) and k cat/K m value (6.35 mg-1 s-1mL) of rGluL toward barley ß-glucan were approximately 1.8- and 2.2-fold higher, respectively, compared to its specific activity (8.3 U mg-1) and k cat/K m value (2.83 mg-1 s-1mL) toward CMC. The enzymatic hydrolysis of CMC, D-cellotetraose, and D-cellohexaose yielded primarily D-cellobiose, accompanied by D-glucose, D-cellotriose, and D-cellotetraose. However, the cleavage of D-cellopentaose by rGluL resulted in the production of only D-cellobiose and D-cellotriose. The findings of the present study imply that rGluL is a novel, acidic, and cold-adapted GH8 endo-ß-1,4-glucanase with high specific activity, which can be exploited as a promising candidate in low-temperature processes including textile and food processes.

Safety evaluation of the food enzyme cellulase from the genetically modified Trichoderma reesei strain AR-852.

The food enzyme cellulase (4-(1,3;1,4)-ß-d-glucan 4-glucanohydrolase; EC 3.2.1.4) is produced with the genetically modified Trichoderma reesei strain AR-852 by AB Enzymes GmbH. The genetic modifications did not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in five food manufacturing processes: baking processes, brewing processes, distilled alcohol production, wine and wine vinegar production, and fruit and vegetable processing for juice production. As residual amounts of total organic solids (TOS) are removed by distillation, dietary exposure was only calculated for the other four food processes. Dietary exposure to the food enzyme TOS was estimated to be up to 0.1 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 1,000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 10,000. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use (other than distilled alcohol production) the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Structural snapshot of a glycoside hydrolase family 8 endo-ß-1,4-glucanase capturing the state after cleavage of the scissile bond.

Bacterial cellulose (BC), which is produced by bacteria, is a biodegradable and biocompatible natural resource. Because of its remarkable physicochemical properties, BC has attracted attention for the development and manufacture of biomedical and industrial materials. In the BC production system, the enzyme endo-ß-1,4-glucanase, which belongs to glycoside hydrolase family 8 (GH8), acts as a cleaner by trimming disordered cellulose fibers to produce high-quality BC. Understanding the molecular mechanism of the endo-ß-1,4-glucanase would help in developing a reasonable biosynthesis of BC. Nevertheless, all of the steps in the reaction of this endo-ß-1,4-glucanase are not clear. This study confirms the BC hydrolytic activity of the endo-ß-1,4-glucanase from the BC-producing bacterium Enterobacter sp. CJF-002 (EbBcsZ) and reports crystal structures of EbBcsZ. Unlike in previously reported GH8 endo-ß-1,4-glucanase structures, here the base catalyst was mutated (D242A) and the structure of this mutant bound to cellooligosaccharide [EbBcsZ(D242A)CPT] was analyzed. The EbBcsZ(D242A)CPT structure showed two cellooligosaccharides individually bound to the plus and minus subsites of EbBcsZ. The glucosyl unit in subsite -1 presented a distorted 5S1 conformation, a novel snapshot of a state immediately after scissile-bond cleavage. In combination with previous studies, the reaction process of endo-ß-1,4-glucanase is described and the ß-1,4-glucan-trimming mechanism of EbBcsZ is proposed. The EbBcsZ(D242A)CPT structure also showed an additional ß-1,4-glucan binding site on the EbBcsZ surface, which may help to accept the substrate.

Interfamily grafting capacity of petunia.

In grafting, an agricultural technique for propagating flower species and fruit trees, two plants are combined to exploit their beneficial characteristics, such as rootstock disease tolerance and vigor. Grafting incompatibility has been observed, however, between distantly related plant combinations, which limits the availability of plant resources. A high grafting capacity has been found in Nicotiana, belonging to Solanaceae, but not in Ipomoea nil, a Convolvulaceae species. Here, we found that Petunia hybrida, another solanaceous species, has similar ability of interfamily grafting, which indicates that interfamily grafting capability in Solanaceae is not limited to the genus Nicotiana. RNA sequencing-based comparative time-series transcriptomic analyses of Nicotiana benthamiana, I. nil, and P. hybrida revealed that N. benthamiana and P. hybrida share a common gene expression pattern, with continued elevated expression of the ß-1,4-glucanase subclade gene GH9B3 observed after interfamily grafting. During self-grafting, GH9B3 expression in each species was similarly elevated, thus suggesting that solanaceous plants have altered regulatory mechanisms for GH9B3 gene expression that allow tissue fusion even with other species. Finally, we tested the effect of the ß-1,4-glucanase inhibitor D-glucono-1,5-lactone, using glucose as a control, on the interfamily grafting usability of P. hybrida with Arabidopsis rootstock. Strong inhibition of graft establishment was observed only with D-glucono-1,5-lactone, thus suggesting the important role of GH9B3 in P. hybrida grafting. The newly discovered grafting compatibility of Petunia with different families enhances the propagation techniques and the production of flower plants.

Identification and characterization of a novel endo-ß-1,4-glucanase from a soil metagenomic library.

A cosmid clone cZFYN1413 with CMCase activity was identified from a soil metagenomic library. The sequence analysis of a subclone of cZFYN1413 revealed an endo-ß-1,4-glucanase gene ZFYN1413 belonging to glycoside hydrolase family 6 and a transmembrane region in the N-terminal of ZFYN1413. Expression of ZFYN1413 in Escherichia coli BL21 (DE3) resulted in ZFYN1413-87, which was a truncated protein cleaved in transmembrane region of ZFYN1413. ZFYN1413-87 was expressed and its enzyme properties were studied. ZFYN1413-87 possessed strong endo-ß-1,4-glucanase activity, and 52% of the activity could be retained after the protein was treated in buffer of pH 3.0 for 2 h. The study provided a special example of endo-ß-1,4-glucanase in GH6 family.

Enhanced hydrolytic efficiency of an engineered CBM11-glucanase enzyme chimera against barley ß-d-glucan extracts.

The ß-d-glucans are abundant cell wall polysaccharides in many cereals and contain both (1,3)- and (1,4)-bonds. The ß-1,3-1,4-glucanases (EC 3.2.1.73) hydrolyze ß-(1,4)-d-glucosidic linkages in glucans, and have applications in both animal and human food industries. A chimera between the family 11 carbohydrate-binding module from Ruminoclostridium (Clostridium)thermocellumcelH (RtCBM11), with the ß-1,3-1,4-glucanase from Bacillus subtilis (BglS) was constructed by end-to-end fusion (RtCBM11-BglS) to evaluate the effects on the catalytic function and its application in barley ß-glucan degradation for the brewing industry. The parental and chimeric BglS presented the same optimum pH (6.0) and temperature (50 °C) for maximum activity. The RtCBM11-BglS showed increased thermal stability and 30% higher hydrolytic efficiency against purified barley ß-glucan, and the rate of hydrolysis of ß-1,3-1,4-glucan in crude barley extracts was significantly increased. The enhanced catalytic performance of the RtCBM11-BglS may be useful for the treatment of crude barley extracts in the brewing industry.

An Aspergillus nidulans endo-ß-1,3-glucanase exhibited specific catalytic features and was used to prepare 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose with high antioxidant activity from barley ß-glucan and laminarin, respectively.

An endo-ß-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of ß-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-ß-gentiobiosyl-d-glucose and hydrolyzed barley ß-glucan to mainly produce 3-O-ß-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley ß-glucan and E. bicyclis laminarin. Further study reveals that 3-O-ß-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley ß-glucan and 3-O-ß-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-ß-cellobiosyl-d-glucose and 3-O-ß-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that ß-1,4-linkage or -1,6-linkage at non-reducing end of ß-glucotrioses had enhancing effect on antioxidant activity compared to ß-1,3-linkage. Furthermore, 3-O-ß-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley ß-glucan, indicating that ß-1,4-linkage at reducing end of ß-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to ß-1,3-linkage.

Structural and biochemical insights into the substrate-binding mechanism of a glycoside hydrolase family 12 ß-1,3-1,4-glucanase from Chaetomium sp.

ß-1,3-1,4-Glucanases are a type of hydrolytic enzymes capable of catalyzing the strict cleavage of ß-1,4 glycosidic bonds adjacent to ß-1,3 linkages in ß-D-glucans and have exhibited great potential in food and feed industrials. In this study, a novel glycoside hydrolase (GH) family 12 ß-1,3-1,4-glucanase (CtGlu12A) from the thermophilic fungus Chaetomium sp. CQ31 was identified and biochemically characterized. CtGlu12A was most active at pH 7.5 and 65 °C, respectively, and exhibited a high specific activity of 999.9 U mg-1 towards lichenin. It maintained more than 80% of its initial activity in a wide pH range of 5.0-11.0, and up to 60 °C after incubation at 55 °C for 60 min. Moreover, the crystal structures of CtGlu12A with gentiobiose and tetrasccharide were resolved. CtGlu12A had a ß-jellyroll fold, and performed retaining mechanism with two glutamic acids severing as the catalytic residues. In the complex structure, cellobiose molecule showed two binding modes, occupying subsites -2 to -1 and subsites + 1 to + 2, respectively. The concave cleft made mixed ß-1,3-1,4-glucan substrates maintain a bent conformation to fit into the active site. Overall, this study is not only helpful for the understanding of the substrate-binding model and catalytic mechanism of GH 12 ß-1,3-1,4-glucanases, but also provides a basis for further enzymatic engineering of ß-1,3-1,4-glucanases.

Molecular cloning and characterization of a thermostable and halotolerant endo-ß-1,4-glucanase from Microbulbifer sp. ALW1.

The bacterium Microbulbifer sp. ALW1 was previously characterized with the capability to break down the cell wall of brown algae into fine pieces. The biological functions of strain ALW1 were yet to be elucidated. In this study, a gene, namely MaCel5A, was isolated from the ALW1 strain genome, encoding an endo-ß-1,4-glucanase. MaCel5A was phylogenetically categorized under the glycoside hydrolase family GH5, with the highest identity to a putative cellulase of Microbulbifer thermotolerans. The recombinant MaCel5A protein purified from heterologous expression in E. coli exhibited maximum activity at 50 °C and pH 6.0, respectively, and functioned selectively toward carboxymethyl cellulose and barley ß-glucan. Recombinant MaCel5A demonstrated considerable tolerance to the exposure to high temperature up to 80 °C for 30 min retaining 49% residual activity. In addition, MaCel5A showed moderate stability against pH 5.0-11.0 and strong stability in the presence of nonionic surfactant. MaCel5A exhibited strong halo-stability and halotolerance. The activity of the enzyme increased about tenfold at 0.5 M NaCl, and about fivefold even at 4.0 M NaCl compared to the enzyme activity without the addition of salt. The two conserved glutamic acid residues in MaCel5A featured the typical catalytic acid/base and nucleophile machinery of glycoside hydrolases. These characteristics highlight the industrial application potential of MaCel5A.

High cellulolytic potential of the Ktedonobacteria lineage revealed by genome-wide analysis of CAZymes.

Traditionally, filamentous fungi and actinomycetes are well-known cellulolytic microorganisms that have been utilized in the commercial production of cellulase enzyme cocktails for industrial-scale degradation of plant biomass. Noticeably, the Ktedonobacteria lineage (phylum Chloroflexi) with actinomycetes-like morphology was identified and exhibited diverse carbohydrate utilization or degradation abilities. In this study, we performed genome-wide profiling of carbohydrate-active enzymes (CAZymes) in the filamentous Ktedonobacteria lineage. Numerous CAZymes (153-290 CAZymes, representing 63-131 glycoside hydrolases (GHs) per genome), including complex mixtures of endo- and exo-cellulases, were predicted in 15 available Ktedonobacteria genomes. Of note, 4-28 CAZymes were predicted to be extracellular enzymes, whereas 3-29 CAZymes were appended with carbohydrate-binding modules (CBMs) that may promote their binding to insoluble carbohydrate substrates. This number far exceeded other Chloroflexi lineages and were comparable to the cellulolytic actinomycetes. Six multi-modular extracellular GHs were cloned from the thermophilic Thermosporothrix hazakensis SK20-1T strain and heterologously expressed. The putative endo-glucanases of ThazG5-1, ThazG9, and ThazG12 exhibited strong cellulolytic activity, whereas the putative exo-glucanases ThazG6 and ThazG48 formed weak but observable halos on carboxymethyl cellulose plates, indicating their potential biotechnological application. The purified recombinant ThazG12 had near-neutral pH (optimal 6.0), high thermostability (60°C), and broad specificity against soluble and insoluble polysaccharide substrates. It also represented described a novel thermostable bacterial ß-1,4-glucanase in the GH12 family. Together, this research revealed the underestimated cellulolytic potential of the Ktedonobacteria lineage and highlighted its potential biotechnological utility as a promising microbial resource for the discovery of industrially useful cellulases.