Cellulose-binding Domain Research Articles

Filter paper disks as a matrix for manipulation of recombinant proteins

Filter paper provides an excellent matrix for retention of proteins containing a cellulose binding domain. To use this capability for manipulating recombinant fusion proteins, binding and elution parameters were explored and procedures developed for small scale purification, modification and assay. Proteins were tagged with the cellulose binding domain from the Clostridium thermocellum CipB gene via a cleavable linker. Filter paper disks of 6 mm diameter were able to bind up to 80 μg protein although there was a substantial dependence on molecular size. Different means of introducing fusion proteins to the disks allow either binding within 20 min from microliter volumes or slower binding from milliliter volumes. Elution with protease in small volumes yielded greater than 10 μg amounts with concentrations in the 1–2 mg/ml range. To demonstrate their utility, disks were used for small scale protein purification, covalent modification of protein, immunoprecipitation, and in a binding assay. These versatile methods allow parallel processing of multiple samples and may find many uses when only small amounts of protein are needed.

Cellulose nanofibril-stabilized Pickering emulsion as a high-performance interfacial biocatalysis system for the synthesis of phosphatidylserine

An efficient and attractive platform for synthesizing phosphatidylserine is indispensable for driving large-scale industrial applications. Given the interfacial effect of phospholipase D (PLD), an adequate oil-water interface surface is essential for the catalytic efficiency of PLD. Herein, a simple ball-milling method with ionic liquid was successfully applied to produce cellulose nanofibrils and immobilized PLD with the cellulose-binding domain through a one-step procedure. Each droplet in the Pickering reaction system was regarded as a microreactor to provide sufficient oil-water interface to the active sites of PLD. The constructed Pickering emulsion system exhibited outstanding catalytic activity, achieving 95.4 % conversion after 2 h with the minimal amount of catalyst (0.8 wt% of CNF-CBD1 addition amount), which was 6-fold higher than the previously reported immobilized MCC-CBD1 and 21-fold higher than free PLD-CBD1. These results demonstrated a sustainable and high-performance biocatalysis method, paving the way for the efficient synthesis of novel phospholipids.

Production and Functional Characterization of a Novel Mannanase from Alteromonadaceae Bacterium Bs31.

Mannans are the main components of hemicellulose in nature and serve as the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their functional characteristics are an important basis for mannan biotechnological applications. In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. Amino acid sequence analysis and protein structural modeling were used to reveal the molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the enzymes were determined by reducing sugar assay and thin-layer chromatography. Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides, and no monosaccharide was generated. Structural analysis suggested that ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases. ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

504 “Chimeric sorting” as an effective tool for selection of genetically modified dermal fibroblasts to be used in fundamental or applied skin research

A technology has been developed for efficient isolation of a stable pool of HIF1a overexpressing mouse fibroblasts using a high-throughput selection of cell clones on a cellulose carrier. The pHIF1a-CBD-PDGFR genetic construct has been generated containing a EYFP reporter, a target sequence of HIF1a gene (a key factor of adaptation to hypoxia in wound healing and regeneration) and a CBD/PDGFR chimeric gene construct. The presence of CBD (cellulose-binding domain) / PDGFR (platelet-derived growth factor receptor) chimeric construct in protein-producing cells enables their specific binding to cellulose carrier thus providing a possibility for cell selection based on their adhesive phenotype (“chimeric sorting”). For genetic modification, a line of immortalized postnatal mouse dermal fibroblasts was used (dp3T3cell line). This cell line has been developed using 3T3 protocol and combines the basic features of primary dermal fibroblasts with the experimental advantages of immortal cells. The high-throughput selection of HIF1a overexpressing cells was performed using BD FACSAria™ Fusion Cell Sorter (with EYFP as a reporter) or by chimeric sorting on day 5th after dp3T3 cell transfection (electroporation) using half of transfected cells for each approach (0.9±0.2x10ˆ6). The CBD-PDGFR driven chimeric sorting was performed on cellulose filter papers, 75g/cm2. In 3 weeks chimeric sorting allowed to obtain 3±1x10ˆ6 stably transfected cells with changed morphology (less stellate and more elongate), while FACS sorting yielded 8 colonies with 0.6±0.3x10ˆ5 cells each with only 3 colonies exhibiting modified phenotype. Thus, “chimeric sorting” of transfected fibroblasts on a cellulose carrier appeared to be more effective in selection of HIF1a overexpressing cells as compared to conventional FACS. The advantageous efficiency of this approach was reflected in both its specificity (low level of contamination with cells of original phenotype) and its productivity (higher yield of stably transfected cells).

Self-cleaved expression of recombinant lysostaphin from its cellulose binding domain fusion.

Mature lysostaphin (mLst) is a glycineglycine endopeptidase, capable of specifically cleaving penta-glycine crosslinker in the peptidoglycan of Staphylococcus aureus cell wall. It is a very effective therapeutic enzyme to kill the multidrug-resistant S. aureus often encountered in hospital acquired infections. Fusing cellulose binding domain (CBD) to mLst significantly reduced the insoluble expression of mLst in E. coli. Employing mLst-cleavable peptides as fusion linkers leaded to an effective self-cleavage expression that CBD and mLst could be completely cleaved off from the fusions during the expression process. The presence of residue linker fragment at N-terminus of the cleaved-off mLst strongly inhibited the cell lytic activity of the recovered recombinant mLst, and only ~ 50% of the wild-type mLst activity could be retained. Intact CBD-Lst fusions were obtained when uncleavable peptide linkers were employed. With CBD at N-terminus of mLst, the intact fusion completely lost its cell lytic activity but the dipeptidase activity still remained. In contrast, approximately 10% cell lytic activity of mLst still could be maintained for the fusion with CBD at C-terminus of mLst. KEY POINTS: • CBD fusion enhanced soluble expression of recombinant lysostaphin. • In vivo self-cleavage of fusion linkers by the expressed lysostaphin fusions. • Self-cleaved lysostaphin fusions retain most of dipeptidase but lose 50% cell lytic activity.

Improvement of saccharification of native grass, Pennisetum sp. using cellulase from isolated Aspergillus fumigatus for bioethanol production: an insight into in silico molecular modelling, docking and dynamics studies

Lignocellulosic grass biomass is potential substrate for economical and sustainable bioethanol production. However, the processing cost of bioethanol that majorly includes the hydrolysis of cellulose by cellulases is still a major concern for its industrial production. Thus, knowledge on the sequence to the structural study of cellulase enzyme with consideration of its catalytic region can give important information for effective enzyme engineering and consequently towards enhanced bioethanol production from Pennisetum sp. Therefore, in this study, sequence conservativeness of different cellulosic site among a group of endoglucanase family of cellulase from previously isolated Aspergillus species has been determined. Furthermore, comparative molecular modeling of the endoglucanase from eight different Aspergillus species including Aspergillus fumigatus was conducted and the obtained structures revealed a high degree of difference in their conformational folds. Analysis from InterProScan revealed that the modeled endoglucanase has similar types of domains and share homology with protein family, such as glycoside hydrolase family-61 and fungal cellulose binding domain. Furthermore, molecular docking and interaction studies demonstrated the presence of residues in the endoglucanase of A. fumigatus viz. His20, His88, Asp96, Ala99, Ser100, Ser101, His102, His169, Glu170, Arg173, Glu178, and Tyr218 that are responsible in forming the substrate interaction. An interesting molecular phenomenon, i.e., catalytic promiscuity has been noted for all the substrate bound complexes of A. fumigatus endoglucanase which also depicts the degree of ligand binding efficacy of the studied enzyme. The molecular interaction study, binding energy analysis and molecular dynamics simulation, demonstrated that heteromeric substrate XylGlc3 is more strongly interacting with the receptor enzyme. Overall, the present findings revealed that important amino acid residues can help in increasing the specificity of endoglucanase from A. fumigatus towards hydrolysis of Pennisetum sp. and other biomass that has an adequate amount of XylGlc3, for possible industrial applications.

Sclerotinia sclerotiorum SsCut1 Modulates Virulence and Cutinase Activity

The plant cuticle is one of the protective layers of the external surface of plant tissues. Plants use the cuticle layer to reduce water loss and resist pathogen infection. Fungi release cell wall-degrading enzymes to destroy the epidermis of plants to achieve the purpose of infection. Sclerotinia sclerotiorum secretes a large amount of cutinase to disrupt the cuticle layer of plants during the infection process. In order to further understand the role of cutinase in the pathogenic process of S. sclerotiorum, the S. sclerotiorum cutinsae 1 (SsCut1) gene was cloned and analyzed. The protein SsCut1 contains the conserved cutinase domain and a fungal cellulose-binding domain. RT-qPCR results showed that the expression of SsCut1 was significantly upregulated during infection. Split-Marker recombination was utilized for the deletion of the SsCut1 gene, ΔSsCut1 mutants showed reduced cutinase activity and virulence, but the deletion of the SsCut1 gene had no effect on the growth rate, colony morphology, oxalic acid production, infection cushion formation and sclerotial development. Complementation with the wild-type SsCut1 allele restored the cutinase activity and virulence to the wild-type level. Interestingly, expression of SsCut1 in plants can trigger defense responses, but it also enhanced plant susceptibility to SsCut1 gene knock-out mutants. Taken together, our finding demonstrated that the SsCut1 gene promotes the virulence of S. sclerotiorum by enhancing its cutinase activity.

Enhanced in situ H2O2 production explains synergy between an LPMO with a cellulose-binding domain and a single-domain LPMO

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze oxidative depolymerization of recalcitrant substrates such as chitin or cellulose. Recent work has shown that LPMOs catalyze fast peroxygenase reactions and that, under commonly used reaction set-ups, access to in situ generated H2O2 likely limits catalysis. Based on a hypothesis that the impact of a cellulose-binding module (CBM) on LPMO activity could relate to changes in in situ H2O2 production, we have assessed the interplay between CBM-containing ScLPMO10C and its truncated form comprising the catalytic domain only (ScLPMO10CTR). The results show that truncation of the linker and CBM leads to elevated H2O2 production and decreased enzyme stability. Most interestingly, combining the two enzyme forms yields strong synergistic effects, which are due to the combination of high H2O2 generation by ScLPMO10CTR and efficient productive use of H2O2 by the full-length enzyme. Thus, cellulose degradation becomes faster, while enzyme inactivation due to off-pathway reactions with excess H2O2 is reduced. These results underpin the complexity of ascorbic acid-driven LPMO reactions and reveal a potential mechanism for how LPMOs may interact synergistically during cellulose degradation.

Application of cellulosic materials as supports for single-step purification and immobilization of a recombinant β-galactosidase via cellulose-binding domain

This study aimed to develop single-step purification and immobilization processes on cellulosic supports of β-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/gsupport, expressed activity values reached 106.88 (microcrystalline cellulose), 115.03 (alkaline nanocellulose), and 108.47 IU/g (acid nanocellulose). The derivatives produced were less sensitive to the presence of galactose in comparison with the soluble purified enzyme. Among the cations assessed (Na+, K+, Mg2+, and Ca2+), magnesium provided the highest increase in the enzymatic activity of β-galactosidases immobilized on cellulosic supports. Supports and derivatives showed no cytotoxic effect on the investigated cell cultures (HepG2 and Vero). Derivatives showed high operational stability in the hydrolysis of milk lactose and retained from 53 to 64% of their hydrolysis capacity after 40 reuse cycles. This study obtained biocatalyzers with promising characteristics for application in the food industry. Biocatalyzers were obtained through a low-cost one-step sustainable bioprocess of purification and immobilization of a β-galactosidase on cellulose via CBD.

Virtual investigation of the biomass conversion efficiency of three Streptomyces species

Global worries about the gradual depletion of the non-renewable fossil fuel reserves, as well as the problem of environmental pollution, and global warming have prompted researchers to opt for more sustainable and environmentally friendly solutions. Converting biomass to bioethanol is a cost effective and inexpensive alternative to produce energy. The lytic polysaccharide monooxygenase (LPMO) enzyme of microbial origin represents an ideal solution because of their catalytic potential for the cleavage of the cellulosic biomass and the production of simple fermentable sugars. Here, we have described the 3D structures, the molecular functioning, as well as the cellulosic biomass conversion efficiency of three LPMOs belonging to three species of Streptomyces, S. albidoflavus, S. flaveus, and S. griseus. Analysis of the conserved domains of the amino acid sequences showed that the three enzymes belong to the LPMO-10 family and include an LPMO domain and a carbohydrate-binding domain. The enzyme of the species S. griseus further comprises two cellulose binding domain and cellulase/cellobiase CelA1 (Carbohydrate transport and metabolism) domain. Homology modeling of the 3D structures of the three enzymes was carried out by the SwissModel program, the models are then checked and validated. The analysis of the interactions at the active site level of the three modeled LPMOs showed the presence of conserved amino acids in all the studied enzymes, specially the His-His brace located around the copper ion. The result of the molecular docking with the cellotetraose substrate shows very close binding energies for the three LPMOs (-5.5 kcal/mol for S. griseus, and −5.3 kcal/mol for S. albidoflavus and S. flaveus) indicating the same affinities of these enzymes to the cellulosic substrates. The visualization of the interactions of cellotetraose within the catalytic sites of the enzymes showed the involvement of the amino acids His, Ala, Trp, Tyr, Phe, and Glu, which are well conserved in the two LPMOs of S. griseus and S. albidoflavus species suggesting a C1 oxidizing activity, whereas for the LPMO of S. flaveus, the Phe and Glu are replaced by Tyr and Gln, respectively suggesting a C1/C4 oxidizing AA10. The molecular information obtained during this study confirms the cellulose degradation potential of Streptomyces LPMOs, as well as their different modes of cellulose oxidation and provides strong evidence of the interest of these enzymes in the biomass conversion.

Bioengineered textiles with peptide binders that capture SARS-CoV-2 viral particles

The use of personal protective equipment (PPE), face masks and ventilation are key strategies to control the transmission of respiratory viruses. However, most PPE provides physical protection that only partially prevents the transmission of viral particles. Here, we develop textiles with integrated peptide binders that capture viral particles. We fuse peptides capable of binding the receptor domain of the spike protein on the SARS-CoV-2 capsid to the cellulose-binding domain from the Trichoderma reesei cellobiohydrolase II protein. The hybrid peptides can be attached to the cellulose fibres in cotton and capture SARS-CoV-2 viral particles with high affinity. The resulting bioengineered cotton captures 114,000 infective virus particles per cm2 and reduces onwards SARS-CoV-2 infection of cells by 500-fold. The hybrid peptides could be easily modified to capture and control the spread of other infectious pathogens or for attachment to different materials. We anticipate the use of bioengineered protective textiles in PPE, facemasks, ventilation, and furnishings will provide additional protection to the airborne or fomite transmission of viruses.

Functional characterization of a GH62 family α-L-arabinofuranosidase from Eupenicillium parvum suitable for monosaccharification of corncob arabinoxylan in combination with key enzymes

Corncob rich in arabinoxylan is an important raw material widely used in bio-refinery. Complete saccharification of arabinoxylan depends on the synergism of different enzymes including α-L-arabinofuranosidase (ABF). This study aimed to investigate the functional characteristics of a new ABF EpABF62A belonging to glycoside hydrolase (GH) 62 family from the fungus Eupenicillium parvum, and to explore its potential in the saccharification of corncob arabinoxylan. The recombinant EpABF62A showed high activity against wheat arabinoxylan and rye arabinoxylan, with the optimal temperature of 55 °C and pH of 4.5. The protein contains an N-terminal cellulose-binding domain family 1 (CBM_1) domain, and displayed a 59.5% absorption rate to phosphoric acid swollen cellulose. Regioselectivity analysis indicated that the enzyme selectively removed α-1,2 or α-1,3 linked arabinofuranosyl residues on mono-substituted xylose residues on arabinoxylan. Corncob arabinoxylans (CAX1 or CAX2) with different (low or high) branching degrees were extracted from the raw material by alkaline hydrogen peroxide pretreatment and graded ethanol precipitation. Single EpABF62A removed 69.5% or 67.1% arabinose from CAX1 or CAX2, respectively. EpABF62A combined with a GH10 xylanase, a GH43 β-D-xylosidase and a GH67 α-glucuronidase released 75.0% or 64.5% xylose from CAX1 or CAX2, respectively. The addition of the four hemicellulases enhanced the saccharification the solid fraction of the pretreated corncob by the commercial cellulase Cellic® CTec2, and the conversion ratios of glucose, xylose and arabinose were up to 94.0%, 91.8% and 82.6%, respectively.

One-step purification of a recombinant beta-galactosidase using magnetic cellulose as a support: Rapid immobilization and high thermal stability

For the first time, this work reported the one-step purification and targeted immobilization process of a β-galactosidase (Gal) with the Cellulose Binding Domain (CBD) tag, by binding it to different magnetic cellulose supports. The process efficiency after β-galactosidase-CBD immobilization on magnetic cellulose-based supports showed values of approximately 90% for all evaluated enzymatic loads. Compared with free Gal, derivatives showed affinity values between β-galactosidase and the substrate 1.2 × higher in the lactose hydrolysis of milk. β-Galactosidase-CBD's oriented immobilization process on supports increased the thermal stability of the immobilized enzyme by up to 7 × . After 15 cycles of reuse, both enzyme preparations showed a relative hydrolysis percentage of 50% of lactose in milk. The oriented immobilization process developed for purifying recombinant proteins containing the CBD tag enabled the execution of both steps simultaneously and quickly and the obtention of β-galactosidases with promising catalytic characteristics for application in the food and pharmaceutical industries.

A rapid simple point-of-care assay for the detection of SARS-CoV-2 neutralizing antibodies

BackgroundNeutralizing antibodies (NAbs) prevent pathogens from infecting host cells. Detection of SARS-CoV-2 NAbs is critical to evaluate herd immunity and monitor vaccine efficacy against SARS-CoV-2, the virus that causes COVID-19. All currently available NAb tests are lab-based and time-intensive.MethodWe develop a 10 min cellulose pull-down test to detect NAbs against SARS-CoV-2 from human plasma. The test evaluates the ability of antibodies to disrupt ACE2 receptor—RBD complex formation. The simple, portable, and rapid testing process relies on two key technologies: (i) the vertical-flow paper-based assay format and (ii) the rapid interaction of cellulose binding domain to cellulose paper.ResultsHere we show the construction of a cellulose-based vertical-flow test. The developed test gives above 80% sensitivity and specificity and up to 93% accuracy as compared to two current lab-based methods using COVID-19 convalescent plasma.ConclusionsA rapid 10 min cellulose based test has been developed for detection of NAb against SARS-CoV-2. The test demonstrates comparable performance to the lab-based tests and can be used at Point-of-Care. Importantly, the approach used for this test can be easily extended to test RBD variants or to evaluate NAbs against other pathogens.

Using recombinant adhesive proteins as durable and green flame-retardant coatings

Current fire retardants are known to be toxic to humans and our environment. As environmental-friendly flame retardants (FRs), protein-based flame retardants have been studied extensively recently, even though they are not durable. In this study, we designed, synthesized and tested a durable protein-based FR through the fusion of the adhesion domain from either mussel foot protein-5 (mfp-5) or cellulose-binding domain (CBD) with flame retardant protein (SR protein and alpha casein). We first verified the expression of the recombinant proteins in Escherichia coli using Western blot. Then, we coated the fusion protein (carrying cell lysates) to cotton fabrics and wood and verified with Infrared (IR) spectroscopy. Using a vertical burning test and wood flammability test, we confirmed the flame retardancy of the materials after the protein coating. In the vertical burning test, the SR protein and alpha casein flame retardant proteins with the CBD adhesion domain showed a 50.0% and 43.3% increase in flame retardancy. The data is also consistent in the wood flame retardancy test. Confocal imaging experiments also suggested these new fire retardants can be preserved on the materials well even after washing. Overall, our results showed that flame-retardant proteins with adhesion domains are high potential candidates of green alternative flame retardants.

Class I hydrophobin fusion with cellulose binding domain for its soluble expression and facile purification

Hydrophobins, highly surface-active proteins, have the ability to reverse surface hydrophobicity through self-assembly at the hydrophilic-hydrophobic interfaces. Their unique structure and interfacial activity lead hydrophobins to have potential applications on surface functional modifications. However, class I hydrophobins are prone to self-assemble into highly insoluble amyloid-like rodlets structure. Recombinant hydrophobins could be produced by Escherichia coli but generally as an insoluble inclusion body. To overcome this insoluble expression limitation, cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of class I hydrophobin HGFI to enhance its soluble expression in E. coli. Approximately, 94% of expressed CBD fused HGFI (CBD-HGFI) was found as soluble protein. The fused CBD could also bind specifically onto bacterial cellulose (BC) nanofibrils produced by Komagataeibacter xylinus to facilitate rapid isolation and purification of HGFI from crude extract. Lysostaphin (Lst), known as GlyGly endopeptidase could successfully cleave the flexible linker (GGGGS)2 between CBD and HGFI to recover HGFI from BC-bound CBD-HGFI. CBD-HGFI purified by immobilized metal-chelated affinity chromatography (IMAC) and Lst cleaved BC-CBD-HGFI still retained interfacial activity of hydrophobin and its effect on accelerating PETase hydrolysis against poly(ethylene terephthalate) (PET) fiber.

Self-assembling protein nanocages for modular enzyme assembly by orthogonal bioconjugation.

The wide variety of enzymatic pathways that can benefit from enzyme scaffolding is astronomical. While enzyme co-localization based on protein, DNA, and RNA scaffolds has been reported, we still lack scaffolds that offer well-defined and uniform three-dimensional structures for enzyme organization. Here we reported a new approach for protein co-localization using naturally occurring protein nanocages as a scaffold. Two different nanocages, the 25 nm E2 and the 34 nm heptatitis B virus, were used to demonstrate the successfully co-localization of the endoglucanase CelA and cellulose binding domain using the robust SpyTag/SpyCatcher bioconjugation chemistry. Because of the simplicity of the assembly, this strategy is useful not only for in vivo enzyme cascading but also the potential for in vivo applications as well.

Enhancing PET hydrolytic enzyme activity by fusion of the cellulose–binding domain of cellobiohydrolase I from Trichoderma reesei

The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETaseS121E/D186H/R280A (IsPETaseEHA) variant. We found that fusing a cellulose–binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of IsPETaseEHA_CBM was increased by 86 % when the protein concentration was increased from 2.5 μg/mL to 20 μg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.

Herbidospora solisilvae sp. nov., a novel cellulose-degrading actinobacterium isolated from forest soil.

An aerobic, non-motile, Gram-stain positive, cellulose-degrading actinobacterium, designated strain NEAU-GS84T, was isolated from a forest soil sample collected from Linchun Ridge Forest Park in Sanya, Hainan Province, China, and characterized using a polyphasic approach. Morphological and chemotaxonomic characteristics indicated that strain NEAU-GS84T could belong to the genus Herbidospora. The 16S rRNA gene sequence analysis indeed confirmed that strain NEAU-GS84T belonged to the genus Herbidospora and was most closely related to Herbidospora yilanensis JCM 18062T (99.2% 16S rRNA gene sequence similarity) and Herbidospora galbida NEAU-GS14T (99.0%). The cell wall of strain NEAU-GS84T contained meso-diaminopimelic acid as the major diamino acid and the whole-cell hydrolysates mainly contained glucose, madurose and ribose. The major polar lipids were diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, hydroxyphosphatidylethanolamin, phosphoglycolipids, two phosphatidylinositol mannosides and one unidentified phospholipid. The predominant menaquinone was MK-10(H4). Major fatty acids were 10-methly C17:0, C17:0 and iso-C16:0. These chemotaxonomic data substantiated the affiliation of strain NEAU-GS84T to the genus Herbidospora. The DNA G+C content was 70.7mol%. The genome size of strain NEAU-GS84T is about 8.37Mb and contained 41 cellulose-binding domain synthesis genes, 13 β-glucosidase synthesis genes, 6 endoglucanase synthesis genes and 9 xylanase synthesis genes. Based on digital DNA-DNA hybridization and average nucleotide identity values, the new strain NEAU-GS84T could be differentiated from its closest relatives. Therefore, the strain represents a novel species of the genus Herbidospora, for which the name Herbidospora solisilvae sp. nov. is proposed. The type strain is NEAU-GS84T (= CCTCC AA 2018041T = JCM 33460T).

Production of beta-galactosidase fused to a cellulose-binding domain for application in sustainable industrial processes

This study aimed to produce and characterize a recombinant Kluyveromyces sp. β-galactosidase fused to a cellulose-binding domain (CBD) for industrial application. In expression assays, the highest enzymatic activities occurred after 48 h induction on Escherichia coli C41(DE3) strain at 20 °C in Terrific Broth (TB) culture medium, using isopropyl β-d-1-thiogalactopyranoside (IPTG) 0.5 mM (108.77 U/mL) or lactose 5 g/L (93.10 U/mL) as inducers. Cultures at bioreactor scale indicated that higher product yield values in relation to biomass (2000 U/g) and productivity (0.72 U/mL.h) were obtained in culture media containing higher protein concentration. The recombinant enzyme showed high binding affinity to nanocellulose, reaching both immobilization yield and efficiency values of approximately 70% at pH 7.0 after 10 min reaction. The results of the present study pointed out a strategy for recombinant β-galactosidase-CBD production and immobilization, aiming toward the application in sustainable industrial processes using low-cost inputs.