•Insights into biochemical characterization of GH-43/subfamily 29 produced by C. thermocellum.•Functional characterization of GH43/subfamily 29 members.•Production of enzymes with potential use in ...biotechnological processes.
An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose deconstruction. Our research group has been working on mapping and characterization of glycoside hydrolases produced by C. thermocellum B8, that are critical for lignocellulosic biomass deconstruction. One of the identified genes expressed during growth on sugar cane bagasse and straw is axb8, which encodes a putative cellulosomal GH43_29 α-arabinofuranosidase (EC 3.2.1.55) that has not previously been characterized at the molecular or kinetic levels. The AxB8 predicted amino acid sequence presented GH43 and dockerin domains, as well as a family 6 carbohydrate-binding module (CBM6). Also, it is a close homologue of Firmicutes putatives α-arabinofuranosidases, including cellulosomal proteins. Multiple alignment analysis grouped AxB8 in a cluster with four uncharacterized putative GH43_29 subfamily enzymes, all containing dockerin type I domain and CBM6 modules. Purified heterologously expressed AxB8 showed activity against the synthetic substrates pNPX (p-nytrophenyl-β-d-xylopyranoside) and pNPA (p-nytrophenyl-α-l-arabinofuranoside), as well as against the natural substrate wheat arabinoxylan (WAX), with maximal activity at 50°C and pH between 5.0 and 6.0. The WAX degradation profile by AxB8 is different from those typically seen for α-arabinofuranosidases, presenting mainly xylose as a hydrolysis product, instead of arabinose. In addition, unlike other GH43_29 enzymes already characterized, AxB8 did not present activity against arabinan. Kinetic parameters using pNPA as a substrate were Km of 23±3mM and kcat of 104±7s−1. Despite its activity against pNPX, we did not observe AxB8 saturation with this substrate. AxB8 is the first member in its clade to be characterized regarding kinetic parameters, and together with its closest homologues could represent a large group of glycoside hydrolases with particular properties within the GH43_29 subfamily.
A putative new virus with sequence similarity to members of the genus
Cavemovirus
in the family
Caulimoviridae
was identified in wild chicory (
Cichorium intybus
) by next-generation sequencing ...(NGS). The putative new virus was tentatively named “chicory mosaic cavemovirus” (ChiMV), and its genome was determined to be 7,775 nucleotides (nt) long with the typical genome organization of cavemoviruses. ORF1 encodes a putative coat protein/movement polyprotein (1,278 aa), ORF2 encodes a putative replicase (650 aa), and ORF3 encodes a putative transactivator factor (384 aa). The first two putative proteins have 46.2% and 68.7% amino acid sequence identity to the CP/MP protein (YP_004347414) and replicase (YP_004347415), respectively, of sweet potato collusive virus (SPCV). ORF3 encodes a protein with 38.5% amino acid sequence identity to the putative transactivator factor (NP_056849) of cassava vein mosaic virus (CsVMV). The new putative viral genome and those of three cavemoviruses (epiphyllum virus 4 EpV-4, SPCV, and CsVMV) differ by 24-27% in the nt sequence of the replicase gene, which exceeds the species demarcation cutoff (>20%) for the family.
The COVID-19 pandemic has demanded a range of biotechnological products for detection of SARS-CoV-2 variants and evaluation of human seroconversion after infection or vaccination. In this work, we ...describe an easy pipeline for expression of SARS-CoV-2 nucleocapsid (N) protein in insect cells followed by its purification via affinity chromatography. The N gene was cloned into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via transposition and the resulting recombinant baculovirus was used for infection of lepidopteran Sf9 cells adapted to high-density suspension. Using Tris−HCl pH 8.0 buffer as mobile phase and eluting bound proteins with 175 mM imidazole as part of a three-step gradient, an average of 1 mg N protein could be purified from each 50 mg of total protein from clarified supernatant. Such protein amount allows the manufacturing of serological tests and the development of basic studies on cellular responses to SARS-CoV-2.
•Identification of cellulosomal proteins produced by C. thermocellum B8.•Lignin derived components as holocellulases inhibitors.•Enzymes assembled in cellulosomes displayed outstanding ...thermal-stability.•Dynamic light scattering technique was used to study poly-cellulosomes.
The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC–MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren’t detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70°C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70°C and PC showed remarkable thermostability at 50 and 60°C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50°C.
Baculoviruses are circular double-stranded DNA viruses that infect insects and are widely used as the baculoviral expression vectors (BEVs), which provide a eukaryotic milieu for heterologous ...expression. The most frequently used vector is based on Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, purification of recombinant proteins produced using BEVs is laborious, time-consuming, and often expensive. Numerous strategies have been explored to facilitate purification of heterologous proteins, such as fusion with occlusion body (OBs)-forming proteins like polyhedrin (Polh). Baculoviruses produce OBs in the late stages of infection to protect the virion in the cellular environment, and the main protein responsible for OB formation is Polh. In this study, we investigated the effect of fusing the gene that encodes the surface antigen (S-HBsAg) of hepatitis B virus (HBV) to either the N- or C-terminus of the AcMNPV Polh. The production of recombinant viruses and recombinant proteins was confirmed, and the ability to form chimeric S-HBsAg-containing OBs was accessed by light and scanning electron microscopy of infected cells. The fusion was found to affect the shape and size of the OBs when compared to wild-type OBs, with the N-terminal fusion producing less-amorphous OBs than the C-terminal construct. In addition, the N-terminal construct gave higher levels of expression than the C-terminal construct. Quantitative and qualitative immunoassays with human serum or plasma antibodies against HBsAg showed that the two forms of the antigen reacted differently. Although both reacted with the antibody, the N-terminal fusion protein reacted with more sensitivity (2.27-fold) and is therefore more suitable for quantitative assays than the C-terminal version. In summary, the BEVs represents a promising tool for the production of reagents for the diagnosis of HBV infection.
The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient ...and low-cost process.
Clostridium thermocellum
has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a
C. thermocellum
strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated
C. thermocellum
gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.
Plant cell wall represents an important source of fermentable sugars for second generation bioethanol production. However, cellulosic biomass hydrolysis still is a bottleneck to bioethanol production ...in an efficient and low cost process. Thermophilic bacteria have been studied as a source of cellulolytic enzymes for cellulosic biomass deconstruction, as their enzymes present unique features compatible with current industrial process conditions. The present study was carried out to evaluate the use of different agro-industrial wastes as suitable carbon sources for growth and enzyme secretion by a strain of Clostridium thermocellum isolated from goat rumen. C. thermocellum B8 was able to grow on/degrade microcrystalline cellulose, Sugar cane bagasse/Straw and Cotton waste, and produced different sets of cellulases and hemicellulases in their presence. The enzymatic mixtures produced by C. thermocellum (B8) showed activities over a broad range of temperatures (50–70 °C). Highest values were obtained between 60 and 70 °C, at a pH range from 5 to 7, decreasing at alkaline pH values. In addition, enzymes displayed thermostability, with CMCase and xylanase activities maintaining maximum values over 12 days at 50 °C.
•Clostridium thermocellum as a source of industrial enzymes.•Lignocellulosic biomasses degradation by C. thermocellum.•Non-cellulossomal enzymes from C. thermocellum.
Penicillium
species have been studied as producers of plant cell wall degrading enzymes to deconstruct agricultural residues and to be applied in industrial processes. Natural environments containing ...decaying plant matter are ideal places for isolating fungal strains with cellulolytic and xylanolytic activities. In the present study, Cerrado soil samples were used as source of filamentous fungi able to degrade xylan and cellulose.
Penicillium
was the most abundant genus among the obtained xylan and carboxymethylcellulose degraders.
Penicillium polonicum
was one of the best enzyme producers in agar-plate assays. In addition, it secretes CMCase, Avicelase, pectinase, mannanase, and xylanase during growth in liquid media containing sugarcane bagasse as carbon source. The highest value for endo-
β
-1,4-xylanase activity was obtained after 4 days of growth. Xyl
PP
, a 20 kDa endo-
β
-1,4-xylanase, was purified and partially characterized. The purified enzyme presented the remarkable feature of being resistant to the lignin-derived phenolic compounds,
p
-coumaric and trans-ferulic acids. This feature calls for its further use in bioprocesses that use lignocellulose as feedstock. Furthermore, future work should explore its structural features which may contribute to the understanding of the relationship between its structure and resistance to phenolic compounds.
PDF
