In Silico Comparative Analysis of the Functional, Structural, and Evolutionary Properties of SARS-CoV-2 Variant Spike Proteins.

Background: A recent global outbreak of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) created a pandemic and emerged as a potential threat to humanity. The analysis of virus genetic composition has revealed that the spike protein, one of the major structural proteins, facilitates the entry of the virus to host cells. Objective: The spike protein has become the main target for prophylactics and therapeutics studies. Here, we compared the spike proteins of SARS-CoV-2 variants using bioinformatics tools. Methods: The spike protein sequences of wild-type SARS-CoV-2 and its 6 variants-D614G, alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), gamma (P.1), and omicron (B.1.1.529)-were retrieved from the NCBI database. The ClustalX program was used to sequence multiple alignment and perform mutational analysis. Several online bioinformatics tools were used to predict the physiological, immunological, and structural features of the spike proteins of SARS-CoV-2 variants. A phylogenetic tree was constructed using CLC software. Statistical analysis of the data was done using jamovi 2 software. Results: Multiple sequence analysis revealed that the P681R mutation in the delta variant, which changed an amino acid from histidine (H) to arginine (R), made the protein more alkaline due to arginine's high pKa value (12.5) compared to histidine's (6.0). Physicochemical properties revealed the relatively higher isoelectric point (7.34) and aliphatic index (84.65) of the delta variant compared to other variants. Statistical analysis of the isoelectric point, antigenicity, and immunogenicity of all the variants revealed significant correlation, with P values ranging from <.007 to .04. The generation of a 2D gel map showed the separation of the delta spike protein from a grouping of the other variants. The phylogenetic tree of the spike proteins showed that the delta variant was close to and a mix of the Rousettus bat coronavirus and MERS-CoV. Conclusions: The comparative analysis of SARS-CoV-2 variants revealed that the delta variant is more aliphatic in nature, which provides more stability to it and subsequently influences virus behavior.

Role of Cel5H protein surface amino acids in binding with clay minerals and measurements of its forces.

Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69 ± 19 pN for wild-type, 58 ± 19 pN for M2, 53 ± 19 pN for M3, and 49 ± 19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

Binding of cloned Cel enzymes on clay minerals related to the pI of the enzymes and database survey of cellulases of soil bacteria for pI.

The Cel genes from Bacillus licheniformis MSB03 were cloned and expressed to investigate binding ability on clay minerals and sea sand at pH ranging 3 to 9. FTIR analysis has been done to characterize bound enzymes on clay minerals. Subsequent, surveying of NCBI database for extracellular enzymes of soil bacteria was carried out. Among the five cloned Cel enzymes assayed for binding to clay minerals, only Cel5H enzyme had the binding ability. Enzyme Cel5H exhibited highest binding to montmorillonite followed by kaolinite and sea sand. Interestingly, Cel5H had higher pI value of 9.24 than other proteins (5.2-5.7). Cel5H binding to montmorillonite was shown to be negatively affected below pH 3 and above pH 9. Infrared absorption spectra of the Cel5H-montmorillonite complexes showed distinct peaks for clay minerals and bound proteins. Furthermore, database survey of soil bacterial extracellular enzymes revealed that Bacillus species enzymes had higher pI than other soil bacterial enzymes.

Modeling the clay minerals-enzyme binding by fusion fluorescent proteins and under atomic force microscope.

In the present study, binding of cellulase protein to different clay minerals were tested using fluorescent-protein complex and microscopic techniques. Cellulase gene (Cel5H) was cloned into three fluorescent vectors and expressed as fusion enzymes. Binding of Cel5H-mineral particles was confirmed by confocal microscopy, and enzyme assay. Among the Cel5H-fusion enzymes, green-fusion enzyme showed higher intensity compared with other red and yellow fusion-proteins. Intensity of fusion-proteins was dependent on the pH of the medium. Confocal microscopy revealed binding of the all three fusion proteins with different clay minerals. However, montmorillonite displayed higher binding capacity than kaolinite clay. Likewise, atomic force microscopy (AFM) image profile analysis showed proteins appeared globular molecules in free-state on mica surface with an average cross sectional diameter of 110 ± 2 nm and rough surface of montmorillonite made protein appear flattened due to structural alteration. Even surface of kaolinite also exerted some strain on protein molecular conformation after binding to surface. Our results provide further evidence for 3D visualization of enzyme-soil complex and encourage furthering study of the force involved interactions. Therefore, our results indicate that binding of proteins to clay minerals was external and provides a molecular method to observe the interaction of clay minerals-enzyme complex.

Defluviimonas aestuarii sp. nov., a marine bacterium isolated from a tidal flat, and emended description of the genus Defluviimonas Foesel et al. 2011.

A novel Gram-staining-negative, strictly aerobic bacterium, designated BS14(T), was isolated from a marine tidal flat of the South Sea in Korea. Colonies were opaque, white, smooth and circular on marine agar. Cells were moderately halophilic, non-motile rods showing catalase- and oxidase-positive reactions. Growth of strain BS14(T) was observed at 5-40 °C (optimum: 30 °C), pH 6.5-9.5 (optimum: 7.0-7.5) and 0-10 % (w/v) NaCl (optimum: 1-1.5 %). The G+C content of the genomic DNA was 61.6 mol%. Strain BS14(T) contained ubiquinone-10 (Q-10) as the sole respiratory quinone and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c), C18 : 0 3-OH, C10 : 0 3-OH and C18 : 0 as the major fatty acids. The polar lipid pattern comprised phosphatidylethanolamine, diphosphatidylglycerol, an unidentified aminolipid, an unidentified phospholipid and an unidentified polar lipid. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BS14(T) formed a tight phylogenetic lineage with Defluviimonas denitrificans D9-3(T) with a bootstrap value of 100 %. The 16S rRNA gene sequence similarity between strain BS14(T) and D. denitrificans D9-3(T) was 97.4 % and their DNA-DNA relatedness was 19.1 ± 3.6 %. Based on the phenotypic and genotypic studies, strain BS14(T) represents a novel species of the genus Defluviimonas, for which the name Defluviimonas aestuarii sp. nov. is proposed. The type strain is BS14(T) (= KACC 16442(T) = JCM 18630(T)). An emended description of the genus Defluviimonas Foesel et al. 2011 is also proposed.

Comparative genomic analysis and benzene, toluene, ethylbenzene, and o-, m-, and p-xylene (BTEX) degradation pathways of Pseudoxanthomonas spadix BD-a59.

Pseudoxanthomonas spadix BD-a59, isolated from gasoline-contaminated soil, has the ability to degrade all six BTEX (benzene, toluene, ethylbenzene, and o-, m-, and p-xylene) compounds. The genomic features of strain BD-a59 were analyzed bioinformatically and compared with those of another fully sequenced Pseudoxanthomonas strain, P. suwonensis 11-1, which was isolated from cotton waste compost. The genome of strain BD-a59 differed from that of strain 11-1 in many characteristics, including the number of rRNA operons, dioxygenases, monooxygenases, genomic islands (GIs), and heavy metal resistance genes. A high abundance of phage integrases and GIs and the patterns in several other genetic measures (e.g., GC content, GC skew, Karlin signature, and clustered regularly interspaced short palindromic repeat [CRISPR] gene homology) indicated that strain BD-a59's genomic architecture may have been altered through horizontal gene transfers (HGT), phage attack, and genetic reshuffling during its evolutionary history. The genes for benzene/toluene, ethylbenzene, and xylene degradations were encoded on GI-9, -13, and -21, respectively, which suggests that they may have been acquired by HGT. We used bioinformatics to predict the biodegradation pathways of the six BTEX compounds, and these pathways were proved experimentally through the analysis of the intermediates of each BTEX compound using a gas chromatograph and mass spectrometry (GC-MS). The elevated abundances of dioxygenases, monooxygenases, and rRNA operons in strain BD-a59 (relative to strain 11-1), as well as other genomic characteristics, likely confer traits that enhance ecological fitness by enabling strain BD-a59 to degrade hydrocarbons in the soil environment.

Mycobacterium yongonense sp. nov., a slow-growing non-chromogenic species closely related to Mycobacterium intracellulare.

A slow-growing non-chromogenic mycobacterium was isolated from a patient with pulmonary disease. Phenotypically, strain 05-1390(T) was similar to Mycobacterium intracellulare ATCC 13950(T). The 16S rRNA gene sequence (1385 bp) of strain 05-1390(T) showed a high degree of similarity to those of the M. intracellulare complex, namely Mycobacterium marseillense 5351974(T) (100 %), M. intracellulare ATCC 13950(T) (99.8 %) and Mycobacterium chimaera DSM 44623(T) (99.9 %). Phylogenetic analysis based on internal transcribed spacer 1 (ITS1) and the hsp65 gene indicated that strain 05-1390(T) was closely related to M. intracellulare ATCC 13950(T), but that it was a distinct phylogenetic entity. Of particular interest, an analysis based on the rpoB gene (701 bp) showed that it is closely related to Mycobacterium parascrofulaceum ATCC BAA-614(T) (99.4 %), a scotochromogenic strain, rather than to the M. intracellulare-related strains. Unique MALDI-TOF MS profiles also supported the taxonomic status of this strain as a distinct species. These data support the conclusion that strain 05-1390(T) represents a novel mycobacterial species, for which the name Mycobacterium yongonense sp. nov. is proposed; the type strain is 05-1390(T) ( = DSM 45126(T) = KCTC 19555(T)).

Comparative genomics reveals adaptation by Alteromonas sp. SN2 to marine tidal-flat conditions: cold tolerance and aromatic hydrocarbon metabolism.

Alteromonas species are globally distributed copiotrophic bacteria in marine habitats. Among these, sea-tidal flats are distinctive: undergoing seasonal temperature and oxygen-tension changes, plus periodic exposure to petroleum hydrocarbons. Strain SN2 of the genus Alteromonas was isolated from hydrocarbon-contaminated sea-tidal flat sediment and has been shown to metabolize aromatic hydrocarbons there. Strain SN2's genomic features were analyzed bioinformatically and compared to those of Alteromonas macleodii ecotypes: AltDE and ATCC 27126. Strain SN2's genome differs from that of the other two strains in: size, average nucleotide identity value, tRNA genes, noncoding RNAs, dioxygenase gene content, signal transduction genes, and the degree to which genes collected during the Global Ocean Sampling project are represented. Patterns in genetic characteristics (e.g., GC content, GC skew, Karlin signature, CRISPR gene homology) indicate that strain SN2's genome architecture has been altered via horizontal gene transfer (HGT). Experiments proved that strain SN2 was far more cold tolerant, especially at 5°C, than the other two strains. Consistent with the HGT hypothesis, a total of 15 genomic islands in strain SN2 likely confer ecological fitness traits (especially membrane transport, aromatic hydrocarbon metabolism, and fatty acid biosynthesis) specific to the adaptation of strain SN2 to its seasonally cold sea-tidal flat habitat.

Kordiimonas aestuarii sp. nov., a marine bacterium isolated from a tidal flat.

A Gram-staining negative, strictly aerobic bacterium, designated 101-1(T), was isolated from a sea tidal flat, Taean, Korea. The strain formed small light-yellow, smooth, and circular colonies on marine agar. Cells were weakly halophilic, motile rods showing catalase- and oxidase-positive reactions. Growth of strain 101-1(T) was observed at 15-40 °C (optimum, 30 °C), pH 5.0-8.0 (optimum, pH 6.5-7.0) and 1.0-9.0% (w/v) NaCl (optimum, 2.0-3.5%). The G+C content of the genomic DNA was 53.3 mol%. Strain 101-1(T) contained ubiquinone-10 (Q-10) as the respiratory quinone and iso-C(17:1)ω9c, iso-C(15:0) and iso-C(17:0) as major fatty acids. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain 101-1(T) formed a tight phylogenetic lineage with members of the genus Kordiimonas and was most closely related to Kordiimonas gwangyangensis GW14-5(T) and Kordiimonas lacus S3-22(T) with 97.3% and 96.3% 16S rRNA gene sequence similarities, respectively. The DNA-DNA relatedness values between strain 101-1(T) and K. gwangyangensis GW14-5(T) and K. lacus S3-22(T) were 24.8 ± 4.4% and 32.2 ± 3.6%, respectively. Based on the data from the phenotypic and genotypic studies, strain 101-1(T) represents a novel species of the genus Kordiimonas, for which the name Kordiimonas aestuarii sp. nov. is proposed. The type strain is 101-1(T) ( = KACC 16184(T) = JCM 17742(T)).

Complete genome sequence of the polycyclic aromatic hydrocarbon-degrading bacterium Alteromonas sp. strain SN2.

Alteromonas sp. strain SN2, able to metabolize polycyclic aromatic hydrocarbons, was isolated from a crude oil-contaminated sea-tidal flat. Here we report the complete 4.97-Mb genome sequence and annotation of strain SN2. These will advance the understanding of strain SN2's adaptation to the sea-tidal flat ecosystem and its pollutant metabolic versatility.

Metagenomic characterization of oyster shell dump reveals predominance of Firmicutes bacteria.

Metagenomic analyses were conducted to evaluate the biodiversity of oyster shell bacteria, under storage conditions, on the basis of 16s rDNA sequences. Temperature was recorded during a one year storage period, and the highest temperature (about 60 degrees C) was observed after five months ofstorage. Bacterial diversity was greatest in the initial stage sample, with 33 different phylotypes classified under seven phyla (Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia and unclassified bacteria), with 42.22% ofphylotypes belonging to Proteobacteria. The lowest diversity was found in the high temperature (fermentation) stage sample, with 10 different phylotypes belonging to Firmicutes (78.57%) and Bacteroidetes. In the final stage sample, bacteria were found belonging to Proteobacteria, Bacteroidetes, and Firmicutes, and some were unclassified bacteria. Of the bacteria constituting the final stage metagenome, 69.70% belonged to Firmicutes. Our results show that bacteria belonging to phylum Firmicutes were predominant during fermentation, and during the final stages of oyster shell storage, which suggests that these bacteria supposed to be the key players for oyster shell biodegradation.

Composted oyster shell as lime fertilizer is more effective than fresh oyster shell.

Physio-chemical changes in oyster shell were examined, and fresh and composted oyster shell meals were compared as lime fertilizers in soybean cultivation. Structural changes in oyster shell were observed by AFM and FE-SEM. We found that grains of the oyster shell surface became smoother and smaller over time. FT-IR analysis indicated the degradation of a chitin-like compound of oyster shell. In chemical analysis, pH (12.3+/-0.24), electrical conductivity (4.1+/-0.24 dS m(-1)), and alkaline powder (53.3+/-1.12%) were highest in commercial lime. Besides, pH was higher in composted oyster shell meal (9.9+/-0.53) than in fresh oyster shell meal (8.4+/-0.32). The highest organic matter (1.1+/-0.08%), NaCl (0.54+/-0.03%), and moisture (15.1+/-1.95%) contents were found in fresh oyster shell meal. A significant higher yield of soybean (1.33 t ha(-1)) was obtained by applying composted oyster shell meal (a 21% higher yield than with fresh oyster shell meal). Thus composting of oyster shell increases the utility of oyster shell as a liming material for crop cultivation.

Organophosphorus hydrolase (OpdB) of Lactobacillus brevis WCP902 from kimchi is able to degrade organophosphorus pesticides.

Lactobacillus brevis WCP902 that is capable of biodegrading chlorpyrifos was isolated from kimchi. The opdB gene cloned from this strain revealed 825 bp, encoding 274 aa, and an enzyme molecular weight of about 27 kDa. OpdB contains the same Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic esterase, lipase, and serine hydrolases, yet it is a novel member of the GDSVG family of esterolytic enzymes. Its conserved serine residue, Ser82, is significantly involved with enzyme activity that may have application for removing some pesticides. Optimum organophosphorus hydrolase (OpdB) activity appeared at pH 6.0 and 35 degrees C and during degradation of chlorpyrifos, coumaphos, diazinon, methylparathion, and parathion.

Effect of plant age on endophytic bacterial diversity of balloon flower (Platycodon grandiflorum) root and their antimicrobial activities.

Balloon flower (Platycodon grandiflorum) is widely cultivated vegetable and used as a remedy for asthma in East Asia. Experiments were conducted to isolate endophytic bacteria from 1-, 3-, and 6-year-old balloon flower roots and to analyze the enzymatic, antifungal, and anti-human pathogenic activities of the potential endophytic biocontrol agents obtained. Total 120 bacterial colonies were isolated from the interior of all balloon flower roots samples. Phylogenetic analysis based on 16S rRNA gene sequences showed that the population of 'low G + C gram-positive bacteria' (LGCGPB) gradually increased 60.0-80.0% from 1 to 6 years balloon flower sample. On the other hand, maximum hydrolytic enzyme activity showing endophytic bacteria was under LGCGPB, among the bacterial strains, Bacillus sp. (BF1-1 and BF3-8), Bacillus sp. (BF1-2 and BF3-5), and Bacillus sp. (BF1-3, BF3-6, and BF6-4) showed maximum enzyme activities. Besides, Bacillus licheniformis (BF3-5 and BF6-6) and Bacillus pumilus (BF6-1) showed maximum antifungal activity against Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum. Moreover, Bacillus licheniformis was found in 3 and 6 years balloon flower roots, but Bacillus pumilus was found only in 6 years sample. It is presumed that older balloon flower plants invite more potential antifungal endophytes for there protection from plant diseases. In addition, Bacillus sp. (BF1-2 and BF3-5) showed maximum anti-human pathogenic activity. So, plant age is presumed to influence diversity of balloon flower endophytic bacteria.

Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells.

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 degrees C, and activity was stimulated with 25 mM Mn2+. In kinetic analysis, Chi18B showed Km values of 9.07 +/- 0.65 microM and 129.27 +/- 0.38 microM with the substrates 4-methylumbelliferyl-N-N'-diacetylchitobiose and alpha-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.

Isolation of a novel gene encoding a 3,5,6-trichloro-2-pyridinol degrading enzyme from a cow rumen metagenomic library.

3,5,6-trichloro-2-pyridinol (TCP) is a major metabolite of the insecticide chlorpyrifos and is hazardous to human and animal health. A gene encoding a TCP degrading enzyme was cloned from a metagenomic library prepared from cow rumen. The gene (tcp3A) is 2.5 kb in length, encoding a protein (Tcp3A) of 599 amino acid residues. Tcp3A has a potential signal sequence, as well as a putative ATP/GTP binding site, and a likely amidation site. The molecular weight of the enzyme is 62 kDa by SDS-PAGE. Comparison of Tcp3A with the NCBI database using BLASTP revealed homology to amidohydrolase proteins. Recombinant Escherichia coli harboring the tcp3A gene could utilize TCP as the sole source of carbon. TLC and HPLC revealed that TCP was degraded by recombinant E. coli harboring tcp3A. This is the first report of a gene encoding a TCP degrading enzyme.

Novel multiplex PCR for the detection of lactic acid bacteria during kimchi fermentation.

We developed a multiplex PCR assay for the detection of lactic acid bacteria (LAB) species, and used it to examine the LAB species involved in kimchi fermentation. The LAB profile during kimchi fermentation varied with pH and acidity. Leuconostoc mesenteroides was observed during early fermentation (pH 5.64-4.27 and acidity 0.48-0.89%), and Lactobacillus sakei become dominant later in fermentation (pH or=0.98%). The efficiency of the multiplex PCR ranged from 86.5% at day 0 (pH 6.17 and acidity 0.24%) to 100% at day 96 (pH 4.16 and acidity 1.14). This multiplex PCR assay will facilitate study of the microbial ecosystem of kimchi and its impact on kimchi fermentation.

Bacterial diversity and structural changes of oyster shell during 1-year storage.

We examined the biodiversity of bacteria associated with oyster-shell waste during a 1-year storage period using 16S ribosomal DNA analysis. Temperature variation and structural changes of oyster shell were observed during storage. Initial and final temperatures were at 16-17 degrees C, but a high temperature of about 60 degrees C was recorded after approximately 6 months of storage. The crystal structure and nanograin of the oyster shell surface were sharp and large in size initially and became gradually blunter and smaller over time. Phylogenetic analysis revealed that Firmicutes were dominant in the oyster-shell waste initially, during the high-temperature stage, and after 1 year of storage (making up >65% of the biodiversity at all three sampling times). Bacillus licheniformis was presumed as the predominate Firmicutes present. These bacteria are likely to have important roles in the biodegradation of oyster shell.

Comparative analysis of the glg operons of Pectobacterium chrysanthemi PY35 and other prokaryotes.

A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Omega mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. gamma-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.

Changes in the activity of the multifunctional beta-glycosyl hydrolase (Cel44C-Man26A) from Paenibacillus polymyxa by removal of the C-terminal region to minimum size.

Paenibacillus polymyxa GS01 secretes Cel44C-Man26A as a multifunctional enzyme with cellulase, xylanase, lichenase, and mannanase activities. Cel44C-Man26A consists of 1,352 amino acids in which present a catalytic domain (CD) of the glycosyl hydrolase family 44 (GH44), fibronectin domain type 3 (Fn3), catalytic domain of glycosyl hydrolase family 26 (GH26), and a cellulose-binding module type 3 (CBM3). A truncated Cel44C-Man26A protein, consisting of 549 amino acid residues, reacted as a multifunctional mature enzyme despite the absence of the 10 amino acids containing GH44, Fn3, GH26, and CBM3. However, the multifunctional activity was not found in the mature Cel44C-Man26A protein truncated to less than 548 amino acids. The truncated Cel44C-Man26A proteins showed the optimum pH for the lichenase activity was pH 7.0, pH 6.0 for the xylanase and mannanase, and pH 5.0 for the cellulase. The truncated Cel44C-Man26A proteins exhibited enzymatic activity 40-120% higher than the full-length Cel44C.