Search Results (7)

To assess the impact of Enchytraeidae (potworms) on the functioning of the decomposer system, knowledge of the feeding preferences of enchytraeid species is required. Different food preferences can be explained by variations in enzymatic activities among different enchytraeid species, as there are no significant differences in the morphology or anatomy of their alimentary tracts. However, it is crucial to distinguish between the contribution of microbial enzymes and the animal’s digestive capacity. Here, we computationally analyzed the endogenous digestive enzyme genes in Enchytraeus albidus. The analysis was based on RNA-Seq of COI-monohaplotype culture (PL-A strain) specimens, utilizing transcriptome profiling to determine the trophic position of the species. We also corroborated the results obtained using transcriptomics data from genetically heterogeneous freeze-tolerant strains. Our results revealed that E. albidus expresses a wide range of glycosidases, including GH9 cellulases and a specific digestive SH3b-domain-containing i-type lysozyme, previously described in the earthworm Eisenia andrei. Therefore, E. albidus combines traits of both primary decomposers (primary saprophytophages) and secondary decomposers (sapro-microphytophages/microbivores) and can be defined as an intermediate decomposer. Based on assemblies of publicly available RNA-Seq reads, we found close homologs for these cellulases and i-type lysozymes in various clitellate taxa, including Crassiclitellata and Enchytraeidae. Full article

In the present paper, we investigated the characteristics of chalky rice grains generated by ripening under high temperature and compared them with whole grains. We evaluated 14 unpolished Japonica rice grains harvested in Japan in 2021, and these samples (original grains) were divided into two groups (a whole grain group and a chalky grain one). We found that not only activities of endogenous amylase and proteinase, but also cell wall-degrading enzymes, such as xylanase and cellulase, changed markedly between chalky grains and whole grains. Using rice grains blended with 30% of chalky grains as the material, we compared the sugar and mineral contents and textural properties of the rice grains soaked and boiled in either ordinary water or hard water, such as Evian or Contrex. It was shown that xylanase, in addition to amylase and proteinase, may play an important role in changing the texture of the boiled chalky rice grains. For the sake of preventing the above-mentioned deterioration in the texture of boiled grains of chalky rice, we tried to use hard water, such as Evian or Contrex, to soak and cook the chalky rice grains. It was shown that the hard water was useful for the prevention of texture deterioration of the boiled rice grains due to inhibition of the activities of endogenous hydrolytic enzymes, such as α-amylase, β-amylase, proteinase, and xylanase. Furthermore, we found that the hard water was useful in increasing the calcium absorption through the meal by 2.6 to 16.5 times. Full article

Trichoderma reesei is a powerful fungal cell factory for the production of cellulolytic enzymes due to its outstanding protein secretion capacity. Endoplasmic reticulum-associated degradation (ERAD) plays an integral role in protein secretion that responds to secretion pressure and removes misfolded proteins. However, the role of ERAD in fungal growth and endogenous protein secretion, particularly cellulase secretion, remains poorly understood in T. reesei. Here, we investigated the ability of T. reesei to grow under different stresses and to secrete cellulases by disrupting three major genes (hrd1, hrd3 and der1) involved in the critical parts of the ERAD pathway. Under the ER stress induced by high concentrations of DTT, knockout of hrd1, hrd3 and der1 resulted in severely impaired growth, and the mutants Δhrd1 and Δhrd3 exhibited high sensitivity to the cell wall-disturbing agents, CFW and CR. In addition, the absence of either hrd3 or der1 led to the decreased heat tolerance of this fungus. These mutants showed significant differences in the secretion of cellulases compared to the parental strain QM9414. During fermentation, the secretion of endoglucanase in the mutants was essentially consistent with that of the parental strain, while cellobiohydrolase and β-glucosidase were declined. It was further discovered that the transcription levels of the endoglucanase-encoding genes (eg1 and eg2) and the cellobiohydrolase-encoding gene (cbh1) were not remarkedly changed. However, the β-glucosidase-encoding gene (bgl1) was significantly downregulated in the ERAD-deficient mutants, which was presumably due to the activation of a proposed feedback mechanism, repression under secretion stress (RESS). Taken together, our results indicate that a defective ERAD pathway negatively affects fungal growth and cellulase secretion, which provides a novel insight into the cellulase secretion mechanism in T. reesei. Full article

The UDP-glycosyltransferase 76G1 (UGT76G1) is responsible for the conversion of stevioside to rebaudioside A. Four single guide RNAs (sgRNAs) were designed from the UGT76G1 proximal promoter region of stevia by using the online-based tool, benchling. The dCas9 fused with VP64 as a transcriptional activation domain (TAD) was produced and purified for the formation of ribonucleoproteins (RNPs) by mixing with the in vitro transcribed sgRNAs. Protoplast yield was the highest from leaf mesophyll of in vitro grown stevia plantlets (3.16 × 10⁶/g of FW) using ES5 (1.25% cellulase R-10 and 0.75% macerozyme R-10). The RNPs were delivered into the isolated protoplasts through the Polyethylene glycol (PEG)-mediated transfection method. The highest endogenous activation of the UGT76G1 gene was detected at 27.51-fold after 24 h of transfection with RNP30 consisting of CRISPR/dCas9-TAD with sgRNA30 and a similar activation level was obtained using RNP18, RNP33, and RNP34, produced using sgRNA18, sgRNA33, and sgRNA34, respectively. Activation of UGT76G1 by RNP18 led to a significant increase in the expression of the rate-limiting enzyme UGT85C2 by 2.37-fold and there was an increasing trend in the expression of UGT85C2 using RNP30, RNP33, and RNP34. Successful application of CRISPR/dCas9-TAD RNP in activating specific genes can avoid the negative integration effects of introduced DNA in the host genome. Full article

The present review examines the factors and variables that should be considered to obtain, design, and evaluate EFEs that might enhance ruminal NDF degradability. Different combinations of words were introduced in Google Scholar, then scientific articles were examined and included if the reported factors and variables addressed the objective of this review. One-hundred-and-sixteen articles were included. The fungal strains and culture media used to grow white-rot fungi induced the production of specific isoforms of cellulases and xylanases; therefore, EFE products for ruminant feed applications should be obtained in cultures that include the high-fibrous forages used in the diets of those animals. Additionally, the temperature, pH, osmolarity conditions, and EFE synergisms and interactions with ruminal microbiota and endogenous fibrolytic enzymes should be considered. More consistent results have been observed in studies that correlate the cellulase-to-xylanase ratio with ruminant productive behavior. EFE protection (immobilization) allows researchers to obtain enzymatic products that may act under ruminal pH and temperature conditions. It is possible to generate multi-enzyme cocktails that act at different times, re-associate enzymes, and simulate natural protective structures such as cellulosomes. Some EFEs could consistently improve ruminal NDF degradability if we consider fungal cultures and ruminal environmental conditions variables, and include biotechnological tools that might be useful to design novel enzymatic products. Full article

Microbial endophytes organize symbiotic relationships with the host plant, and their excretions contain diverse plant beneficial matter such as phytohormones and bioactive compounds. In the present investigation, six bacterial and four fungal strains were isolated from the common bean (Phaseolus vulgaris L.) root plant, identified using molecular techniques, and their growth-promoting properties were reviewed. All microbial isolates showed varying activities to produce indole-3-acetic acid (IAA) and different hydrolytic enzymes such as amylase, cellulase, protease, pectinase, and xylanase. Six bacterial endophytic isolates displayed phosphate-solubilizing capacity and ammonia production. We conducted a field experiment to evaluate the promotion activity of the metabolites of the most potent endophytic bacterial (Bacillus thuringiensis PB2 and Brevibacillus agri PB5) and fungal (Alternaria sorghi PF2 and, Penicillium commune PF3) strains in comparison to two exogenously applied hormone, IAA, and benzyl adenine (BA), on the growth and biochemical characteristics of the P. vulgaris L. Interestingly, our investigations showed that bacterial and fungal endophytic metabolites surpassed the exogenously applied hormones in increasing the plant biomass, photosynthetic pigments, carbohydrate and protein contents, antioxidant enzyme activity, endogenous hormones and yield traits. Our findings illustrate that the endophyte Brevibacillus agri (PB5) provides high potential as a stimulator for the growth and productivity of common bean plants. Full article

The purpose of this study was to characterize the endogenous cellulase gene MaCel1 of Monochamus alternatus, which is an important vector of Bursaphelenchus xylophilus, a pine wood nematode, which causes pine wilt disease (PWD). In this study, MaCel1 was cloned by rapid amplification of cDNA end (RACE), and its expression analyzed by RT-qPCR (real-time quantitative PCR detecting). A total of 1778 bp of cDNA was obtained. The encoding region of this gene was 1509 bp in length, encoding a protein containing 502 amino acids with a molecular weight of 58.66 kDa, and the isoelectric point of 5.46. Sequence similarity analysis showed that the amino acids sequence of MaCel1 had high similarity with the β-Glucosinolate of Anoplophoraglabripennis and slightly lower similarity with other insect cellulase genes (GH1). The β-D-Glucosidase activity of MaCel1 was 256.02 ± 43.14 U/L with no β-Glucosinolate activity. MaCel1 gene was widely expressed in the intestine of M. alternatus. The expression level of MaCel1 gene in male (3.46) and female (3.51) adults was significantly higher than that in other developmental stages, and the lowest was in pupal stage (0.15). The results will help reveal the digestive mechanism of M. alternatus and lay the foundation for controlling PWD by controlling M. alternatus. Full article