Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, beta-xylosidases, endomannanases, and beta-mannosidases, when grown on cellulose or hemicellulose as carbon sources. beta-Mannosidases (EC 3.2.1.25), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for beta-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a beta-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative beta-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53 degrees C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl beta-D-mannopyranoside (pNP-betaM) substrate were as follows: K(m) = 180 micro M and V(max) = 5.96 micro mol min(-1) mg(-1); the inhibition constant for mannose was K(i) = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-alphaM and pNP-betaM; under these conditions mannosyl groups were transferred by the enzyme from pNP-betaM to pNP-alphaM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.
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http://dx.doi.org/10.1128/AEM.69.4.1944-1952.2003 | DOI Listing |
Adv Sci (Weinh)
January 2025
College of Pharmacy, Key Laboratory of Protection, Development and Utilization of Medicinal Resources in Liupanshan Area, Ministry of Education, Ningxia Medical University, Yinchuan, 750004, P.R. China.
Hispidin (1) is a polyphenolic compound with a wide range of pharmacological activities that is distributed in both plants and fungi. In addition to natural extraction, hispidin can be obtained by chemical or enzymatic synthesis. In this study, the identification and characterization of an undescribed enzyme, PheG, from Phellinus igniarius (P.
View Article and Find Full Text PDFNucleic Acids Res
January 2025
School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3JH, United Kingdom.
The growing demand for biological products drives many efforts to maximize expression of heterologous proteins. Advances in high-throughput sequencing can produce data suitable for building sequence-to-expression models with machine learning. The most accurate models have been trained on one-hot encodings, a mechanism-agnostic representation of nucleotide sequences.
View Article and Find Full Text PDFBreed Sci
September 2024
Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi, Ishikawa 921-8836, Japan.
The introgression of heterologous genomes through interspecific hybridization offers a great opportunity to expand the gene pool of crops, thereby broadening the traits that can be targeted for improvement. The introgression of C genomic regions carrying desirable traits from (AACC) into the diploid (AA) via homoeologous recombination (HR) has been commonly used. However, the precise identification of HR sites remains a significant challenge, limiting the practical application of genome introgression via HR in breeding programs.
View Article and Find Full Text PDFTranspl Int
January 2025
Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States.
An overview is provided of the evolution of strategies towards xenotransplantation during the past almost 40 years, focusing on advances in gene-editing of the organ-source pigs, pre-transplant treatment of the recipient, immunosuppressive protocols, and adjunctive therapy. Despite initial challenges, including hyperacute rejection resulting from natural (preformed) antibody binding and complement activation, significant progress has been made through gene editing of the organ-source pigs and refinement of immunosuppressive regimens. Major steps were the identification and deletion of expression of the three known glycan xenoantigens on pig vascular endothelial cells, the transgenic expression of human "protective" proteins, e.
View Article and Find Full Text PDFEpilepsia
January 2025
Atalanta Therapeutics, Boston, Massachusetts, USA.
Objective: Gain-of-function variants in the KCNT1 gene, which encodes a sodium-activated potassium ion channel, drive severe early onset developmental epileptic encephalopathies including epilepsy of infancy with migrating focal seizures and sleep-related hypermotor epilepsy. No therapy provides more than sporadic or incremental improvement. Here, we report suppression of seizures in a genetic mouse model of KCNT1 epilepsy by reducing Kcnt1 transcript with divalent small interfering RNA (siRNA), an emerging variant of oligonucleotide technology developed for the central nervous system.
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