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Advances in Microbial Molecular Biology
Published in Gustavo Molina, Zeba Usmani, Minaxi Sharma, Abdelaziz Yasri, Vijai Kumar Gupta, Microbes in Agri-Forestry Biotechnology, 2023
Deborah Catharine de Assis Leite, Naiana Cristine Gabiatti
The Restriction Analysis of Amplified Ribosomal DNA (ARDRA) is a type of RFLP based on the conservation degree of rDNA restriction sites reflecting phylogenetic patterns. Despite ARDRA being useful for a quick analysis of the diversity of an environment (Sklarz et al. 2009), it is necessary caution when choosing the rDNA fragment to be amplified and analyzed by this method. When considering intraspecific diversity analysis or accessing microbial isolated groups with high phylogenetic affinity, the amplified fragment must include the 16S–23S rDNA intergenic spacer. This intergenic region presents greater variability in both its base composition and its size when compared to 16S or 23S rDNAs. When the study in question deals with the diversity between distant isolates, phylogenetically, the amplified fragments can be 16S or 23S rDNAs (Hoffmann et al. 2010).
Heterologous Protein Production by Yeast Host-Vector Systems
Published in Yoshikatsu Murooka, Tadayuki Imanaka, Recombinant Microbes for Industrial and Agricultural Applications, 2020
A circular plasmid, pKDl, has been subsequently isolated from K. drosphilarum [64] and employed as a stable vector for high-level production of recombinant proteins in a variety of Kluyveromyces species. The copy number of pKDl is about 70 per cell, and the stability of pKDl-based vectors is remarkably high, even in the absence of selection pressure. For instance, after 40 generations of nonselective growth, over 90% of cells of the transformants of a particular strain of K. lactis still harbor the vector, although the stability is indeed species- and strain-dependent. Bergkamp et al. [65] have recently developed vector pMIRKl for high-copy-number integration into ribosomal DNA of K. lactis (Fig. 5). This system is analogous to the pMIRY system developed for S. cerevisiae, described previously (see Sec. II. A. 1). Plasmid pMIRKl was present in about 60 copies per cell and was stably maintained during growth under nonselective conditions. When a heterologous gene was introduced into this vector, the copy number was reduced fourfold, but the stability remained the same. When compared with extrachromosomal K.lactis vectors harboring the same fusion construct, the multicopy integrant showed a much higher production level and a considerably higher stability under nonselective cultural conditions.
Treatment of Paper and Pulp Industry Effluents
Published in Mihir Kumar Purkait, Piyal Mondal, Chang-Tang Chang, Treatment of Industrial Effluents, 2019
Mihir Kumar Purkait, Piyal Mondal, Chang-Tang Chang
Although several mesophilic resin acid degrading microbes have been isolated and characterized, there are few reports regarding the use thermophilic species. Recently, Mohn et al. (1998) obtained five isolates, three from a thermophilic (55°C) bioreactor and two from forestry waste compost. Of these, three were found to use abietanes, abietic acid, and dehydroabietic acid as the sole organic substrate, but were unable to grow on pimeranes, pimaric acid, and isopimaric acid. These isolates were found to grow between pH values of 6 and 8 and temperatures of 30°C–60°C. The 16S ribosomal DNA of these isolates has been sequenced for phylogenic analysis. In an extension of the same study, a semicontinuous enrichment method was used to isolate two thermophilic Rubrivivax spp. strains, namely DhA-73 and DhA-71. These were found to completely degrade dehydroabietic acid (Yu and Mohn, 1999). The use of thermophilic bacteria is an attractive option for the treatment of forest industry wastewaters, since they are released at high temperatures and can thus support the growth of thermophilic species.
Enzymatic diversity of filamentous fungi isolated from forest soil incremented by sugar cane solid waste
Published in Environmental Technology, 2022
Lidiane M. S. Lima, Debora N. Okamoto, Michel R. Z. Passarini, Sarah S. Gonçalves, Gustavo H. Goldman, Marghuel A. V. Silveira, Patrícia L. Ramos, João B. Cruz, Maria Juliano, Marcelo F. M. Marcondes, Suzan P. Vasconcellos
Morphological identification of fungi was based on the characters of colony diameter, colour, texture, topology and pigmentation on MEA or PDA media. Molecular characterization was based on the analysis of conserved gene sequences, internal transcribed spacer (ITS) region of ribosomal DNA. Mycelium fungal was collected from YES agar cultures by sterile scalpel and processed with PrepMan kit (Applied Biosystems, EUA). From the total genomic DNA (40 ng) it was performed the PCR amplification of ITS-5.8S-ITS2 rRNA using ITS1 primer (5′ TCCGTAGGTGAACCTGCG 3') and ITS4 primer (5′ TCCTCCGC TTATTGATAT3') according to [27]. Amplified products were purified using GFX PCR DNA or QIAquick PCR (Quiagen). The sequence analysis was performed using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystem, Foster City, CA, USA). Subsequently, PCR product was reacted with 2.0 μL BigDye v. 3.1 (Applied Biosystems) and 2 pmol/μL of primers. The sequences were edited in the Sequencher version 4.5 program (Gene Codes Corporation). Consensus sequences obtained were compared to the existing ones, deposited in databases such as Genbank (http://www.ncbi.nem.nih.gov) and CBS (http://www.cbs.knaw.nl/ index.htm).