scholarly journals Development of Improved Cellulase Variants for the Conversion of Spent Mushroom Substrate Supplemented with Wheat Straw by Directed Evolution

2021 ◽  
Author(s):  
Valentina Mauriello ◽  
Anna Pennacchio ◽  
Irantzu Alegria Dallo ◽  
Laura Garcia Saez ◽  
Petri Ihalainen ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Wheat Straw ◽  
Recombinant Expression ◽  
Scale Up ◽  
Wild Type ◽  
Spent Mushroom Substrate ◽  
Glucose Yield ◽  
Hydrolysis Of ◽  
Selection Of

Abstract To improve the Spent mushroom substrate (SMS) saccharification, cloning, recombinant expression in Escherichia coli and characterization of two new GH5 family cellulases (Cel1 and Cel2) were performed. Based on enzymes properties, Cel2 was selected for the generation of 30,000 random mutants by directed evolution in order to develop improved biocatalysts. Error-prone Polymerase Chain Reaction was used for diversity generation in cel2 gene and the screening for activity of mutants allowed selection of 63 improved variants that were subjected to a scale up production. Among these, 13 clones exhibited two-fold higher activity than Cel2 and a higher thermoresistance after 72h. The performances of these mutants in the hydrolysis of pretreated SMS/ wheat straw (40/60) were compared to the wild type Cel2 in conjunction with a commercial enzymatic mixture (MetZyme® SUNO™ BOOSTER 144). All the mutants exhibited a glucose yield two-fold or four fold higher than wild-type Cel2 after 72h of incubation.

scholarly journals Genome mining of novel rubiginones from Streptomyces sp. CB02414 and characterization of the post-PKS modification steps in rubiginone biosynthesis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jingyan Zhang ◽  
Ying Sun ◽  
Yeji Wang ◽  
Xin Chen ◽  
Lu Xue ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Pathway ◽  
Scale Up ◽  
Genome Mining ◽  
Gene Clusters ◽  
Combinatorial Biosynthesis ◽  
Wild Type ◽  
Aromatic Polyketides ◽  
A Genome

Abstract Background Rubiginones belong to the angucycline family of aromatic polyketides, and they have been shown to potentiate the vincristine (VCR)-induced cytotoxicity against VCR-resistant cancer cell lines. However, the biosynthetic gene clusters (BGCs) and biosynthetic pathways for rubiginones have not been reported yet. Results In this study, based on bioinformatics analysis of the genome of Streptomyces sp. CB02414, we predicted the functions of the two type II polyketide synthases (PKSs) BGCs. The rub gene cluster was predicted to encode metabolites of the angucycline family. Scale-up fermentation of the CB02414 wild-type strain led to the discovery of eight rubiginones, including five new ones (rubiginones J, K, L, M, and N). Rubiginone J was proposed to be the final product of the rub gene cluster, which features extensive oxidation on the A-ring of the angucycline skeleton. Based on the production profiles of the CB02414 wild-type and the mutant strains, we proposed a biosynthetic pathway for the rubiginones in CB02414. Conclusions A genome mining strategy enabled the efficient discovery of new rubiginones from Streptomyces sp. CB02414. Based on the isolated biosynthetic intermediates, a plausible biosynthetic pathway for the rubiginones was proposed. Our research lays the foundation for further studies on the mechanism of the cytochrome P450-catalyzed oxidation of angucyclines and for the generation of novel angucyclines using combinatorial biosynthesis strategies.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. IV. DIRECTED EVOLUTION OF THE EBG REPRESSOR

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 673-681
Author(s):  
Barry G Hall
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzyme Synthesis ◽  
Regulatory Function ◽  
Wild Type ◽  
Selection For ◽  
Selection Of

ABSTRACT In Escherichia coli, the wild-type repressor of ebg (evolved β-galactosidase) enzyme synthesis, specified by the ebgR  + gene, responds very weakly to lactulose (fructose-β-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 x 10-10. ebgR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

scholarly journals Pharmacogenomics characterization of the MDM2 inhibitor MI-773 reveals candidate tumours and predictive biomarkers

2021 ◽  
Vol 5 (1) ◽  
Author(s):  
Vincent Vuaroqueaux ◽  
Hans R. Hendriks ◽  
Hoor Al-Hasani ◽  
Anne-Lise Peille ◽  
Samayita Das ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Predictive Biomarkers ◽  
Wild Type ◽  
Mutation Status ◽  
Anti Tumour Activity ◽  
Molecule Inhibitor ◽  
Tumour Activity ◽  
Selection Of

AbstractMI-773 is a recently developed small-molecule inhibitor of the mouse double minute 2 (MDM2) proto-oncogene. Preclinical data on the anti-tumour activity of MI-773 are limited and indicate that tumour cell lines (CLs) with mutated TP53 are more resistant to MI-773 than wild type TP53. Here, we explored the compound’s therapeutic potential in vitro using a panel of 274 annotated CLs derived from a diversity of tumours. MI-773 exhibited a pronounced selectivity and moderate potency, with anti-tumour activity in the sub-micromolar range in about 15% of the CLs. The most sensitive tumour types were melanoma, sarcoma, renal and gastric cancers, leukaemia, and lymphoma. A COMPARE analysis showed that the profile of MI-773 was similar to that of Nutlin-3a, the first potent inhibitor of p53–MDM2 interactions, and, in addition, had a superior potency. In contrast, it poorly correlates with profiles of compounds targeting the p53 pathway with another mechanism of action. OMICS analyses confirmed that MI-773 was primarily active in CLs with wild type TP53. In silico biomarker investigations revealed that the TP53 mutation status plus the aggregated expression levels of 11 genes involved in the p53 signalling pathway predicted sensitivity or resistance of CLs to inhibitors of p53–MDM2 interactions reliably. The results obtained for MI-773 could help to refine the selection of cancer patients for therapy.

scholarly journals Fungal Phytotoxins with Potential Herbicidal Activity to Control Chenopodium album

2015 ◽  
Vol 10 (6) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Alessio Cimmino ◽  
Marco Masi ◽  
Marco Evidente ◽  
Antonio Evidente
Keyword(s):  
Chenopodium Album ◽  
Scale Up ◽  
Field Trials ◽  
Herbicidal Activity ◽  
Biological Characterization ◽  
Fungal Toxin ◽  
Structure Activity ◽  
Ascochyta Caulina ◽  
Selection Of

This review deals with the isolation and chemical and biological characterization of phytotoxins produced by Ascochyta caulina and Phoma chenopodiicola proposed as mycoherbicides for the biological control of Chenopodium album, a worldwide spread weed which causes serious problems to some agrarian crops, including sugar beet and maize. Studies on the structure activity relationships and on the modes of actions of toxins isolated are also described, as well as the optimization of analytical methods focused on selection of the best fungal toxin producers. The attempts to scale up production of these phytotoxins aimed to obtain sufficient amounts for their application in greenhouse and field trials are also reported.

scholarly journals Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

2019 ◽  
Vol 20 (19) ◽  
pp. 4701 ◽  
Author(s):  
Shahar Yoav ◽  
Johanna Stern ◽  
Orly Salama-Alber ◽  
Felix Frolow ◽  
Michael Anbar ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Random Mutagenesis ◽  
Cellulose Hydrolysis ◽  
Inhibitory Effect ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glucose Yield ◽  
Multiple Alignments ◽  
Subsequent Hydrolysis ◽  
Hydrolysis Of

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

scholarly journals Preliminary Characterization of Yeasts from Bombino Bianco, a Grape Variety of Apulian Region, and Selection of an Isolate as a Potential Starter

Fermentation ◽  
2019 ◽  
Vol 5 (4) ◽  
pp. 102 ◽  
Author(s):  
Barbara Speranza ◽  
Daniela Campaniello ◽  
Leonardo Petruzzi ◽  
Milena Sinigaglia ◽  
Maria Rosaria Corbo ◽  
...  
Keyword(s):  
Reference Strain ◽  
Southern Italy ◽  
Small Scale ◽  
Grape Variety ◽  
Pectolytic Activity ◽  
Ethanol Resistance ◽  
Moderate Resistance ◽  
Hydrolysis Of ◽  
Selection Of

Eighty-seven yeasts were isolated from Bombino bianco, a white grape variety from Apulian Region (Southern Italy). The isolates were characterized for the splitting of arbutin, the hydrolysis of pectins, sulphite production, the resistance to acetic acid, SO2, and ethanol. An enhanced arbutin splitting (β-glucosidase) and a moderate pectolytic activity were found. Concerning ethanol resistance, the most of yeast population showed a low-to-moderate resistance, but some isolates, identified as Saccharomyces cerevisiae, were able to grow in presence of 15% v/v of ethanol. Four isolates were selected (coded as 43D, 44D, 45D, and 46D), studied for their ability to decarboxylate amino acids and used in small-scale fermentation trial; for this last experiment a reference strain was used (S. cerevisiae EC1118). This experiment suggested the existence of an isolate (S. cerevisiae 46D) with interesting traits and performances, which could be potentially proposed as a starter for Bombino bianco.

scholarly journals Hydrolysis of wheat straw hemicelluloses for maximum xylose extraction

2021 ◽  
Vol 939 (1) ◽  
pp. 012006
Author(s):  
Zh Makhatov ◽  
Zh Yelemanova ◽  
R Aitkulova ◽  
Z Narymbayeva ◽  
A Dairabayeva ◽  
...  
Keyword(s):  
Wheat Straw ◽  
Atmospheric Pressure ◽  
Reaction Conditions ◽  
Glucose Yield ◽  
Process Duration ◽  
Mild Conditions ◽  
Dilute Sulfuric Acid ◽  
Hydrolysis Process ◽  
Initial Content ◽  
Hydrolysis Of

Abstract The aim of the study is to select reaction conditions for hydrolysis of wheat straw with dilute sulfuric acid for maximum xylose extraction under mild conditions (at atmospheric pressure and temperature of 100°C). The authors found that maximum glucose yield (72.4-77.1 weight % of the initial content of hemicelluloses in wheat straw) is achieved at a concentration of H2SO4 2-3 weight % and the hydrolysis process duration of 5 hours. Analysis of the obtained hydrolysates showed that they contain cellulose (56.8-70.4 weight %), lignin (19.8-28.8 weight %) and hemicelluloses (2.8-15.3 weight %).

scholarly journals Recombinant expression and characterization of Solanum tuberosum aspartic protease

SURG Journal ◽  
2009 ◽  
Vol 3 (1) ◽  
pp. 19-25
Author(s):  
Lauren Agro ◽  
Brian Bryska ◽  
Rickey Yada
Keyword(s):  
Solanum Tuberosum ◽  
Disulfide Bonds ◽  
Inclusion Bodies ◽  
Signal Sequence ◽  
Recombinant Expression ◽  
Aspartic Proteinase ◽  
Wild Type ◽  
Sds Page ◽  
Hemoglobin Degradation

Unique to aspartic proteinases from plants are the presence of approximately 100 amino acid regions, which are usually excised during activation of the zymogen. These sequences are termed ‘Plant-Specific Inserts’ and are implicated in membrane interactions of their parent enzymes, including vacuolar targeting and host defense. The need to further characterize the structure-function rol (s) of plant-specific inserts stimulated the current study of the characterization of Solanum tuberosum AP (StAP). Recombinant expression of wild-type StAP resulted in the 54 kDa protein being visualized by SDS-PAGE analysis with a protein yield of 0.03%. A protein purification factor could not be established since activation of the protein at pH 2.2, 3.0, 3.7 and 5.5 was not achieved as evidenced by a lack of change in band patterns on SDS-PAGE as well as acidification and hemoglobin degradation assays. To potentially improve enzyme folding and activation ability, two mutants, (1) lacking the pre-signal sequence and (2) lacking both the signal sequence and the prosegment, were designed, sub-cloned, and expressed. Both products proved to be insoluble and inactive. New constructs were designed for the expression of StAP inclusion bodies for insoluble expression and subsequent re-folding of the protein. Additionally, CysAla mutations for each PSI Cys residue were made to investigate the rol (s) of plant-specific insert disulfide bonds in plant aspartic proteinase enzyme folding and structure. All PSI cysteine mutations (eight mutants) were successfully created using QuickChange MutagenesisTM

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2931-2939 ◽  
Author(s):  
Alex K. Jaques ◽  
Tamo Fukamizo ◽  
Diana Hall ◽  
Richard C. Barton ◽  
Gemma M. Escott ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Wild Type Strain ◽  
Chitinase Activity ◽  
Gene Replacement ◽  
Wild Type ◽  
Gene Encoding ◽  
Batch Cultures ◽  
Putative Active Site ◽  
Hydrolysis Of

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the β-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc4, GlcNAc5 and GlcNAc6 indicated that the predominant reaction involved hydrolysis of GlcNAc2 from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

Sign in / Sign up

Export Citation Format

Share Document