Identification and Characterization of Three Previously Undescribed Crystal Proteins from Bacillus thuringiensis subsp.jegathesan

ABSTRACTThe total protoxin complement in the parasporal body of mosquitocidal strain,Bacillus thuringiensissubsp.jegathesan367, was determined by use of a polyacrylamide gel block coupled to mass spectrometry. A total of eight protoxins were identified from this strain, including five reported protoxins (Cry11Ba, Cry19Aa, Cry24Aa, Cry25Aa, and Cyt2Bb), as well as three previously undescribed (Cry30Ca, Cry60Aa, and Cry60Ba) in this isolate. It was interesting that the encoding genes of three new protoxins existed ascry30Ca-gap-orf2andcry60Ba-gap-cry60Aa. Thecry30Caand a downstreamorf2gene were oriented in the same direction and separated by 114 bp, andcry60Bawas located 156 bp upstream from and in the same orientation tocry60Aa. The three new protoxin genes were cloned fromB. thuringiensissubsp.jegathesanand expressed in an acrystalliferous strain under the control ofcyt1Agene promoters and the STAB-SD stabilizer sequence. Recombinant strain containing onlycry30Cadid not produce visible inclusion under microscope observation, while that containing bothcry30Caandorf2could produce large inclusions. Cry60Aa and Cry60Ba synthesized either alone or together in the acrystalliferous host could yield large inclusions. In bioassays using the fourth-instar larvae ofCulex quinquefasciatus, Cry60Aa and Cry60Ba alone or together had estimated 50% lethal concentrations of 2.9 to 7.9 μg/ml; however, Cry30Ca with or without ORF2 was not toxic to this mosquito.

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A Novel Colletotrichum graminicola Raffinose Oxidase in the AA5 Family

Applied and Environmental Microbiology ◽

10.1128/aem.01383-17 ◽

2017 ◽

Vol 83 (20) ◽

Cited By ~ 9

Author(s):

Martina Andberg ◽

Filip Mollerup ◽

Kirsti Parikka ◽

Sanna Koutaniemi ◽

Harry Boer ◽

...

Keyword(s):

Mass Spectrometry ◽

Uronic Acid ◽

Modular Structure ◽

Substrate Preference ◽

Galactose Oxidase ◽

Ionization Mass ◽

Colletotrichum Graminicola ◽

Content Type ◽

Identification And Characterization

ABSTRACT We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5_2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola (CgRaOx; EC 1.1.3.−) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5_2 galactose oxidase. CgRaOx was expressed in Pichia pastoris, and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of CgRaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. CgRaOx presented a novel activity pattern in the AA5 family. IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola. As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing.

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Identification and characterization of ketapang seeds oil (Terminalia catappa L.) using the gas chromatography-mass spectrometry (GC-MS)

Journal of Physics Conference Series ◽

10.1088/1742-6596/1763/1/012075 ◽

2021 ◽

Vol 1763 (1) ◽

pp. 012075

Author(s):

M Napitupulu ◽

Muhammad Firdaus ◽

D K Walanda

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Gas Chromatography Mass Spectrometry ◽

Terminalia Catappa ◽

Chromatography Mass Spectrometry ◽

Identification And Characterization

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Identification and Characterization of a Halotolerant, Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota

Applied and Environmental Microbiology ◽

10.1128/aem.01194-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4958-4967 ◽

Cited By ~ 35

Author(s):

Marjolaine Martin ◽

Sophie Biver ◽

Sébastien Steels ◽

Tristan Barbeyron ◽

Murielle Jam ◽

...

Keyword(s):

Metagenomic Library ◽

Functional Screening ◽

Sample Collection ◽

Prolonged Incubation ◽

Content Type ◽

Cold Active ◽

Esterase Loci ◽

Collection Period ◽

Identification And Characterization

ABSTRACTA metagenomic library was constructed from microorganisms associated with the brown algaAscophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.

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