Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi

Kamijo, Junya; Sakai, Kiyota; Suzuki, Hiromitsu; Suzuki, Kengo; Kunitake, Emi; Shimizu, Motoyuki; Kato, Masashi

doi:10.1016/j.foodchem.2018.10.059

Cited by 29 publications

(18 citation statements)

References 39 publications

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“…Isomaltase activity was assayed in 0.5 mL reaction mixtures containing 50 mM sodium phosphate (pH 6.8), 1.0% isomaltose and cell extracts at a final protein concentration of 0.5 mg/mL or purified recombinant IMAs. Reactions were incubated at 30 °C for 3 h, and proteins were removed from the reaction using a Nanosep® Centrifugal Device (Pall Corporation, Port Washington, NY, USA) 40 . Flow-through fractions were boiled at 100 °C for 5 min, and the glucose produced from isomaltose by IMA was determined using the reducing-sugar HPLC analytical system (Shimadzu) and TLC Silica gel 60 plates (Merck-Millipore) using n-butanol:water:pyridine:toluene (10:6:6:1) visualized by staining with Aniline-phthalic acid solution (2 mL of aniline containing 3.25 g of phthalic acid).…”

Section: Methodsmentioning

confidence: 99%

Ability of Saccharomyces cerevisiae MC87-46 to assimilate isomaltose and its effects on sake taste

Tsutsumi

Mochizuki

Sakai

et al. 2019

Sci Rep

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Recently, wild strains of Saccharomyces cerevisiae isolated from a variety of natural resources have been used to make bread, beer, wine, and sake. In the current study, we isolated wild S. cerevisiae MC strain from the carnation (Dianthus caryophyllus L) flower and produced sake using its cerulenin-resistant mutant strain MC87-46. Then, we characterized the components, including ethanol, amino acids, organic acids, and sugars, in the fermented sake. Sake brewed with MC87-46 is sweet owing to the high content of isomaltose, which was at a concentration of 44.3 mM. The low sake meter value of −19.6 is most likely due to this high isomaltose concentration. The genomic DNA of MC87-46 encodes for isomaltases IMA1, IMA2, IMA3, IMA4 and IMA5, as well as the isomaltose transporter gene, AGT1. However, these genes were not induced in MC87-46 by isomaltose, and the strain did not possess isomaltase activity. These results show that MC87-46 cannot utilize isomaltose, resulting in its accumulation in the fermented sake. Isomaltose concentrations in sake brewed with MC87-46 were 24.6-fold more than in commercial sake. These findings suggest that MC87-46 may be useful for commercial application in Japanese sake production because of its unique flavour and nutrient profile.

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Section: Methodsmentioning

confidence: 99%

Ability of Saccharomyces cerevisiae MC87-46 to assimilate isomaltose and its effects on sake taste

Tsutsumi

Mochizuki

Sakai

et al. 2019

Sci Rep

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“…An approximately 35-kDa protein band exhibiting pectinolytic activity has been detected using PGA. The enzyme has been purified from Aspergillus luchuensis mut (Kamijo et al, 2019). A 53 kDa endopolygalacturonase of Bacillus paralicheniformis has been identified by SDS-PAGE and pectic zimography (Khan et.…”

Section: Enzyme Identification By Sds Pagementioning

confidence: 99%

Bir Bacillus clausii İzolatı Dört Farklı Pektinolitik Aktiviteye Sahip Görünmektedir

Berber

Çetinkaya

2021

Iğdır Üniversitesi Fen Bilimleri Enstitüsü Dergisi

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For the bacterial source, Koyulhisar (Sivas, Turkey) apple orchard soil samples were used. Obtained isolates were identified at the species level by partial protein-and 16S rRNA gene sequence homology All of them were determined to be Bacillus clausii. Cultures were prepared with these isolates and after removing the cells, the proteins in the upper fluid were precipitated with the help of an organic solvent. While performing pectinase screening, it was noticed that an existing protease digested other proteins. This proteolytic activity was eliminated by increasing the incubation temperature to 55 o C. Four different pectinase activities, polygalacturonase, pectin lyase, pectate lyase, and pectin methylesterase, were then demonstrated. Four protein bands corresponding to these activities visualised by SDS-PAGE were 80 kDa, 58 kDa, 46 kDa and ~ 28 kDa, respectively. Spectroscopic methods were employed for the identification of the enzymes. The smallest protein band (28 kDa) was found to be pectin methylesterase. Km and Vmax values of this enzyme were 0.4 and 217U, respectively.

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“…The PCR product was digested by EcoRI and then ligated into pPICZα-A (Novagen, Darmstadt, Germany) that had been digested with the same restriction enzyme. The resulting plasmid was introduced into P. pastoris KM71H (Invitrogen, Carlsbad, CA), and the resulting strain was cultured to produce recombinant GH12 protein, as previously described (Kamijo et al 2019). The culture supernatant was concentrated using an Amicon Ultra filter unit (Merck-Millipore, Massachusetts, USA).…”

Section: Preparation Of Recombinant Proteinmentioning

confidence: 99%

Characterization of FsXEG12A from the cellulose-degrading ectosymbiotic fungus Fusarium spp. strain EI cultured by the ambrosia beetle

et al. 2020

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Despite the threat of Fusarium dieback posed due to ambrosia fungi cultured by ambrosia beetles such as Euwallacea spp., the wood-degradation mechanisms utilized by ambrosia fungi are not fully understood. In this study, we analyzed the 16S rRNA and 18S rRNA genes of the microbial community from the Ficus tree tunnel excavated by Euwallacea interjectus and isolated the cellulose-degrading fungus, Fusarium spp. strain EI, by enrichment culture with carboxymethyl cellulose as the sole carbon source. The cellulolytic enzyme secreted by the fungus was identified and expressed in Pichia pastoris, and its enzymatic properties were characterized. The cellulolytic enzyme, termed FsX-EG12A, could hydrolyze carboxymethyl cellulose, microcrystalline cellulose, xyloglucan, lichenan, and glucomannan, indicating that the broad substrate specificity of FsXEG12A could be beneficial for degrading complex wood components such as cellulose, xyloglucan, and galactoglucomannan in angiosperms. Inhibition of FsXEG12A function is, thus, an effective target for Fusarium dieback caused by Euwallacea spp.

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Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi

Cited by 29 publications

References 39 publications

Ability of Saccharomyces cerevisiae MC87-46 to assimilate isomaltose and its effects on sake taste

Ability of Saccharomyces cerevisiae MC87-46 to assimilate isomaltose and its effects on sake taste

Bir Bacillus clausii İzolatı Dört Farklı Pektinolitik Aktiviteye Sahip Görünmektedir

Characterization of FsXEG12A from the cellulose-degrading ectosymbiotic fungus Fusarium spp. strain EI cultured by the ambrosia beetle

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