Purification and Characterization of Six Kiwifruit Proteases Isolated with Two Ion-exchange Resins, Toyopearl-SuperQ and Bakerbond WP-PEI

Sugiyama, Sumi; Ohtsuki, Kozo; Satô, Kenji; Kawabata, Makoto

doi:10.1271/bbb.60.1994

Cited by 21 publications

(9 citation statements)

References 7 publications

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“…The actinidin clade belongs to the C1A papain-like subfamily, which includes enzymes such as caricain, bromelain, ficin, and phytolacain (Beers et al, 2004) that accumulate to high concentrations in fruit of other species. Most genetic and biochemical characterization of actinidin has been carried out in the major commercial green-fleshed kiwifruit (Actinidia deliciosa var Hayward), with multiple cDNA sequences (Praekelt et al, 1988;Podivinsky et al, 1989), genomic/promoter sequences (Keeling et al, 1990;Snowden and Gardner, 1990), and protein isoforms (Tello-Solis et al, 1995;Sugiyama et al, 1996Sugiyama et al, , 1997 being reported. Varietal differences in CP activity have been reported in the fruit of four A. deliciosa varieties (Prestamo, 1995), in a range of noncommercial Actinidia genotypes (Boyes et al, 1997), as well as in fruit juices extracted from Actinidia varieties and genotypes (Nishiyama, 2007).…”

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confidence: 99%

Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit

et al. 2011

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Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.

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confidence: 99%

Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit

et al. 2011

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“…Protease extraction from pear, fig, or kiwifruit Cysteine proteases from pear, fig, and kiwifruit powder were extracted with extraction buffer (10 mM cysteine, 1 mM EDTA, 50 mM sodium phosphate, pH 6.5) followed by a 60% ethanol precipitation as described previously (14,16). Here, cysteine in the extraction buffer is important to keep the activity of cysteine proteases because these proteases use a catalytic cysteine residue as a nucleophile during proteolysis and are activated by thiol compounds (20).…”

Section: Resultsmentioning

confidence: 99%

“…Protease extracts were assayed for proteolytic activity using the substrate succinylated casein as described previously (16). Briefly, caseinolytic activities of the extracted proteases from pear, fig, or kiwifruit (100 µg) were determined using a Quanti-protease Assay kit (Protease assay kit Quanticleave; Thermo Fisher).…”

Section: Methodsmentioning

confidence: 99%

“…Dried samples were then further ground by a pulverizer (FM-681C; Hanil Electric Co., Ltd., Seoul, Korea) and stored at −20 o C in polyethylene bags. Proteolytic enzyme from pear, fig, or kiwifruit was extracted as described previously with slight modifications (14,16). Each powder (10 g) was mixed with extraction buffer (1 mM cysteine, 1 mM EDTA, 50 mM sodium phosphate, pH 6.5) at a ratio of 1:9 (w/v) and then incubated with gentle shaking at 4 o C for 1 h. The extract was centrifuged at 4 o C for 30 min at 15,000xg to remove insoluble materials (Avan-Tr J-E; Beckman Coulter, Brea, CA, USA).…”

Section: Introductionmentioning

confidence: 99%

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Functional characterization of purified pear protease and its proteolytic activities with casein and myofibrillar proteins

Nam

Kim

Walsh

et al. 2016

Food Sci Biotechnol

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This study was performed to characterize pear protease proteolytic activity and investigate the use of pear protease as a meat tenderizer. Pear protease was purified and stabilized by 5% dextrin during lyophilization (dry) or concentration (liquid). Pear protease was further characterized with respect to pH, thermodynamics, and enzyme kinetics. Pear protease was stable at a pH range of 5-8 with an optimum pH of 6.5. From Arrhenius plots, liquid protease showed higher temperature dependency (23.49 kJ/mol) than dry protease (18.62 kJ/mol) due to its higher activation energy. The kcat/Km, catalytic efficiency of enzyme, was similar with 2.9 and 2.7 µM/min with dry and liquid proteases. Pear protease was evaluated for its proteolytic activities with casein and beef myofibrillar proteins by individually and combination with fig and kiwifruit proteases. These result indicated that pear and kiwifruit proteases could be complementary to be a desirable product for meat tenderization.

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“…The substrate Z-Phe-Arg-NHMec on the other hand is a general substrate for C1 family members and has been used for characterization of papain-like cysteine proteases in plants (Pardo et al, 2000;Rogers et al, 1985;Sugiyama et al, 1996;Yamada et al, 2000). These proteases express maximal activity towards the substrate at acidic pH (Popovic et al, 1998).…”

Section: Fluorimetric Assays Of Protease Activitiesmentioning

confidence: 99%

Characterization of cysteine proteases in Malian medicinal plants

Bah

Paulsen

Diallo

et al. 2006

Journal of Ethnopharmacology

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Purification and Characterization of Six Kiwifruit Proteases Isolated with Two Ion-exchange Resins, Toyopearl-SuperQ and Bakerbond WP-PEI

Cited by 21 publications

References 7 publications

Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit

Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit

Functional characterization of purified pear protease and its proteolytic activities with casein and myofibrillar proteins

Characterization of cysteine proteases in Malian medicinal plants

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