Expression and characterization of honeybee, Apis mellifera, larva chymotrypsin-like protease

Matsuoka, Takuma; Takasaki, Akihiko; Mishima, Tomoyuki; Kawashima, Takuji; Kanamaru, Yoshihiro; Nakamura, T; Yabe, Tomio

doi:10.1007/s13592-014-0313-2

Cited by 8 publications

(12 citation statements)

References 31 publications

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“…Dahlmann and coauthors observed chymotryptic proteases in both larval and adult bees, while tryptic proteases only in adult bees, which is not in accordance with our results. Recently, a chymotrypsin‐like protease from honeybee larvae was found and characterized , suggesting a role in food digestion. The absence of these proteinases in the prepupae from infected bees could be caused by the degradation of the peritrophic matrix and the attack of the epithelial cells.…”

Section: Discussionmentioning

confidence: 99%

“…Among proteins, the proteases, mainly the proteinases (endopeptidases) are involved in several physiological and regulatory processes . Since proteases expression could be a mechanism by which honeybee react to the infection, the aim of this work was to compare production of proteases in worker prepupae originating from healthy colonies and from colonies affected by AFB.…”

Section: Introductionmentioning

confidence: 99%

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Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography

et al. 2018

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American foulbrood disease (AFB) is the main devastating disease that affects honeybees' brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin-1 and Chymotrypsin-1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography

et al. 2018

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“…The characteristics of HCLPase expressed in SF9 were the same as that expressed in E. coli, except for its specific activity. Although we previously expressed HCLPase in E. coli as a GST-tagged protein, 9) the characteristics of HCLPase expression were different from the present HCLPase expressed as SUMO-tagged protein in several respects. In the GST-tagged HCLPase, the enzyme had lower specific activity, showed optimal activity at 40°C and pH 9, and was not inhibited by TPCK.…”

mentioning

confidence: 60%

“…8) The recombinant HCLPase expressed in Escherichia coli produced a 26-kDa protein that had chymotryptic activity. 9) In this study, a recombinant HCLPase expressed in SF9 insect cells as eukaryotic cells was produced, and its characteristics were compared to the recombinant protein expressed in E. coli.…”

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confidence: 99%

Characterization and comparison of recombinant honeybee chymotrypsin-like protease (HCLPase) expressed in Escherichia coli and insect cells

Matsuoka¹,

Kawashima²,

Nakamura³

et al. 2017

Bioscience, Biotechnology, and Biochemistry

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We previously found a novel chymotrypsin-like protease in honeybee, designated as HCLPase. The recombinant enzyme expressed in insect cells was produced and compared to that in Escherichia coli. Both enzymes showed equivalent molecular size and specificity. However, HCLPase produced in insect cells showed higher specific activity. The C-terminal cleavage sites of HCLPase were phenylalanine, leucine, and tyrosine residues.

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“…TPCK and chymostatin, being potent inhibitors of chymotrypsin, inhibited the chymotrypsin‐like enzyme activity in the present study. In contrast, TPCK and TLCK did not affect the chymotrypsin‐like enzyme activity in T. molitor larvae and Apis mellifera (Elpidina et al., ; Matsuoka et al., ). Serine protease inhibitors, PMSF, aprotonin, leupeptin, pefabloc, and antipain completely inhibited the activities of both the enzymes.…”

Section: Discussionmentioning

confidence: 99%

Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications

Akbar

Sharma

2016

Arch Insect Biochem Physiol

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We characterized trypsin- and chymotrypsin-like serine alkaline proteases from cotton bollworm, Helicoverpa armigera, for their probable potential application as additives in various bio-formulations. Purification was achieved by using hydroxylapatite, DEAE sephadex and CM sephadex columns, which resulted in increased enzyme activity by 13.76- and 14.05-fold for trypsin and chymotrypsin, respectively. Michaelis-Menten constants (K ) for substrates of trypsin and chymotrypsin, BApNA and SAAPFpNA, were found to be 1.25 and 0.085 mM, correspondingly. Fluorescent zymogram analysis indicated the presence of five trypsin bands with molecular masses of ∼21, 25, 38, 40, and 66 kDa and two chymotrypsin bands with molecular masses of ∼29 and 34 kDa in SDS-PAGE. The optimum pH was 10.0 and optimum temperature was 50°C for proteolytic activity for the purified proteases. The proteases were inhibited by synthetic inhibitors such as PMSF, aprotonin, leupeptin, pefabloc, and antipain. TLCK and TPCK inhibited about 94 and 90% of trypsin and chymotrypsin activity, respectively, while EDTA, EGTA, E64, pepstatin, idoacetamide, and bestatin did not affect the enzymes. The purified enzymes exhibited high stability and compatibility with metal ions; oxidizing, reducing, and bleaching agents; organic solvents; and commercial detergents. Short life cycles, voracious feeding behavior, and production of multiple forms of proteases in the midgut with rapid catalytic activity and chemostability can serve H. armigera as an excellent alternative source of industrially important proteases for use as additives in stain removers, detergents, and other bio-formulations. Identification of enzymes with essential industrial properties from insect species could be a bioresource.

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Expression and characterization of honeybee, Apis mellifera, larva chymotrypsin-like protease

Cited by 8 publications

References 31 publications

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography

Characterization and comparison of recombinant honeybee chymotrypsin-like protease (HCLPase) expressed in Escherichia coli and insect cells

Alkaline serine proteases from Helicoverpa armigera: potential candidates for industrial applications

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