Angiotensin-converting enzyme inhibitor activity of peptides derived from Kacang goat skin collagen through thermolysin hydrolysis

Background and Aim: Angiotensin-converting enzyme (ACE) is one of the inhibitory enzymes isolated from animals for the treatment of hypertension. ACE inhibitor (ACE-I) peptides can be obtained by hydrolyzing proteins from various animal tissues, including muscle and connective tissues. However, the study on ACE-I activity from collagen of Kacang goat skin has not been conducted. This study explores the potency of collagen from Kacang goat skin as a source of an antihypertensive agent through ACE inhibition. Thermolysin will hydrolyze collagen and produce the peptide classified antihypertensive bioactive peptides. This study aimed to determine the potential of thermolysin to hydrolyze collagen of Kacang goat skin for ACE-I peptide production and to identify the production of ACE-I peptides. Materials and Methods: Collagen from Kacang goat skin was hydrolyzed with thermolysin and incubated at 37°C for 1 h. Molecular weight (MW) evaluation was performed by SDS PAGE; fractionation peptides at <5 kDa, 3-5 kDa, and <3 kDa were performed by ultrafiltration and ACE-I activity determined by IC50 measurement. Results: Collagen was hydrolyzed by thermolysin, resulting in protein with MW of 117.50-14.60 kDa. The protein content of fractionation at >5 kDa was 3.93±0.72 mg/mL, content of 3-5 kDa was 3.81±0.68 mg/mL, and that of <3 kDa was 2.33±0.38 mg/mL. Fractionation was performed 3 times and one of the results was selected for the ACE-I test. The selected fraction was tested by IC50 measurement with three repetitions and it showed an average enzyme activity at 0.83 μg/mL or 82.94 mg/mL. Conclusion: Thermolysin hydrolysis of collagen from Kacang goat skin showed the potential to produce bioactive peptides, such as ACE-I.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

Potensi Aktinidin sebagai Pelunak Daging

This study aimed to discuss the activity of actinidin in meat tenderization and to understand the ability of actinidin as a substitute for other tenderizing enzymes such as papain. Actinidin could be found in kiwi fruits with a similar structure and proteolytic activity characteristics to papain. The proteolytic activity of actinidin was found to be able to substitute papain in meat tenderizing without creating an off flavor side effect. However, increasing the actinidin concentration could enhance its proteolytic activity. Besides, actinidin also has the ability to hydrolyze collagen proteins and fibrinogens perfectly compared to papain activity.

Aplikasi Pendayagunaan Asam In-Situ pada Kulit Pikel Terbuang untuk Pembuatan Gelatin Pangan

Skinswaste at pre-tanning operations can be processed into food grade gelatin. The degradation of collagen using acid, base, or enzymes produced gelatin. Pickle skins is skins that acidified, the results of the final phase of the pre-tanning operations. The addition of salt on the skin makes the skins pickle not swollen, produced a wide space between collagen fibers and collagen can not be degraded. Thereby directly extract pickle skins or waste will not be obtained gelatin.This study discussed the processing of food gelatin type A pickle skins through the utilization of waste acid it contains. The discussion includes the components of animal skins, pre-tanning waste, acidification of skins, processing gelatin and gelatin from skins picklewaste and usefulness for the food industry. Salt hydrate collagen fibers in the skin pickle including waste can be separated by washing, to a certain extent still acidic skins waste. The remaining acid on the skins pickle waste can be utilized to hydrolyze collagen into gelatin. The resulting gelatin is gelatin type A, that can be used for food industry.ABSTRAKKulit limbah pada operasi pra-penyamakan dapat diolah menjadi gelatin pangan. Pemecahan kolagen menggunakan asam, basa, atau enzim dihasilkan gelatin. Kulit pikel merupakan kulit yang diasamkan, hasil dari tahap akhir operasi pra-penyamakan. Penambahan garam pada kulit pikel menjadikan kulit tidak bengkak, menghasilkan ruang lebar diantara serat kolagen dan menjadikan kolagen tidak dapat terdegradasi. Hal ini berarti ekstrak secara langsung kulit pikel atau limbahnya tidak akan diperoleh gelatin. Dalam kajian ini dibahas pengolahan gelatin pangan tipe A dari kulit pikel limbah melalui pendayagunaan asam yang dikandungnya. Bahasan mencakup komponen kulit hewan, limbah pra-penyamakan, pengasaman kulit, pengolahan gelatin, dan pengolahan gelatin dari kulit pikel limbah melalui pendayagunaan asam yang dikandungnya serta kegunaannya untuk industri pangan. Garam yang menghidrasi serat kolagen pada kulit pikel termasuk limbahnya dapat dipisahkan dengan cara pencucian, sampai batas tertentu kulit limbah masih bersifat asam. Asam yang tersisa pada kulit pikel limbah tersebut dapat didayagunakan untuk menghidrolisis kolagen menjadi gelatin. Gelatin yang dihasilkan adalah gelatin tipe A, dapat digunakan untuk keperluan industri pangan. Kata kunci : Kulit pikel limbah, gelatin, pengasaman, pangan.