Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and Human Trypsins

Deng, Yongkai; Gruppen, Harry; Wierenga, Peter A.

doi:10.1021/acs.jafc.8b00679

Cited by 27 publications

(12 citation statements)

References 27 publications

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“…Trypsin works specifically by hydrolyzing the peptide bonds on the carboxylic terminal of lysines and arginines. According to the trypsin concentration and the amino acid sequence of the protein, the maximum degree of hydrolysis varies . In case of Aug, gelatin is the main reducing and capping agent for AuNCs.…”

Section: Resultsmentioning

confidence: 99%

NIR‐Emitting Gold Nanoclusters–Modified Gelatin Nanoparticles as a Bioimaging Agent in Tissue

El‐Sayed

Trouillet

Clasen

et al. 2019

Adv Healthcare Materials

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Gold nanocluster (AuNC) synthesis using a well‐distinguished polymer for nanoparticle‐mediated drug delivery paves the way for developing efficient theranostics based on pharmaceutically accepted materials. Gelatin‐stabilized AuNCs are synthesized and modified by glutathione for tuning the emission spectra. Addition of silver ions enhances the fluorescence, reaching also high quantum yield (26.7%). A simplified model can be proposed describing the nanoclusters' properties–structure relationship based on X‐ray photoelectron spectroscopy data and synthesis sequence. Furthermore, these modifications improve fluorescence stability toward pH changes and enzymatic degradation, offering different AuNCs for various applications. The impact of nanocluster formation on gelatin structure integrity is investigated by Fourier transform infrared spectrometry and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy, being important to further formulate gelatin nanoparticles (GNPs). The 218 nm‐sized NPs show no cytotoxicity up to 600 µg mL−1 and are imaged in skin, as a challenging autofluorescent tissue, by confocal microscopy, when transcutaneously delivered using dissolving microneedles. Linear unmixing allows simultaneous imaging of AuNCs–GNPs and skin with accurate signal separation. This underlines the great potential for bioimaging of this system to better understand nanomaterials' behavior in tissue. Additionally, it is drug delivery system also potentially serving as a theranostic system.

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Section: Resultsmentioning

confidence: 99%

NIR‐Emitting Gold Nanoclusters–Modified Gelatin Nanoparticles as a Bioimaging Agent in Tissue

El‐Sayed

Trouillet

Clasen

et al. 2019

Adv Healthcare Materials

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show abstract

“…Consequently, the engineered SGT tbcf (K101A, R201V) displayed the same hydrolysis capacity as commercial porcine trypsin. Future studies should investigate the application of SGT in the manufacturing of insulin and other pharmaceuticals [45].…”

Section: Discussionmentioning

confidence: 99%

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor

Zhang

Liang

Zhang³

et al. 2020

Microb Cell Fact

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Background: Trypsin has many applications in food and pharmaceutical manufacturing. Although commercial trypsin is usually extracted from porcine pancreas, this source carries the risks of infectivity and immunogenicity. Microbial Streptomyces griseus trypsin (SGT) is a prime alternative because it possesses efficient hydrolysis activity without such risks. However, the remarkable hydrolysis efficiency of SGT causes autolysis, and five autolysis sites, R21, R32, K122, R153, and R201, were identified from its autolysate. Results: The tbcf (K101A, R201V) mutant was screened by a directed selection approach for improved activity in flask culture (60.85 ± 3.42 U mL −1 , increased 1.5-fold). From the molecular dynamics simulation, in the K101A/R201V mutant the distance between the catalytical residues D102 and H57 was shortened to 6.5 Å vs 7.0 Å in the wild type, which afforded the improved specific activity of 1527.96 ± 62.81 U mg −1. Furthermore, the production of trypsin was increased by 302.8% (689.47 ± 6.78 U mL −1) in a 3-L bioreactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. Conclusions: SGT protein could be a good source of trypsin for insulin production. As a result of the hydrolysates analysis and direct selection, the activity of the tbcf (K101A, R201V) mutant increased 1.5-fold. Furthermore, the production of trypsin was improved threefold by overexpressing chaperone protein in Pichia pastoris. Future studies should investigate the application of SGT to insulin and pharmaceutical manufacturing.

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“…Consequently, the engineered SGT tbcf (K101A, R201V) showed the same hydrolysis capacity with commercial poricne trypsin. The future study should be emphasized on the application of SGT in insulin manufacture and pharmaceutical [44].…”

Section: Discussionmentioning

confidence: 99%

Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

Zhang

Liang

Zhang³

et al. 2019

Preprint

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Background: Trypsin has a plenty application in food and pharmaceutical manufacture. While, the commercial trypsin is usually extracted from pork pancreas, which has the risk of infectious and immunogenicity. Therefore, the microbial Streptomyces griseus trypsin (SGT) is a prior alternation because it processes efficient hydrolysis activity without the aforementioned risk. The remarkable hydrolysis efficiency of SGT caused its autolysis, and five autolysis sites R21, R32, K122, R153, and R201 were identified from its' autolysate. Results: The tbcf (K101A, R201V) mutant was screened by directed selection approach for improved activity in flask culture (60.85 ± 3.42 U·mL -1 , increased 1.5-fold). From the molecular dynamics simulation, the K101A/R201V mutation shortened the distant between catalytical residues D102 and H57 from 6.5 Å vs 7.0 Å, which afforded the improved specific activity 1527.96 ± 62.81 U·mg -1 . Further, the production of trypsin was increased 302.8% (689.47 ± 6.78 U·mL −1 ) in 3-L bio-reactor, with co-overexpression of chaperones SSO2 and UBC1 in Pichia pastoris. Conclusions: The SGT protein could be an adequate trypsin for insulin production. When working with hydrolysates analysis and direction selection, the tbcf (K101A, R201V) mutant increased 1.5-fold activity. Further, the production of trypsin was improved 3-fold by overexpressing chaperone protein in Pichia pastoris . The future study should be emphasized on the application of SGT in insulin manufacture and pharmaceutical.

show abstract

Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and Human Trypsins

Cited by 27 publications

References 27 publications

NIR‐Emitting Gold Nanoclusters–Modified Gelatin Nanoparticles as a Bioimaging Agent in Tissue

NIR‐Emitting Gold Nanoclusters–Modified Gelatin Nanoparticles as a Bioimaging Agent in Tissue

Improving production of Streptomyces griseus trypsin for enzymatic processing of insulin precursor

Improving production of Streptomyces griseus trypsin for its application in processing insulin precursor

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