Coagulation of soy proteins induced by thermolysin and comparison of the coagulation reaction with that induced by subtilisin Carlsberg

Asaoka, K.; Yasukawa, Kiyoshi; Inouye, Kuniyo

doi:10.1016/j.enzmictec.2008.10.001

Cited by 7 publications

(3 citation statements)

References 25 publications

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“…Previous studies also reported only one mutation that considerably affects substrate specificity. 45 Protease PT121 and its mutant Y114S might be useful in the degradation and engineering of food proteins 46 and in the synthesis of bioactive peptides, such as aspartame, respectively. Further work will be conducted to engineer proteases PT121 and Y114S for enhancing yield in the synthesis of other active peptides.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis

Zhu

Liu

et al. 2016

J. Agric. Food Chem.

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Metalloprotease PT121 and its mutant Y114S (Tyr114 was substituted to Ser) are effective catalysts for the synthesis of Z-aspartame (Z-APM). This study presents the selection of a suitable signal peptide for improving expression and extracellular secretion of proteases PT121 and Y114S by Escherichia coli. Co-inducers containing IPTG and arabinose were used to promote protease production and cell growth. Under optimal conditions, the expression levels of PT121 and Y114S reached >500 mg/L, and the extracellular activity of PT121/Y114S accounted for 87/82% of the total activity of proteases. Surprisingly, purer protein was obtained in the supernatant, because arabinose reduced cell membrane permeability, avoiding cell lysis. Comparison of Z-APM synthesis and caseinolysis between proteases PT121 and Y114S showed that mutant Y114S presented remarkably higher activity of Z-APM synthesis and considerably lower activity of caseinolysis. The significant difference in substrate specificity renders these enzymes promising biocatalysts.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis

Zhu

Liu

et al. 2016

J. Agric. Food Chem.

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“…This variant and others presented as well in this study might be useful not only for elucidating the structure-function relationship of thermolysin but also for food science and technology in degradation and engineering of food proteins (Asaoka et al, 2009) and synthesis of bioactive peptides such as aspartame or l-aspartyl-l-phenylalanine methyl ester (Inouye, 1992(Inouye, , 2003Kusano et al, 2010).…”

Section: Figmentioning

confidence: 94%

Effects of the mutational combinations on the activity and stability of thermolysin

Kusano

Yasukawa

Inouye

2010

Journal of Biotechnology

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We have previously indicated that three single mutations (Leu144-->Ser, Asp150-->Glu, and Ile168-->Ala) in the site-directed mutagenesis of thermolysin increase the activity and two single (Ser53-->Asp and Leu155-->Ala) and one triple (Gly8-->Cys/Asn60-->Cys/Ser65-->Pro) mutations increase the stability. In the present study, aiming to generate highly active and stable thermolysin variants, we combined these mutations and analyzed the effect of combinations on the activity and stability of thermolysin. The combination of the mutations of Leu144-->Ser and Asp150-->Glu yielded the most significant increase in the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide (FAGLA) and N-carbobenzoxy-L-Asp-L-Phe methyl ester (ZDFM), while that of Leu144-->Ser and Ile168-->Ala abolished the activity. The combination of Ser53-->Asp and Leu155-->Ala yielded the greatest increase in the thermal stability, while that of Ser53-->Asp and Gly8-->Cys/Asn60-->Cys/Ser65-->Pro increased the stability as high as the individual mutations do. The combination of three mutations of Leu144-->Ser, Asp150-->Glu, and Ser53-->Asp yielded a variant L144S/D150E/S53D with improved activity and stability. Its k(cat)/K(m) values in the hydrolysis of FAGLA and ZDFM were 8.6 and 10.2 times higher than those of wild-type thermolysin (WT), respectively, and its rate constant for thermal inactivation at 80 degrees C was 60% of that of WT.

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“…To further expand the use of collagen in industry, a better understanding of the pepsin digestion mechanism is required, as this would allow better control over the reaction. Previously we characterized the digestion of soy proteins with subtilisin Carlsberg and with thermolysin using SDS‐PAGE, fluorescence, and circular dichroism (CD) (Asaoka and others ; Inouye and others ). In this study, we devised a method for the kinetic analysis of the digestion of bovine type I collagen with pepsin by measuring the reaction rates for the decrease in collagen's variant β11 chain by SDS‐PAGE, and examined the effects of temperatures and pH on the kinetic parameters.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin

Qian

Okada

Ogura

et al. 2015

Journal of Food Science

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Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N- and C-terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N- and in the C-terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N-terminal and in the C-terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS-PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the Km and kcat values increased with increasing temperature (30 to 65 °C), although the kcat /Km values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the Km and kcat values increased with decreasing pH, and the kcat /Km values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.

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Coagulation of soy proteins induced by thermolysin and comparison of the coagulation reaction with that induced by subtilisin Carlsberg

Cited by 7 publications

References 25 publications

Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis

Efficient Extracellular Expression of Metalloprotease for Z-Aspartame Synthesis

Effects of the mutational combinations on the activity and stability of thermolysin

Kinetic Analysis of the Digestion of Bovine Type I Collagen Telopeptides with Porcine Pepsin

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