Ficin: A protease extract with relevance in biotechnology and biocatalysis

Due to the problems raised by the use of animal or microbial recombinant proteases, the use of proteases from vegetable origin is becoming increasingly popular.. Among them, sulfidryl proteases have a special interest. Ficin is an outstanding example of this kind of proteases. This paper aims to be to make a comprehensive review of the recent uses of this enzyme, including for example protein hydrolysis, the production of bioactive peptides and antibodies fragments (researchers point that ficin results are more reproducible than using other proteases), meat tenderization, milk coagulations in cheese making or peptide synthesis. Efforts to get industrial immobilized biocatalysts of the enzyme will be also described. The review shows the huge potential and brilliant prospect that this enzyme can have in the near future.We gratefully recognize the support from the Ministerio de Ciencia e Innovación from Spanish Government (project number CTQ2017-86170-R). The FPU fellowship (Ministerio de Educacion) for Mr. Morellon–Sterling and the fellowship for Mr. Siar from the Algerian Ministry of Higher Education and Scientific Research are also thanked

Isolation, solubility and in vitro hydrolysis of chickpea vicilin-like protein

The chickpea vicilin-like globulin was isolated and chromatographed on Sepharose CL-6B and Sephacryl S-300. The native globulin with a molecular weight of 140 kDa was resolved in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in seven polypeptide bands in the range of 12.4-67 kDa. The solubility profile of the protein in water and NaCl solutions was typical of a legume globulin. The purified vicilin-like globulin, native and heated, was hydrolyzed by pepsin, trypsin and chymotrypsin. The hydrolysis patterns indicated that the native vicilin-like protein was only partially degraded by the enzymes in comparison with casein. Heating increased its susceptibility to hydrolysis relative to the native form, for all the enzymes. However, the results obtained by the pH-drop method revealed that the in vitro digestibility of the vicilin-like protein was not altered by heating, while 11 S-like and total globulins suffered a small increase, indicating that the structural characteristics of storage globulins may be important factors limiting the protein digestion. (c) 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved

Influência da germinação e do processamento térmico na digestibilidade proteica e atividade de inibição de tripsina de grãos de quinoa The effect of germination and heat treatment on the protein digestibility and trypsin inhibition activity of quinoa grains

Em função de sua versatilidade e indicativos de alto valor nutritivo, a quinoa tem despertado crescente interesse dos pesquisadores das áreas de ciências nutricionais e de alimentos, bem como dos consumidores, que visam cada vez mais ao consumo de produtos associados à promoção da saúde ou alternativos para aqueles com necessidades específicas, como os celíacos, que encontram na quinoa uma possibilidade de consumo. Neste trabalho, avaliaram-se alterações relativas à qualidade proteica dos grãos, nos seguintes aspectos: a atividade de inibição de proteases e a digestibilidade proteica in vitro, em função de modificações sofridas por processo de germinação de 2, 4 e 6 dias, além de diferentes tipos de processamentos térmicos, incluindo-se aquecimentos brandos, a 40 ºC e 45 ºC, e cozimento sob fervura. O processo de germinação não proporcionou melhorias na digestibilidade proteica dos grãos de quinoa, embora tenha sido possível verificar uma redução na atividade de inibição de tripsina ao longo da germinação. Diversamente, os processos envolvendo tratamento térmico se mostraram efetivos em melhorar a qualidade proteica dos grãos, ainda quando as temperaturas de 40 ºC e 45 ºC foram utilizadas. Utilizando-se temperatura de apenas 45 ºC para tratamento dos grãos, seus valores de digestibilidade proteica foram aumentados a ponto de serem equivalentes ao observado para o cozimento tradicional dos grãos, realizado sob fervura, o que pode ser uma observação positiva aos que optam por consumo de grãos minimamente processados.Due its versatility and indications concerning its high nutritive value, quinoa has attracted growing interest from food and nutrition researchers, as also from consumers who seek healthier or alternative food products. These foods are of particular relevance for people with specific needs such as those suffering from celiac disease. In this study changes occurring in some of the nutritional characteristics of the quinoa seed proteins, such as protease inhibition and in vitro protein digestibility, were evaluated during the germination process (2, 4 and 6 days) and after different heat treatments, including mild heating at 40 ºC and 45 ºC, and boiling. The germination processes evaluated here caused a significant decrease in the trypsin inhibition activity, but did not increase protein digestibility. However all the heat treatments used caused improvements in protein digestibility, even at low temperatures. The heat treatment at 45 ºC for 30 minutes was sufficient to increase the protein digestibility to the same level as that produced by boiling, which could be a positive observation for those who consume minimally processed grains

Optimization of the immobilization of sweet potato amylase using glutaraldehyde-agarose support. Characterization of the immobilized enzyme

A simplified procedure for the preparation of immobilized beta-amylase using non-purified extract from fresh sweet potato tubers is established in this paper, using differently activated agarose supports. Beta-amylase glutaraldehyde derivative was the preparation with best features, presenting improved temperature and pH stability and activity. The possibility of reusing the amylase was also shown, when this immobilized enzyme was fully active for five cycles of use. However, immobilization decreased enzyme activity to around 15%. This seems to be mainly due to diffusion limitations of the starch inside the pores of the biocatalyst particles. A fifteen-fold increase in the Km was noticed, while the decrease of Vmax was only 30% (10.1 U mg−1 protein and 7.03 U mg−1 protein for free and immobilized preparations, respectively).We would like to thank CNPq and PADC-FCFAr for financial support. We gratefully recognize the support from the Spanish Government, grant CTQ2009-07568.Peer Reviewe

Biotechnological Applications of Proteases in Food Technology

This review presents some of the hottest topics in biotechnological applications: proteases in biocatalysis. Obviously, one of the most relevant areas of application is in the hydrolysis of proteins in food technology, and that has led to a massive use on proteomics. The aim is to identify via peptide maps the different proteins obtained after a specific protease hydrolysis. However, concepts like degradomics are also taking on a more relevant importance in the use and study of proteases and will also be discussed. Other protease applications, as seem in cleaning (detergent development), the pharmaceutical industry, and in fine chemistry, will be analyzed. This review progresses from basic areas such as protease classification to a discussion of the preparation of protease‐immobilized biocatalysts, considering the different problems raised by the use of immobilized proteases due to the peculiar features of the substrates, usually large macromolecules. Production of bioactive peptides via limited hydrolysis of proteins will occupy an important place in this review.The authors are grateful to Univ. Federal de Alfenas – MG- Brazil. This work was partially supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) projects number CTQ2013-41507-R and CTQ2017-86170-R. A.B.M. thanks MINECO, Generalitat Valenciana and FEDER (CTQ2015-66080-R MINECO/FEDER and PROMETEOII/2014/010) for financial support

The Immobilization and Stabilization of Trypsin from the Porcine Pancreas on Chitosan and Its Catalytic Performance in Protein Hydrolysis

In this study, trypsin from the porcine pancreas was immobilized on a heterofunctional support prepared by activating chitosan (Chit) hydrogel with glutaraldehyde (GA), then functionalizing it with glycine (Chit–GA–Gly). The catalytic performance of the immobilized trypsin in the hydrolysis reactions was compared with the catalytic performance of the immobilized enzyme on glutaraldehyde-activated chitosan (Chit–GA) and chitosan hydrogel (Chit). The maximum concentration of immobilized protein on Chit–GA–Gly was approximately 16 mg·g−1 at pH 9.0 (5 mmol·L−1 buffer sodium carbonate) at 25 °C from an offered protein loading of 20 mg·g−1. This biocatalyst exhibited maximum specific activity (SA) of 33.1 ± 0.2 nmol·min−1·mg−1 for benzoyl-DL-arginine-p-nitroanilide (BAPNA) hydrolysis, twice as high as the enzyme immobilized on the classic Chit–GA support (SA values ranging between 6.7 ± 0.1 nmol·min−1·mg−1 and 8.1 ± 0.1 nmol·min−1·mg−1). The Elovich kinetic model was used to describe the adsorption process using low (3 mg·g−1) and high (20 mg·g−1) initial protein loadings. The optimum temperature for BAPNA hydrolysis catalyzed by the immobilized trypsin (60 °C) was 10 °C higher than that of its soluble form. Additionally, the immobilized enzyme was 16 to 20 times more stable than its soluble form at 50–55 °C. Thermodynamic studies were conducted to elucidate the kinetics of the thermal inactivation process of soluble and immobilized forms. Complete hydrolysis of bovine serum albumin (BSA) at 37 °C was achieved after 2 h using a soluble enzyme, while for its immobilized form, the hydrolysis yield was 47%. Reuse tests revealed that this biocatalyst retained 37% of its original activity after 10 successive hydrolysis batches. Based on these results, this support could be used as an interesting alternative for producing heterogeneous biocatalysts with high catalytic activity and thermal stability when producing protein hydrolysates

Nutritional Responses of Rats to Diets Based on Chickpea (Cicer arietinum L.) Seed Meal or Its Protein Fractions

The aim of this study was to isolate the protein fractions from chickpea, var. IAC-Marrocos, as well as to evaluate its in vivo nutritional protein quality. Among the proteins, albumins showed better nutritional value in the in vivo assays and amino acid contents, despite their higher trypsin inhibitor contents. Trypsin inhibitors were found to be heat labile in all samples, but the digestibility results for unheated and heated flour and albumins suggest that their contents are not very decisive. The PER values for casein (not supplemented) were very similar to those of heated flour and unheated or heated albumin and total globulins. The albumin and glutelin fractions showed the best results for PDCAAS, however, lower than those of casein. Despite the high digestibility of the globulin the very low essential amino acid content lowered its PDCAAS, and it had the lowest values

Effect of chickpea (Cicer arietinum L.) germination on the major globulin content and in vitro digestibility

A germinação das sementes de grão-de-bico foi acompanhada por um período de 6 dias, no qual pequenas variações nos teores de nitrogênio e globulina total foram registradas. A globulina majoritária (tipo 11 S) apresentou maiores variações após o quarto dia de germinação. A natureza e distribuição da fração globulina majoritária isolada na cromatografia em Sepharose CL-6B mostrou pequenas modificações ao final do período de germinação. A eletroforese em gel de poliacrilamida com dodecilssulfato de sódio do pico eluído na cromatografia em Sepharose CL-6B demonstra modificações nas bandas de proteínas entre os pesos moleculares de 20 e 30 kDa e acima de 60 kDa, indicando degradação protéica durante o período. Atividade proteolítica foi detectada na fração albumina da semente que aumentou até o quarto dia, seguido de queda até o sexto dia de germinação, quando da utilização de globulina total isolada da semente e caseína como substratos. Farinha de grão-de-bico, frações albumina e globulina total isoladas não apresentaram aumento na digestibilidade in vitro; entretanto, a fração globulina majoritária isolada foi mais suscetível à hidrólise após germinação.Chickpea seed germination was carried out over a period of 6 days. Little variation in the nitrogen and total globulin content was observed. The major globulin (11 S type) showed higher variation after the 4th day of germination. The elution behaviour and distribution of the isolated major globulin fraction on Sepharose CL-6B chromatography showed little modification at the end of germination. on SDS-PAGE the peak eluted from Sepharose CL-6B showed changes in protein bands between 20 and 30 kDa and above 60 kDa, indicating protein degradation during the period. Proteolytic activity was detected in the albumin fraction of the seeds, which increased up to the fourth and then decreased up to the sixth day, when isolated chickpea total globulin and casein were used as substrates. Chickpea flour, isolated albumin and total globulin fractions did not show an increase for in vitro digestibility; however, the isolated major globulin was more susceptible to hydrolysis after germination