Simultaneous extraction of phycobiliproteins and carbonic Anhydrase from Spirulina Platensis Leb-52

C-phycocyanin (C-PC), allophycocyanin (APC) and carbonic anhydrase (CA) are intracellular bioproducts produced by Spirulina platensis LEB-52. In order to extract them, a cell rupture step is required. The influence of extraction time on ultrasound-assisted extraction (UAE) was evaluated at two biomass concentrations (0.2 and 23 g/L). At biomass concentration of 0.2 g/L, 12-min UAE was sufficient to simultaneously obtain phycobiliproteins (C-PC and APC) and esterase-expressed CA. Enzyme activity of hydratase-expressed CA in cyanobacteria is very low. Therefore, to determine the activity of hydratase-expressed CA, biomass concentration of 23 g/L and 8-min extraction time were needed. UAE is an efficient method of rapid determination of phycobiliproteins in wet biomass, even though it is not a selective method which extracts an array of contaminants from biomass

Immobilization of protease extracted from Bacillus sp. P45 on different supports

Immobilization is a direct tool which not only improves the activity and stability of enzymes, but also enables their reuse. Therefore, selecting methods and supports to obtain biocatalysts with high selectivity and thermal stability is an important step. This study aimed at evaluating the immobilization of partially purified protease from Bacillus sp. P45, by different methods with the use of the following carriers: Amberlite IR® 120, montmorillonite clay, chitosan, glutaraldehyde-activated chitosan, Eupergit® C, DEAE-Cellulose® and QAE-Sephadex®. It also aimed at determining the operational stability of the derivative. The best results were obtained with the use of glutaraldehyde-activated chitosan, Amberlite IR® 120 and montmorillonite clay, with loading capacities of 25.4, 6.2 and 2.3 U/g support, respectively. Regarding the operational stability of the derivative glutaraldehyde-activated chitosan, the enzyme was found to keep 53.5% of its residual activity after being reused four times

Two-step purification and partial characterization of keratinolytic proteases from feather meal bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 ◦C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 ◦C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes

Utjecaj sastava i početne pH-vrijednosti podloge na proizvodnju karotenoida iz otpada dobivenog pri proizvodnji biodizela i parene riže

Search for naturally grown food has stimulated the biotechnological production of carotenoids. Therefore, the use of the yeast Xanthophyllomonas dendrorhous has been researched due to its abilities to assimilate different sources as substrates and to produce high amounts of carotenoids. Furthermore, alternative sources have been used as the culture medium to reduce costs and environmental impact. A potent carotenoid is astaxanthin in view of its health-promoting and antioxidative properties. It consists of different geometrical isomers with trans and cis configuration. In X. dendrorhous this carotenoid is mostly found in the trans form, but cis isomers can also be found. Carotenoid production was investigated in culture medium containing by-products such as raw glycerol (from biodiesel) and parboiled rice effluent. The effects of the culture medium components on biomass concentration and specific and volumetric productions of carotenoids were verified by the Plackett-Burman design. Cultivations were carried out with yeast Xanthophyllomonas dendrorhous NRRL Y-17268 at 25 °C and 150 rpm for 168 h. In this study, maximum production of carotenoids was obtained under the following conditions (in g/L): raw glycerol 10, glucose 10, yeast extract 10, malt extract 10 and peptone 1 at pH=6. Resulting specific and volumetric productions of carotenoids were 326.8 and 4.1 μg/g, respectively.Sve veća potražnja za organskom hranom potaknula je razvoj biotehnološke proizvodnje karotenoida. Stoga je ispitana mogućnost proizvodnje velike količine karotenoida iz različitih supstrata pomoću kvasca Xanthophyllomonas dendrorhous. Osim toga, upotrijebljeni su alternativni izvori hranjivih tvari za rast kvasca, s ciljem smanjenja troškova proizvodnje i negativnog utjecaja na okoliš. Astaksantin je karotenoid koji ima snažan pozitivni učinak na zdravlje zbog svojih antioksidativnih svojstava, a javlja se u različitim oblicima cis- i trans-izomera. U kvascu X. dendrorhous prisutan je uglavnom u trans-obliku, ali može se pronaći i u obliku cis-izomera. Ispitana je mogućnost proizvodnje karotenoida na podlozi koja sadržava nusprodukte, kao što su otpadni glicerol nastao pri proizvodnji biodizela i otpad dobiven pri proizvodnji parene riže. Pomoću Plackett-Burmanovog dizajna potvrđeno je da sastav podloge utječe na dobivenu koncentraciju biomase te specifičnu i volumetrijsku proizvodnju karotenoida. Za proizvodnju karotenoida upotrijebljen je soj kvasca Xanthophyllomonas dendrorhous NRRL Y-17268 pri temperaturi od 25 °C i brzini vrtnje od 150 rpm tijekom 168 h. Najveći je prinos karotenoida postignut uzgojem kvasca u podlozi sljedećeg sastava: otpadni glicerol 10 g/L, glukoza 10 g/L, ekstrakt kvasca 10 g/L, ekstrakt slada 10 g/L i pepton 1 g/L, pri pH=6. Pritom je specifična proizvodnja karotenoida bila 326,8 μg/g, a volumetrijska 4,1 μg/g

Optimiranje podloge za proizvodnju β-galaktozidaze iz soja kvasca Kluyveromyces marxianus CCT 7082

Seven strains of the genus Kluyveromyces were screened for β-galactosidase activity in a synthetic medium. Kluyveromyces marxianus CCT 7082 was selected as the best enzyme producer for optimization of the culture medium. A fractional factorial design 2^(4-1) was used to determine the most relevant variables of the culture medium composition for the enzyme production. The parameters studied were the concentrations of lactose, yeast extract and (NH4)2SO4 as well as pH, and all were shown to have significant effects on the production of the enzyme. Based on the results from the first factorial design, lactose, yeast extract and (NH4)2SO4 concentrations were selected to be utilized in a 2³ central composite rotatable design (CCRD). This led to a further optimization of the fermentation conditions to achieve higher enzyme activities, which reached 10.6 U/mL.Ispitana je aktivnost β-galaktozidaze u sedam sojeva vrste Kluyveromyces u sintetskoj podlozi. Za optimiranje podloge odabran je soj Kluyveromyces marxianus CCT 7082 koji je proizveo najviše enzima. Pri određivanju najznačajnijih varijabla sastava podloge za proizvodnju enzima primijenjen je reducirani faktorski plan 2^(4-1). Istraženi su sljedeći parametri: koncentracija laktoze, kvaščeva ekstrakta i (NH4)2SO4 te pH-vrijednost, pa je utvrđeno da svi parametri značajno utječu na proizvodnju enzima. Prema rezultatima prvoga faktorskog plana, za izradu 2³ centralno složenog rotacijskog plana odabrani su koncentracija laktoze, kvaščeva ekstrakta i (NH4)2SO4. Daljnjim optimiranjem uvjeta fermentacije postignuta je veća aktivnost enzima, i to čak do 10,6 U/mL

Kinetička i termička svojstva sirove i pročišćene β-galaktozidaze, upotrijebljenih za proizvodnju galaktooligosaharida

β-Galactosidase is an enzyme that catalyzes the hydrolysis of lactose. It has potential importance due to various applications in the food and dairy industries, involving lactose-reduced ingredients. The properties of two β-galactosidase enzymes, crude and purified, from different sources, Kluyveromyces marxianus CCT 7082 and Kluyveromyces marxianus ATCC 16045, were analyzed. The pH and temperature optima, deactivation energy, thermal stability and kinetic and thermodynamic parameters were determined, as well as the ability to hydrolyze lactose and produce galactooligosaccharides. Purification process improved the properties of the enzymes, and the results showed that purified enzymes from both strains had a higher optimum temperature, and lower values of Km, thus showing greater affinity for o-nitrophenyl-β-D-galactopiranoside than the crude enzymes. The production of galactooligosaccharides was also greater when using purified enzymes, increasing the synthesis by more than 30 % by both strains.β-Galaktozidaza je enzim koji katalizira hidrolizu laktoze. Primjenjuje se u prehrambenoj i mliječnoj industriji za dobivanje proizvoda sa smanjenim udjelom laktoze. U ovom su radu ispitana svojstva dvaju enzima, sirove i pročišćene β-galaktozidaze, dobivenih iz sojeva kvasca Kluyveromyces marxianus CCT 7082 i Kluyveromyces marxianus ATCC 16045. Određeni su sljedeći parametri: optimalna pH-vrijednost i temperatura, energija deaktivacije, termička stabilnost, kinetički i termodinamički parametri, te sposobnost enzima da hidrolizira laktozu i proizvede galaktooligosaharide. Pročišćavanjem su poboljšana svojstva enzima, što je vidljivo iz rezultata; pročišćeni enzimi iz oba soja imali su višu optimalnu temperaturu i niže vrijednosti Km, te veću specifičnost prema o-nitrofenil-β-D-galaktopiranozidu od sirovih enzima. Također je povećana proizvodnja galaktooligosaharida, i to za 30 % pomoću oba soja

Hydrolysates containing xylooligosaccharides produced by different strategies: structural characterization, antioxidant and prebiotic activities

" Available online 22 May 2022"This study explores the structural characterization, antioxidant and prebiotic activities of hydrolysates containing xylooligosaccharides (XOS) produced by different strategies: direct fermentation of beechwood xylan (FermBX) and enzymatic treatment of beechwood (EnzBX) and rice husk (EnzRH) xylans. EnzBX and EnzRH showed XOS with a backbone of (1 4)-linked-xylopyranosyl residues and branches of arabinose, galactose, and uronic acids. FermBX presented the highest content of total phenolic compounds (14 mg GAE/g) and flavonoids (0.6 mg QE/g), which may contribute to its antioxidant capacity 39.1 mol TE/g (DPPH), 45.7 mol TE/g (ABTS), and 79.9 mol Fe II/g (FRAP). The fermentation of hydrolysates decreased the abundance of microorganisms associated with intestinal diseases from Eubacteriales, Desulfovibrionales and Methanobacteriales orders, while stimulating the growth of organisms belonging to Bacteroides, Megamonas and Limosilactobacillus genera. The production of short-chain fatty acids, ammonia, and CO2 suggested the prebiotic potential. In conclusion, hydrolysates without previous purification and obtained from non-chemical approaches demonstrated promising biological activities for further food applications.This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001; by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) – Grant numbers 423285/2018-1 and 304857/2018-1; by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit; by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte; by the COMPETE 2020 (POCI-01-0145- FEDER-006684); by the FoSynBio (POCI-01-0145-FEDER-029549); and LAQV/REQUIMTE (UIDB/50006/2020, UIDP/50006/2020) through national founds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement. GG acknowledges the Universidade Federal do Rio Grande for the financial support from Postgraduate Student Mobility Program (PROPESP/FURG). BC, CA, and SPS acknowledge their grants (SFRH/BD/132324/2017, 2020.00293 CEECIND, and SFRH/BD/136471/2018) from FCT. LB also acknowledge FCT for the junior research contract (CEECIND/03280/2020). EC thanks the research contract (CDL-CTTRI-88-ARH/2018 – REF. 049-88-ARH/2018) funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.info:eu-repo/semantics/publishedVersio

Produção de inulinase por Kluyveromyces marxianus e purificação da enzima por cromatografia de troca ionica em coluna de leito expandido

Orientador: Maria Isabel RodriguesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Frutose é o açúcar natural mais doce, sendo uma alternativa segura para substituir a sacarose. A lnulinase tem um importante papel na produção de uutose através da hidrólise enzimática da inulina, que pode ser realizada em uma única etapa e render até 95% de conversão. Este trabalho teve por objetivos otimizar as condições de produção da enzima inulinase produzida por Kluyveromyces marxianus ATCC 16045, otimizar as condições de reação sobre a sacarose e a inulina e recuperar a enzima em coluna de leito expandido utilizando a resina de troca iônica Strearnline SP. Técnicas de planejamento experimental e análise de superficie de resposta foram utilizadas para otimizar o meio de cultura para produção da enzima e as condições de determinação da atividade enzimática. Na produção da enzima foram estudadas as variáveis concentração de sacarose, extrato de levedura, peptona, K2HP04 e pH. Para tanto utilizou-se um planejamento fatorial uacionário (25-1)para determinar as variáveis mais importantes, sendo selecionadas três para serem utilizadas em um planejamento fatorial completo. A enzima obtida foi utilizada nos ensaios de determinação enzimática para verificar os efeitos da concentração de substrato (sacarose ou inulina), pH e temperatura na atividade de inulinase. Usando-se sacarose como substrato, verificou-se que a temperatura e o pH são as variáveis mais importantes e no caso da inulina somente a temperatura, dentro das faixas estudadas. A atividade enzimática passou de 9 para Ii OU/rnL após o estudo de otimização da produção da enzima. O caldo bruto clarificado foi utilizado para determinar a isoterma e cinética de adsorção da inulinase extracelular em resina de troca iônica Strearnline SP. Através dos dados experimentais de adsorção e mediante modelagem matemática, obteve-se os parâmetros cinéticos intrínsecos e os de transporte de massa. Os valores encontrados foram: kl=6,25.1O-3rnLlU.mÍn. e k2=2,09.1O-3mÍn.-1 independentemente da concentração de meio na solução. Os parâmetros coeficiente de transferência de massa (Ks) e difusividade efetiva (Def) foram 5,03.10-2, 5,03.10-4, 5,03.10-4cm/s e 1,105.10-6, 6,58.10-7 , 3,25.10-7 cm2/s para 45%, 60% e 100% de caldo bruto na solução estudada respectivamente. Os ensaios no leito expandido com o meio não clarificado permitiram determinar o expoente de Richardson -Zaki (5,3) e a velocidade terminal (17,6 cm/min.). Através dos ensaios de adsorção obteve-se as curvas de ruptura e determinou-se o grau de expansão mais adequado para a purificação da enzima (expansão de 2 vezes). Através dos testes de purificação da inulinase diretamente do caldo bruto, obteve-se uma recuperação de 74% e um fator de purificação de 10,4 vezes para a eluição do tipo degrau.Abstract: Fructose is the sweetest sugar, being a safe alternative for sucrose. Inulinase may play an important role in fiuctose production, by enzymatic hydrolysis of inulin. The reaction occurs in a single step and it is possible to obtain up to 95% of conversion. In this work the optimization of the conditions for both inulinase production by Kluyveromyces marxianus ATCC 16045 and inulinase reaction on sucrose and inulin are proposed, as well as, the enzyme purification using an expanded bed with the ion exchanger Strearnline SP is studied. Factorial design and surface response analysis were used to optimize the culture conditions for inulinase production and the enzymatic reaction parameters. In the enzyme production the following variables were studied: sucrose, yeast extract, peptone and K2HPO4concentration and pH. The technique of factorial fiactional design (25-1)was used, resulting in the selection of three variables utilization in a complete factorial designo The inulinase produced in fermentation for Kluyveromyces marxianus was used to verify the effects of substrate concentration, pH and temperature on enzymatic reaction. Using sucrose as substrate it was verified that temperature and pH were the most important variables. However, when inulin was used as substrate only the temperature was selected. The enzymatic activity increased fiom 9 U/rnL to 110 U/rnL after production optimizationby Surface Response Analysis. In terms of enzyme purification, the filtrate of crude broth was used for the determination of the adsorption isotherm and the adsorption kinects of inulinase on the ion exchanger Strearnline SP. The kinetics parameters were: kl=6.25.1O-3rnL/U.min. and k2=2.09.1O-3min.-1. For 45%,60% and 100% of crude broth of the solution, the film coefficient (Ks) of 5.03.10-2, 5.03.10-4, 5.03.10-4 cm/s and the effective diffusion coefficient (Def) of 1.105.10-6,6.58.10-7 , 3.25.10-7 cm% were determined using the experimental results and mathematical modeling. The trials on expanded bed with crude broth were carried out to determine the Richarson - Zaki exponent (5.3) and terminal settling velocity (17.6 cm/min.). The studies of inulinase adsorption performance were used to obtain the breakthrough curves and established the best degree of bed expansion for enzyme recuperation. The inulinase was purified directly fiom an unclarified broth in 74% with a purification factor of 10.4 for elution step.DoutoradoDoutor em Engenharia de Alimento