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RESEARCH BACKGROUND: Microbial ß-fructofuranosidases are widely employed in food industry to produce inverted sugar or fructooligosaccharides. In this study, a newly isolated Aspergillus carbonarius PC-4 strain was used to optimize the ß-fructofuranosidase production in a cost-effective process and the sucrose hydrolysis was evaluated to produce inverted sugars. EXPERIMENTAL APPROACH: Optimization of nutritional components of culture medium was carried out using simplex lattice mixture design for 72 and 120 h at 28 °C. One-factor-at-a-time methodology was used to optimize the physicochemical parameters. Crude enzyme was used for sucrose hydrolysis at different concentrations. RESULTS AND CONCLUSIONS: The optimized conditions of enzyme production were achieved from cultivations containing pineapple crown waste (1.3%, m/V) and yeast extract (0.3%, m/V) after 72 h with an enzyme activity of 9.4 U/mL, obtaining R2=91.85%, R2 adjusted=85.06%, highest F-value (13.52) and low p-value (0.003). One-factor-at-a-time used for optimizing the physicochemical conditions showed optimum temperature (20 °C), pH (5.5), agitation (180 rpm) and time course (72 h) with a 3-fold increase of enzyme production. The invertase-induced sucrose hydrolysis showed the maximum yield (3.45 mmol of reducing sugars) using 10% of initial sucrose concentration. Higher sucrose concentrations caused the inhibition of invertase activity, possibly due to the saturation of substrate or formation of sucrose aggregates, making it difficult for the enzyme to access sucrose molecules within the created clusters. Therefore, a cost-effective method was developed for the invertase production using agroindustrial waste and the produced enzyme can be used efficiently for inverted sugar production at high sucrose concentration. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study presents an efficient utilization of pineapple crown waste to produce invertase by a newly isolated Aspergillus carbonarius PC-4 strain. This enzyme exhibited a good potential for inverted sugar production at high initial sucrose concentration, which is interesting for industrial applications.

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The Cunninghamella echinulata PA3S12MM fungus is a great producer of invertases in a growth medium supplemented by apple peels. The enzyme was purified 4.5 times after two chromatographic processes, and it presented a relative molecular mass of 89.2 kDa. The invertase reached maximum activity at pH of 6 and at 60°C, in addition to presenting stability in alkaline pH and thermal activation at 50°C. The enzymatic activity increased in the presence of Mn2+ and dithiothreitol (DTT), while Cu2+ and Z2+ ions inhibited it. Also, DTT showed to protect enzymatic activity. The apparent values for Km , Vmáx , and Kcat for the sucrose hydrolysis were, respectively, 173.8 mmol/L, 908.7 mmol/L min-1 , and 1,388.79 s-1 . The carbohydrate content was of 83.13%. The invertase presented hydrolytic activity over different types of glycosidic bonds, such as α1 â†” 2ß (sucrose), α1 â†’ 4 (polygalacturonic acid), α1 â†’ 4 and α1 â†’ 2 (pectin), and α1 â†” 1 (trehalose), indicating that the enzyme is multifunctional. Thus, the biochemical properties showed by the C. echinulata PA3S12MM suggest a broad industrial application, such as in the biomass hydrolysis or in the food industry. PRACTICAL APPLICATIONS: Invertases are hydrolytic enzymes employed in several industrial sectors. Given their great importance for the economy and several industrial sectors, there is a growing interest in microorganisms producing this enzyme. The analysis of the biochemical properties of invertase in C. echinulata PA3S12MM suggest applications in the food industry. Due to its increased hydrolytic activity, the hydrolysis process of the sucrose may employ invertase for the production of invert sugar. The stability at alkaline pH suggests an application in the development of enzymatic electrodes for the quantification of sucrose in food and beverage. The multifunctional activity may work in the biomass hydrolysis or saccharification of by-products for the extraction of fermentable sugars. The high level of invertase N-linked glycosylation of invertase grants this enzyme thermal stability at high temperatures, in addition to resistance against the action of proteases, which are desirable characteristics for the application of this enzyme in industrial processes.

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Sucrose hydrolysis by invertase [EC.3.2.1.26] produces inverted sugar syrup, an ingredient mainly used in the food industry. To properly catalyze hydrolysis, the enzyme should be reused after this reaction. It is advisable to maintain constant activity over a considerable period. Thus, sucrose hydrolysis was performed in a membrane bioreactor - a continuously stirred tank reactor coupled with an ultrafiltration membrane (UFM) which provides good diffusion and high activity per unit volume. Molecular weight cut-off for soluble invertase UFMs was up to 100kDa. This study focused on the role of UFM invertase cut-off as it is the main element in the process. We demonstrated that both the cut-off and chemical nature of the UFM affected specific invertase activity.

A hidrólise da sacarose através da invertase [EC.3.2.1.26] gera xarope de açúcar invertido, que é usado principalmente como ingrediente na indústria alimentícia. Para ter-se uma hidrólise satisfatória, a enzima deve ser reaproveitada após a reação. É desejável que sua atividade seja mantida constante durante um período considerável de reação. A hidrólise da sacarose foi realizada em um reator com membrana (RM) - que é um reator continuamente agitado acoplado a uma membrana de ultrafiltração (MUF) -, porque apresenta efeitos mínimos de difusão e elevada atividade por unidade de volume. O corte molecular da MUF utilizada para reter a invertase solúvel dentro do MR foi de até 100kDa. Como a invertase é o elemento principal deste processo, este trabalho foi focado no papel do corte molecular da MUF na sua atividade. O corte molecular e a natureza química da membrana-UF mostraram afetar a atividade específica da invertase.

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