Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation / Otimização da produção de asparaginase de Zymomonas mobilis por fermentação contínua

Asparaginase is an enzyme used in clinical treatments as a chemotherapeutic agent and in food technology to prevent acrylamide formation in fried and baked foods. Asparaginase is industrially produced by microorganisms, mainly gram-negative bacteria. Zymomonas mobilis is a Gram-negative bacterium that utilizes glucose, fructose and sucrose as carbon source and has been known for its efficiency in producing ethanol, sorbitol, levan, gluconic acid and has recently aroused interest for asparaginase production. Current assay optimizes the production of Z. mobilis asparaginase by continuous fermentation using response surface experimental design and methodology. The studied variables comprised sucrose, yeast extract and asparagine. Optimized condition obtained 117.45 IU L-1 with dilution rate 0.20 h-1, yeast extract 0.5 g L-1, sucrose 20 g L-1 and asparagine 1.3 g L-1. Moreover, carbon:nitrogen ratio (1:0.025) strongly affected the response of asparaginase activity. The use of Z. mobilis by continuous fermentation has proved to be a promising alternative for the biotechnological production of asparaginase.(AU)

A asparaginase é uma enzima usada em tratamento clínico como agente quimioterapêutico e em tecnologia de alimentos na prevenção de formação de acrilamida em alimentos fritos e assados. Asparaginase é industrialmente produzida por micro-organismos, principalmente bactérias gram negativas. Zymomonas mobilis é uma bactéria gram negativa que utiliza glicose, frutose e sacarose como fonte de carbono e é conhecida por sua eficiência para produzir etanol, sorbitol, levana, ácido glicônico, e mais recentemente, tem despertado interesse no uso desse micro-organismo na produção de asparaginase. Este trabalho teve como objetivo otimizar a produção de asparaginase de Z. mobilis por fermentação contínua, pelo uso do delineamento experimental e da metodologia da superfície de resposta, testando as variáveis: sacarose, extrato de levedura e asparagina. A condição ótima alcançada, com produção de 117,45 UI L-1 foi na taxa de diluição 0,20 h-1, utilizando 0,5 g L-1 de extrato de levedura, 20 g L-1 de sacarose e 1,3 g L-1 de asparagina. Observou-se que a relação carbono:nitrogênio (1:0,025) exerceu forte influência na resposta da atividade de asparaginase. A utilização de Z. mobilis por fermentação contínua demonstrou ser uma alternativa promissora na produção biotecnológica da asparaginase.(AU)

Naringinase de Aspergillus niger: otimização da produção por metodologia de superfície de resposta e uso do ultrassom para extração / Nariginase of Aspergillus niger: production optimization by response surface methodology and utilisation of ultrasound for extraction

O Brasil é o maior produtor mundial de laranja e de suco concentrado destinado à exportação. Sucos cítricos concentrados para exportação com níveis elevados de naringina são excessivamente amargos, o que reduz a qualidade e o valor comercial do produto. A redução do amargor pode ser obtida pela naringinase, um complexo enzimático que degrada a naringina. Neste trabalho Aspergillus niger 426 foi usado como produtor de naringinase utilizando matérias-prima da agroindústria, melaço como fonte de carbono e extrato de levedura como fonte de nitrogênio. Naringina foi usada como indutor. Dentre as cinco fontes de nitrogênio pesquisadas, extrato de levedura apresentou o melhor resultado de 225,6 mU/mL de naringinase em 120 horas de cultivo. Para otimização da produção de naringinase, foi aplicada a metodologia de superfície de resposta, com planejamento fatorial incompleto 22, obtendo 354,26 mU/mL de atividade de naringinase, tendo como variáveis independentes o extrato de levedura (14,0g/L) e naringina (0,2g/L). Quando aplicadas ondas de ultrassom com intensidade de 20 kHz por 2 minutos na cultura, a atividade de naringinase atingiu o valor máximo de 473,6 mU/mL. Assim, a naringinase, enzima de grande potencial de aplicação biotecnológica, teve a produção aumentada pelo uso do planejamento estatístico e ultrassom.

Brazil is the world's largest producer of orange and concentrated juice for export. Concentrated juice with high levels of naringin has excessive bitterness, which reduces the quality and value on the market. The debittering can be obtained by using naringinase, an enzymatic complex that degrades naringin. This study reports the production of naringinase by Aspergillus niger 426 utilizing both sugar cane molasses as carbon source and yeast extract as nitrogen source. Naringin was used as inducer. Five nitrogen sources were studied and yeast extract was found as the best one as 225.6 mU/mL of naringinase activity at 120 hours of fermentation was achivied. For optimization of naringinase production it was applied response-surface methodology, with 22 incomplete factorial design. An activity value of 354.26 mU/mL of naringinase was achivied when as independent variables yeast extract (14.0g/L) and naringin (0.2g/L) were used. When applied ultrasound waves at 20 kHz of intensity for 2 minutes in the fermented broth, the activity reached the highest value of 473.6 mU/mL. Thus, naringinase, this enzyme of great potential for biotechnological applications, has its production increased by using statistical design and ultrasound.

Evaluation of supplementation of sucrose medium on the synthesis of Zymomonas mobilis bio-products / Avaliação da suplementação do meio de sacarose na formação de bio–produtos por Zymomonas mobilis

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

A influência das variáveis: ácido pantotênico, extrato de levedura, cloreto de sódio, e a técnica de permeabilização celular foram investigadas na formação de levana, sorbitol, etanol e biomassa de Zymomonas mobilis utilizando um delineamento estatístico fatorial fracionado 24-1. A biomassa foi determinada por turbidimetria, Os açúcares redutores foram quantificados por Somogy e Nelson, açúcar total por Fenol Sulfúrico, sorbitol por HPLC e etanol por micro-destilação. A levana produzida foi precipitada com etanol absoluto e determinada como unidade de frutose. Na biossíntese de levana, a variável que mais contribuiu foi a condição celular. Os resultados sugerem que, para a formação da biomassa e etanol, os fatores que mais interferiram foram a concentração de cloreto de sódio e a condição celular que influencia negativamente a produção. Para o sorbitol, a variável que teve efeito significativo foi a permeabilização celular que atuou diminuindo a sua síntese. Estudos que ampliam a faixa de variação dos fatores estabelecidos são interessantes.

Evaluation of supplementation of sucrose medium on the synthesis of Zymomonas mobilis bio-products - doi: 10.4025/actascibiolsci.v32i3.1519

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

Evaluation of supplementation of sucrose medium on the synthesis of Zymomonas mobilis bio-products / Avaliação da suplementação do meio de sacarose na formação de bio–produtos por Zymomonas mobilis

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.(AU)

A influência das variáveis: ácido pantotênico, extrato de levedura, cloreto de sódio, e a técnica de permeabilização celular foram investigadas na formação de levana, sorbitol, etanol e biomassa de Zymomonas mobilis utilizando um delineamento estatístico fatorial fracionado 24-1. A biomassa foi determinada por turbidimetria, Os açúcares redutores foram quantificados por Somogy e Nelson, açúcar total por Fenol Sulfúrico, sorbitol por HPLC e etanol por micro-destilação. A levana produzida foi precipitada com etanol absoluto e determinada como unidade de frutose. Na biossíntese de levana, a variável que mais contribuiu foi a condição celular. Os resultados sugerem que, para a formação da biomassa e etanol, os fatores que mais interferiram foram a concentração de cloreto de sódio e a condição celular que influencia negativamente a produção. Para o sorbitol, a variável que teve efeito significativo foi a permeabilização celular que atuou diminuindo a sua síntese. Estudos que ampliam a faixa de variação dos fatores estabelecidos são interessantes.(AU)

Uso de matérias primas da agroindústria: garapa e extrato de levedura: na produção de asparaginase por Zymomonas mobilis CP4 / Utilization of raw materials from agroindustry: sugar cane juice and yeast extract: for asparaginase production by Zymomonas mobilis CP4

Garapa e extrato de levedura foram usados na produção de asparaginase por Zymomonas mobilis CP4. Na otimização utilizou metodologia de superfície de resposta com 2 variáveis (extrato de levedura e asparagina) em 3 níveis (1,0; 5,5 e 10,0 g/L) e uma repetição do ponto central. A fermentação em batelada utilizou garapa diluída a 8 % (P/V) de Açúcares Totais e inóculo de Zymomonas mobilis CP4 na concentração de 2 mg/mL. Após a fermentação de 18 horas, a maior produção obtida de asparaginase foi de 9,75 U/L em extrato de levedura em 5,5 g/L e asparagina em 1,0 g/L.(AU)

Sugar cane juice and yeast extract have been used for asparaginase production by Z. mobilis CP4. A complete factorial design of two variables (yeast extract and asparagin) at three levels (1.0; 5.5 and 10.0 g/L) with one replication at the central point was used. Batch fermentation utilised sugar cane juice diluted at 8 % (W/V) of Total Sugars and an inoculum of 2 mg of cells/mL. After fermentation time of 18 hours, the highest production of asparaginase was 9.75 U/L using both yeast extract (5.5 g/L) and asparagin (1.0 g/l).(AU)

Evaluation of supplementation of sucrose medium on the synthesis of Zymomonas mobilis bio-products - doi: 10.4025/actascibiolsci.v32i3.1519

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

Mixotrophic growth of Nostoc sp. on glucose, sucrose and sugarcane molasses for phycobiliprotein production / Crescimento mixotrófico de Nostoc sp. Glucose, sacarose e melaço de cana-de-açúcar foram testados como substratos para produção de biomassa e ficobiliproteinas - DOI: 10.4025/actascibiolsci.v29i1.121

Glucose, sacarose, and sugarcane molasses were tested as substrates for production of biomass and phycobiliproteins by Nostoc sp., varying their concentrations in relation to a mineral medium, BG11. All substrates increased the biomass and phycobiliproteins when compared with the control. Sugarcane molasses showed to be the best substrate for production of both biomass and phycobiliproteins. Greater biomass production occurred in sugarcane molasses 1.0 g L-1 and it was 5.7 times greater than the control. With glucose, it was in 2.5 g L-1 and sucrose, in 1.5 g L-1, reaching 2.5 and 4.8 times greater than the control, respectively. For phycobiliproteins, the major production was in sugarcane molasses 1.0 g L-1, 12.5 times greater than the control. With glucose, it was in 1.0 g L-1 and sucrose, in 0,5 g L-1, reaching 3.0 and 4.5 times greater than the control, respectively. The Nostoc sp. assayed can grow mixotrophically, using glucose, sucrose, and sugarcane molasses as organic substrates, and a greater production of biomass and phycobiliproteins can be reached when compared with the autotrophic growth.

Todos os substratos aumentaram a biomassa e ficobiliproteinas em relação ao controle, meio mineral BG11. Melaço de cana-de-açúcar foi o melhor substrato tanto para a produção de biomassa como de ficobiliproteinas. A maior produção de biomassa ocorreu usando melaço de cana-de-açúcar 1,0 g L-1 sendo 5,7 vezes maior que o controle. Com glucose foi em 2,5 g L-1 e sacarose 1,5 g L-1, sendo 2,5 e 4,8 vezes maior que o controle, respectivamente. A maior produção de ficobiliproteinas ocorreu usando melaço de cana-de-açúcar 1,0 g L-1 sendo 12,5 vezes maior que o controle. Com glucose foi em 1,0 g L-1 e sacarose 0,5 g L-1, 3,0 e 4,5 vezes maior que o controle, respectivamente. Nostoc sp. testado pode crescer mixotroficamente, usando glucose, sacarose e melaço de cana-deaçúcar como substratos orgânicos, uma maior produção de biomassa e ficobiliproteinas podendo ser alcançada nessas condições quando comparadas com o crescimento autotrófico.