Production of yeast extract from whey using Kluyveromyces marxianus

The yeast Kluyveromyces marxianus CBS 6556 was grown on whey to produce nucleotide-rich yeast extracts. Thermal treatments of cells at 35 or 50ºC for 15-30h resulted in yeast extracts containing about 20 g/L protein, with only the second treatment resulting in the presence of small amounts of RNA. In contrast, autolysis in buffered solution was the unique treatment that resulted in release of high amounts of intracellular RNA, being, therefore, the better procedure to produce 5'-nucletide rich extract with K. marxianus

Combi-lipase for heterogeneous substrates- a new approach for hydrolysis and transesterification using mixtures of biocatalysts

Trabajo presentado en el VII Workshop on Biocatalysis and Biotransformations - 1º Simposio Latinoamericano de Biocatalisis y Biotransformaciones, celebrado en Búzios (Brasil) del 23 al 26 de septiembre de 2014.[Introduction]: Combination of different enzymes is mainly used for cascade or sequential reactions, however, in the present work, is being proposed the design of a combi-lipase biocatalyst strategy for the simultaneous hydrolysis or transesterification of a mixture of different substrates.1,2 It is based on the combination of different lipases as biocatalysts in reactions using heterogeneous substrates, such vegetable oils.[Results and discussion]: The hydrolysis and the transesterification of different vegetable oils (soybean oil, olive oil and palm oil) were evaluated, and Novozym 435 (CALB), Lipozyme TL-IM (TLL), and Lipozyme RM-IM (RML) were used as biocatalysts. For each reaction and oil, it was evaluated the best mixture of lipase, and thereafter the reaction parameters (substrate molar ratio, temperature and biocatalyst content) were optimized using an experimental design. For hydrolysis of soybean oil, a mixture of 80 % of RML and 20 % of CALB was the best biocatalyst. Reaction parameters were optimized, allowing to obtain more than 80 % of hydrolysis in 24 h using the combi-lipase, up from less than 50 % when any individual lipase, as shown in Figure 1. In the transesterification, using olive oil and ethanol the best mixture was composed by 30 % TLL, 12.5 % of RML and 57.5 % of CALB, reaching to 98 % of conversion after 18 h, while using palm oil and ethanol the best mixture was 52.5 % of TLL and 47.5 % of RML, yielding 80 % after 18 h. Reusability of the combi-lipase showed that it could be used for at least 15 cycles without any significant decrease in hydrolysis, and for 7 cycles in transesterification.[Conclusion]: The concept of combi-biocatalyst might be a useful technology for reactions including full modification of heterogeneous substrates. Further studies are granted to test these preparations in continuous flow reactors.This work was supported by grants from FAPERGS and CNPq (Brazil) and CTQ2013-41507-R from Spanish MINECO. The authors would like to thank Mr. Ramiro Martínez (Novozymes, Spain) for kindly supplying the enzymes used in this research.Peer Reviewe

Optimization of ethyl ester production from olive and palm oils using mixtures of immobilized lipases

Although reactions of transesterification are generally catalyzed by one specific lipase preparation, the concept of “combi-lipase” could be better explored for the production of biodiesel, since oils are heterogeneous substrates. In this research, we tested this concept by evaluating the enzymatic transesterification of olive and palm oils, two diverse fatty acid compositions, using standalone or mixtures of three immobilized lipases as biocatalysts: Novozym 435 (CALB), Lipozyme TL-IM (TLL), and Lipozyme RM-IM (RML). For olive oil, the combination of 29.0% of TLL, 12.5% of RML, and 58.5% of CALB was the best, allowing for 95% conversion efficiency in 18 h of reaction, up from 50% for the best individual lipase (CALB). For palm oil, the best enzyme combination was 52.5% of TLL and 47.5% of RML, resulting in 80% of conversion of ethyl esters in 18 h, compared to only 44% when standalone TLL was used. Repeated batches of reaction were carried out in order to test the operational stability of the combi-lipase systems, with results showing that they could be used for at least seven cycles keeping higher than 80% of their initial activities.This work was supported by grants from Brazilian Coordenação de Aperfoiçoamento de Pessoal de Nível Superior (CAPES) and CTQ2013-41507-R from Spanish MINECO.Peer Reviewe

Comparison of the performance of commercial immobilized lipases in the synthesis of different flavor esters

In this work, it is compared the performance of three commercial lipase preparations (Novozym 435, Lipozyme TL-IM, and Lipozyme RM-IM) in the synthesis of flavor esters obtained by esterification of acetic, propionic, and butyric acids using ethanol, isopropyl alcohol, butanol, or pentanol. A comprehensive comparison was performed verifying activities of these three enzyme preparations versus the different couples of substrates, checking the obtained yields. In general, the longer the acid chain, the higher the reaction yields. Novozym 435 was the most efficient enzyme in most cases, and only Lipozyme RM-IM offered better results than Novozym 435 in the production of ethyl butyrate. Reactions with butyric acid showed the highest conversion rates using all biocatalysts. Using optimal substrates, the reactions catalyzed by the three enzymes were optimized using the response surface methodology, and the catalytic performance of the biocatalysts in repeated batches was assessed. After optimization, yields higher than 90% were obtained for all three enzymes, but Lipozyme TL-IM needed four-times more biocatalyst content than the other two preparations. Novozym 435 kept over 80% of its activity when reused in 9 successive batches, whereas Lipozyme RM-IM can be reused 5 times and Lipozyme TL-IM only 3 times. In general, Novozym 435 showed to be more suitable for these reactions than the other two enzyme preparations.This work was supported by grants and scholarships (first and second authors) from FAPERGS (Fundação de Amparo a Pesquisa do Rio Grande do Sul process 11/0485-0 ARD/2011), CNPq (Brazilian Bureau of Science and Technology process 478169/2011-6 Universal 2011), and CTQ2009-07568 (Ministerio de Ciencia y Innovación, Spain).Peer Reviewe

Combi-lipase for heterogeneous substrates: A new approach for hydrolysis of soybean oil using mixtures of biocatalysts

The concept of the combi-lipase biocatalyst has been proposed. It is based on the combination of different lipases as biocatalysts in reactions using heterogeneous substrates. The hydrolysis of soybean oil was evaluated as a model substrate, and Novozym 435 (CALB), Lipozyme TL-IM (TLL), and Lipozyme RM-IM (RML) were used as biocatalysts. Results showed that, although individually TLL was the most active enzyme, whereas CALB was the less active one, the combination of 80% of RML and 20% of CALB was the best biocatalyst. Reaction parameters were optimized, allowing to obtain more than 80% hydrolysis in 24 h using the combi-lipase, up from less than 50% with any individual lipase. Reusability of the combi-lipase showed that it could be used for at least 15 cycles without any significant decrease. The concept of the combi-biocatalyst might be a useful technology for reactions including full modification of heterogeneous substrates.This work was supported by grants from Fundaçao de Amparo a Pesquisa do Rio Grande do Sul (FAPERGS; ARD/2011, Brazil), from CNPq (Brazilian Bureau of Science and Technology), and CTQ2009-07568 from Spanish Ministerio de Ciencia e Innovación.Peer Reviewe