Production Of Lactobionic Acid Research Articles

Inline monitoring of lactobionic acid production from cheese whey by Pseudomonas taetrolens in a stirred bioreactor using electrical conductivity

AbstractIn this study, we introduce a novel experimental approach and present a simplified mathematical model for a quick monitoring of a biotec process producing lactobionic acid (LBA). It relies on monitoring the electrical conductivity of the fermentation broth and it is designed to predict the concentration of LBA throughout the microbial cheese whey valorization via LBA production. Following a systematic series of experiments conducted to refine the mathematical model, we performed conductivity monitoring during LBA production from “caciotta” and “squacquerone” wheys by Pseudomonas taetrolens in a 3 L stirred tank bioreactor. Throughout the bioproduction process, the conductivity values exhibited an upward trend corresponding to the increase in LBA concentration. Our findings underscore the feasibility and advantages of employing inline conductivity monitoring during LBA production from various cheese wheys. The results emphasize that conductivity measurements can effectively estimate product concentration in a fermentation process, particularly when there is a shift in ionic concentration. Furthermore, these conductivity measurements offer valuable insights for monitoring and optimizing the working conditions in a fermentation process.

All lactose-oxidizing enzymes of Pseudomonas taetrolens, a highly efficient lactobionic acid-producing microorganism, are pyrroloquinoline quinone-dependent enzymes.

In previous and present studies, four enzymes (GCD1, GCD3, GCD4, and MQO1) have been found to act as lactose-oxidizing enzymes of Pseudomonas taetrolens. To investigate whether the four enzymes were the only lactose-oxidizing enzymes of P. taetrolens, we performed the inactivation of gcd1, gcd3, gcd4, and mqo1 genes in P. taetrolens. Compared to the wild-type strain, the lactobionic acid (LBA)-producing ability of P. taetrolens ∆gcd1 ∆gcd3 ∆gcd4 ∆mqo1 was only slightly decreased, implying that P. taetrolens possesses more lactose-oxidizing enzymes. Interestingly, the four lactose-oxidizing enzymes were all pyrroloquinoline quinone (PQQ)-dependent. To identify other unidentified lactose-oxidizing enzymes of P. taetrolens, we prevented the synthesis of PQQ in P. taetrolens by inactivating the genes related to PQQ synthesis such as pqqC, pqqD, and pqqE. Surprisingly, all three knocked-out strains were unable to convert lactose to LBA, indicating that all lactose-oxidizing enzymes in P. taetrolens were inactivated by eliminating PQQ synthesis. In addition, external PQQ supplementation restored the LBA production ability of P. taetrolens ∆pqqC, comparable to the wild-type strain. These results indicate that all lactose-oxidizing enzymes in P. taetrolens are PQQ-dependent.

Conversion of Deproteinized Cheese Whey to Lactobionate by an Engineered Neurospora crassa Strain F5.

We report a novel production process for lactobionic acid (LBA) production using an engineered Neurospora crassa strain F5. The wild-type N. crassa strain produces cellobiose dehydrogenase (CDH) and uses lactose as a carbon source. N. crassa strain F5, which was constructed by deleting six out of the seven β-glucosidases in the wild type, showed a much slower lactose utilization rate and produced a much higher level of cellobiose dehydrogenase (CDH) than the wild type. Strain N. crassa F5 produced CDH and laccase simultaneously on the pretreated wheat straw with 3µM of cycloheximide added as the laccase inducer. The deproteinized cheese whey was added directly to the shake flasks with the fungus present to achieve LBA production. Strain F5 produced about 37g/L of LBA from 45g/L of lactose in 27h since deproteinized cheese whey addition. The yield of LBA from consumed lactose was about 85%, and the LBA productivity achieved was about 1.37g/L/h.

Production of Lactobionic Acid Using Gold Nanoparticles Synthesized with Fruit Myrciaria dubia Extract

Lactobionic acid (LBA) is a polyhydroxy acid with attractive properties in the pharmaceutical, cosmetic, food, medical, and chemical industries, making it a versatile product with multiple applications, which supports the various studies aimed at its production by increasingly more simple, efficient, and environmentally friendly processes. For this reason, the purpose of this research was to synthesize gold nanoparticles (AuNPs) by a synthesis process using Myrciaria dubia (Camu camu) fruit extract. Subsequently, AuNPs were used to produce LBA from lactose. The results demonstrate that the Myrciaria dubia extract manages to synthesize AuNPs that were characterized by UV/vis spectrophotometry, energy-dispersive X-ray spectroscopy (EDX), and Zetasizer. LBA was quantified by FTIR-ATR spectroscopy and ion chromatography. The results showed that AuNPs succeeded in producing LBA from lactose showing the highest LBA production efficiency at a dose of 0.5 g/L and a temperature of 60°C. It has been shown that the AuNPs obtained by synthesis using the Myrciaria dubia extract efficiently catalyze the production of LBA from lactose, with a yield of 45.24%, which can be used to produce LBA for industrial or research purposes.

Lactobionic acid production via mutant cellobiose dehydrogenase/laccase continuous enzymatic regeneration of electron acceptors

Lactobionic acid (LBA) has diverse number of applications in pharmaceutical, food, chemical and nanotechnology fields. Bioproduction of LBA by bi-enzymatic cellobiose dehydrogenase (CDH)/laccase (LAC) is a system where CDH catalyses oxidation of lactose to LBA, whilst LAC has, at the same time, ability to regenerate the redox mediator as electron acceptor for CDH. In this study, CDH mutants derived from Phanerochaete chrysosporium produced in Pichia pastoris were used to determine LBA production rate with varying number of redox mediators. The highest specific productivity was observed for triple mutant CDH (tmCDH) for all redox mediators used in this study. The notable redox mediator was hydroquinone where specific productivity was 29.14 g L−1 kU−1 for tmCDH while specific productivity with DCIP was 27.2 g L−1 kU−1, with ABTS showed 19.1 g L−1 kU−1.

Valorization of cheese whey to lactobionic acid by a novel strain Pseudomonas fragi and identification of enzyme involved in lactose oxidation

BackgroundEfficient upgrading of inferior agro-industrial resources and production of bio-based chemicals through a simple and environmentally friendly biotechnological approach is interesting Lactobionic acid is a versatile aldonic acid obtained from the oxidation of lactose. Several microorganisms have been used to produce lactobionic acid from lactose and whey. However, the lactobionic acid production titer and productivity should be further improved to compete with other methods.ResultsIn this study, a new strain, Pseudomonas fragi NL20W, was screened as an outstanding biocatalyst for efficient utilization of waste whey to produce lactobionic acid. After systematic optimization of biocatalytic reactions, the lactobionic acid productivity from lactose increased from 3.01 g/L/h to 6.38 g/L/h in the flask. In batch fermentation using a 3 L bioreactor, the lactobionic acid productivity from whey powder containing 300 g/L lactose reached 3.09 g/L/h with the yield of 100%. Based on whole genome sequencing, a novel glucose dehydrogenase (GDH1) was determined as a lactose-oxidizing enzyme. Heterologous expression the enzyme GDH1 into P. putida KT2440 increased the lactobionic acid yield by 486.1%.ConclusionThis study made significant progress both in improving lactobionic acid titer and productivity, and the lactobionic acid productivity from waste whey is superior to the ever reports. This study also revealed a new kind of aldose-oxidizing enzyme for lactose oxidation using P. fragi NL20W for the first time, which laid the foundation for further enhance lactobionic acid production by metabolic engineering.Graphical

Acid Whey Valorization for Biotechnological Lactobionic Acid Bio-production

The dairy industry is facing a problem associated with 1.6 billion tons of acid whey per year as a waste stream. The extended amount of acid whey has encouraged studies for novel approaches of acid whey utilization. The production of lactobionic acid (LBA) using dairy waste has been in rapid demand as an economically feasible and environmentally friendly approach. The composition of acid whey makes lactose conversion into LBA by Pseudomonas taetrolens complicated. Therefore, the aim of the current research was to evaluate factors (quality of whey (salts, protein concentration, pH), volume of inoculum, and cultivation time) with the purpose of increasing the suitability of acid whey for biotechnological LBA production. LBA production was performed in a 4L bioreactor, which was equipped with a pH electrode and a dissolved oxygen electrode. The whole experiment was performed at a temperature of 30 °C under 350 rpm agitation. The continuous aeration was set at 0.5 L/min. The current study presents the study of acid and sweet whey combinations in different ratios (100:0; 50:50; 60:40, 70:30, 80:20, respectively) inoculated with 10% or 30% v/v of fresh P. taetrolens inoculum reaching up to 59.9 ± 1% LBA yield during cultivation. Increasing protein and pH in a substrate slows down the lactose converting ability of P. taetrolens. Results demonstrated that increasing the acid whey amount in a substrate can affect the LBA yield, and a combination of sweet and acid whey could be a good solution for biotechnological LBA production using dairy waste. Doi: 10.28991/HEF-SP2022-01-04 Full Text: PDF

Production of lactobionic acid at high salt concentrations by Acinetobacter halotolerans isolated from seaside soil.

A lactobionic acid (LBA)-producing bacterium isolated from seaside soils was identified as Acinetobacter halotolerans and designated as strain KRICT-1. We determined whether KRICT-1 can produce LBA at high salt concentrations. The KRICT-1 strain grew on a nutrient broth (NB) agar plate with up to 7.0% NaCl, indicating high NaCl tolerance, and 30°C was the optimum growth temperature for LBA production. We produced LBA using the KRICT-1 strain in NB medium containing various concentrations of NaCl. While Pseudomonas taetrolens, an efficient LBA-producing bacterium, could produce LBA with up to 5.5% NaCl, the KRICT-1 strain could produce LBA at up to 7.0% NaCl and produced more LBA than P. taetrolens with over 5.5% NaCl. We produced LBA using NB medium containing 7.0% NaCl by batch fermentation of the KRICT-1 strain in a 5 L fermenter. The LBA production titer and productivity of the KRICT-1 strain were 32.1g/L and 0.22g/L/h, respectively, which were approximately 1.35- and 1.38-fold higher than those (23.7g/L and 0.16g/L/h) obtained from flask culture. Additionally, quinoprotein glucose dehydrogenase is an LBA-producing enzyme in A. halotolerans. We demonstrated that the A. halotolerans KRICT-1 strain is appropriate for LBA production at high salt concentrations.

The antimicrobial and bioactive properties of lactobionic acid.

Lactobionic acid (LBA) is a bioactive molecule that has generated keen interest in different industries. However, its future application in the food area is one of the most promising. Chemically, it is a polyhydroxy acid formed by the union of two molecules (galactose and gluconic acid) linked by an ether-bond, showing many interesting and unusual properties due to its structure and composition, although it is traditionally known in the food industry for its chelating, moisturizing, gelling, and antioxidant properties. There has been much research into the production of LBA, either by microbial fermentation or biocatalytic approaches such as enzymatic synthesis, but its use in foodstuffs, to produce new functional products and to evaluate its antimicrobial activity against food-borne pathogens, is a relatively new topic that has attracted the interest of the international research community recently. Furthermore, in spite of the potential of LBA, it has been approved only by the US Food and Drug Administration, and for its use as the salt form, but the publication of new comprehensive studies, able to agglutinate all the new food-related LBA research results, could disseminate knowledge about this compound and have an influence on its current regulation status. The aim of the present review is to describe the most recent advances and research on its antimicrobial potential, as well as summarizing the significant aspects that make LBA a promising bioactive compound for the food sector. © 2022 Society of Chemical Industry.

Downstream Approach Routes for the Purification and Recovery of Lactobionic Acid.

The successful development of a lactobionic acid (LBA) bioconversion process on an industrial scale demands the selection of appropriate downstream methodological approaches to achieve product purification once the bioconversion of LBA is completed. These approaches depend on the nature of the substrate available for LBA production, and their necessary implementation could constitute a drawback when compared to the lesser effort required in downstream approaches in the production of LBA obtained by chemical synthesis from refined lactose. Thus, the aim of this research is to separate LBA from an acid whey substrate after bioconversion with Pseudomonas taetrolens. Freeze drying, crystallization, adsorption with activated carbon, microfiltration, centrifugation, and precipitation with 96% (v/v) ethanol were carried out to separate and purify LBA. The closest product to commercial LBA was obtained using precipitation with ethanol, obtaining a white powder with 95 ± 2% LBA concentration. The procedure described in this paper could help to produce LBA on an industrial scale via microbial bioconversion from acid whey, developing a promising biotechnological approach for lactose conversion.

Efficient Production of Lactobionic Acid Using Escherichia coli Capable of Synthesizing Pyrroloquinoline Quinone.

Lactobionic acid (LBA) is an emerging chemical that has been widely utilized in food, cosmetic, and pharmaceutical industries. We sought to produce LBA using Escherichia coli. LBA can be produced from lactose in E. coli, which is innately unable to produce LBA, by coexpressing a heterologous quinoprotein glucose dehydrogenase (GDH) and a pyrroloquinoline quinone (PQQ) synthesis gene cluster. Using a recombinant E. coli strain, we successfully produced LBA without additional supplementation of PQQ, and changing the type of heterologous GDH improved the LBA production titer and productivity. To further enhance LBA production, culture conditions, such as growth temperature and isopropyl-β-d-1-thiogalactopyranoside concentration, were optimized. Using optimized culture conditions, batch fermentation of the recombinant E. coli strain was performed using a 5 L bioreactor. After fermentation, this strain produced an LBA titer of 209.3 g/L, a yield of 100%, and a productivity of 1.45 g/L/h. To our best knowledge, this is the first study to produce LBA using heterologous GDH in an E. coli strain without any additional cofactors. Our results provide a simple method to produce LBA from lactose in a naturally non-LBA-producing bacterium and lay the groundwork for highly efficient LBA production in E. coli, which is one of the most versatile metabolite-producing bacterial hosts.

Lactobionic acid production by Pseudomonas taetrolens in a fed‐batch bioreactor using acid whey as substrate

Acid whey causes a major disposal problem for the dairy industry due to its low pH and high salt concentration. Lactobionic acid (LBA) production by whey fermentation is an inexpensive process. The goal of this work was to employ acid whey for LBA production. Acid whey was fermented in a bioreactor, adding Pseudomonas taetrolens LMG 2336 at 30°C and 6.5 pH, with 1.5‐Lpm oxygen aeration and 350‐rpm agitation. Three fermentation tests were carried out with a different amount of inoculum (10%, 30% and 10% three times within 24 h). Results indicated that repeatedly adding P. taetrolens inoculum to the acid whey substrate allows a complete lactose conversion into LBA, while the lactose oxidation process was insufficient in the sample where the inoculum was only added at the beginning of the fermentation process (only 29.7% LBA production with 10% inoculum). The physiological heterogeneity of P. taetrolens was determined by multiparametric flow cytometry, and results showed that there was a phenotypic adaptation of the microorganism due to the changes observed in its heterogeneity and physiological state. The results achieved will help to recycle acid whey for value‐added product production such as LBA production.

Efficient isolation of new lactobionic acid-producing microorganisms from environmental samples by colloidal calcium carbonate agar plate-based screening.

Lactobionic acid (LBA) has recently emerged as an important substance in various industries, such as cosmetics, foods, and pharmaceuticals. In this study, we developed a simple, efficient, and high-throughput method for screening LBA-producing microorganisms. First, an agar plate was prepared to isolate LBA-producing microorganisms by utilizing the property of LBA to solubilize colloidal calcium carbonate (CaCO3), resulting in the formation of a clear halo around colonies on a nutrient broth agar plate containing CaCO3. Subsequently, LBA production from the isolated microorganisms was confirmed using high-performance liquid chromatography (HPLC). Approximately 560 colonies from soil samples in Ulsan, Korea were screened and a clear halo was observed around three colonies on the prepared LBA-screening agar plate. The culture supernatants of these three colonies were analyzed by HPLC and it was found that these strains could produce LBA from lactose. Phylogenetic analysis by comparing their 16S rRNA nucleotide sequences revealed that these strains were Pseudomonas spp. and Alcaligenes faecalis. This is the first report highlighting that A. faecalis can produce LBA. As per the aforementioned results, the LBA-screening method that we devised here is highly effective for isolating and identifying new LBA-producing microorganisms.

Isolation and Characterization of a New Superior Lactobionic Acid-Producing Bacterium, Enterobacter cloacae KRICT-1, from Environmental Soil Samples

Lactobionic acid (LBA) is a specialty aldonic acid that has been broadly employed in various industries, including food, cosmetics, and pharmaceuticals. In this study, we isolated a new excellent LBA-producing bacterium from soil, designated as strain KRICT-1. A phylogenetic analysis revealed that this strain was identified as Enterobacter cloacae. Previously, we successfully produced LBA using Pseudomonas taetrolens, an excellent LBA-producing bacterium. In a flask culture at 30 °C, E. cloacae KRICT-1 showed the same LBA production concentration (209.3 g/L) and yield (100%) from 200 g/L lactose, compared with those obtained from P. taetrolens cultivated at 25 °C, the growth temperature of which showed the highest LBA production ability. E. cloacae KRICT-1 exhibited an approximately 1.08-fold higher LBA productivity (8.37 g/L/h) than that (7.75 g/L/h) of P. taetrolens, indicating that E. cloacae KRICT-1 was a more efficient LBA-producing bacterium than P. taetrolens. The LBA productivity of E. cloacae KRICT-1 further improved to 8.72 g/L/h at 35 °C. Furthermore, the LBA productivity (9.97 g/L/h) improved at 35 °C in a 5 L fermenter. After whole-genome sequencing of E. cloacae KRICT-1, we found that two quinoprotein glucose dehydrogenases (GDH1 and GDH2) from this strain could produce LBA from lactose by recombinantly expressing these genes in Escherichia coli. Our present results clearly demonstrated that E. cloacae KRICT-1 can be a new promising host for the industrial production of LBA.

Isolation of new lactobionic acid-producing microorganisms and improvement of their production ability by heterologous expression of glucose dehydrogenase from Pseudomonas taetrolens

Lactobionic acid (LBA) is a specialty organic acid that is widely employed in the food, cosmetic, and pharmaceutical industries. In the present study, we screened new LBA-producing bacteria from the soil of a poultry farm. Among the 700 bacterial colonies, five that exhibited LBA-producing ability were successfully isolated. Phylogenetic analysis based on 16 S rRNA sequences identified strain 2–15 as an Acinetobacter sp., strains 3–13 and 3–15 as Pseudomonas spp., and strains 7–7 and 7–8 as Psychrobacter spp. The LBA-producing abilities of the five strains were compared in flask culture, whereupon Psychrobacter sp. 7–8 showed the highest LBA titer (203.7 g/L), LBA yield from lactose (97.3%), and LBA productivity (2.83 g/L/h). To our best knowledge, this is the first study showing that Acinetobacter and Psychrobacter spp. can produce LBA from lactose. Our results would help broaden the spectrum of workhorse bacteria available for the industrially important microbial production of LBA. In addition, we improved the LBA-production ability of the three isolated bacteria, namely Acinetobacter sp. 2–15, Pseudomonas spp. strains 3–13 and 3–15, by heterologously expressing quinoprotein glucose dehydrogenase from Pseudomonas taetrolens. In particular, the LBA-production ability of the recombinant Pseudomonas sp. 3–13 were highly improved that the LBA titer and productivity were 19.2- (205.6 vs. 10.7 g/L, respectively) and 17.8-fold (1.07 vs. 0.06 g/L/h, respectively) higher, respectively, than those of the wild-type strain. These values were almost identical to those of the wild-type Psychrobacter sp. 7–8, which showed the highest LBA productivity among the five isolated strains. This result demonstrated that the expression of lactose-oxidizing enzyme in LBA-producing microorganisms was highly effective to enhance their LBA-production ability. Our study presents a practical method to screen for efficient LBA-producing microorganisms and to improve their production ability by genetic engineering for industrial LBA production.

Identification of a lactose-oxidizing enzyme in Escherichia coli and improvement of lactobionic acid production by recombinant expression of a quinoprotein glucose dehydrogenase from Pseudomonas taetrolens

Lactobionic acid (LBA), an aldonic acid prepared by oxidation of the free aldehyde group of lactose, has been broadly used in cosmetic, food, and pharmaceutical industries. Although Escherichia coli is unable to produce LBA naturally, a wild-type E. coli strain successfully produced LBA from lactose upon pyrroloquinoline quinone (PQQ) supplementation, indicating that E. coli contains at least one lactose-oxidizing enzyme as an apo-form. By inactivating the candidate genes in the E. coli chromosome, we found that the lactose-oxidizing enzyme of E. coli was the quinoprotein glucose dehydrogenase (GCD). To improve the LBA production ability of the E. coli strain, quinoprotein glucose dehydrogenase (GDH) from Pseudomonas taetrolens was recombinantly expressed and culture conditions such as growth temperature, initial lactose concentration, PQQ concentration, and isopropyl-β-D-1-thiogalactopyranoside induction concentration were optimized. We performed batch fermentation using a 5-L bioreactor under the optimized culture conditions determined in flask culture experiments. After batch fermentation, the LBA production titer, yield, and productivity of the recombinant E. coli strain were 200 g/L, 100 %, and 1.28 g/L/h, respectively. To the best our knowledge, this is the first report to identify the lactose-oxidizing enzyme of E. coli and to produce LBA using a recombinant E. coli strain as the production host. Because E. coli is one of the most easily genetically manipulated bacteria, our result provides the groundwork to further enhance LBA production by metabolic engineering of LBA-producing E. coli.

Biosynthesis of Lactobionic Acid in Whey-Containing Medium by Microencapsulated and Free Bacteria of Pseudomonas taetrolens

The aim of this research was to develop a method of its production from whey using bacteria of the species Pseudomonas taetrolens. Analyses of the lactobionic acid production method from whey showed that the following factors have a significant effect on its efficiency: the frequency of whey batch feeding, pH and the type of bacteria application, i.e. microencapsulated vs. free. Lactose and lactobionic acid were assayed using high performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI–MS). The highest concentration of lactobionic acid of 22.03 mg/cm3 was obtained when whey was batch fed at 72-h intervals, pH was maintained at 6.25 and bacteria were enclosed in alginate microcapsules.

Lactobionic Acid Production from Acid Whey under Different Fermentative Conditions

Lactobionic Acid Production from Acid Whey under Different Fermentative Conditions

Purification and Characterization of a Malate:Quinone Oxidoreductase from Pseudomonas taetrolens Capable of Producing Valuable Lactobionic Acid.

In this study, we successfully purified a novel lactose-oxidizing enzyme in Pseudomonas taetrolens for the first time. The purified enzyme was identified as malate:quinone oxidoreductase (MQO, EC 1.1.5.4), which showed the malate-oxidizing activity converting malate into oxaloacetate. We characterized the enzymatic properties of this interesting MQO from P. taetrolens, such as the substrate specificity toward various saccharides and the effects of temperature, pH, and metal ions on the activity and stability of MQO. MQO exhibited unique substrate specificity, as it only oxidized disaccharides with reducing-end glucosyl residues, such as lactose, but not monosaccharides. Using the high oxidizing activity of MQO toward lactose, we successfully produced lactobionic acid (LBA), a valuable organic acid used in the cosmetic, food, and pharmaceutical industries, from lactose in Escherichia coli in which the quinoprotein glucose dehydrogenase gene was inactivated, the LBA nonproducing strain, by heterologously expressing MQO with pyrroloquinoline quinone. At 37 h cultivation in a 300 mL flask culture, the LBA production, yield, and productivity of the recombinant E. coli strain were 23 g/L, 100%, and 0.62 g/L/h, respectively. This study is the first to reveal the lactose-oxidizing activity of MQO, which could be used for producing LBA in heterologous bacteria.

Enhancement of Lactobionic Acid Productivity by Homologous Expression of Quinoprotein Glucose Dehydrogenase in Pseudomonas taetrolens.

This is the first study on improving lactobionic acid (LBA) production capacity in Pseudomonas taetrolens by genetic engineering. First, quinoprotein glucose dehydrogenase (GDH) was identified as the lactose-oxidizing enzyme of P. taetrolens. Of the two types of GDH genes in P. taetrolens, membrane-bound (GDH1) and soluble (GDH2), only GDH1 showed lactose-oxidizing activity. Next, the genetic tool system for P. taetrolens was developed based on the pDSK519 plasmid for the first time, and GDH1 gene was homologously expressed in P. taetrolens. Recombinant expression of the GDH1 gene enhanced intracellular lactose-oxidizing activity and LBA production of P. taetrolens in flask culture. In batch fermentation of the recombinant P. taetrolens using a 5 L bioreactor, the LBA productivity of the recombinant P. taetrolens was approximately 17% higher (8.70 g/(L h)) than that of the wild type (7.41 g/(L h)). The LBA productivity in this study is the highest ever reported using bacteria as production strains for LBA.