Production and characterization of recombinant Aliivibrio fischeri L-Asparaginase with low L-Glutamine affinity: a potential antileukemic drug obtained by genetic engineering

L-Asparaginase has been successfully applied in the treatment of lymphoid malignancies. Some limitations in the use of the commercial preparations of this drug include several side effects that may be correlated to L-Glutaminase activity, as immunosuppressive effects (Castro et al., 2021). The objective of this study was to evaluate the characteristics of a novel engineered Aliivibrio fischeri L-asparaginase type II expressed by Bacillus subtilis. Cultivations were carried out in shaken flasks at 30 ºC, 200 rpm, 24 h, using Luria-Bertani medium. Intracellular enzyme was recovered by sonication and enzymatic activities were evaluated by Nessler colorimetric method (Mashburn; Wriston, 1963). Recovered enzymatic extracts achieved L-Asparaginase activity up to 1.43 U.mL-1 at optimum pH 7.5. Substrate affinity was much higher for L-Asparagine than for L-Glutamine (Km= 1.226 mmol.L-1 and Km= 28.584 mmol.L-1, respectively), which indicate the potential application of the recombinant enzyme as biopharmaceutical.publishe

L-asparaginase production review: bioprocess design and biochemical characteristics

In the past decades, production of biopharmaceuticals has gained high interest due to its high sensitivity, specificity and lower risk of negative effects to patients. Biopharmaceuticals are mostly therapeutic recombinant proteins produced through biotechnological processes. In this context, L-Asparaginase (L-Asparagine amidohydrolase, L-ASNase (E.C. 3.5.1.1)) is a therapeutic enzyme that has been abundantly studied by researchers due to its antineoplastic properties. As a biopharmaceutical, L-ASNase has been used in the treatment of acute lymphoblastic leukemia (ALL), acute myeloblastic leukemia (AML) and other lymphoid malignancies, in combination with other drugs. Besides its application as a biopharmaceutical, this enzyme is widely used in food processing industries as an acrylamide mitigation agent and as a biosensor for the detection of L-Asparagine in physiological fluids at nano-levels. The great demand for L-ASNase is supplied by recombinant enzymes from Escherichia coli and Erwinia chrysanthemi. However, production processes are associated to low yields and proteins associated to immunogenicity problems, which leads to the search for a better enzyme source. Considering the L-ASNase pharmacological and food importance, this review provides an overview of the current biotechnological developments in L-ASNase production and biochemical characterization aiming to improve the knowledge about its production.publishe

Vinasse Treatment within the Sugarcane-Ethanol Industry Using Ozone Combined with Anaerobic and Aerobic Microbial Processes

The production of ethanol from sugarcane or molasses generates vinasse, a residue rich in organic matter and minerals. Vinasse is often used in fertilization and irrigation practices, which may be linked to negative environmental outcomes if excess is applied. Herein, we introduce a novel alternative to the treatment of vinasse promoting the reduction in Chemical Oxygen Demand (COD) levels, phenolic compounds, and its mineral content through the coupling of ozone treatment, anaerobic digestion, and the aerobic growth of fungi. The ozone treatment is able to remove about 30% of the total COD, and deplete the concentration of phenolic compounds, while anaerobic digestion produces biogas and generates vinasse digestate, which is less biorecalcitrant than raw vinasse. The aerobic fungal growth generates oleaginous fungal biomass and promotes over 80% of Kjeldahl-Nitrogen in the vinasse. If vinasse were treated following the sequence of anaerobic digestion, aerobic fungal growth, and ozone treatment, the effluent would have about 95% of the COD decreased, complete removal of phenolic compounds, and over 80% of Kjeldahl-Nitrogen

Xylanase Production by Talaromyces amestolkiae Valuing Agroindustrial Byproducts

In general, agroindustrial byproducts can be easily assimilated by several microorganisms due to their composition, which is rich in carbohydrates. Therefore, they could be appropriate for use as raw materials in a sustainable refinery concept, including the production of hydrolytic enzymes with industrial applicability. In this work, xylanase production by the filamentous fungi Talaromyces amestolkiae in submerged culture was evaluated using five agroindustrial byproducts, namely, wheat bran, citrus pulp, rice bran, peanut skin, and peanut shell. Firstly, the aforementioned byproducts were characterized in terms of cellulose, xylan, lignin, and extractives. Next, production studies were performed, and wheat bran generated the highest enzymatic activity (5.4 U·mL−1), probably because of its large amount of xylan. Subsequently, a factorial design was performed to evaluate the independent variables yeast extract, wheat bran, K2HPO4, and pH, aiming to improve the variable response, xylanase activity. The condition that promoted the highest production, 13.02 U·mL−1 (141% higher than the initial condition), was 20 g·L−1 wheat bran, 2.5 g·L−1 yeast extract, 3 g·L−1 K2HPO4, and pH 7. Thus, industrial byproducts with a high content of xylan can be used as a culture medium to produce xylanase enzymes with a Talaromyces strain through an economical and sustainable approach

Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.publishe

In Situ Transesterification of Microbial Biomass for Biolubricant Production Catalyzed by Heteropolyacid Supported on Niobium

Lubricants are substances of the foremost importance in the modern world, as they are essential to the proper functioning of various mechanisms. Most lubricants, however, are still made from petroleum fractions. I light of this, and due to various environmental problems, the search for feasible biolubricants has become essential. This study obtained biolubricants through the in situ transesterification of microbial biomass, containing at least 20 wt% of lipids. The following two distinct biomasses were evaluated: the marine microalgae, Dunaliella salina, and the consortium of microalgae-fungi, Scenedesmus obliquus and Mucor circinelloides. Microbial oil from both biomasses presented a fatty acid profile with high amounts of oleic acid. The oil of D. salina had a lower content of polyunsaturated fatty acids relative to the microbial consortium profile, which indicates that this is a good configuration for increasing biolubricant oxidation resistance. The catalyst used was a Keggin-structure heteropolyacid supported on niobium, H3PMo12O40/Nb2O5, activated at 150 °C, which had high transesterification yields, notwithstanding the feedstocks, which were rich in free fatty acids. The performed transesterification reactions resulted in excellent yields, up to 97.58% and 96.80%, for marine microalgae and the consortium, respectively, after 6 h at 250 °C, with 10 wt% of catalyst (related to the lipid amount). As such, the (H3PMo12O40/Nb2O5) catalyst could become an attractive option for producing biolubricants from microbial biomass

In Situ Transesterification of Microbial Biomass for Biolubricant Production Catalyzed by Heteropolyacid Supported on Niobium

Lubricants are substances of the foremost importance in the modern world, as they are essential to the proper functioning of various mechanisms. Most lubricants, however, are still made from petroleum fractions. I light of this, and due to various environmental problems, the search for feasible biolubricants has become essential. This study obtained biolubricants through the in situ transesterification of microbial biomass, containing at least 20 wt% of lipids. The following two distinct biomasses were evaluated: the marine microalgae, Dunaliella salina, and the consortium of microalgae-fungi, Scenedesmus obliquus and Mucor circinelloides. Microbial oil from both biomasses presented a fatty acid profile with high amounts of oleic acid. The oil of D. salina had a lower content of polyunsaturated fatty acids relative to the microbial consortium profile, which indicates that this is a good configuration for increasing biolubricant oxidation resistance. The catalyst used was a Keggin-structure heteropolyacid supported on niobium, H3PMo12O40/Nb2O5, activated at 150 °C, which had high transesterification yields, notwithstanding the feedstocks, which were rich in free fatty acids. The performed transesterification reactions resulted in excellent yields, up to 97.58% and 96.80%, for marine microalgae and the consortium, respectively, after 6 h at 250 °C, with 10 wt% of catalyst (related to the lipid amount). As such, the (H3PMo12O40/Nb2O5) catalyst could become an attractive option for producing biolubricants from microbial biomass

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved