Lactose obtained from cheese whey is a low value commodity despite its great potential as raw material for the production of bioactive compounds. Among them, prebiotics stand out as valuable ingredients to be added to food matrices to build up functional foods, which currently represent the most active sector within the food industry. Functional foods market has been growing steadily in the recent decades along with the increasing awareness of the World population about healthy nutrition, and this is having a strong impact on lactose-derived bioactives. Most of them are produced by enzyme biocatalysis because of molecular precision and environmental sustainability considerations. The current status and outlook of the production of lactose-derived bioactive compounds is presented with special emphasis on downstream operations which are critical because of the rather modest lactose conversion and product yields that are attainable. Even though some of these products have already an established market, there are still several challenges referring to the need of developing better catalysts and more cost-effective downstream operations for delivering high quality products at affordable prices. This technological push is expected to broaden the spectrum of lactose-derived bioactive compounds to be produced at industrial scale in the near future.
White biotechnology uses enzymes and microorganisms to produce value-added chemicals from renewable sources. White biotechnology provides valuable components for the food, pharmaceutical, agricultural sectors as well as other industries. Metabolic diversity in fungi, yeast, and bacteria can be exploited to produce food additives and other industrial products. This is an interesting topic for those interested in screening and metabolic testing of microorganisms, industrial biotechnology, fermentation technology, and the biological products research community. The use of microbial-derived compounds has a long history in the food industry, and compounds such as flavorings, essential amino acids, poly-unsaturated fatty acids, organic acids, gelling, etc. can be obtained from microbial sources. Also, the role of microbes in human health and well-being cannot be ignored. Microbes produce primary metabolites such as vitamins, nucleotides, and amino acids, as well as secondary metabolites. These secondary metabolites are used to make many drugs. In agriculture, microbes are also used to make fertilizers and biological pesticides. This paper reviews the types of bio-products obtained through biotechnology and the barriers and challenges of white biotechnology.
Human activity pollution has been shown to harm the environment, ecology, and health impacts. The polycyclic aromatic hydrocarbons (PAHs) produced by the industries such as tannery, distillery, pulp paper, and oil refineries are a major source of contaminant. PAHs are found all across the world, owing to long-term human pollution sources. PAHs' physico-chemical features, such as hydrophobicity and electrochemical stability, contribute to their environmental persistence and contribute to their carcinogenic and health effects. Numerous analytical and biological techniques for the qualitative and quantitative assessment of PAHs have been proposed. Bioaccumulation, adsorption, chemical oxidation, photolysis, volatilization, and microbiological degradation are the principal breakdown pathways of PAHs in the environment. Microbial populations, such as bacteria, fungi, and algae, play a crucial role in the biological elimination of PAHs. Oxidase, manganese peroxidases, lipases, and laccases are the enzymes involved in PAHs breakdown. The synthesis of surfactants by bacteria increases PAHs bioavailability and improves the elimination process of PAHs. Temperature, pH, aeration, moisture content, nutrition availability, absence of hazardous chemicals, and the kind and number of degrading microbial populations are all factors that influence PAHs decomposition. Microbial degradation mechanisms result in intermediate metabolites and carbon dioxide mineralization. The elimination of PAHs is improved by molecular approaches such as gene engineering and protein engineering. This review discussed the benefits of bioremediation strategies that were investigated for precise evaluation and were trusted at both the regulatory and scientific studies levels.
Trehalose is a non-reducing disaccharide connected by α-1,1-glycosidic bonds; it is widely distributed in bacteria, fungi, yeast, insects, and plant tissues and plays various roles. It can be hydrolyzed by trehalase into two glucose molecules. Trehalases from different sources have been expressed in Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, baculovirus-silkworm, and other expression systems; however, it is most common in E. coli. The structural characteristics of different glycoside hydrolase (GH) family trehalases and the sources of trehalase have been analyzed. The catalytic mechanism of GH37 trehalase has also been elucidated in detail. Moreover, the molecular modification of trehalase has mainly focused on directed evolution for improving enzyme activity. We comprehensively reviewed the current application status and adaptable transformations was comprehensively overviewed in the context of industrial performance. We suggest that the level of recombinant production is far from meeting industrial requirements, and the catalytic performance of trehalase needs to be improved urgently. Finally, we discuss developmental prospects and future trends.
Consumption of insects as food has a long history and has been documented by many researches. Globally, it is extensively practiced in Africa, Asia, Australia, and Latin America as traditional food providing nutritional, economic and ecological benefit for rural communities. In today’s world, edible insects are limelight by many researchers and industrialist due to the presence of enormous nutrient potential as well as bioactive compounds. This review mainly focuses on the potential of edible insect for its diverse nutraceutical properties, production and processing as a functional food and its acceptance and boost in the market trends in global scenario. The consumption of edible insects is purely based on ethnic traditional knowledge of the local communities. Previous studies on edible insects have shown their potential as source of nutraceuticals with promote health benefit and can be an alternative source of protein. Most of the edible insects are rich source of protein, energy, vitamins, essential fatty acids and minerals. Beyond these insects can be a source of bioactive compounds especially peptides, which can be applied in functional food industry. In many countries, the traditional knowledge of entomophagy is being applied for production of value-added products using modern technology.
Phospholipase A1 (PLA1) is a member of the hydrolase family with applications in various fields, especially in the food industry. A calcium-independent PLA1 from Streptomyces albidoflavus was expressed in E. coli BL21 (DE3) in this study. The results indicated that the soluble expression of PLA1 was at low level, which was possibly due to the toxicity of PLA1 to the host. In contrast, the expression of the enzyme as inclusion bodies exhibited a high-level expression and 0.3 mg inclusion bodies protein could be derived from 1 mL culture medium. Furthermore, the renaturation of PLA1 was achieved through a direct dilution method, yielding 29.6 U/mL PLA1 activity after 16 h of renaturation at 4 °C. For improving the efficiency of the dilution refolding process, a continuous refolding strategy was established, and 155 U/mL PLA1 activity was derived from the continuous refolding process. With soybean lecithin as the substrate, the specific activity of purified renatured PLA1 was 1380 U/mg and the optimal temperature and pH was found to be 60 °C and 6.5. In addition, the renatured PLA1 was observed with better activity towards phosphatidyl inositol, whilst lipase activity was detected when the catalyzing temperature was below 55 °C. Overall, this study provides a possible solution to obtain calcium-independent PLA1 with high yield by heterologous expression in E. coli and hence to promote its further application in the field of food industry.
To address the deficient activity of TrCel5A in naturally secreted cellulase preparation, this study used the GAP promoter to induce constitutive expression of Trichoderma reesei TrCel5A in Pichia pastoris. A recombinant TrCel5A was screened out after gene optimization, synthesis, and expression. The biochemical and enzymatic properties of the new recombinant were characterized. As a result, optimization of shake-flask fermentation of the recombinant was obtained at 28 °C, 2% inoculum volume, an initial pH of 6.0, as well as glycerol and Tween-80 additions of 30 g/L and 6 g/L, respectively. Under the above-optimized conditions, the recombinant produced 14.8 U/mL of the enzyme activity at 96 h of fermentation. To further enhance enzyme production, pilot-scale cultivation was evaluated using 5-L bioreactors. Using high-cell-density fermentation, the recombinant strain increased enzyme activity to 130.4 U/ml and protein content to 2.49 g/L. In addition, the kinetic factors, including K m and V max values for TrCel5A, were detected to be 5.1 mg/mL and 265.9 μmol/(min.mg), respectively. Thus, TrCel5A was effectively expressed in P.pastoris under the GAP promoter, and it demonstrated its potential in commercially relevant enzyme hydrolysis of lignocellulosic biomass.
N-methyl-d-aspartic acid (NMDA), an amino acid existing in human and animal central nervous system, exerts agonist action on one of the glutamate receptor subtypes and is clinically used for treatment of diabetes, Parkinson’s and Alzheimer’s syndrome. In this study, an enzymatic protocol for the chiral resolution of N-methyl-d,l-aspartic acid was built with a predicted N-demethylase (GenBank ID: OJV90073.1) from the genome of Chloroflexi bacterium 54-19. Through sequence alignment, the enzyme shares an identity of 32.19% to 2UZZ (PDB ID) with a conserved catalytic center. Recombinantly expressed in Bacillus subtilis WB600, the N-demethylase was characterized with optimal temperature and pH at 55 ℃ and 7.5, and adaptive temperature and pH were 40–60 ℃ and 6–8. The effects of organic solvents and metal ions were investigated as well. Compared with other previously reported sarcosine oxidases, the enzyme showed a specific N-demethylation activity against N-methyl-l-aspartic acid according to the analysis by chiral liquid chromatography, LC–MS/MS and a detection by polarimeter. The results demonstrated 76% of 4.5 mM N-methyl-d,l-aspartic acid could be chirally separated by 7 mg·L−1 enzyme after reaction of 80 min. This work provided a foundation for mild synthesis of NMDA in industry.
The bacterium Rhodococcus erythropolis MI2 uses 4,4´-dithiodibutyric acid (DTDB) as carbon source to synthesize polythioesters (PTE). The first step for the production of PTE using DTDB is catalyzed by an NADH:flavin oxidoreductase (nox) as it was previously shown in our laboratory, and the second step is catabolized by a putative luciferase-like monooxygenase (Llm). In the current study, experiments were carried out to identify the function of Llm. Hence, the llm gene, which encodes for the Llm protein, was amplified from the genomic DNA of MI2 using polymerase chain reaction and expressed in Escherichia coli BL21 cells. Protein purification was done using His Spin Trap affinity columns. Enzyme assay was carried out using the purified protein and p-coumaric acid as substrate giving a specific activity of 1.6 U/mg protein for the purified Llm. The responsible gene (llm) was deleted in the genome of MI2, and a single deletion mutant was subsequently generated. Finally, growth of the wild-type strain (MI2) and the mutant strain (MI2Δllm) were compared using DTDB or succinate as carbon sources. Whereas the wild type was successfully grown with DTDB or succinate, the llm-negative mutant exhibited low grow with DTDB although it grows very well with succinate.
Due to the selective permeability of the cytomembrane, high-yield fumaric acid strains form a steep difference between intra- and extracellular concentrations. Intracellular biosensors cannot detect the real concentration change of extracellular fumaric acid. To overcome this limitation, a two-component biosensor (TCB) that could respond to extracellular fumaric acid was designed based on the DcuS-DcuR two-component system. The two-component system consists of a histidine kinase (SK) and response regulator. SK is a transmembrane histidine kinase sensor that can detect concentration changes in extracellular compounds. To improve the dynamic range of the constructed fumaric acid TCB, we optimized the expression ratio and expression intensity of dcuS and dcuR. We found that the optimum expression ratio of dcuS:dcuR was 46:54. Under this ratio, the higher was the expression level, the greater the dynamic range. In addition, we modified the ATP-binding site on the DcuS, and the final dynamic range of the TCB reached 6.6-fold. Overall, the obtained fumaric acid-responsive TCB with a high dynamic range is reported for the first time, providing a synthetic biology tool for high-throughput screening and dynamic metabolic regulation of fumaric acid cell factories.
4-hydroxyisoleucine (4-HIL) is a potential drug for diabetes and weight control. 4-HIL was produced by expressing ido gene in L-isoleucine (Ile)-producing Corynebacterium glutamicum. But L-lysine (Lys) was also accumulated as the main by-product in this recombinant strain SN02. To attenuate Lys synthesis, two genes in Lys synthetic pathway, i.e., ddh encoding the diaminopimelic acid dehydrogenase and lysE encoding the specific Lys exporter were deleted in SN02. However, the deletion of ddh increased 4-HIL titer by 28.1%, but did not decrease Lys content; while the deletion of lysE significantly reduced Lys content by 66.7%, but 4-HIL titer also decreased by 19.3%. Therefore, we carried out transcriptome analysis to reveal the global variation in these mutants. Deletion of ddh and lysE (especially lysE) enhanced the transcription of key enzymes in succinylase branch of Lys synthesis pathway (DapD and DapC) and several enzymes involved in succinyl-CoA accessibility (SucC, SucD and OdhI), suggesting the compensatory synthesis of Lys via succinylase branch. In addition, the transcription of ilvBN in Ile synthesis pathway was improved, while the transcription of some genes in the 2-methylcitrate cycle and inositol metabolism pathway was weakened in these mutants. Mere deletion of ddh enhanced the transcription of aceA, ppc and pck, thus promoting oxaloacetate supply and 4-HIL synthesis. Deletion of lysE affected the transcription of some stress-related genes and transporter genes, suggesting that this mutant would be under stress, thus attenuating its 4-HIL synthesis. These findings will be helpful for systematic microbiology and bio-manufacturing of C. glutamicum.
Alginate lyase mainly produces active alginate oligosaccharides (AOS) by degrading alginate via β-elimination process. In this study, the Pseudoalteromonas sp. Alg6B alginate lyase-encoding gene alg6B-7 from polysaccharide lyase (PL)-7 family was successfully cloned, sequenced, expressed in Escherichia coli. Based on rational design and amino acid sequence alignment of the alginate lyase from various sources, four positive mutants were obtained. The specific enzyme activities of four mutants I62A, A99K, V132S, and L157T were 38.84%, 42.85%, 75.8% and 51.83% higher than that of the wild enzyme, respectively. The K cat/K m values of the four mutants were both increased, and the catalytic efficiency of V132S was 1.92-fold higher than that of the wild enzyme, especially. The rational design that was employed in this study achieved the dramatic improvement of catalytic activity, which may provide the application potential in industrial production.
In this study, an alkaline protease BaApr1 from the Bacillus altitudinis W3 was chosen to hydrolysis grass carp (Ctenopharyngodon idella) scales. The hydrolysate of alkaline protease BaApr1 exhibited the best antioxidant activity compared to other protease hydrolysates. The optimal hydrolysis conditions for BaApr1 were an enzyme dosage of 1250 U/g, a hydrolysis time of 7 h, a pH of 9.5 and a temperature of 50 °C. Three novel peptides were purified using ultrafiltration, anion exchange chromatography, gel filtration chromatography and ultra-performance liquid chromatography, and their sequences were identified as Tyr-Val-Gln-Ala-Gly-Ala-Ala-Gly-Ala-Ala-Ala-His (SHP2), Val-Lys-Leu-Tyr-Val-Leu-Leu-Val-Pro (SHP4), and Val-Gln-Val-Leu-Ala-Gly-Pro-Val-Val-Lys-Leu-Tyr (SHP5) with molecular weights of 1086.53 Da, 1043.69 Da and 1285.79 Da, respectively. Among them, SHP2 exhibited the highest scavenging activity on DPPH· (EC50 4.08 mg/mL), ABTS+· (EC50 0.23 mg/mL) and HO· (EC50 2.78 mg/mL), and the strongest reducing power. Additionally, SHP5 can significantly inhibit lipid peroxidation in the linoleic acid system. In conclusion, three peptides isolated from scales of hydrolysate of grass carp showed great antioxidant activity and might be used as potential food ingredients and pharmaceuticals.
An efficient ammonia nitrogen degrading bacterial strain was isolated from a fish and shrimp pond and identified as Bacillus subtilis (Ab03). Firstly, the strain was continuously domesticated in ammonium solution to improve its nitrogen removal capacity. The performance of the strain in terms of nitrogen removal efficiency under different culture conditions was then examined. Finally, the nitrogen removal process and related strain mechanisms were analyzed by nitrogen balance. The results showed the strain Ab03 could remove 91.67% of NH4 +-N at 300 mg/L under the conditions of glucose as the single carbon source, C/N of 15, pH of 7.5, the temperature of 35 ℃ and dissolved oxygen of 7–8 mg/L. It was also found that under conditions where ammonia nitrogen was the only nitrogen source, strain Ab03 could also undergo aerobic denitrification for simultaneous conversion, with a final gas conversion rate of 74.81%.