Preparative Biochemistry & Biotechnology最新文献

Tyrosinase, an enzyme involved in browning reactions in plants/crops exposed to mechanical injury, was isolated from the pulp of some different locally available bananas (M. cavendish, M. acuminata, and M. paradisiaca). Tyrosinase from the pulps was extracted, purified, immobilized, and characterized. Thereafter, the potentials of the immobilized tyrosinase in the possible production of l-3,4-dihydroxyphenylalanine (L-DOPA) in an improvised batch reactor was exploited using tyrosine and ascorbate as the substrates. L-DOPA production was monitored via thin-layer chromatography and spectrophotometry (Arnow's method). L-DOPA is a drug that is used in the treatment of Parkinson's disease. Hence, this study exploited a non-chemical route for its synthesis using the tyrosinase obtained from the banana pulps. The purified tyrosinase had an optimum pH and temperature of 6.5 and 7.0, respectively. The molecular weight of the purified tyrosinase was 45 kDa. Quercetin and resorcinol both competitively inhibited the purified tyrosinase from the three cultivars. Immobilized M. cavendish tyrosinase produced the highest concentration (0.60 mM) of L-DOPA after 8 h in an improvised batch reactor. The tyrosinase in the banana pulps serves as a cheap and readily available green route for the possible production of L-DOPA.

Probiotic bacteria continue to receive increasing attention in the food and feed industries. However, the production of Bifidobacterium and Lactobacillus at an industrial scale is challenging because of specific nutrient requirements and conditions, which are complicated and costly. We developed low-cost culture media by modifying the carbon and nitrogen sources for Bifidobacterium animalis subsp. lactis KMP-H9-01 and Lactobacillus reuteri KMP-P4-S03 from available food grade components. Sucrose (15 g/l) was selected as a suitable carbon source for both strains because it was the most economic and facilitated bacterial growth that was equal to that of glucose. The Bifidobacterium strain required beef extract as a nitrogen source to multiply. The fermentation of both strains using the modified media formula in 5-L and 50-L bioreactors showed that the highest cell counts of L. reuteri and B. animalis subsp. lactis were 9 and 9.8 log CFU/ml after 12-15 h, respectively. The concentration (g/l) ratio between lactate and acetate obtained from B. animalis subsp. lactis was 7:7.4 at 12 h and 11.4:10.6 at 40 h; the ratio was similar at both time points (6.9: 1.1) for L. reuteri. Thus, this economically modified food-grade medium for the large-scale fermentation of two probiotic bacteria was efficient.

Synthetic genes for the two subunits of phenylalanyl-tRNA synthetase (PheRS) from wheat were expressed in Escherichia coli. When each gene was induced individually, the α subunit with a cleavable 6 × His tag at the amino terminus was largely soluble, while the β subunit was almost completely insoluble. When the two subunits were co-expressed, a soluble fraction containing the two subunits were obtained. This was purified by a standard method in which the tag was cleaved off with a specific protease after affinity purification. As the sample contained slightly more PheRSα than PheRSβ, we further resolved the sample by gel filtration to obtain the fraction that showed the size of the conventional α₂β₂ tetrameric complex and contains an almost equal amount of the two subunits. The final yield was 0.6 mg per 1 liter of the culture medium, and the specific activity was 28 nmol min^-1mg^-1, which was higher than that of a fraction purified from wheat germ. This recombinant PheRS was used, along with purified samples of the elongation factors and the ribosomes from wheat germ, for a poly(U)-dependent poly(Phe) synthesis reaction. The reaction was dependent on the added components and lasted for more than several hours.

With the industrialization and development of modern science, the application of enzymes as green and environmentally friendly biocatalysts in industry has been increased widely. Among them, lipase (EC. 3.1.1.3) is a very prominent biocatalyst, which has the ability to catalyze the hydrolysis and synthesis of ester compounds. Many lipases have been isolated from various sources, such as animals, plants and microorganisms, among which microbial lipase is the enzyme with the most diverse enzymatic properties and great industrial application potential. It therefore has promising applications in many industries, such as food and beverages, waste treatment, biofuels, leather, textiles, detergent formulations, ester synthesis, pharmaceuticals and medicine. Although many microbial lipases have been isolated and characterized, only some of them have been commercially exploited. In order to cope with the growing industrial demands and overcome these shortcomings to replace traditional chemical catalysts, the preparation of new lipases with thermal/acid-base stability, regioselectivity, organic solvent tolerance, high activity and yield, and reusability through excavation and modification has become a hot research topic.

Terminalia bellirica (T. bellirica) (Gaertn.) Roxb. is a well-known traditional medicinal plants that show promising treatment because of fewer side effects in humans. In the present study, the total phenol, flavonoid, condensed and hydrolyzable tannins extracted and analyzed from cold macerated (CM) T. bellirica (Gaertn.) Roxb. fruit (TBF) and leaves (TBL) extract with the identification of bioactive compounds using GC-MS/MS technique. The highest amount of bioactive content was found in ethanolic extract than toluene. Current experimental data of TBF extract shows the maximum and significant biological activity like free radical scavenging activity against DPPH and FRAP assays with IC₅₀ values of 51.07 ± 0.52 μg/ml and 63.14 ± 0.59 μg/ml respectively. However, IC₅₀ cytotoxicity values of TBF extract on MCF-7 cells for 24 hrs was found to be 6.34 ± 0.72 μg/ml. Minimum inhibitory concentration (MIC) for infectious pathogens Escherichia coli and Bacillus cereus was >12.5 μg/ml and >100 μg/ml respectively, however, anti-inflammatory activity was demonstrated as an IC₅₀ value of 509.1 ± 1.72 μg/ml. Cold macerated fruit extract revealed threatening inhibitory potential against the α-amylase and α-glucosidase enzymes, with IC₅₀ of 50.98 ± 0.23 μg/ml and 46.70 ± 1.38 μg/ml respectively. Finally, the outcome of this study showed that T. bellirica (Gaertn.) Roxb. fruit extract could be an effective source of bioactives with efficient biomedical properties.

Ginger (Zingiber officinale Roscoe, Zingeberaceae) is a medicinal plant widely used as food, spice, or flavoring agent worldwide. 6-Shogaol is a compound of prime interest in exhibiting anti-inflammatory, antioxidant and chemopreventive effects. The objective of the study is to investigate the effect of microwave-assisted drying (MAD) followed by microwave-assisted extraction (MAE) so as to produce 6-Shogaol enriched Ginger with improved therapeutic benefits. Various drying techniques viz. shade drying, tray drying, microwave-assisted drying and osmotic dehydration as a pretreatment were used for drying Ginger rhizomes. The dried rhizomes were extracted by conventional solvent extraction and microwave-assisted extraction techniques and tested for content of 6-Shogaol using the newly developed HPLC method whereas total flavonoid and polyphenol content were determined using the UV spectrophotometric method. Subjecting the microwave dried Ginger to microwave-assisted extraction for 45 min at constant power level of 284 W resulted in a significant rise in the extractability of 6-Shogaol (1.660 ± 0.018), total polyphenols (855.46 ± 5.33) and flavonoids (617.97 ± 6.40) compared to the conventional method of extraction. The proposed Ginger processing method of microwave drying followed by microwave extraction outperforms traditional methods in terms of speed, convenience, and performance thus can be scaled up to industrial levels.

Polysaccharides derived from Auricularia auricula exhibit diverse biological activities and hold significant potential for commercial utilization as functional food ingredients. In this investigation, polysaccharides from A. auricula were obtained using six extraction techniques (ammonium oxalate solution extraction, sodium hydroxide solution extraction, hot water extraction, pectinase and cellulase-assisted extraction, ultrasonic-assisted extraction, and microwave-assisted extraction). Subsequently, a comprehensive comparison was conducted to evaluate their physicochemical properties and biological functionalities. The ammonium oxalate solution extraction method yielded a higher extraction rate (11.76%) and polysaccharide content (84.12%), as well as a higher uronic acid content (10.13%). Although the six Auricularia polysaccharides had different molecular weight distributions, monosaccharide molar ratios, similar monosaccharide compositions, and characteristic functional groups of polysaccharides, they exhibited different surface morphology. In vitro assays showed that polysaccharides extracted by ammonium oxalate solution possessed good scavenging ability against DPPH free radical, hydroxyl free radical and superoxide anion free radical as well as reduction power of iron ion. At the same time, both polysaccharides extracted by ammonium oxalate solution and sodium hydroxide solution promoted NO production in mouse macrophages along with the secretion of cytokines TNF-α, IL-1β, and IL-6. These results indicated significant differences in the structure and characteristics among Auricularia polysaccharides prepared by various extraction methods, which may be related to the variety or origin of A. auricula; furthermore, their bioactivities varied accordingly in vitro assays where the ammonium oxalate solution extraction method was found more beneficial for obtaining high-quality bioactive Auricularia polysaccharides.

Recent SARS-CoV-2 pandemic elevated research interest in microorganism-related diseases, and protective health application importance such as vaccination and immune promoter agents emerged. Among the production methods for proteins, recombinant technology is an efficient alternative and frequently preferred method. However, since the production and purification processes vary due to the protein nature, the effect of these differences on the cost remains ambiguous. In this study, brucellosis and its two important vaccine candidate proteins (rOmp25 and rEipB) with different properties were selected as models, and industrial-scale production processes were compared with the SuperPro Designer^® for estimating the unit production cost. Simulation study showed raw material cost by roughly 60% was one of the barriers to lower-cost production and 52.5 and 559.8 $/g were estimated for rEipB and rOmp25, respectively.

Proteases are the main enzymes traded worldwide-comprising 60% of the total enzyme market-and are fundamental to the degradation and processing of proteins and peptides. Due to their high commercial demand and biological importance, there is a search for alternative sources of these enzymes. Crotalaria stipularia is highlighted for its agroecological applications, including organic fertilizers, nematode combat, and revegetation of areas contaminated with toxic substances. Considering the pronounced biotechnological functionality of the studied species and the necessity to discover alternative sources of proteases, we investigated the extraction, purification, and characterization of a protease from seeds of the C. stipularia plant. Protease isolation was achieved by three-phase partitioning and single-step molecular exclusion chromatography in Sephacryl S-100, with a final recovery of 47% of tryptic activity. The molecular mass of the isolated enzyme was 40 kDa, demonstrating optimal activities at pH 8.0 and 50 °C. Enzymatic characterization demonstrated that the protease can hydrolyze the specific trypsin substrate, BApNA. This trypsin-like protease had a K_m, V_max, K_cat, and catalytic efficiency constant of 0.01775 mg/mL, 0.1082 mM/min, 3.86 s^-1, and 217.46, respectively.

In this study, the wild-type Bacillus cereus ATA179 was mutagenized by random UV mutagenesis to increase lipase production. The mutant with maximum lipolytic activity was named Bacillus cereus EV4. The mutant strain (10.6 U/mL at 24 h) produced 60% more enzyme than the wild strain (6.6 U/mL at 48 h). Nutritional factors on lipase production were investigated. Sucrose was the best carbon source, (NH₄)₂HPO₄ was the best nitrogen source and CuSO₄ was the best metal ion source. Mutant EV4 showed a 32% increase in lipase production in the modified medium. The optimum temperature and pH were found to be 60 °C and 7.0, respectively. CuSO₄, CaCl₂, LiSO₄, KCl, BaCl₂, and Tween 20 had an activating effect on the enzyme. V_max and K_m values were found to be 17.36 U/mL and 0.036 mM, respectively. The molecular weight was determined as 28.2 kDa. The activity of lipase was found to be stable up to 60 days at 20 °C, 75 days at 4 °C, and 90 days at -20 °C. The potential of lipase in the detergent industry was investigated. The enzyme was not affected by detergent additives but was effective in removing stains in fabrics contaminated with oily substances.