Esterases catalyze the hydrolysis and transesterification of short-chain fatty acid esters, and microbial esterases are used in the production of biofuels, cosmetics, food, and pharmaceuticals. The soil strain Bacillus paralicheniformis T7 secretes enzymes with esterase activity; however, many bacterial enzymes remain insufficiently studied. Therefore, this study aimed to identify and characterize novel GDSL esterases produced by B. paralicheniformis. Protein mass spectrometry, combined with proteomics and genomics, identified genes encoding two GDSL esterases, which were cloned into the pET-28c(+) vector. The resulting proteins were obtained in Escherichia coli BL21(DE3) as the recombinant esterases rEST-24 and rEST-28. These recombinant GDSL esterases showed maximum activity at 40 °C and pH 7.0. Moreover, Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ ions inhibited their activity, and rEST-28 was resistant to the detergents Tween-20, Tween-80, and Triton X-100. High-yield esterase activity was detected in bacteria cultured on feather medium and nutrient broth, and submerged fermentation of the B. paralicheniformis T7 strain on feather medium enabled the production of an esterase extract exhibiting activity of 17,618 ± 610 U/g. These results suggest that the B. paralicheniformis T7 strain can produce esterases and shows promising potential for application in technologies that degrade fatty acid esters using hydrolytic enzymes.

Phytases are widely used as feed additives because they improve phosphorus utilization from plant-derived feed ingredients and reduce phosphorus excretion into the environment. Most recombinant AppA phytases produced in Komagataella phaffii have been obtained using methanol-inducible expression systems, whereas information on constitutive methanol-free production remains limited. In this study, Escherichia coli AppA phytase was expressed in K. phaffii under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Cultivation temperature and initial glucose concentration were evaluated in shake-flask cultures, while the selected conditions were assessed in a 2 L stirred-tank bioreactor. Extracellular phytase activity reached 114.17 ± 2.71 U/mL in shake flasks at 27 °C with 3% initial glucose. The K. phaffii-produced enzyme was N-glycosylated, with an apparent molecular weight of approximately 54 kDa compared with approximately 45 kDa after deglycosylation. Compared with purified rAppA-E, the rAppA-P preparation showed a lower temperature optimum, a narrower pH–activity profile, and lower apparent thermostability. In a single bioreactor run, extracellular phytase activity reached 186.81 ± 0.25 U/mL, approximately 1.6-fold higher than the maximum mean shake-flask value. These findings demonstrate constitutive, methanol-free extracellular production of AppA in K. phaffii and support further development of controlled cultivation strategies.

Esterases are hydrolytic enzymes that catalyze the hydrolysis and transesterification of short-chain fatty acid esters. They are widespread in nature and can be found in plants, animals, and microorganisms. Microbial esterases are used in the production of biofuels, and in cosmetic, food, and pharmaceutical industries. Recently, microbial esterases have attracted interest due to their ability to hydrolyze plastics, particularly polyethylene terephthalate (PET). It was established that Bacillus paralicheniformis T7 secretes an enzyme with esterase activity of 18.32 ± 2.38 U/mL. Protein mass spectrometry in combination with proteomic and genomic analysis identified a bacillary esterase with a molecular weight of 26.8 kDa. Based on the complete genome sequence of the strain, oligonucleotides were designed to amplify the esterase gene, which was subsequently cloned into the pET-28c(+) vector. Transformation of Escherichia coli BL-21(DE3) cells with the esterase-containing vector resulted in a strain that produced recombinant esterase with a yield of 614 mg/L. The recombinant esterase was purified using Ni-NTA affinity chromatography on a Ni²⁺ column. Studies revealed that the recombinant esterase exhibits maximum activity at 40 °C and pH 7.0. The enzyme remains active within the temperature range of 30–55 °C and pH 5.0–8.0. Thermostability assays demonstrated that the esterase is stable after 15 minutes of incubation at 30–50 °C and within the pH range of 8.0–10.0. Submerged fermentation of B. paralicheniformis T7 followed by drying of the culture supernatant yielded a preparation with esterase activity of 15,328.1 ± 528.6 U/g. The results indicate that B. paralicheniformis T7 is a promising esterase-producing strain, and the enzyme itself holds potential for use as a hydrolytic biocatalyst in the degradation of fatty acid esters.

Esterases represent an important class of hydrolase enzymes widely utilized in biotechnology due to their high catalytic activity, substrate specificity, and stability against various physico-chemical factors. The sporogenic strain Bacillus paralicheniformis T7 produces extracellular enzymes, including esterase, which are associated with sporulation processes and adaptation to stressful conditions. In this study, the substrate specificity and catalytic properties of a recombinant esterase from the sporogenic strain B. paralicheniformis T7, expressed in Escherichia coli cells, were investigated. Structural analysis revealed that the esterase belongs to the GDSL/SGNH-hydrolase family and contains a conserved Ser–Asp–His catalytic triad. Substrate specificity analysis using p-nitrophenyl esters (C2–C18) showed that the enzyme exhibits maximum activity toward short-chain substrates, specifically p-nitrophenyl acetate (250.0 ± 0.1 U/mg), classifying it as a true esterase. The study of various factors demonstrated that divalent metal ions Ca2+, Mg2+, Mn2+, Cu2+, Zn2+, Fe2+ inhibit enzyme activity, while the reducing agents DTT and β-mercaptoethanol completely suppress its catalytic function. At the same time, the enzyme shows relative stability toward non-ionic detergents and sodium dodecyl sulfate (SDS). Kinetic analysis revealed that the Km value for p-nitrophenyl acetate is 26.18 mM, Vmax is 52.37 U/mg, and the catalytic constant kcat is 1.74 s-1. The results indicate a distinct affinity of the enzyme for the substrate and moderate catalytic efficiency compared to other bacillar esterases. Thus, the esterase from B. paralicheniformis T7 is characterized by a pronounced specificity for short-chain esters and resistance to a range of surfactants, determining its potential for application in the biocatalytic synthesis of esters used in the food and fragrance industries.

На территории ТОО «Экополигон» в г. Астана в местах скопления бытовых отходов были собраны образцы почвы, из которых было выделено 10 изолятов микроорганизмов. Идентификация выделенных изолятов была проведена на основании культурально-морфологических признаков, протеомного профиля рибосомальных белков, и молекулярно-генетических данных. Исследования показали, что все изоляты способны к утилизации Tween-20 и Tween-80, что свидетельствует о наличии эстеразной активности. Штамм Aeromonas veronii Tw8.1 на основании скрининга по утилизации трибутирина показал наибольшую эстеразную активность. Экстрацеллюлярная эстераза из Aeromonas veronii Tw8.1 проявляет субстратную специфичность относительно п-нитрофенил октаноата и п-нитрофенил ацетата, активна в диапазоне температур 40-60°C и pH 8-12. Максимальная активность эстеразы из Aeromonas veronii Tw8.1 установлена при 50°C и pH 11. Полученые результаты свидетельствуют о перспективности использования штамма Aeromonas veronii Tw8.1 в качестве продуцента эстеразы.

Phytases are industrial enzymes that enhance the availability of phytate-bound phosphorus and can reduce the environmental burden associated with undigested phytate. Bacillus paralicheniformis T7 is a spore-forming strain exhibiting multiple hydrolytic activities, including phytase activity. The phyL gene encoding β-propeller phytase was cloned from B. paralicheniformis T7 and expressed in Escherichia coli, followed by purification and biochemical characterization of the recombinant enzyme—rPhyL. The molecular mass of rPhyL was 41.0 kDa, and maximal activity was observed at pH 8.0 and 60 °C. The enzyme displayed alkaline stability, retaining 90% and 93% of its initial activity after 5 h at pH 8.0 and 10.0, respectively. Following 5 h of incubation at 50 °C and 55 °C, residual activities were 83% and 46%, respectively. rPhyL was strongly Ca²⁺-dependent, with maximal activity at 0.6–1.0 mM CaCl₂. Kinetic analysis using sodium phytate as the substrate yielded an apparent K_m of 1.21 mM, an apparent V_max of 328.6 μM·min⁻¹, and an apparent K_i of 1.34 mM under the specified assay conditions. Under the tested conditions, rPhyL exhibited detectable activity toward sodium phytate, whereas no detectable activity was observed toward ATP, sodium pyrophosphate, β-glycerophosphate, or p-nitrophenyl phosphate. These findings characterize rPhyL as a Ca²⁺-dependent bacillary β-propeller phytase with alkaline stability, expanding the enzymatic repertoire of the spore-forming B. paralicheniformis T7 strain.