Enzymatic Properties Research Articles

Entrapment of protease from Bacillus sp. in polyvinyl alcohol hydrogels

This study highlights the effective immobilization of protease from Bacillus sp. in polyvinyl alcohol hydrogels and its characterization. Both free and entrapped proteases exhibited optimal activity at pH 8.0 and 55°C, indicating that the immobilization did not significantly alter the enzyme's fundamental properties. The entrapment in polyvinyl alcohol hydrogels significantly enhanced thermal stability. After 24 hours at 55°C, the free protease retained only 19% of its initial activity, whereas the entrapped protease retained 72%. The entrapped protease showed a longer half-life of 53.3 hours compared to 10.6 hours for the free protease. The Km and Vmax values of free protease were determined to be 0.5 mg/mL and 23.3 U/mg protein, respectively, for casein. These values were found to be 0.2 mg/mL and 23.8 U/mg protein, respectively for the entrapped protease. The entrapped protease retained 58% of its initial activity after 5 reuses in a batch reactor. As a result, the entrapment of Bacillus sp. protease in polyvinyl alcohol is an effective immobilization method due to its simplicity, low cost, and ability to provide a 5-fold increase in thermal stability.

Engineering the Non-catalytic Domain to Enhance Catalytic Activity and Thermal Stability of a Nκ2-Producing κ-Carrageenase.

κ-Carrageenases play an important role in achieving the high-value utilization of carrageenan polysaccharides. They can be used in the preparation of even-numbered κ-neocarrageenan oligosaccharides by degrading κ-carrageenan (KC). We previously identified and characterized a κ-carrageenase, CaKC16B, with high specificity for producing a single κ-neocarrabiose. It can produce a single κ-neocarrabiose from degrading KC. However, they also exhibited poor thermal stability and catalytic efficiency. To improve these properties, we introduced noncatalytic domains (nonCDs) from a heat-resistant κ-carrageenase, MtKC16A, into the C-terminus of CaKC16B to construct CaKC16BUN and CaKC16BUNB. Compared to the original CaKC16B, both of them exhibited improved enzymatic properties, including optimal reaction temperature, thermal stability, substrate affinity, and catalytic efficiency. Remarkably, the kcat/Km values of 16BUN and 16BUNB increased by 127 and 290 times, respectively. Importantly, the addition of nonCDs did not alter the final products of degrading KC, retaining the high product specificity of CaKC16B. Interestingly, the addition of nonCDs changed CaKC16B's substrate specificity for hydrolyzing KC and βκ-carrageenan, with mutants exhibiting higher relative activity for βκ-carrageenan. We further observed through biolayer interferometry that the binding and dissociation process between MtUN nonCD and βκ-carrageenan is faster compared to that of KC. This may explain the change in the substrate specificity observed in the mutants of CaKC16B.

Structural and Functional Biology of Mammalian ALOX Isoforms with Particular Emphasis on Enzyme Dimerization and Their Allosteric Properties

The human genome involves six functional arachidonic acid (AA) lipoxygenase (ALOX) genes, and the corresponding enzymes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) have been implicated in cell differentiations and in the pathogenesis of inflammatory, hyperproliferative, metabolic, and neurological disorders. Humans express two different AA 15-lipoxygenating ALOX isoforms, and these enzymes are called ALOX15 (15-LOX1) and ALOX15B (15-LOX2). Chromosomal localization, sequence alignments, and comparison of the enzyme properties suggest that pig and mouse ALOX15 orthologs (leukocyte-type 12-LOX) on the one hand and rabbit and human ALOX15 orthologs on the other (reticulocyte-type 15-LOX1) belong to the same enzyme family despite their different reaction specificities with AA as a substrate. In contrast, human ALOX12 (platelet-type 12-LOX), as well as pig and mouse ALOX15 (leukocyte-type 12-LOX), belong to different enzyme families, although they exhibit a similar reaction specificity with AA as a substrate. The complex multiplicity of mammalian ALOX isoforms and the controversial enzyme nomenclatures are highly confusing and prompted us to summarize the current knowledge on the biological functions, enzymatic properties, and allosteric regulation mechanisms of mammalian ALOX15, ALOX15B, and ALOX12 orthologs that belong to three different enzyme sub-families.

Appraisal of Heavy Metal Risk Hazards of Eisenia fetida-Mediated Steel Slag Vermicompost on Oryza sativa L.: Insights from Agro-Scale Inspection and Machine Learning Analytics

The steel industry drives world economic growth, yet it generates heavy metal-rich steel slag, which jeopardizes the environment. The utilization of vermi-technology is essential for the sustainable transformation of toxic steel waste slag (SW) into organic amendments, although field-scale use of vermiprocessed SW remains unexplored. To bridge the gap, this study evaluated the efficacy of vermiprocessed SW as an organic supplement for rice field cultivation, focusing on heavy metal (HM) bioavailability, human health risk, and yield in comparison to raw slag and NPK fertilizer. The results indicated a considerable decrease in the bioavailable fraction of heavy metals in T4 (1:1 SW vermicompost 50% + 50% fertilizer). In treatments, T9 (100% SW) and T10 (50% SW + 50% fertilizer) (FIAM) free ion activity modeling confirmed grain absorption of HMs, and the FIAM HQ values indicated the health risk for the direct application of steel slag waste on the field. The risk factor evaluation of HMs’ presence in treatments T9 and T10 established the possible cancer risk for living beings. Similarly, machine learning models like SOBOL sensitivity analysis and artificial neural networks revealed potential threats associated with HMs on different treatments, respectively. The correlation coefficient revealed the negative effects of bioavailable HMs on various soil microbial and enzymatic properties. Moreover, the abundant yield of rice was attributed to the combination treatment (1:1 50% + NPK 50%), which paved the way for an alternative agronomic approach based on the utilization of vermicomposted steel waste slag.

Microbial Decomposer for Crop Residue Management in Rice-Wheat Cropping System.

Crop residue management is vital in the Rice-Wheat cropping system, influencing soil health and crop productivity. This study examined the effects of organic and inorganic fertilizers and microbial decomposers on rice growth and yield. We evaluated seven treatments: 100% recommended dose fertilizer (RDF); 50% residue + 50% RDF; 50% residue + 50% RDF + Pusa decomposer; 50% residue + 50% Green Manuring (GM)/Green Leaf Manuring (GLM); 50% residue + 50% GM/GLM + Pusa decomposer; Residue @ 2.5 tons per acre + Pusa decomposer; Residue @ 2.5 tons per acre + no Pusa decomposer; and absolute control. Results indicate that integrating organic and inorganic fertilizers with microbial decomposers positively affects rice growth and yield parameters. While adding microbial decomposer to RDF did not consistently enhance rice yield, it improved soil enzymatic properties. This suggests that the effectiveness of microbial decomposers may vary based on specific soil and crop conditions. Therefore, microbial decomposers present a promising approach to boost soil health and fertility. Further research is needed to optimize conditions for their use and systematically assess their impact on crop yields.

Exploring Enzyme Encapsulation Efficiency in MOFs Using Eco-Friendly Approaches.

The encapsulation of protein enzymes in metal-organic frameworks (MOFs) has been recognized as an effective enzyme immobilization approach. In this study, we demonstrated the influence of enzyme amount and the isoelectric points (pI) of different enzymes on the enzyme loading capacity in both mechanochemical (ball-milling) and water-based approaches. We found that increasing enzyme amounts enhances MOF enzyme loading without compromising activity, while the MOF shell protects encapsulated enzymes from proteinase K degradation through its size-sheltering mechanism. However, an excess of enzymes can hinder the formation of ZIF-90. Moreover, enzymes with low pI values (e. g., catalase, pI 5.4) facilitate encapsulation in MOFs, whereas enzymes with high pI values (e. g., lysozyme, pI 11.35) are more challenging to encapsulate. The simulation results revealed that increasing the enzyme amounts and pI values raises the activation energy necessary for MOF formation. This study highlights the crucial role of enzyme properties in the encapsulation process within MOFs, providing valuable insights for fabricating enzyme-MOF biocomposites for diverse applications, such as protein drug delivery.

Immobilization and characterization of β-galactosidase from Aspergillus oryzae in polyvinyl alcohol hydrogels.

One of the main goals of contemporary biotechnology has been the development of novel immobilized enzyme formulations. In the present study, the industrially important β-galactosidase was trapped in a polyvinyl alcohol (PVA) gel to immobilize it. The optimization of immobilization method and characterization of the immobilized enzyme were studied. The results were compared with free enzymes. The results indicate that the optimal temperature range for the enzyme to be at following immobilization is between 40°C and 50°C. At pH 7, the optimal pH, the activity increased, the Vmax value increased from 1.936 to 2.495 Umg‒1, and the Km value decreased from 4.861 to 0.982mM.Depending on how stable the immobilized enzyme when stored, β-galactosidases immobilized on PVA gels showed 52.87% activity at the end of the seventh week and 58.86% activity at the end of the fifth week. Their initial activity subsided after three reuses. The final result was 66%. Therefore, one may argue that it increases the catalytic effect of the enzyme. As a result, it has been found that immobilized β-galactosidase has more potent enzymatic properties than free β-galactosidase, which may make it more advantageous for industrial processes. Further studies could delve deeper into the mechanistic aspects of the immobilization process in an effort to improve optimization and tailor the immobilized enzyme to specific industrial needs.

Advances in the biosynthesis of D-allulose.

D-allulose is a rare monosaccharide and a C-3 epimer of D-fructose. It has physiological functions, such as antihyperglycemic, obesity-preventing, neuroprotective, and reactive oxygen species (ROS) scavenging effects, making it an ideal sugar substitute. The synthesis methods for D-allulose include chemical synthesis and biosynthesis. Chemical synthesis requires strict reaction conditions and tends to produce byproducts. Biosynthesis is mainly an enzymatic process. Enzymatic catalysis for the conversion of starch or glycerol to D-allulose is performed mainly by enzymes such as isoamylase (IA), glucose isomerase (GI), D-allulose 3-epimerase (DPE), D-allulose-6-phosphate 3-epimerase (A6PE), D-allulose 6-phosphate phosphatase (A6PP), ribitol 2-dehydrogenase (RDH), glycerophosphate kinase (GK), glycerophosphate oxidase (GPO), and dihydroxyacetone phosphate (DHAP)-dependent aldolase. Biosynthesis is a more energy-efficient process, producing fewer harmful by-products and pollutants, and significantly reducing negative environmental impacts. Furthermore, the specific catalytic activity of enzymes facilitates the production of compounds of higher purity, thereby facilitating the isolation and purification of the products. It has thus become the main method for producing D-allulose. This article reviews the progress in research on the biosynthetic production of D-allulose, focusing on the enzymes involved and their enzymatic properties, and discusses the production prospects for D-allulose.

Trehalose-6-phosphate phosphatase expression and enzymatic properties of Fusariumgraminearum

This study presents an exhaustive characterization of the enzymatic attributes and structural properties of trehalose-6-phosphate phosphatase (TPP) derived from Fusarium graminearum. Enzyme activity was evaluated through a meticulously designed enzymatic assay. The findings indicate that the molecular weight of the enzyme is approximately 99.8 kDa, with an optimal reaction temperature and pH of 40 °C and 6.5, respectively. Magnesium ions (Mg2+) markedly enhance the enzymatic activity, resulting in a specific activity of 1.795 U/μg. Kinetic analysis revealed a Km value of 0.96 μmol/L and a Vmax of 15.79 μmol/L/min. Subsequent computational analysis elucidated the three-dimensional architecture of the enzyme and identified the binding site for the substrate trehalose-6-phosphate (T6P). T6P was found to form hydrogen bonds with TPP at residues Lys754, Arg720, His665, Glu758, and Asn756. Additionally, hydrophobic interactions were observed between T6P and residues Phe802, Ile610, Asp801, Pro752, and Gly753. The binding energy calculated for the T6P-TPP complex stood at −5.7 kcal/mol.

Eukaryotic expression of chitinase from dark sleeper (Odontobutis potamophila) and its effects on growth and immunity

Chitinase, an enzyme that hydrolyzes β-1,4-glycosidic bonds to degrade chitin, is essential for the digestion of chitin in fish. In this study, the chitinase OpCht from Odontobutis potamophila was expressed in Pichia pastoris, and its enzymatic properties and functional effects were evaluated. The findings revealed that OpCht exhibited optimal activity at pH 6.0 and 50 °C, with stability in the pH range of 4–8 and temperatures from 4 to 40 °C. K+, Na+, Ca2+, Mg2+, Mn2+, Hg2+, and Al3+ showed varying degrees of activation on the enzyme. At the end of the 8-week trial, the addition of OpCht significantly increased the height of intestinal villi and the thickness of the muscular layer, leading to significantly weight in the treated groups. The alleviation of intestinal inflammation also resulted in an increased survival rate (SR) of O. potamophila. High concentration treatment groups (2, 4 μg/g) showed significantly elevated digestive enzyme activities, as well as increased antioxidant enzyme activities and immune parameters. These results demonstrate that the P. pastoris expression system has successfully produced the chitinase OpCht from O. potamophila, and the addition of a certain concentration of OpCht can promote fish growth and enhance immune functions, offering a promising enzyme preparation for the aquaculture industry.

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest (∼10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both α-helix and β-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a Gx3Sx4G motif homologous to canonical ‘GxSxG’ motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid- and/or monounsaturated fatty acid-containing diacylglycerol

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) (EC 3.1.4.3) and phosphatidylethanolamine (PE)-specific PLC (PE-PLC) (EC 3.1.4.62), which generate diacylglycerol (DG) and are tricyclodecan-9-yl-xanthogenate (D609)-sensitive, were detected in detergent-insoluble fractions of mammalian tissues approximately 70 and 35 years ago, respectively. However, the genes and proteins involved in PC-PLC and PE-PLC activities remain unknown. In a recent study, we observed that mammalian sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr) display PC-PLC and PE-PLC activities in vitro. In the present study, we showed that human SMS2, which is located in detergent-insoluble fractions of the plasma membrane, also possesses PC-PLC activity (approximately 41% of SMS activity), PE-PLC activity (approximately 4%), ceramide phosphoethanolamine synthase (CPES) activity (approximately 46%), and SMS activity in the presence of phospholipid-detergent mixed micelles. Moreover, purified SMS2 reconstituted in detergent-free proteoliposomes (near-native environments) showed PC-PLC, PE-PLC, and CPES activities. Notably, in the presence of approximately 2 mol% ceramide and 4 mol% PC (1:2 ratio), PC-PLC activity was almost equal to SMS activity. SMS2 as PC/PE-PLC showed substrate selectivity for saturated fatty acid- and/or monounsaturated fatty acid-containing PC and PE species. The PC-PLC/SMS inhibitor D609 inhibited all enzyme activities (SMS, PC-PLC, PE-PLC, and CPES) of SMS2. Moreover, Zn2+ strongly inhibited all the enzymatic activities of SMS2. Interestingly, DG inhibited the SMS activity of SMS2 (feedback control). These results indicate that mammalian SMS2 has unique enzymatic properties and is a candidate for a long-sought mammalian PC/PE-PLC.

Immobilization β-glucosidase from Dictyoglomus thermophilum on UiO-66-NH2: An efficient catalyst for enzymatic synthesis of kinsenoside via reverse hydrolysis reaction

Kinsenoside is a rare and valuable glycoside with extensive bioactivities. However, the enzymatic synthesis of kinsenoside has been a challenging task due to the limited enzyme toolbox and unsatisfactory yield. Herein, the β-glucosidase from Dictyoglomus thermophilum (DtBGL) was heterologously expressed, purified and enzymatically characterized. The purified DtBGL was successfully immobilized on the metal-organic frameworks of UiO-66-NH2. The DtBGL@UiO-66-NH2 was fully characterized using SEM, XRD, TGA and FTIR. The studies on enzymatic properties demonstrated that DtBGL@UiO-66-NH2 exhibited increased catalytic activity and stability compared to the free DtBGL. Particularly, DtBGL@UiO-66-NH2 could catalyze the synthesis of kinsenoside via the reverse hydrolysis reaction and the kinsenoside yield was 34.12 % under the optimized catalytic system, which was 1.9-fold higher compared with the free DtBGL. Moreover, DtBGL@UiO-66-NH2 displayed good reusability with a kinsenoside yield of 27.02 % after reuse for 3 times. The present work not only identifies and characterizes a highly active β-glucosidase with reverse hydrolysis activity, but also proposes the immobilized enzyme as an effective catalyst for the industrial production of glycosides.

Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Enhancing Cellular and Enzymatic Properties Through In Vivo Continuous Evolution.

Directed evolution seeks to evolve target genes at a rate far exceeding the natural mutation rate, thereby endowing cellular and enzymatic properties with desired traits. In vivo continuous directed evolution achieves these purposes by generating libraries within living cells, enabling a continuous cycle of mutant generation and selection, enhancing the exploration of gene variants. Continuous evolution has become powerful tools for unraveling evolution mechanism and improving cellular and enzymatic properties. This review categorizes current continuous evolution into three distinct classes: non-targeted chromosomal, targeted chromosomal, and extra-chromosomal hypermutation approaches. It also compares various continuous evolution strategies based on different principles, providing a reference for selecting suitable methods for specific evolutionary goals. Furthermore, this review discusses the two primary limitations for further widespread application of in vivo continuous evolution, which are lack of general applicability and insufficient mutagenic capability. We envision that developing generally applicable mutagenic components and methods to enhance mutation rates for in vivo continuous evolution are promising future directions for wide range applications of continuous evolution.

Nickel-doped cuprous oxide nanocauliflowers with specific peroxidase-like activity for sensitive detection of hydrogen peroxide and uric acid

Copper-based nanomaterials have the properties of mimetic enzymes and can be used as excellent candidates for colorimetric sensing due to their environmental friendliness, low cost, and high abundance. In this paper, Ni-doped Cu2O nano cauliflower (Ni-Cu2O) was synthesized for the first time and applied to the detection of H2O2 and uric acid (UA) in human serum and urine. It was found that the proportion of Ni incorporation controls the morphology and the catalytic effect of Ni-Cu2O. The catalytic mechanism was studied by X-ray photoelectron spectroscopy, free radical capture experiments, photoluminescence spectroscopy, and steady-state kinetic analysis, which verified the redox reactions involving electron transfer and active substances. The results showed that Ni-Cu2O could catalyze the formation of reactive oxygen species (•OH, O2•−, 1O2, h+) from H2O2, which could oxidize 3,3′, 5,5′-tetramethylbenzidine (TMB) to oxTMB, and the color changed from colorless to blue. The Michaelis-Menten constant (Km) and the maximum initial velocity (Vmax) of Ni-Cu2O were 1.8 mM and 15.2×10−8 M/s, respectively. Based on the excellent peroxidase-like (POD) activity of Ni-Cu2O, a colorimetric sensing platform combined with TMB was proposed to sensitively detect H2O2 and UA in a wide range, and the detection limits were as low as 0.17 μM and 0.22 μM, respectively. This study creates a platform for using the Cu-based cauliflowers as a biosensor to detect UA in the medical and biomedicine fields.

Mutations of methionine 444 interacting with T1Cu-coordinating amino acids affect the structure and function of multicopper oxidase CopA.

Manganese is an essential trace element for humans, animals, and plants, but excessive amounts of manganese can cause serious harm to organisms. The biological manganese oxidation process mainly oxidizes Mn(II) through the secretion of unique manganese oxidase by manganese-oxidizing bacteria. The T1 Cu site of multicopper oxidase is the main site for substrate oxidation, and its role is to transfer electrons to TNC, where dioxygen reduction occurs. In this study, methionine (Met) No. 444 interacting with the T1Cu-coordinating amino acid in the multicopper oxidase CopA from Brevibacillus panacihumi MK-8 was mutated to phenylalanine (Phe) and leucine (Leu) by the enzyme. Based on the analysis of enzymatic properties and the structural model, the mutant protein M444F with 4.58 times the catalytic efficiency of the original protein CopA and the mutant protein M444L with 1.67 times the catalytic efficiency of the original protein CopA were obtained. The study showed that the manganese removal rate of the manganese-oxidizing engineered bacterium Rosetta-pET-copAM444L cultured for 7 days was 88.87%, which was 10.77% higher than that of the original engineered bacterium. Overall, this study provides a possibility for the application of genetic engineering in the field of biological manganese removal.

A Novel Approach for Colorimetric Detection of Glyphosate in Food Based on a Split Aptamer Nanostructure and DNAzyme Activity.

Glyphosate has become the most widely used herbicide worldwide in recent years. There are many concerns about toxicity and mutagenicity from long-term use of glyphosate in humans and animals. Therefore, the methods that can help in easy and quick detection of this chemical compound in food and water are critical. In this work, a biosensor was fabricated by combining the enzymatic properties of a specific DNA G-quadruplex and selectivity of a split aptamer to detect glyphosate in foods and water in a quick and simple colorimetric manner. The color change in this method is based on the oxidation of TMB by the G-quadruplex enzyme and the function of aptamer to trap glyphosate, which is visible to the naked eye in the presence and absence of the herbicide. The biosensor showed its high performance in various real samples of water and foods and provided a detection limit of 1.37 nM (R² = 0.9899) with a linear response range of 100 to 400 nM of glyphosate. This biosensor can provide an innovative, cheap and fast approach for the detection and monitoring of glyphosate in various foods and water.

Enhanced Production, Enzymatic Activity, and Thermostability of an α-Amylase from Bacillus amyloliquefaciens in Lactococcus lactis.

A novel α-amylase gene (BAA) from Bacillus amyloliquefaciens was cloned into Lactococcus lactis, designing two recombinant α-amylases to facilitate extracellular secretion. Following optimizing the expression conditions, the highest yield of BAA (88.12 mmol/L) was achieved upon 36 h induction and 5 ng/mL nisin concentration. Determining the enzymatic properties of BAA revealed its poor stability and activity at high temperatures, hindering its widespread application. Therefore, we used computer-aided design to generate a mutant, S275L, which exhibited significantly improved activity and thermostability: an 18.7% increase in enzymatic activity (3767.38 U/mg), a 10 °C increase in optimal temperature, and a 49.2% improvement in stability at 60 °C. Molecular dynamics simulations and force analysis confirmed these enhancements. Finally, the mutant S275L's potential was further analyzed for starch hydrolysis on poultry feed. Therefore, the mutant S275L holds promising as an enzyme agent for enhanced feed digestibility and quality.

Characterization of Pectinase Enzyme Produced from Indigenous Yeast Isolates During Fermentation of Robusta Coffee Beans

The following research aims to understand indigenous yeast isolates that are pectinolytic, the effect of incubation time on pectinase enzyme activity, and the pH and temperature properties of pectinase enzymes obtained from indigenous yeasts from spontaneous fermentation of robusta coffee beans. The following type of research is an experiment through RAL (Completely Randomized Design). The results showed that testing of seven indigenous yeast isolates was pectinolytic and that the highest pectinolytic ability was isolate K24I7 with a clear zone diameter of 3.13 mm. The characterization results of the pectinase enzyme produced from isolate K24I7 were optimum at pH 5.5 with an enzyme specific activity value of 10.09 U/mg and optimum temperature in the range of 40oC-60oC with an average enzyme specific activity of 10.08 U/mg. The pectinase enzyme produced from isolate K24I7 is thermophile because it is still active at 70°C