D-allulose 3-epimerase for low-calorie D-allulose synthesis: microbial production, characterization, and applications.

D-allulose, an epimer of D-fructose at C-3 position, is a low-calorie rare sugar with favorable physiochemical properties and special physiological functions, which displays promising perspectives in the food and pharmaceutical industries. Currently, D-allulose is extremely sparse in nature and is predominantly biosynthesized through the isomerization of D-fructose by D-allulose 3-epimerase (DAEase). In recent years, D-allulose 3-epimerase as the key biocatalyst for D-allulose production has received increasing interest. The current review begins by providing a summary of D-allulose regarding its characteristics and applications, as well as different synthesis pathways dominated by biotransformation. Then, the research advances of D-allulose 3-epimerase are systematically reviewed, focusing on heterologous expression and biochemical characterization, crystal structure and molecular modification, and application in D-allulose production. Concerning the constraint of low yield of DAEase for industrial application, this review addresses the various attempts made to promote the production of DAEase in different expression systems. Also, various strategies have been adopted to improve its thermotolerance and catalytic activity, which is mainly based on the structure-function relationship of DAEase. The application of DAEase in D-allulose biosynthesis from D-fructose or low-cost feedstocks through single- or multi-enzymatic cascade reaction has been discussed. Finally, the prospects for related research of D-allulose 3-epimerase are also proposed, facilitating the industrialization of DAEase and more efficient and economical bioproduction of D-allulose.

Partitioning purification, biochemical characterization, and milk coagulation efficiency of protease from a newly Streptomyces sp. isolate.

An actinobacteria strain was isolated from an olive waste mill and tested for protease production on skimmed milk media. The strain identification was achieved through both 16 S rDNA sequencing and phenotypic characterization. The enzyme was purified using the ammonium sulfate/t-butanol three-phase partitioning (TPP) method, followed by characterization to investigate the effect of pH, temperature, and various chemical agents. Subsequently, the enzyme was assessed for its milk coagulation activity. The strain belonging to the Streptomyces genera, exhibits significant phylogenetic and phenotypic differences from the aligned species, suggesting its novelty as a new strain. The enzyme was best separated in the TPP aqueous phase with a 5.35 fold and 56.25% yield. Optimal activity was observed at pH 9.0 and 60 °C, with more than half of the activity retained within the pH range of 7-10 over one hour. The protease demonstrated complete stability between 30 and 60 °C. While metallic ions enhanced enzyme activity, EDTA acted as an inhibitor. The enzyme displayed resistance to H2O2, SDS, Tween 80, and Triton X-100. Notably, it was activated in organic solvents (ethyl acetate, petroleum ether, and xylene), maintaining > 75% of its original activity in butanol, ethanol, and methanol. Additionally, the enzyme yielded high milk coagulant activity of 11,478 SU/mL. The new Streptomyces sp. protease revealed high activity and stability under a wide range of biochemical conditions. Its use in the dairy industry appears particularly promising. Further industrial process investigations will be valuable in determining potential uses for this enzyme.

Detergent-resistant α-amylase derived from Anoxybacillus karvacharensis K1 and its production based on whey.

In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg-1. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg-1) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg-1). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5'-dithio-bis-[2-nitrobenzoic acid (DNTB), ß-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme's activity, and the presence of CaCl2 (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (Km) and maximum reaction rate (Vmax), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL-1 and 1580.3 ± 183.7 µmol mg-1 protein min-1, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with ß-glucosidase for 24 h.

Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

Construction and characterization of a novel fusion alginate lyase with endolytic and exolytic cleavage activity for industrial preparation of alginate oligosaccharides.

Alginate lyases with high activity and good thermostability are lacking for the preparation of alginate oligosaccharides (AOS) with various biological activities. We constructed a fusion alginate lyase with both endo-and exo-activities. AlyRm6A-Zu7 was successfully constructed by connecting the highly thermostable AlyRm6A to a new exotype lyase, AlyZu7. The fusion enzyme exhibited high catalytic activity and thermostability. It transformed sodium alginate into oligosaccharides with degrees of polymerization (DP) of 2-4 while producing 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). The maximum reducing sugar, AOS, and DP1 + DEH yields were 75 %, 45 %, and 40 %, respectively. Molecular docking confirmed the formation of a stable complex between the substrate and AlyRm6A-Zu7. Protein interactions increased the thermostability of AlyZu7. This work provides new insights into the industrial formation of AOS and monosaccharide DEH using thermally stable fusion enzymes, which has a positive effect in the fields of functional oligosaccharide production and biofuel formation.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

Various Strategies for the Immobilization of a Phospholipase C from Bacillus cereus for the Modulation of Its Biochemical Properties.

In this study, the effect of various immobilization methods on the biochemical properties of phospholipase C (PLC) from Bacillus cereus obtained from the oily soil located in Sfax, Tunisia, was described. Different supports were checked: octyl sepharose, glyoxyl agarose in the presence of N-acetyl cysteine, and Q-sepharose. In the immobilization by hydrophobic adsorption, a hyperactivation of the PLCBc was obtained with a fold of around 2 times. The recovery activity after immobilization on Q-sepharose and glyoxyl agarose in the presence of N-acetyl cysteine was 80% and 58%, respectively. Furthermore, the biochemical characterization showed an important improvement in the three immobilized enzymes. The performance of the various immobilized PLCBc was compared with the soluble enzyme. The derivatives acquired using Q-sepharose, octyl sepharose, and glyoxyl agarose were stable at 50 °C, 60 °C, and 70 °C. Nevertheless, the three derivatives were more stable in a large range of pH than the soluble enzyme. The three derivatives and the free enzyme were stable in 50% (v/v) ethanol, hexane, methanol, and acetone. The glyoxyl agarose derivative showed high long-term storage at 4 °C, with an activity of 60% after 19 days. These results suggest the sustainable biotechnological application of the developed immobilized enzyme.

Insights into Chitin-Degradation Potential of Shewanella khirikhana JW44 with Emphasis on Characterization and Function of a Chitinase Gene SkChi65.

Chitin, a polymer of ß-1,4-linked N-acetylglucosamine (GlcNAc), can be degraded into valuable oligosaccharides by various chitinases. In this study, the genome of Shewanella khirikhana JW44, displaying remarkable chitinolytic activity, was investigated to understand its chitin-degradation potential. A chitinase gene SkChi65 from this strain was then cloned, expressed, and purified to characterize its enzymatic properties and substrate hydrolysis. Genome analysis showed that, of the 14 genes related to chitin utilization in JW44, six belonged to glycoside hydrolase (GH) families because of their functional domains for chitin binding and catalysis. The recombinant chitinase SkChi65, consisting of 1129 amino acids, was identified as a member of the GH18 family and possessed two chitin-binding domains with a typical motif of [A/N]KWWT[N/S/Q] and one catalytic domain with motifs of DxxDxDxE, SxGG, YxR, and [E/D]xx[V/I]. SkChi65 was heterologously expressed as an active protein of 139.95 kDa best at 37 °C with 1.0 mM isopropyl-ß-d-thiogalactopyranoside induction for 6 h. Purified SkChi65 displayed high stability over the ranges of 30-50 °C and pH 5.5-8.0 with optima at 40 °C and pH 7.0. The kinetic parameters Km, Vmax, and kcat of SkChi65 towards colloidal chitin were 27.2 µM, 299.2 µMs-1, and 10,203 s-1, respectively. In addition to colloidal chitin, SkChi65 showed high activity towards glycol chitosan and crystalline chitin. After analysis by thin-layer chromatography, the main products were N,N'-diacetylchitobiose, and GlcNAc with (GlcNAc)2-6 used as substrates. Collectively, SkChi65 could exhibit both exo- and endochitinase activities towards diverse substrates, and strain JW44 has a high potential for industrial application with an excellent capacity for chitin bioconversion.

Characterization and in-vitro plant-based control of hindgut bacteria isolated from Odontotermes obesus Rambur (Termitidae) and Heterotermes indicola Wasmann (Rhinotermitidae).

Termites cause a serious menace to wooden structures all over the world. They rely mostly on entozoic fauna residing in their hindgut for the digestion of cellulosic and hemicellulosic materials. One of the ways to control termites is through their gut symbionts. The present study was designed to characterize the hindgut bacteria isolated from Odontotermes obesus and Heterotermes indicola. Furthermore, the growth inhibitory effect of eight tropical plant extracts was investigated to find out potential control agents for these bacterial isolates. The characterization of bacteria was carried out based on their morphology, Gram staining, biochemical and amplification of 16SrRNA gene. Amplified products were sequenced to confirm their relationship with bacterial isolates from termites of other regions. The growth inhibitory effect of ethanolic leaf extracts of eight plants was evaluated in an invitro agar well diffusion method. Qualitative and quantitative phytochemical analysis of the most effective plant was carried out to learn about bioactive agents. The results confirmed the presence of five bacteria from each termite species. The Bacillus cereus, Escherichia coli, and Lysinibacillus fusiformis were common to both termites whereas Lysinibacillus xylanilyticus and Lysinibacillus macrolides were found in O. obesus only and H. indicola harbor Bacillus subtilis and Shigella sonnei in addition to common three ones. Among the plant extracts of Carica papaya, Eucalyptus camaldulensis, Osmium basilicum, Grevillea robusta, Eucalyptus globulus, Pongamia pinnata, Mentha longifolia, and Melia azedarach, the G. robusta > E. camaldulensis > O. basilicum were found to have growth inhibitory effects with increasing concentrations from 100 to 2000 µg/mL. The biodiversity of the bacterial fauna is important for the biological control of termites. Leaf extracts of these medicinal plants can be used to control termite infestation in an environment-friendly manner to save huge economic loss.

Purification and Biochemical Characterization of Polyphenol Oxidase Extracted from Wheat Bran (Wan grano).

Currently, little is known about the characteristics of polyphenol oxidase from wheat bran, which is closely linked to the browning of wheat product. The wheat PPO was purified by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column, and Superdex G-75 chromatography column. Purified wheat PPO activity was 11.05-fold higher, its specific activity was 1365.12 U/mg, and its yield was 8.46%. SDS-PAGE showed that the molecular weight of wheat PPO was approximately 21 kDa. Its optimal pH and temperature were 6.5 and 35 °C for catechol as substrate, respectively. Twelve phenolic substrates from wheat and green tea were used for analyzing the substrate specificity. Wheat PPO showed the highest affinity to catechol due to its maximum Vmax (517.55 U·mL-1·min-1) and low Km (6.36 mM) values. Docking analysis revealed strong affinities between catechol, gallic acid, EGCG, and EC with binding energies of -5.28 kcal/mol, -4.65 kcal/mol, -4.21 kcal/mol, and -5.62 kcal/mol, respectively, for PPO. Sodium sulfite, ascorbic acid, and sodium bisulfite dramatically inhibited wheat PPO activity. Cu2+ and Ca2+ at 10 mM were considered potent activators and inhibitors for wheat PPO, respectively. This report provides a theoretical basis for controlling the enzymatic browning of wheat products fortified with green tea.

Biochemical Characterization of a Novel Thermostable 1,4-α-Glucoamylase from Aspergillus brasiliensis Strain Isolated in the Brazilian Atlantic Forest.

Glucoamylases are exo-enzymes that cleave the ends of the starch chain, releasing glucose units. In the current work, we described a novel 1,4-α-glucoamylase from an A. brasiliensis strain isolated from an environmental sample. The purified glucoamylase, GlaAb, has a molecular mass of 69 kDa and showed a starch binding domain. GlaAb showed a similar sequence to other fungal glucoamylases, and the molecular 3D model analysis of GlaAb suggests an overall structure as described in the literature, except by elongation in the loop connecting the 4th and 5th α-helices. The enzyme showed activity over a wide range of pH and temperature, with maximum activity at pH 4.5 and 60 °C. GlaAb was stable at 50 °C for 7 h, maintaining 67% residual activity, and it was not inhibited by glucose up to 0.1 M. The glucoamylase was 65% more active in the presence of Mn2+ and showed a Km of 2.21 mg mL-1, Vmax of 155 U mg-1, Kcat 179 s-1, and Kcat/Km 81.06 mg mL-1 s-1 using potato starch as substrate. The results obtained are promising and provide the basis for the development of applications of GlaAb in the industrial process.

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides.

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0-6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides. KEY POINTS: • An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii. • TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin. • TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins.

Percentage Incidences of Bacteria in Mahseer (Tor putitora), Silver carp (Hypophthalmichthys molitrix) Fish Collected from Hatcheries and Local Markets of District Malakand and Peshawar of Khyber Pakhtunkhwa, Pakistan / Incidências percentuais de bactérias em Mahseer (Tor putitora), carpa-prateada (Hypophthalmichthys molitrix), peixes coletados em incubatórios e mercados locais do distrito de Malakand e Peshawar de Khyber Pakhtunkhwa, Paquistão

Abstract Fish is the main source of animal protein for human diet. The aim of this study was to find out prevalence of pathogenic bacteria of two selected economically important fish of Pakistan namely Mahseer (Tor putitora) and Silver carp (Hypophthalmichthys molitrix). Live fish samples from hatcheries and dead fish samples from different markets of study area were randomly collected. The fish samples were analyzed for isolation, identification and prevalence of bacteria. The isolated bacteria from study fish were identified through biochemical test and about 10 species of pathogenic bacteria were identified including the pathogenic bacteria to human and fish namely, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. and Salmonella spp. The bacterial percentage frequency of occurrence in Silver carp and Mahseer fish showed Pseudomonas aeruginosa 21.42%, Staphylococcus epidermidis 17.85%, Escherichia coli 11.90%, Staphylococcus aureus 9.52%, Citrobacter spp. 9.52%, Serratia spp. 8.33%, Streptococcus iniae 7.14%, Stenotrophomonas spp. 5.95%, Bacillus spp. 4.76% and Salmonella spp. 3.57%. The study revealed that Fish samples of Mahseer and Silver carp that were collected from markets have found more isolates (10 bacterial species) than did the fresh fish pond samples (03 bacterial species) of hatcheries. The occurrence of pathogenic bacteria in study fish showed risk factor for public health consumers.

Resumo O peixe é a principal fonte de proteína animal para a alimentação humana. O objetivo deste estudo foi descobrir a prevalência de bactérias patogênicas de dois peixes economicamente importantes selecionados do Paquistão, nomeadamente Mahseer (Tor putitora) e carpa prateada (Hypophthalmichthys molitrix). Amostras de peixes vivos de incubatórios e amostras de peixes mortos de diferentes mercados da área de estudo foram coletadas aleatoriamente. As amostras de peixes foram analisadas quanto ao isolamento, identificação e prevalência de bactérias. As bactérias isoladas dos peixes do estudo foram identificadas através de testes bioquímicos e cerca de 10 espécies de bactérias patogênicas foram identificadas incluindo as bactérias patogênicas para humanos e peixes, nomeadamente, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. e Salmonella spp. A porcentagem de freqüência de ocorrência bacteriana em carpa prateada e peixes Mahseer mostrou Pseudomonas aeruginosa 21,42%, Staphylococcus epidermidis 17,85%, Escherichia coli 11,90%, Staphylococcus aureus 9,52%, Citrobacter spp. 9,52%, Serratia spp. 8,33%, Streptococcus iniae 7,14%, Stenotrophomonas spp. 5,95%, Bacillus spp. 4,76% e Salmonella spp. 3,57%. O estudo revelou que as amostras de peixes de Mahseer e carpa prateada coletadas nos mercados encontraram mais isolados (10 espécies bacterianas) do que as amostras de peixes frescos (03 espécies bacterianas) de incubatórios. A ocorrência de bactérias patogênicas nos peixes do estudo apresentou fator de risco para consumidores de saúde pública.

Purification and biochemical characterization of novel α-amylase and cellulase from Bacillus sp. PM06.

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg, 1.154 ± 0.1 mM, and 589 ± 3.5/(s mM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(s mM) of Vmax, Km, Kcat/Km, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes.

Percentage Incidences of Bacteria in Mahseer (Tor putitora), Silver carp (Hypophthalmichthys molitrix) Fish Collected from Hatcheries and Local Markets of District Malakand and Peshawar of Khyber Pakhtunkhwa, Pakistan

Abstract Fish is the main source of animal protein for human diet. The aim of this study was to find out prevalence of pathogenic bacteria of two selected economically important fish of Pakistan namely Mahseer (Tor putitora) and Silver carp (Hypophthalmichthys molitrix). Live fish samples from hatcheries and dead fish samples from different markets of study area were randomly collected. The fish samples were analyzed for isolation, identification and prevalence of bacteria. The isolated bacteria from study fish were identified through biochemical test and about 10 species of pathogenic bacteria were identified including the pathogenic bacteria to human and fish namely, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. and Salmonella spp. The bacterial percentage frequency of occurrence in Silver carp and Mahseer fish showed Pseudomonas aeruginosa 21.42%, Staphylococcus epidermidis 17.85%, Escherichia coli 11.90%, Staphylococcus aureus 9.52%, Citrobacter spp. 9.52%, Serratia spp. 8.33%, Streptococcus iniae 7.14%, Stenotrophomonas spp. 5.95%, Bacillus spp. 4.76% and Salmonella spp. 3.57%. The study revealed that Fish samples of Mahseer and Silver carp that were collected from markets have found more isolates (10 bacterial species) than did the fresh fish pond samples (03 bacterial species) of hatcheries. The occurrence of pathogenic bacteria in study fish showed risk factor for public health consumers.

Resumo O peixe é a principal fonte de proteína animal para a alimentação humana. O objetivo deste estudo foi descobrir a prevalência de bactérias patogênicas de dois peixes economicamente importantes selecionados do Paquistão, nomeadamente Mahseer (Tor putitora) e carpa prateada (Hypophthalmichthys molitrix). Amostras de peixes vivos de incubatórios e amostras de peixes mortos de diferentes mercados da área de estudo foram coletadas aleatoriamente. As amostras de peixes foram analisadas quanto ao isolamento, identificação e prevalência de bactérias. As bactérias isoladas dos peixes do estudo foram identificadas através de testes bioquímicos e cerca de 10 espécies de bactérias patogênicas foram identificadas incluindo as bactérias patogênicas para humanos e peixes, nomeadamente, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. e Salmonella spp. A porcentagem de freqüência de ocorrência bacteriana em carpa prateada e peixes Mahseer mostrou Pseudomonas aeruginosa 21,42%, Staphylococcus epidermidis 17,85%, Escherichia coli 11,90%, Staphylococcus aureus 9,52%, Citrobacter spp. 9,52%, Serratia spp. 8,33%, Streptococcus iniae 7,14%, Stenotrophomonas spp. 5,95%, Bacillus spp. 4,76% e Salmonella spp. 3,57%. O estudo revelou que as amostras de peixes de Mahseer e carpa prateada coletadas nos mercados encontraram mais isolados (10 espécies bacterianas) do que as amostras de peixes frescos (03 espécies bacterianas) de incubatórios. A ocorrência de bactérias patogênicas nos peixes do estudo apresentou fator de risco para consumidores de saúde pública.

Biochemical characterization and key catalytic residue identification of a novel alpha-agarase with CBM2 domain.

Agarooligosaccharides have great potential in food industry because of their various bio-activities, while the limited availability and diversity of α-agarases hinder agarooligosaccharides' broader application. To overcome this limitation, a computer-assisted method was used to screen and identify novel agarases. Firstly, one novel α-agarase, AgaB, with an N-terminal CBM2 domain (the first report of this domain in agarases), was discovered. Purified agarases only exhibited activity against agarose, with optimum activity at 40℃ and pH 8.0. Analysis of hydrolysis products indicated that AgaB is an endo-type α-agarase, producing agarotetraose and agarohexaose. Secondly, AgaB truncated CBM2 showed increased Km values, suggesting that CBM2 aids in substrate binding. Thirdly, E468 and D333 are possibly catalytic amino acids, which was supported by molecular docking results and mutants. Biochemical characterization of first reported CBM2-containing agarase and catalytic mechanism study lay the foundation for the exploration and development of α-agarases in the future.

Chicken viscera meal as substrate for the simultaneous production of antioxidant compounds and proteases by Aspergillus oryzae.

The use of chicken waste can contribute to the development of new processes and obtaining molecules with high added value. An experimental design was applied to evaluate the effect of moisture, temperature, and inoculum size on the production of antioxidant peptides and proteases by A. oryzae IOC3999 through solid-state fermentation (SSF) of chicken viscera meal. As a result, the process conditions strongly influenced protease production and antioxidant activity of the fermented products. A global analysis of the results indicated that the most adequate conditions for SSF were (assay 9): 40% initial moisture, 30 °C as the incubation temperature, 5.05 × 106 spores/g as the inoculum size, and 48-h fermentation as the fermentation time. Under this condition, the antioxidant activities for the ABTS- and DPPH-radicals inhibition and ferric reducing antioxidant power (FRAP) methods were 376.16, 153.29, and 300.47 (µmol TE/g), respectively, and the protease production reached 428.22 U/g. Ultrafiltration of the crude extract obtained under optimized fermentation conditions was performed, and the fraction containing peptides with molecular mass lower than 3 kDa showed the highest antioxidant activity. The proteases were biochemically characterized and showed maximal activity at pH values ranging from 5.0 to 6.0 and a temperature of 50 °C. The thermodynamic parameters indicated that the process of thermal protease inactivation is not spontaneous (ΔG*d > 88.78 kJ/mol), increasing with temperature (ΔH*d 27.01-26.88 kJ/mol), and with reduced disorder in the system (ΔS*d < - 197.74 kJ/mol) probably caused by agglomeration of partially denatured enzymes.

Cloning, expression, and biochemical characterization of ß-etherase LigF from Altererythrobacter sp. B11.

Lignin, a complex heteropolymer present in plant cell walls, is now recognized as a valuable renewable resource with potential applications in various industries. The lignin biorefinery concept, which aims to convert lignin into value-added products, has gained significant attention in recent years. ß-etherases, enzymes that selectively cleave ß-O-4 aryl ether bonds in lignin, have shown promise in lignin depolymerization. In this study, the ß-etherase LigF from Altererythrobacter sp. B11 was cloned, expressed, purified, and biochemically characterized. The LigF-AB11 enzyme exhibited optimal activity at 32 °C and pH 8.5 when catalyzing the substrate PNP-AV. The enzyme displayed mesophilic behavior and demonstrated higher activity at moderate temperatures. Stability analysis revealed that LigF-AB11 was not thermostable, with a complete loss of activity at 60 °C within an hour. Moreover, LigF-AB11 exhibited excellent pH stability, retaining over 50 % of its activity after 1 h under pH conditions ranging from 3.0 to 11.0. Metal ions and surface impregnation agents were found to affect the enzyme's activity, highlighting the importance of considering these factors in enzymatic processes for lignin depolymerization. This study provides valuable insights into the biochemical properties of LigF-AB11 and contributes to the development of efficient enzymatic processes for lignin biorefineries. Further optimization and understanding of ß-etherases will facilitate their practical application in the valorization of lignin.

Characterization of a Type II L-Asparaginase from the Halotolerant Bacillus subtilis CH11.

L-asparaginases from bacterial sources have been used in antineoplastic treatments and the food industry. A type II L-asparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited an activity of 234.38 U mg-1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme showed a homotetrameric form with an estimated molecular weight of 155 kDa through gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of its initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL-1 min-1 and 4.75 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.

Role of Mutant Phosphate Solubilizing Microorganism on Growth of Brassica juncea (L.) cv RH 30.

Phosphorus is the second major plant nutrient and the availability of this element to plants is a major challenge due to conversion to insoluble form by chemical reactions with metal cations depending upon the soil pH. Total of sixty eight isolates of phosphate solubilizing bacteria from rhizosphere of mustard grown in different region of Haryana was studied. The isolates were mutagenised by giving treatment of Nitrosoguanidine (50 ug/ml). Three PSB strains (15M, 22M and 25M) and six mutants (15M2, 15M6, 22M28, 22M29, 25M11 and 25M30) were evaluated for their establishment in the rhizosphere, their effect on biomass production in mustard (Brassica juncea). Total bacterial count in rhizosphere increased after 30 and 40 days of sowing while decrease in growth was observed at 60 days of sowing. The phosphate solubilizing bacterial count in the rhizosphere varied from 1 to 24, 2 to 20 and 1 to 11 at 30, 45 and 60 days after sowing. Phosphate uptake also increased upto 11-21% which shows thatmutants had significant effect on increase in plant dry biomass and P-uptake under pot house conditions.