Evidence for Zoonotic Potential of Enterocytozoon bieneusi in Its First Molecular Characterization in Captive Mammals at Bangladesh National Zoo

To determine the occurrence and genotypes of Enterocytozoon bieneusi in captive mammals at Bangladesh National Zoo and to assess their zoonotic significance, 200 fecal samples from 32 mammalian species were examined using a nested PCR and sequencing of internal transcribed spacer (ITS) gene. Enterocytozoon bieneusi was detected in 16.5% (33/200) of the samples. Seven different ITS genotypes were identified, including two known genotypes (D and J) and five new ones (BAN4 to BAN8). Genotype D was the most common genotype being observed in 19 isolates. In phylogenetic analysis, four genotypes (D, BAN4, BAN5, and BAN6), detected in 30 isolates (90.9%), belonged to Group 1 having zoonotic potential. The sequence of genotype J found in a Malayan pangolin was clustered in so‐called ruminant‐specific Group 2. The other two genotypes BAN7 and BAN8 were clustered in primate‐specific Group 5. To our knowledge, this is the first report of molecular characterization of E. bieneusi in Bangladesh, particularly in captive‐bred wildlife in this country. The potentially zoonotic genotypes of E. bieneusi are maintained in zoo mammals that may transmit among these animals and to the humans through environmental contamination or contact.     

Production,optimization, purification,characterization, and anti-cancer application of extracellular L-glutaminase produced from the marine bacterial isolate

Abstract

L-glutaminase from bacterial sources has been proven to be effective and economical agents in cancer therapy, food industry and high-value chemicals like threonine. In the present study, a newly isolated bacterial strain was potentially producing extracellular L-glutaminase, it identified as Bacillus subtilis OHEM11 (MK389501) using the 16S rRNA gene. L-glutaminase production optimized and the optimum factors for production under submerged fermentation were at pH 6.5–7.0 and 35?°C after 28?hr using rhamnose and glutamine as carbon and nitrogen sources, respectively, while bagasse was the best inducer for the production under solid-state fermentation. Ethanol precipitation and ion-exchange chromatography using QFF are the purification steps. L-glutaminase was purified to 2-fold with specific activity 89.78?U/mg and its molecular weight about 54.8?kDa with the alkaline property of the enzyme makes it clear having carcinostatic property; maximum enzyme activity at pH 8.2 and 40?°C and retained about 90% activity for 1?hr. The cytotoxicity effect of L-glutaminase indicated a significant safety on Vero cells with high anticancer activity against NFS-60, HepG-2, and MCF-7 cancer cell lines. The outcomes demonstrated that L-glutaminase could be applied in many biotechnological applications such as pharmaceutical and food processing.     

Fur Seal Feces-Associated Circular DNA Virus Identified in Pigs in Anhui,China

Fur seal feces-associated circular DNA virus(FSfa CV) is an unclassified circular replication-associated protein(Rep)-encoding single-stranded(CRESS) DNA virus that has been detected in mammals(fur seals and pigs). The biology and epidemiology of the virus remain largely unknown. To investigate the virus diversity among pigs in Anhui Province,China, we pooled 600 nasal samples in 2017 and detected viruses using viral metagenomic methods. From the assembled contigs, 12 showed notably high nucleotide acid sequence similarities to the genome sequences of FSfa CVs. Based on these sequences, a full-length genome sequence of the virus was then obtained using overlapping PCR and sequencing, and the virus was designated as FSfa CV-CHN(Gen Bank No. MK462122). This virus shared 91.3% and 90.9% genome-wide nucleotide sequence similarities with the New Zealand fur seal strain FSfa CV-as50 and the Japanese pig strain FSfa CVJPN1, respectively. It also clustered with the two previously identified FSfa CVs in a unique branch in the phylogenetic tree based on the open reading frame 2(ORF2), Rep-coding gene, and the genome of the reference CRESS DNA viruses.Further epidemiological investigation using samples collected in 2018 showed that the overall positive rate for the virus was 56.4%(111/197) in Anhui Province. This is the first report of FSfa CVs identified in pigs in China, and further epidemiological studies are warranted to evaluate the influence of the virus on pigs.     

Quality Control Testing,Characterization and Critical Quality Attributes of Adeno-Associated Virus Vectors Used for Human Gene Therapy

For adeno-associated virus (AAV)-based human gene therapy, challenges for the translation of promising research results to successful clinical development include optimization of vector design and manufacturing processes to ensure that vectors prepared for administration to human subjects have attributes consistent with safe and durable expression. This article briefly reviews quality control methods for routine testing and supplemental characterization of AAV vectors for investigational product development. The relationship of vector and manufacturing process design with product critical quality attributes is discussed.     

Production and characterization of a cold-active and n-hexane activated lipase from a newly isolated Serratia grimesii RB06-22

Abstract

A lipase-producing bacterium isolated from raw milk was identified as Serratia grimesii based on 16S rRNA sequence analysis. The extracellular lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Maximal activity was observed at 10°C, the optimum pH was 8.0 and the enzyme was stable at 5–30°C for 1 h. The K_m and V_max values were 1.7 mM and 0.3 mM/min respectively. It was found that the lipase had the highest hydrolytic activity towards sunflower oil and soybean oil. CaCl₂ had a stimulatory effect on lipase activity, while EDTA and iodoacetic acid slightly inhibited the lipase activity and the enzyme was strongly inhibited by PMSF. The enzyme was compatible with various non-ionic surfactants as well as sodium cholate and saponin. In addition, the enzyme was relatively stable towards oxidizing agents. This lipase exhibited maximum activity in 35% n-hexane retaining about 2191% activity for 1 h.     

Purification and characterization of a novel β-glucosidase from Aspergillus flavus and its application in saccharification of soybean meal

Abstract

Aspergillus flavus has been regarded as a potential candidate for its production of industrial enzymes, but the details of β-glucosidase from this strain is very limited. In herein, we first reported a novel β-glucosidase (AfBglA) with the molecular mass of 94.2?kDa from A. flavus. AfBglA was optimally active at pH 4.5 and 60?°C and is stable between pH 3.5 and 9.0 and at a temperature of up to 55?°C for 30?min remaining more than 90% of its initial activity. It showed an excellent tolerance to Trypsin, Pepsin, Compound Protease, and Flavourzyme and its activity was not inhibited by specific certain cations. AfBglA displayed broad substrate specificity, it acted on all tested pNP-glycosides and barley glucan, indicating this novel β-glucosidase exhibited a β-1, 3-1, 4-glucanase activity. Moreover, the AfBglA could effectively hydrolyze the soybean meal suspension into glucose and exhibit a strong tolerance to the inhibition of glucose at a concentration of 20.0?g/L during the saccharification. The maximum amount of the glucose obtained by AfBglA corresponded to 67.0?g/kg soybean meal. All of these properties mentioned above indicated that the AfBglA possibly attractive for food and feed industry and saccharification of cellulolytic materials.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

PURIFICATION AND CHARACTERIZATION OF PHOSPHODIESTERASE I FROM Walterinnesia aegyptia VENOM

A phosphodiesterase I (EC 3.1.4.1; PDE-I) was purified from Walterinnesia aegyptia venom by preparative native polyacrylamide gel electrophoresis (PAGE). A single protein band was observed in analytical native PAGE and sodium dodecyl sulfate (SDS)-PAGE. PDE-I was a single-chain glycoprotein with an estimated molecular mass of 158 kD (SDS-PAGE). The enzyme was free of 5′-nucleotidase and alkaline phosphatase activities. The optimum pH and temperature were 9.0 and 60°C, respectively. The energy of activation (Ea) was 96.4, the V_max and K_m were 1.14 µM/min/mg and 1.9 × 10^?3 M, respectively, and the K_cat and K_sp were 7 s^?1 and 60 M ^?1 min^?1 respectively. Cysteine was a noncompetitive inhibitor, with K_i = 6.2 × 10^?3 M and an IC₅₀ of 2.6 mM, whereas adenosine diphosphate was a competitive inhibitor, with K_i = 0.8 × 10^?3 M and an IC₅₀ of 8.3 mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg²⁺ slightly potentiated the activity. PDE-I hydrolyzed thymidine-5′-monophosphate p-nitrophenyl ester most readily, whereas cyclic 3′-5′-AMP was least susceptible to hydrolysis. PDE-I was not lethal to mice at a dose of 4.0 mg/kg, ip, but had an anticoagulant effect on human plasma. These findings indicate that W. aegyptia PDE-I shares various characteristics with this enzyme from other snake venoms.