Heterologous Expression of Extracellular Proteinase pAsPs of Aspergillus pseudotamarii in Komagataella phaffii.

Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from Aspergillus pseudotamarii. pAsPs was for the first time integrated in the genome of yeast strain Komagataella phaffii T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu2+. Optimal activity pH was shown in the range of pH 6.5-8.0, and optimal temperature-50-60 °C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry.

Heterologous Expression of Xylanase xAor from Aspergillus oryzae in Komagataella phaffii T07

Xylanases (EC 3.2.1.8) hydrolyze the hemicellulose of plant cell walls. Xylanases are used in the food and paper industries and for bioconversion of lignocellulose to biofuel. In this work, the producer-strain with four copies of the xAor xylanase gene was organized in two tandem copies for optimal expression in Komagataella phaffii T07 yeast. The secreted 35 kDa xylanase was purified from culture medium by gel filtration on Sephadex G-25 and anion exchange chromatography on DEAE-Sepharose 6HF. Tryptic peptides of the recombinant enzyme were analyzed by liquid chromatography-tandem mass spectrometry where the amino acid sequence corresponded to Protein Accession # O94163 for Endo-1,4-beta-xylanase from Aspergillus oryzae RIB40. The recombinant xylanase was produced in a bioreactor where the secreted enzyme hydrolyzed oat xylane with an activity of 258240 IU/mL. High activity in the culture medium suggested xylanase could be used for industrial applications without being purified or concentrated. The pH optimum for xylanase xAor was 7.5, though the enzyme was active from pH 2.5 to pH 10. Xylanase was active at temperatures from 35 °C to 85 °C with a maximum at 60 °C. In conclusion, this protocol yields soluble, secreted xylanase suitable for industrial scale production.

Clinical evaluation of commercial PCR assays for antimicrobal resistance in Mycoplasma genitalium and estimation of resistance-mediated mutation prevalence in Moscow and Moscow region.

Mycoplasma genitalium is a widespread sexually transmitted infection (STI) with growing rate of antimicrobials resistance. In our study, 137 vaginal and 131 urethral M. genitalium-positive swabs were sequentially collected through the work of Reference Center for STI during 2019. For prevalence evaluation of macrolide-resistance mutations three commercially available kits were used: AmpliSens® M. genitalium-ML/FQ-Resist-FL (Central Research Institute of Epidemiology, Russia), ResistancePlus® MG (SpeeDx, Australia), and S-DiaMGRes™ (Diagenode, Belgium). Macrolide resistance mutations were detected in 16% (43 of 268) of samples. Diagnostic characteristics were evaluated against Sanger sequencing. For AmpliSens® M. genitalium-ML/FQ-Resist-FL specificity was shown to be 100% (CI 95%, 98.4-100), and sensitivity was 90.7% (CI 95%, 77.9-97.4). ResistancePlus® MG specificity was 100% (CI 95%, 98.3-100), and sensitivity was 92.1% (CI 95%, 78.6-98.3). S-DiaMGRes™ specificity was shown to be 88.6% (CI 95%, 83.9-92.4), and sensitivity was 100% (CI 95%, 84.4-100). Mutations of parC gene region were detected in 14.5% (38 of 268) using AmpliSens® M. genitalium-ML/FQ-Resist-FL with further validation by Sanger sequencing. Of studied samples, 6.3% (17 of 268) contained both antimicrobials of class resistance mutations. Prevalence of macrolide-resistant M. genitalium in Moscow was 21.7% (23 of 106) and of fluoroquinolone-resistant M. genitaliuim was 20.8% (22 of 106). In Moscow region, macrolide-resistant M. genitalium were 12.3% (20 of 162) and 9.9% (16 of 162) of fluoroquinolone-resistant M. genitalium. All three kits can be used both for epidemiological monitoring of M. genitalium presence and mutation prevalence estimation. In Moscow, macrolide- and fluoroquinolone-resistant mutant prevalence increased in 3.9 and 2.7 times in 3 years.