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1.
Extremophiles ; 27(2): 10, 2023 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-37071215

RESUMO

An acid-active exo/endo-chitinase; comprising a GH18 catalytic domain and substrate insertion domain; originating from the thermophilic filamentous fungus Rasamsonia emersonii, was expressed in Pichia pastoris. In silico analysis including phylogenetic analysis, and recombinant production, purification, biochemical characterisation, and industrial application testing, was carried out. The expressed protein was identified by SDS-PAGE as a smear from 56.3 to 125.1 kDa, which sharpens into bands at 46.0 kDa, 48.4 kDa and a smear above 60 kDa when treated with PNGase F. The acid-active chitinase was primarily a chitobiosidase but displayed some endo-chitinase and acetyl-glucosamidase activity. The enzyme was optimally active at 50 °C, and markedly low pH of 2.8. As far as the authors are aware, this is the lowest pH optima reported for any fungal chitinase. The acid-active chitinase likely plays a role in chitin degradation for cell uptake in its native environment, perhaps in conjunction with a chitin deacetylase. Comparative studies with other R. emersonii chitinases indicate that they may play a synergistic role in this. The acid-active chitinase displayed some efficacy against non-treated substrates; fungal chitin and chitin from shrimp. Thus, it may be suited to industrial chitin hydrolysis reactions for extraction of glucosamine and chitobiose at low pH.


Assuntos
Quitina , Quitinases , Filogenia , Quitina/química , Quitinases/genética , Quitinases/química , Quitinases/metabolismo , Especificidade por Substrato
2.
Appl Microbiol Biotechnol ; 105(20): 7769-7783, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34581845

RESUMO

Rasamsonia emersonii (previously Talaromyces emersonii) is a thermophilic filamentous fungus displaying optimum growth at 45 °C. It has a history of use in commercial food enzyme production. Its unfractionated chitinolytic secretome was partially characterised in the early 1990s; however, no individual chitinase from this source has been described in literature previously. This study describes two GH18 chitinases originating from the R. emersonii genome, expressed in the methylotrophic yeast P. pastoris. Chit1 comprises of a GH18 catalytic domain and Chit2 comprises of a GH18 catalytic domain and a chitin-binding motif at the C-terminal. The chitinases were expressed as glycoproteins. The apparent molecular weight of Chit1 was 35.8-42.1 kDa with a smearing tail associated with glyco-sidechains visible up to 72.2 kDa. This became two bands of 30.8 and 29.0 kDa upon de-glycosylation. The apparent molecular weight of Chit2 was 50.4 kDa, reducing to 48.2 kDa upon de-glycosylation. Both chitinases displayed endo-chitinase and chitobiosidase activity, temperature optima of 50-55 °C and low pH optima (pH 4.5 or lower); Chit1 displayed a pH optimum of 3.5, retaining > 60% maximum activity at pH 2.2, a pH range lower than most enzymes of fungal origin. Chit2 displayed the highest chitin-degrading ability at 3456 µmol/mg on 4-NP-triacetylchitotriose, but lost activity faster than Chit1, which displayed 403 µmol/mg on the same substrate. The predicted D values (time required to reduce the enzyme activity to 10% of its original value at 50 °C) were 19.2 and 2.3 days for Chit1 and Chit2, respectively. Thus, Chit1 can be considered one of few hyperthermostable chitinase enzymes described in literature to date. Their physicochemical properties render these chitinases likely suitable for shrimp chitin processing including one-step chitin hydrolysis and alternative sustainable protein processing and the attractive emerging application of mushroom food waste valorisation.Key points• Two GH18 chitinases originating from the industrially relevant thermophilic fungus R. emersonii were cloned and expressed in P. pastoris.• The purified recombinant chitinases showed low pH and high temperature optima and appreciable thermostability at industrially relevant temperatures.• The chitinases displayed characteristics that indicate their likely suitability to several industrial applications including sustainable alternative protein processing, food waste valorisation of commercial mushroom production and one-step shrimp chitin processing.


Assuntos
Quitinases , Eurotiales/enzimologia , Eliminação de Resíduos , Quitina , Quitinases/biossíntese , Quitinases/genética , Alimentos , Microbiologia Industrial , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
3.
J Pediatr Pharmacol Ther ; 9(2): 110-6, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23118697

RESUMO

OBJECTIVES: Patients are assuming responsibility for their own health by self-medicating with dietary supplements, often without physician knowledge or oversight. The objectives of this study were to determine: 1) pediatric dietary supplement use by surveying parents of children who were hospitalized in a university institution; 2) if any health care professional inquired about supplement use at the time the child was hospitalized; 3) whether the use of a supplement was documented in the patient's medical record; and 4) parents' attitudes about dietary supplements. STUDY DESIGN: Parents of 100 hospitalized pediatric patients (<18 years of age) were randomly selected to complete a survey about their child's use of dietary supplements prior to and during hospitalization. They were also asked if they intended to use these products after hospitalization. The purpose of the study was explained, informed consent was obtained, and parents were given ample time to complete the survey. RESULTS: Fifty percent of parents reported giving their child a dietary supplement prior to hospitalization; 17% reported use of an herbal supplement. Only 24% of parents reported being asked about supplement use by a health care professional upon admission or during the hospital stay. The response to only five of these queries was documented in the child's medical record. CONCLUSIONS: Increasing dietary supplement use mandates that all health care professionals elicit this information as part of the routine History and Physical Examination at the time a child is hospitalized. This information should also be documented in the patient's medical record. Likewise, parents should be encouraged to discuss the use of these products with their physician and pharmacist.

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