Molecular modification and highly efficient expression of L-asparaginase from Rhizomucor miehei / 生物工程学报

L-asparaginase hydrolyzes L-asparagine to produce L-aspartic acid and ammonia. It is widely distributed in microorganisms, plants and serum of some rodents, and has important applications in the pharmaceutical and food industries. However, the poor thermal stability, low catalytic efficiency and low yield hampered the further application of L-asparaginase. In this paper, rational design and 5' untranslated region (5'UTR) design strategies were used to increase the specific enzyme activity and protein expression of L-asparaginase derived from Rhizomucor miehei (RmAsnase). The results showed that among the six mutants constructed through homology modeling combined with sequence alignment, the specific enzyme activity of the mutant A344E was 1.5 times higher than the wild type. Subsequently, a food-safe strain Bacillus subtilis 168/pMA5-A344E was constructed, and the UTR strategy was used for the construction of recombinant strain B. subtilis 168/pMA5 UTR-A344E. The enzyme activity of B. subtilis 168/pMA5 UTR-A344E was 7.2 times higher than that of B. subtilis 168/pMA5-A344E. The recombinant strain B. subtilis 168/pMA5 UTR-A344E was scaled up in 5 L fermenter, and the final yield of L-asparaginase was 489.1 U/mL, showing great potential for industrial application.

Biosorption potential of lead tolerant fungi isolated from refuse dumpsite soil in Nigeria

Metals are non-biodegradable and recurrent in the environs. Heavy metals tolerant fungiwere isolated from refuse dumpsite soil using pour plate method. These fungiwere identified as Aspergillus niger, Penicillium chrysogenumandRhizomucor sp. The fungal isolates were screened for cadmium (Cd), lead (Pb) and zinc (Zn) with concentration of 200ppm, 400ppm and 600ppm. Aspergillus nigerand Penicillium chrysogenumshowed high tolerance for the metals in contrast to the control. The fungiwith high tolerance were used for biosorption study. However, Penicillium chrysogenumshowed higher lead removal or biosorption potential of 1.07ppm, 3.35ppm and 4.19ppm as compared with Aspergillus nigerwith lead removal of 0.67ppm, 3.11ppm and 3.79ppm at 5th, 10thand 15thday respectively. One-way Analysis of Variance was used to interpret the data generated from the biosorption study which revealed that there was no significant different (p>0.05)between the lead removal of Aspergillus nigerandPenicillium chrysogenumon the 5thday but there was significant difference (p<0.05)in the lead removal of Aspergillus nigerand Penicillium chrysogenumon the 10thand 15thday. This study suggests the use of these fungal isolates for removal and biotreatment of heavy metal contaminated and polluted environment.

A Case of Cutaneous Mucormycosis Occurring after Systemic Steroid Therapy / 대한의진균학회지

Cutaneous mucormycosis is a rare disease caused by zygomycetes such as Rhizomucor, Mucor, Absidia, and Rhizopus. The disease usually occurs in immunocompromised individuals, and the organism is rarely pathogenic in an immunocompetent host. Herein, we report a 77-year-old female patient who had multiple erythematous papules and pustules on the left 3rd finger. She had received systemic steroid therapy prior to the occurrence of the skin lesions. The histopathological examination of Periodic Acid Schiff stained section showed chronic granulomatous inflammation and fungal hyphae. Rhizopus species was isolated on the fungal culture of the tissue specimen. The patient was finally diagnosed with cutaneous mucormycosis and was treated with itraconazole.

Mucormicosis rinoorbitocerebral por Rhizomucor pusillus en una paciente diabética / Rhino-orbito-cerebral mucormycosis caused by Rhizomucor pusillus in a diabetic patient

La mucormicosis define a un grupo de infecciones oportunistas poco frecuentes pero potencialmente letales que puede presentarse en pacientes inmunodeprimidos, tales como diabéticos no controlados o pacientes con enfermedad hematológica maligna, entre otros. La forma infecciosa rinoorbitocerebral (ROC) es la más frecuente en pacientes diabéticos. Se presenta el caso de una paciente de 63 años, diabética mal controlada que desarrolló cetoacidosis y mucormicosis de tipo ROC. Evolucionó con rápido deterioro clínico, no respondió al tratamiento medicoquirúrgico ofrecido y falleció el quinto día de hospitalización. El estudio histológico y micológico de la muestra extraída confirmaron el diagnóstico de mucormicosis. Se aisló Rhizomucor pusillus, un agente mucoral raro y de muy baja frecuencia en la patogénesis de la mucormicosis.

Mucormycosis defines a group of opportunistic infections not frequent but potentially lethal that it may occur in immunocompromised patients, such as not controlled diabetics patients or patients with malignant hematologic disease, among others. The rhino-orbito-cerebral infectious form (ROC) is the most common in diabetic patients. This was the case of a 63 year-old poor controlled diabetic patient who developed ketoacidosis and mucormycosis type ROC. She evolved with rapid clinical deterioration, did not respond to the medical and surgical treatment and died on the fifth day of hospitalization. Mycological and histological study confirmed the diagnosis of mucormycosis. It was isolated Rhizomucor pusillus, a rare mucoral agent and an infrequent causative organism of mucormycosis.

Chemical modification studies of Rhizomucor miehei protease: evidence for the role of basic amino acids in enzyme catalysis.

The effect of chemical modification on milk clotting and proteolytic activities of aspartyl protease obtained from Rhizomucor miehei NRRL 3500 was examined in the absence and the presence of its specific inhibitor pepstatin A. The effect on the ratio of milk clotting activity (MC) to proteolytic activity (PA), an index of the quality of milk clotting proteases was also determined. Modification of the enzyme with trinitrobenzenesulfonic acid, diethylpyrocarbonate and phenylglyoxal produced an increase in the ratio of MC/PA, while modification with 2- hydroxy-5-nitrobenzyl bromide did not affect the ratio. Modification with N-acetylimidazole resulted in a marginal increase in MC/PA ratio. Protection using pepstatin A during modification with phenylglyoxal, N-acetylimidazole and 2-hydroxy-5-nitrobenzyl bromide, protected both MC and PA. In the case of modification by diethylpyrocarbonate, pepstatin A protected only MC. Pepstatin A did not protect both the activities on the modification of the enzyme by trinitrobenzene sulfonic acid. These observations indicate the presence of arginine, tyrosine and tryptophan at the catalytic site of the enzyme, for eliciting MC and PA of the enzyme. In general, modification of the positively charged residues increases the MC/PA ratio of the enzyme. In addition the modified lysine residues responsible for the inactivation of the enzyme were not involved in the active site of the enzyme. Thus the lysine residues might have a secondary role in enzyme catalysis. Further, histidine at the catalytic site was found to be exclusively involved in milk clotting activity. The enzyme with modified histidine residues were more susceptible to autocatalysis, indicating that histidine residues protect the enzyme against autolysis.