Case Report: Metagenomics Next-Generation Sequencing for Diagnosing Cerebral Infarction and Infection Caused by Hematogenous Disseminated Mucormycosis in a Patient With Acute Lymphoblastic Leukemia

Disseminated mucormycosis, a serious complication, is associated with high mortality in patients with acute leukemia after chemotherapy. Blood cultures are always negative because of recurrent empirical antifungal treatments. The identification of pathogens is important for diagnosis and therapy. In this case report, we diagnosed culture-negative disseminated mucormycosis with Rhizomucor miehei infection leading to cerebral infarction in a patient with leukemia using metagenomics next-generation sequencing (mNGS) form peripheral blood, cerebral spinal fluid, and bronchoalveolar lavage fluid. mNGS technology can be applied to precisely diagnose culture-negative disseminated mucormycosis.

Heterologous Expression and Rational Design of l-asparaginase from Rhizomucor miehei to Improve Thermostability

l-asparaginase (EC 3.5.1.1) hydrolyzes l-asparagine to produce l-aspartate and ammonia and is widely found in microorganisms, plants, and some rodent sera. l-asparaginase used for industrial production should have good thermostability. We heterologously expressed l-asparaginase from Rhizomucor miehei, selected nine loci for site-directed mutagenesis by rational design, and obtained two mutants with significantly improved thermostability. The optimal temperature of mutants S302I and S302M was 50 °C. After incubating the mutant and wild-type enzymes at 45 °C for 35 h, the residual activity of the wild-type enzyme (WT) was only about 10%. In contrast, the residual activity of S302I and S302M was more than 50%. After combination mutagenesis, Bacillus subtilis 168-pMA5-A344E/S302I was constructed using the food-safe host strain B. subtilis 168. Additionally, a 5′ untranslated region (UTR) modification strategy was adopted to enhance the expression level of R. miehei-derived l-asparaginase in B. subtilis. In a 5-L fermenter scale-up experiment, the enzyme activity of recombinant B. subtilis 168-pMA5-UTR-A344E/S302I reached 521.9 U·mL−1 by fed-batch fermentation.

Improved methanol tolerance of Rhizomucor miehei lipase based on N‑glycosylation within the α-helix region and its application in biodiesel production

Background Liquid lipases are widely used to convert oil into biodiesel. Methanol-resistant lipases with high catalytic activity are the first choice for practical production. Rhizomucor miehei lipase (RML) is a single-chain α/β-type protein that is widely used in biodiesel preparation. Improving the catalytic activity and methanol tolerance of RML is necessary to realise the industrial production of biodiesel. Results In this study, a semi-rational design method was used to optimise the catalytic activity and methanol tolerance of ProRML. After N-glycosylation modification of the α-helix of the mature peptide in ProRML, the resulting mutants N218, N93, N115, N260, and N183 increased enzyme activity by 66.81, 13.54, 10.33, 3.69, and 2.39 times than that of WT, respectively. The residual activities of N218 and N260 were 88.78% and 86.08% after incubation in 50% methanol for 2.5 h, respectively. In addition, the biodiesel yield of all mutants was improved when methanol was added once and reacted for 24 h with colza oil as the raw material. N260 and N218 increased the biodiesel yield from 9.49% to 88.75% and 90.46%, respectively. Conclusions These results indicate that optimising N-glycosylation modification in the α-helix structure is an effective strategy for improving the performance of ProRML. This study provides an effective approach to improve the design of the enzyme and the properties of lipase mutants, thereby rendering them suitable for industrial biomass conversion.

Characterization of a Novel Aspartic Protease from Rhizomucor miehei Expressed in Aspergillus niger and Its Application in Production of ACE-Inhibitory Peptides

Rhizomucor miehei is an important fungus that produces aspartic proteases suitable for cheese processing. In this study, a novel aspartic protease gene (RmproB) was cloned from R. miehei CAU432 and expressed in Aspergillus niger. The amino acid sequence of RmproB shared the highest identity of 58.2% with the saccharopepsin PEP4 from Saccharomyces cerevisiae. High protease activity of 1242.2 U/mL was obtained through high density fermentation in 5 L fermentor. RmproB showed the optimal activity at pH 2.5 and 40 °C, respectively. It was stable within pH 1.5–6.5 and up to 45 °C. RmproB exhibited broad substrate specificity and had Km values of 3.16, 5.88, 5.43, and 1.56 mg/mL for casein, hemoglobin, myoglobin, and bovine serum albumin, respectively. RmproB also showed remarkable milk-clotting activity of 3894.1 SU/mg and identified the cleavage of Lys21-Ile22, Leu32-Ser33, Lys63-Pro64, Leu79-Ser80, Phe105-Met106, and Asp148-Ser149 bonds in κ-casein. Moreover, duck hemoglobin was hydrolyzed by RmproB to prepare angiotensin-I-converting enzyme (ACE) inhibitory peptides with high ACE-inhibitory activity (IC50 of 0.195 mg/mL). The duck hemoglobin peptides were further produced at kilo-scale with a yield of 62.5%. High-level expression and favorable biochemical characterization of RmproB make it a promising candidate for cheese processing and production of ACE-inhibitory peptides.

Influence of different milk-coating enzymes on the process of producing soft cheeses

The effect of the type of milk clotting enzyme (MCE) on the duration of milk coagulation, parameters of the composition of whey and cheeses, and the output of cheeses in the production of soft cheese such as “Lyubitel'skiy” were investigated. Three brands of MCE of different origins were investigated: Marzyme® (MCE of microbial origin based on Rhizomucor miehei protease), Naturen® (calf rennet) and Chy-max® M (recombinant camel chymosin). It was established that MCEs had different ratios of milk clotting activity (MCA) to total proteolytic activity (PA). It was determined that the MCA/PA ratio, which characterizes the degree of specificity of the MCE action with regard to kappa-casein, in Chy-max M 1000 is ~7 times higher than that of Naturen and ~50 times higher than that of Marzyme. Such differences did not lead to a negative effect when using the Marzyme preparation in the production of soft cheeses. There were no statistically significant differences in the amount of dry matter loss of the curd into the whey, physicochemical parameters and output between the variants of cheeses made with the studied brands of MCE. Shorter duration of milk clotting (16.5 min) was observed with Marzyme than with MCE of Naturen (20.5 min) and Chy-max M (22.5 min). The results of the coagulation duration were explained by the stimulation of the activity of MCE of microbial origin, by the pH level of milk before coagulation (below pH 6.4). It was shown that modern MCEs of microbial origin could be recommended as a cost-effective replacement for more expensive rennet and recombinant chymosins in the production of soft and fresh cheeses.

Изучение in silico структурных особенностей липаз из различных продуцентов как потенциальных агентов для иммобилизации на заряженных и гидрофобных носителях

Липаза – гидролитический фермент, получаемый из многих организмов, таких как животные, растения, грибы и бактерии. Она осуществляет расщепление триглицеридов до моноглицеридов и жирных кислот, при этом обладает широкой субстратной специфичностью. Липазы применяются в пищевой промышленности и других сферах человеческой деятельности. Характерная особенность многих липаз – явление поверхностной активации, обуславливающее свойственную им зависимость скорости каталитической реакции от концентрации и агрегатного состояния субстрата. Доказано, что функционирование ферментов зависит от их структурных особенностей. Внутренние полости, туннели и поры являются неотъемлемыми компонентами нативной конформации белка. Они играют важную роль в транспорте субстрата, кофакторов и ионов к активному и регуляторным центрам фермента. Кроме того, их конфигурация может влиять на термостабильность энзимов, в связи с чем изучение вышеперечисленных структур является необходимым для понимания механизмов функционирования биокатализаторов. Также важным элементом структуры ферментов являются скопления заряженных и гидрофобных аминокислотных остатков на их поверхности. Данное свойство необходимо учитывать при планировании путей адсорбционной иммобилизации биокатализаторов на различных носителях для использования в промышленности и медицине. В работе исследованы состав, локализация и конфигурация внутренних полостей, туннелей, пор, а также поверхностных скоплений заряженных и гидрофобных аминокислотных остатков в молекулах липаз из Rhizopus niveus (PDB ID: 1LGY), Rhizomucor miehei (PDB ID: 3TGL), Burkholderia cepacia (PDB ID: 1OIL), панкреатических липаз Homo sapiens (PDB ID: 1N8S) и Equus caballus (PDB ID: 1HPL). Для расчета параметров и визуализации данных структур использовались программы MOLE и Maestro. Показано наличие соответственно 2, 1, 5, 5 и 2 туннелей и отсутствие пор для данных ферментов; а также наличие по 6 внутренних полостей для липаз из R. niveus и E. caballus, 5 внутренних полостей для молекулы из R. miehei и по 2 внутренних полости для энзимов из B. cepacia и H. sapiens. Приведены аминокислотный состав и профили туннелей изучаемых липаз. Выявлено, что данные структуры не сообщаются друг с другом общими пустотами. Установлено преобладание гидрофобных аминокислотных остатков в большинстве туннелей данных ферментов. Изучены структура, локализация и состав скоплений заряженных и гидрофобных аминокислотных остатков на поверхностях макромолекул. Выявлено возможное влияние расположения данных скоплений на связывание липаз с носителем при их адсорбционной иммобилизации.

Effect of biosurfactant sophorolipids on Rhizomucor miehei lipase fermentation by Aspergillus oryzae

In this study, the effect of biosurfactant sophorolipids (SLs) on Rhizomucor miehei lipase (RML) fermentation by Aspergillus oryzae was investigated. With the exogenous addition of 0.3% (w/v) SLs in the initial medium, the RML activity reached 430.0 U/mL, an increase of 25.0% compared to the control group. Subsequently, the physiological metabolic responses of A. oryzae to the addition of SLs were further explored. The results showed that though SLs had almost no effect on the RML secretion, it would affect the morphology of the cells. During the late phase of the fermentation, the proportion of middle pellets, which was generally considered as an energetic and stable state for enzyme production was increased with the addition of SLs. Simultaneously, the viscosity of fermentation broth was reduced, which facilitated the increase of oxygen transfer, thereby improving the RML production. Finally, it could be found that the addition of SLs significantly increased the contents of precursor amino acids, especially for those rank first and second of the RML composition, and it could promote the synthesis of RML.