To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.
DNA, Recombinant ; Electroporation ; Genetic Engineering ; Genetic Vectors ; Hygromycin B ; Protoplasts ; Real-Time Polymerase Chain Reaction ; Rhizopus ; genetics ; Transformation, Genetic

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.
Absidia ; Aged ; Female ; Fingers ; Humans ; Hyphae ; Inflammation ; Itraconazole ; Mucor ; Mucormycosis* ; Periodic Acid ; Rare Diseases ; Rhizomucor ; Rhizopus ; Skin

The study aimed to evaluate the probiotic properties, antioxidant activity and fermentative capacity of Acremonium charticola and Rhizopus oryzae isolated from the Indonesian fermented dried cassava, with particular application on poultry. A. charticola inhibited the growth of Escherichia coli and Aspergillus flavus. A. charticola and R. oryzae grew in potato dextrose agar (PDA) adjusted to pH 3 and 8 or in PDA supplemented with bile salt up to 0.8%. After soaking for 8 hr, the survival rate of A. charticola in the simulated gastric juice (pH 2) and bile solutions (2% bile salt) was lower than that of R. oryzae. A. charticola and R. oryzae exhibited strong antioxidant activities. Compared to unfermented cassava pulp (control), the fibre content of cassava pulp tended to be lower after fermentation with A. charticola for 14 days. The populations of A. charticola and R. oryzae were significantly higher in fermented cassava pulp than in unfermented one. Coliform was higher in cassava pulp fermented with R. oryzae or A. charticola + R. oryzae compared to control after 7 days of fermentation, however, the bacteria were not different between A. charticola-fermented cassava pulp and control. Lactic acid bacteria (LAB) were higher in A. charticola- and R. oryzae-fermented cassava pulp than those in control, however, no difference of LAB was observed between A. charticola + R. oryzae-fermented cassava pulp and control. In conclusion, A. charticola exhibited antibacterial, antifungal and antioxidant activity, gastrointestinal persistence and fermentative capacity that may be beneficial for poultry industry.
Acremonium ; Agar ; Aspergillus flavus ; Bacteria ; Bile ; Escherichia coli ; Fermentation ; Fungi* ; Gastric Juice ; Glucose ; Hydrogen-Ion Concentration ; Lactic Acid ; Manihot* ; Oryza ; Poultry* ; Probiotics ; Rhizopus ; Solanum tuberosum ; Survival Rate

No abstract available.
Aged, 80 and over ; DNA, Ribosomal/chemistry ; Diabetes Mellitus, Type 2/*complications/diagnosis ; Fatal Outcome ; Humans ; Male ; Mucormycosis/*complications/diagnosis/*microbiology ; Rhizopus/*isolation & purification ; Sequence Analysis, DNA ; Sequence Homology ; Tomography, X-Ray Computed

BACKGROUND/OBJECTIVES: Soy isoflavones are structurally similar to estrogen and bind to estrogen receptors, suggesting that they exhibit estrogenic activities; therefore, they are referred to as phytoestrogens. Fermentation may affect the bioavailability of isoflavones altering soy isoflavone glycosides in the form of aglycones. Thus, this study investigated the effects of fermented soybeans by Rhizopus oligosporus on bone metabolism in both young rats as a pilot test and in ovariectomized (ovx) old rats as a model of menopause. MATERIALS/METHODS: In the pilot test, a total of 24 seven-week-old female Sprague-Dawley (SD) rats were fed one of three diets for a period of four weeks: casein, unfermented soybean product, or fermented soybean product by R. oligosporus. In the ovx rat model, 20-week-old SD rats weighing 260-290 g underwent either sham-operation (n = 10) or bilateral ovariectomy (n = 30) and were then fed the AIN-93M diet for one week. Thereafter, rats were fed sham-casein, ovx-casein, ovx-soybean, or ovx-fermented soybean diet for five weeks. After decapitation, femoral bones were isolated and preserved in 9% formalin for assessment of bone mineral density (BMD), bone mineral content (BMC), and bone-breaking strength (BBS). RESULTS: Ovx rats showed significantly increased weight gain and decreased uterine wet weight. Of particular interest, ovx rats fed fermented soybeans showed increased uterine wet weights compared to control rats. Fermented soybean diet caused a significant increase in plasma 17-beta estradiol concentrations in young rats, and 17-beta estradiol levels were enhanced in ovx rats to match those of sham-operated ones. Significantly lower femoral BMD and BMC were observed in ovx rats compared to sham-operated controls, whereas bone areas did not differ statistically among the groups. In addition, BBS tended to be increased in ovx rats fed soybeans and fermented soybeans. CONCLUSIONS: Supplementation of fermented soybeans could have preventive and therapeutic effects against osteoporosis in postmenopausal women.
Animals ; Biological Availability ; Bone Density ; Caseins ; Decapitation ; Diet ; Estradiol ; Estrogens ; Female ; Fermentation ; Formaldehyde ; Glycosides ; Humans ; Isoflavones ; Menopause ; Metabolism ; Models, Animal ; Osteoporosis ; Ovariectomy ; Phytoestrogens ; Plasma ; Rats* ; Rats, Sprague-Dawley ; Receptors, Estrogen ; Rhizopus* ; Soybeans* ; Weight Gain ; Weights and Measures

Directed evolution was conducted to improve the thermostability of lipase from Rhizopus chinensis CCTCC M201021. Mutations were introduced by two rounds of error-prone PCR and mutant lipase was selected by fast-blue RR top agar screening. Two positive variants were selected in the first-round and four in the second-round screening process. Ep2-4 was proved as the most thermostable lipase and its DNA sequencing revealed three amino acid substitutions: A129S, P168L and V329A. Compared with the parent, its half-life at 60 degrees C was 5.4- times longer and T50 was 7.8 degrees higher. Purified lipase of Ep2-4 was characterized and the result shows that its thermostability improved without compromising enzyme activity. According to the mimicked protein structure, mutation A129S formed a hydrogen bond with Gln133 and improved the thermostability by increasing the hydrophilicity and polarity of protein; mutation P168L by forming a hydrophobic bond with the nearby Leu164.
Cloning, Molecular ; Directed Molecular Evolution ; methods ; Enzyme Stability ; genetics ; Hot Temperature ; Industrial Microbiology ; Lipase ; chemistry ; genetics ; Mutation ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Protein Engineering ; methods ; Rhizopus ; enzymology

Soft rot on banana fruit caused by Rhizopus oryzae was identified for the first time in Korea. Colonies were white to light brown and formed numerous sporangiospores. Optimum temperature for mycelial growth was 30degrees C. Sporangia were globose and 30~200 microm. Sporangiophores were usually straight, 8~20 microm, and rhizoids usually in groups of 3~5. Columella were globose to sub-globose and 90~110 microm. Sporangiospores were sub-globose or oval and 4~10 microm. Based on its mycological characteristics, molecular analysis, and pathogenicity to host plants, this fungus was identified as Rhizopus oryzae Went & Prisen Geerligs. This is the first report of soft rot on banana caused by Rhizopus oryzae in Korea.
Fruit ; Fungi ; Humans ; Korea ; Light ; Musa ; Oryza ; Rhizopus ; Sporangia

AIM: To observe the anti-oxidative activity and adverse laxative effect of raw, traditional processed and fermented products of Polygoni Multiflori Radix (PMR), and furthermore, to evaluate the fermentation method used in the processing procedure of PMR. METHODS: In vitro ferric reducing antioxidant power (FRAP) assay was carried out to evaluate the anti-oxidative activity. Modulation of normal defecation and effect on gastrointestinal motility in mice were carried out to investigate their adverse laxative effect. RESULTS: Fermented PMR induced less severe laxative adverse effect than Polygoni Multiflori Radix Praeparata (PMRP). PMR fermented with Rhizopus sp. (FB) could modulate the defecation significantly. The gastrointestinal motility was inhibited by PMRP and PMR fermented with Rhizopus oryzae (FA). FA and FB showed better antioxidant activity than PMRP in 50% and 95% ethanol group. Contents of 2, 3, 5, 4'-tetrahydroxy-stilbene-2-O-β-D-glucoside (TSG) were reduced significantly after traditional processing but maintained after fermentation. Emodin and physcion were increased after traditional processing and fermented with Rhizopus oryzae. CONCLUSION All processing procedure, including fermentation, might reduce its anti-oxidative activity. However, most of the processed products could lessen the adverse effect on gastrointestinal tract compared to PMR. Fermentation with Rhizopus oryzae was considered as a promising processing method of PMR.
Animals ; Antioxidants ; pharmacology ; Defecation ; drug effects ; Emodin ; adverse effects ; analogs & derivatives ; pharmacology ; Female ; Fermentation ; Gastrointestinal Motility ; drug effects ; Gastrointestinal Tract ; drug effects ; Laxatives ; adverse effects ; Male ; Mice ; Mice, Inbred Strains ; Plant Extracts ; adverse effects ; pharmacology ; Plant Roots ; chemistry ; Polygonum ; adverse effects ; chemistry ; Rhizopus

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.
Candida ; genetics ; isolation & purification ; metabolism ; Genetic Vectors ; genetics ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; biosynthesis ; Metabolic Engineering ; Recombination, Genetic ; Rhizopus ; enzymology ; genetics ; Transformation, Bacterial

Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
Codon ; Enzyme Precursors ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; Lipase ; biosynthesis ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Protein Engineering ; methods ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; Rhizopus ; enzymology ; genetics ; Substrate Specificity