Rhizopus Delemar Research Articles

Anti-CotH3 antibodies protect mice from mucormycosis by prevention of invasion and augmenting opsonophagocytosis.

Mucorales are fungal pathogens that cause mucormycosis, a lethal angioinvasive disease. Previously, we demonstrated that Rhizopus, the most common cause of mucormycosis, invades endothelial cells by binding of its CotH proteins to the host receptor GRP78. Loss of CotH3 renders the fungus noninvasive and attenuates Rhizopus virulence in mice. Here, we demonstrate that polyclonal antibodies raised against peptides of CotH3 protected diabetic ketoacidotic (DKA) and neutropenic mice from mucormycosis compared to mice treated with control preimmune serum. Passive immunization with anti-CotH3 antibodies enhanced neutrophil inlfux and triggered Fc receptor-mediated enhanced opsonophagocytosis killing of Rhizopus delemar. Monoclonal antibodies raised against the CotH3 peptide also protected immunosuppressed mice from mucormycosis caused by R. delemar and other Mucorales and acted synergistically with antifungal drugs in protecting DKA mice from R. delemar infection. These data identify anti-CotH3 antibodies as a promising adjunctive immunotherapeutic option against a deadly disease that often poses a therapeutic challenge.

In vitro interactions between isavuconazole and tacrolimus, cyclosporin A or sirolimus against Mucorales.

To evaluate the in vitro interactions of isavuconazole with immune suppressors (tacrolimus, cyclosporin A or sirolimus) against 30 Mucorales isolates belonging to the most common species responsible for mucormycosis in humans (Rhizopus arrhizus, Rhizopus delemar, Rhizopus microsporus, Lichtheimia corymbifera, Lichtheimia ramosa, Mucor circinelloides and Rhizomucor pusillus). In vitro interaction was evaluated by a microdilution chequerboard technique. Combination of isavuconazole with tacrolimus, cyclosporin A or sirolimus, was synergistic for 50%, 46% and 7% of the isolates, respectively. Antagonistic interaction was observed for 4% of the isolates for the combination with cyclosporin A (one R. arrhizus isolate) and for 32% of the isolates for the combination with sirolimus (six R. arrhizus isolates and three R. pusillus isolates). These in vitro data show that calcineurin inhibitors are more likely than inhibitors of the mTOR pathway to enhance the activity of isavuconazole against Mucorales. These in vitro results warrant further animal experiments.

Rhizopus delemar로 발효된 지황의 이화학적 성분 분석

Background: This study was carried out to determine the physicochemical components of Rehmannia glutinosa (RG) fermented with Rhizopus delemar.BR Methods and Results: Physicochemical components such as changes in moisture content, pH value, total acidity, amount of reducing sugars as well as quantity of free sugars, free amino acids, and catalpol were investigated. Result showed that, the moisture content ranged from 64.26 to 65.51%. The pH and total acidity of the fermented RG decreased significantly during fermentation. The reducing sugar content ranged from 0.10 to 1.34%. The most abundant main free sugars were identified as raffinose, xylose, glucose, fructose, and sucrose. The sucrose content in 80% ethanol and in water extracts increased during RG fermentation. In total, 26 free amino acids were detected, including seven essential amino acids. In addition, the quantity of free amino acids decreased significantly during fermentation. Finally, the catalpol content of the fermented RG was highest on the 2SUPnd/SUP day of fermentation at 2,028.67㎎/ 100 g.BR Conclusions: These results indicated that fermentation of Rhizopus delemar could be used to enhance biological activity, and that fermented RG could be used as a functional material and as an edible resource in food and functional materials industries.

CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar

Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.

Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal Pathogen Rhizopus delemar.

Rhizopus delemar is an invasive fungal pathogen responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which infectious spores of Rhizopus delemar cause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. The molecular mechanisms that underpin this transformation may be key to controlling mucormycosis; however, the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes that take place over the course of germination. This process is characterized by four distinct stages: dormancy, isotropic swelling, germ tube emergence, and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton, and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transforming Rhizopus delemar from a single-cellular to multicellular organism.IMPORTANCE Germination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis. Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis, and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.

Genetic tools for investigating Mucorales fungal pathogenesis.

Mucormycosis is an emerging opportunistic fungal infection whose causative agents are found within the Mucorales family. A recent increase in immunocompromised cohorts with solid organ transplants, diabetes mellitus, and other medical conditions have resulted in increased fungal infections including mucormycosis. Our current knowledge about Mucoralean fungi is in its infancy compared to other fungal pathogens, which may be due to lack of robust genetic tools for Mucorales. In this review we summarize recent advances in genetic tools to study the two most prevalent and genetically amenable Mucoralean fungi, Mucor circinelloides and Rhizopus delemar. There have been advances made in the study of Mucorales family genetics. These findings include the construction of recyclable markers to manipulate the genome, as well as silencing vectors, and the adaptation of the CRISPR/Cas9 gene editing system. We present how these genetic methods have been applied to understand basic biology, morphogenesis, pathogenesis, and host-pathogen interactions in the two Mucoralean fungi, M. circinelloides and R. delemar. With these advances in Mucorales the opportunity to further understand the pathogenesis of these organisms is opened.

Comparative genome-wide analysis of extracellular small RNAs from the mucormycosis pathogen Rhizopus delemar

Rhizopus delemar is an emerging fungal pathogen causing devastating mucormycosis in immunocompromised individuals. The organism remains understudied and there are urgent needs for new methods of rapid disease diagnosis for timely therapy. Extracellular vesicles with encapsulated RNAs have recently been discovered to have great potential applications for disease diagnoses and treatments. To explore the utilization of ex-RNA in studies of mucormycosis, we have performed RNA-Seq of ex-sRNAs from two clinical strains of R. delemar. Approximately 3.3 and 3.2 million clean reads were obtained from FGSC-9543 and CDC-8219 strains, respectively. The median sequence length of the sRNAs was 22 nts, with a minimum of 18 and a maximum of 30 nts. Further annotation identified 560 and 526 miRNAs from FGSC-9543 and CDC-8219 strains, respectively. miRNA target prediction and analysis of GO and KEGG pathways have revealed that the regulation of metabolism, secondary metabolite biosynthesis, and two-component system signaling are important during growth. We have also validated RNA-Seq by qRT-PCR and Northern blotting analysis of randomly selected miRNAs. Our results show that R. delemar has a rich reservoir of secreted ex-sRNAs and our studies could facilitate the development of improved diagnostic methods as well as elucidating virulence mechanisms for R. delemar infection.

Identification of sucA, Encoding β-Fructofuranosidase, in Rhizopus microsporus.

Rhizopus microsporus NBRC 32995 was found to hydrolyze fructooligosaccharides (FOS), as well as sucrose, almost completely into monosaccharides through the production of sufficient amounts of organic acids, indicating that the complete hydrolysis of FOS was caused by the secretion of β-fructofuranosidase from fungal cells. Thus, the sucA gene, encoding a β-fructofuranosidase, was amplified by degenerate PCR, and its complete nucleotide sequence was determined. The total length of the sucA gene was 1590 bp, and the SucA protein of R. microsporus NBRC 32995 belonged to clade VIa, which also contains Rhizopus delemar and is closely related to Saccharomycotina, a subdivision of the Ascomycota.

Utilization of horticultural waste (Apple Pomace) for multiple carbohydrase production from Rhizopus delemar F2 under solid state fermentation.

The brown rot fungus Rhizopus delemar F2 was shown to produce extracellular thermostable and multiple carbohydrase enzymes. The potential of Rhizopus delemar F2 in utilizing apple pomace under solid state fermentation (SSF) is the purpose of the study. Solid state fermentation (SSF) is a very effective technique opposed to submerged fermentation in various aspects. Enhanced production of multiple carbohydrases 18.20 U g−1 of cellulose, 158.30 U g−1 of xylanase, 61.50 U g−1 of pectinase and amylase 21.03 U g−1 was released by microwave pretreatment of apple pomace at 450 W for 1 min and then by incubation the culture thus obtained at 30 °C for 6 days with moisture content of 1:4.5. Apple pomace can serve as a potential source of raw material for the production of multiple carbohydrases. Besides, it can find great commercial significance in production of bioethanol and various industries like textile, fruit juice, paper and pulp industry.

Production of cellulosic organic acids via synthetic fungal consortia.

Consolidated bioprocessing (CBP) is a potential breakthrough technology for reducing costs of biochemical production from lignocellulosic biomass. Production of cellulase enzymes, saccharification of lignocellulose, and conversion of the resulting sugars into a chemical of interest occur simultaneously within a single bioreactor. In this study, synthetic fungal consortia composed of the cellulolytic fungus Trichoderma reesei and the production specialist Rhizopus delemar demonstrated conversion of microcrystalline cellulose (MCC) and alkaline pre-treated corn stover (CS) to fumaric acid in a fully consolidated manner without addition of cellulase enzymes or expensive supplements such as yeast extract. A titer of 6.87 g/L of fumaric acid, representing 0.17 w/w yield, were produced from 40 g/L MCC with a productivity of 31.8 mg/L/hr. In addition, lactic acid was produced from MCC using a fungal consortium with Rhizopus oryzae as the production specialist. These results are proof-of-concept demonstration of engineering synthetic microbial consortia for CBP production of naturally occurring biomolecules.

Biofuel production from spent coffee grounds via lipase catalysis

ABSTRACTLipases namely Mucor miehei, Pseudomonas cepacia, Rhizopus delemar, Geotrichum candidum, Candida rugosa, Porcine pancreas-II, Pseudomonas fluorescence, and Candida antarctica lipase-B (Novozyme-435) were employed for biodiesel synthesis from spent coffee oil. Around 96% oil-to-biodiesel conversion was obtained using Novozyme-435 as a catalyst at 1:5 oil-to-methanol molar ratio and 40ºC. Total spent coffee grounds generated at the North-West University, Potchefstroom Campus (NWU PC) was estimated which could be used to produce 162 L of biodiesel. A waste valorization strategy was devised for converting organic wastes produced at the NWU PC to bioenergy.

Rhizopus delemar 발효 백수오의 이화학적 성분 분석

This study carried out to determine the physicochemical components of Cynanchi wilfordii Radix (CWR) fermented with Rhizopus delemar. The physicochemical components such as changes on moisture content, pH, total acidity, reducing sugars, and the contents of free sugars, free amino acid, cynandione A were investigated. The moisture content of fermented CWR was increased after 4 days of fermentation. The pH value of fermented CWR was decreased after 4 days of fermentation. The reducing sugars contents were in the range of 1.42-1.69%. The major free sugars were identified as rhamnose, glucose, fructose and sucrose. The content of free sugars was decreased during fermentation process of CWR while the glucose contents in 80% ethanol and water extracts were increased. In addition, the free amino acid increased significantly during fermentation. Finally, The content of cynandion A of the fermented CWR was highest at the level of 14.09 mg/g on the 8th day. These results are indicated that fermentation with Rhizopus strains could be used to increase the bioactivity of CWR and the fermented CWR can be used as a functional material and edible resource in the food industry.

Sustainable carbon sources for microbial organic acid production with filamentous fungi

BackgroundThe organic acid producer Aspergillus oryzae and Rhizopus delemar are able to convert several alternative carbon sources to malic and fumaric acid. Thus, carbohydrate hydrolysates from lignocellulose separation are likely suitable as substrate for organic acid production with these fungi.ResultsBefore lignocellulose hydrolysate fractions were tested as substrates, experiments with several mono- and disaccharides, possibly present in pretreated biomass, were conducted for their suitability for malic acid production with A. oryzae. This includes levoglucosan, glucose, galactose, mannose, arabinose, xylose, ribose, and cellobiose as well as cheap and easy available sugars, e.g., fructose and maltose. A. oryzae is able to convert every sugar investigated to malate, albeit with different yields. Based on the promising results from the pure sugar conversion experiments, fractions of the organosolv process from beechwood (Fagus sylvatica) and Miscanthus giganteus were further analyzed as carbon source for cultivation and fermentation with A. oryzae for malic acid and R. delemar for fumaric acid production. The highest malic acid concentration of 37.9 ± 2.6 g/L could be reached using beechwood cellulose fraction as carbon source in bioreactor fermentation with A. oryzae and 16.2 ± 0.2 g/L fumaric acid with R. delemar.ConclusionsWe showed in this study that the range of convertible sugars for A. oryzae is even higher than known before. We approved the suitability of fiber/cellulose hydrolysate obtained from the organosolv process as carbon source for A. oryzae in shake flasks as well as in a small-scale bioreactor. The more challenging hemicellulose fraction of F. sylvatica was also positively evaluated for malic acid production with A. oryzae.

The rhizoferrin biosynthetic gene in the fungal pathogen Rhizopus delemar is a novel member of the NIS gene family

Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99–880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD, that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC–MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi.

Prophylaxis with Isavuconazole or Posaconazole Protects Immunosuppressed Mice from Pulmonary Mucormycosis.

We assessed prophylactic or continuous therapy of isavuconazole, posaconazole, or voriconazole in treating pulmonary murine mucormycosis. In the prophylaxis studies, only isavuconazole treatment resulted in significantly improved survival and lowered tissue fungal burden of immunosuppressed mice infected with Rhizopus delemar. In the continuous treatment studies, isavuconazole and posaconazole, but not voriconazole, equally prolonged survival time and lowered tissue fungal burden compared to placebo-treated mice. These results support the use of isavuconazole and posaconazole in prophylaxis treatment.

Optimization of the CRISPR/Cas9 System to Manipulate Gene Function in Rhizopus delemar

Background The genus Rhizopus is the main cause of mucormycosis, a life-threatening infection that affects predominantly hosts with an impaired immune system. However, patients with severe trauma and burns, without prior immune deficiency, are also at increased risk of developing mucormycosis. Despite aggressive treatment that involves disfiguring surgery and antifungal therapy, mortality rates range from ~50 to 100%. Genetic manipulation of Rhizopus is critical for identifying fungal targets to develop more effective therapies. However, Rhizopus genetics are challenging because of lack of dominant selection markers, low efficiency of transformation, and rarity of chromosomal integration. Here we attempted to adapt the CRISPR/Cas9 technology to disrupt genes in R. delemar.Methods We used the Gibson cloning strategy to assemble all necessary elements of the CRISPR/Cas9 system in one plasmid using the pyrF as a selection marker. The targeted gene for disruption was a toxin-encoding gene with similarity to ricin. This disruption cassette was transformed using biolistic delivery system into R. delemar pyrF - strain (M16). Recombination events were studied by Southern blot analysis and ricin gene expression was analyzed by qRT-PCR. Furthermore, damage to alveolar epithelial cells (A549) and nasal epithelial cells (CCL30) was studied with 51Cr-release assay.ResultsFive stable transformants were obtained with the CRISPR/Cas9 construct. Southern blot analysis and nucleotide sequencing confirmed a partial deletion of the ricin gene, in the region where the guide RNA was designed. Moreover, gene disruption was confirmed by abrogation of ricin expression in comparison to reference strains (wild type or mutant with the CRISPR/Cas9 plasmid void of ricin gene sequence). Finally, ricin-mutants showed significant reduction in damage to A549 cells and CCL30 cells when compared with the reference strains (20–30% reduction, P < 0.01 by t-test).Conclusion We have successfully adapted the CRISPR/Cas9 system to disrupt the ricin-like gene in R. delemar. This tool will enable us to better understand the pathogenesis of mucormycosis and ultimately aid in designing novel and more successful strategies to manage this lethal fungal infection.Disclosures All authors: No reported disclosures.

APX001A Protects Immunosuppressed Mice from Rhizopus delemar Infection

BackgroundMucormycosis is a life-threatening infection with high mortality that occurs predominantly in immunocompromised patients. APX001A is an antifungal agent that targets Gwt1, an early step in the conserved glycosylphosphotidyl inositol (GPI) post-translational modification pathway of surface proteins in eukaryotic cells. Inhibition of inositol acylation by APX001A results in pleiotropic effects such as inhibition of maturation of GPI-anchored proteins necessary for growth and virulence and results in lethality. APX001A has in vitro activity against Mucorales. Here we assessed the in vivo activity of APX001A against Rhizopus delemar (MIC = 0.25 µg/mL).MethodsICR mice were immunosuppressed with cyclophosphamide (200 mg/kg) and cortisone acetate (500 mg/kg) on days -2, +3, and +8 relative to intratracheal infection with 2.5 x 105 cells of R. delemar 99–880. For survival studies, treatment with APX001 (prodrug) at 52, 104, or 156 mg/kg (twice daily, po), was compared with liposomal amphotericin B (LAmB) at 15 mg/kg (once daily, iv). Treatment started on day +1 through day +8 for APX001 and through day +4 for LAmB. Placebo mice received vehicle control. For fungal burden studies, dosing started 8 hours post infection through day +3. Mice were sacrificed on day +4. Survival time, and tissue fungal burden (by qPCR) served as efficacy endpoints.ResultsAPX001 treatment at either 52 or 104 mg/kg prolonged survival of mice vs. placebo (n = 20 per arm) (21-day survival of 0% for placebo, 30% for 52 mg/kg, 45% for 104 mg/kg, P < 0.05 by Log Rank test). APX001 at 104 mg/kg was as good as LAmB treatment (21-day survival of LAmB-treated mice [n = 20] =50%). APX001 at 156 mg/kg did not enhance survival vs. placebo. Further, APX001 at 104 mg/kg and LAmB reduced pulmonary and brain fungal burden by ~1 log and 1.5 log vs. placebo, respectively (P < 0.05, by Wilcoxon rank-sum). The 52 and the 156 mg/kg APX001 doses also reduced tissue fungal burden vs. placebo mice (0.5–1.0 log).ConclusionAPX001 protected immunosuppressed mice from R. delemar infection with efficacy similar to that of LAmB. Higher doses of APX001 were not protective despite lowering fungal burden. Continued investigation of APX001 as a novel antifungal agent against mucormycosis is warranted.Disclosures K. J. Shaw, Amplyx Pharmaceuticals Inc.: Employee, Salary; Linnaeus: Consultant, Consulting fee

Metagenomics analysis of microbial communities associated with a traditional rice wine starter culture (Xaj-pitha) of Assam, India.

This is the first report on the microbial diversity of xaj-pitha, a rice wine fermentation starter culture through a metagenomics approach involving Illumine-based whole genome shotgun (WGS) sequencing method. Metagenomic DNA was extracted from rice wine starter culture concocted by Ahom community of Assam and analyzed using a MiSeq® System. A total of 2,78,231 contigs, with an average read length of 640.13 bp, were obtained. Data obtained from the use of several taxonomic profiling tools were compared with previously reported microbial diversity studies through the culture-dependent and culture-independent method. The microbial community revealed the existence of amylase producers, such as Rhizopus delemar, Mucor circinelloides, and Aspergillus sp. Ethanol producers viz., Meyerozyma guilliermondii, Wickerhamomyces ciferrii, Saccharomyces cerevisiae, Candida glabrata, Debaryomyces hansenii, Ogataea parapolymorpha, and Dekkera bruxellensis, were found associated with the starter culture along with a diverse range of opportunistic contaminants. The bacterial microflora was dominated by lactic acid bacteria (LAB). The most frequent occurring LAB was Lactobacillus plantarum, Lactobacillus brevis, Leuconostoc lactis, Weissella cibaria, Lactococcus lactis, Weissella para mesenteroides, Leuconostoc pseudomesenteroides, etc. Our study provided a comprehensive picture of microbial diversity associated with rice wine fermentation starter and indicated the superiority of metagenomic sequencing over previously used techniques.

Expression and characterization of a novel 1,3-regioselective cold-adapted lipase from Rhizomucor endophyticus suitable for biodiesel synthesis.

BackgroundThe biodiesel production can be carried out by transesterification using either chemical or enzymatic process. The enzymatic transesterification is more promising as it offers an environmental friendly option compared to the chemical process, where the lipases with high catalytic efficiency and good stability play a key role. Hence, it is of great value to identify novel lipases which are suitable for biodiesel production.ResultsA lipase gene (ReLipA) from Rhizomucor endophyticus was cloned and heterologously expressed in Pichia pastoris. ReLipA shared the highest identity of 61 % with the lipases from Rhizopus delemar, Rhizopus oryzae, and Saccharomyces cerevisiae. The recombinant lipase (ReLipA) was secreted as an active protein with the highest activity of 1961 U mL−1 in a 5-L fermentor by high cell-density fermentation. ReLipA was purified to homogeneity with a recovery yield of 75.7 %. The purified enzyme was most active at pH 6.0 and 40 °C, respectively, and it was stable up to 55 °C. ReLipA displayed 75 % of its maximal activity at 0 °C, indicating that it is a cold-adapted lipase. It exhibited broad substrate specificity toward various p-nitrophenyl esters and triglycerides. ReLipA hydrolyzed triolein to release mainly 1,2-diolein without the formation of 1,3-diolein, suggesting that it is a sn-1,3 regiospecific lipase. Furthermore, ReLipA synthesized different types of oleates by esterification using oleic acid and short chain alcohols (e.g., methanol, ethanol, and butanol) as the substrates with the highest conversion yield of 82.2 %. Therefore, the cold-adapted lipase may be a good biocatalyst in ester synthesis in biodiesel industry.ConclusionsA novel cold-adapted lipase was identified and characterized. The high yield and excellent properties may confer the enzyme with great potential for biodiesel production in bioenergy industry. This is the first report on a cold-adapted lipase from Rhizomucor species.

Evaluation of gas supply configurations for microbial product formation involving multiple gaseous substrates

Gaseous substrates such as O2 and CO2 are often required in fermentation processes. However, a simple methodology to compare different gas supply strategies using gaseous substrates from different sources is missing. In this study, we present a methodology to identify and theoretically compare different configurations to supply mixtures of gaseous compounds to fermentations that consume these gases. For the different configurations that were identified, all gas flow rates can be calculated in terms of other process parameters such as optimal concentrations of the gaseous compounds in the liquid phase, top pressures of the fermentation, and consumption/production rates. The approach is demonstrated for fumaric acid fermentation with Rhizopus delemar, which consumes O2 and can theoretically produce or consume CO2. Three different gas supply configurations were identified: Air supplemented with O2, a mixture of O2 and CO2, and air supplemented with CO2. All three configurations lead to gas supply costs in the same order of magnitude. O2 and CO2 prices and consumption rates determine which configuration is best. However, the overall production costs will not be dominated by the gas costs, but by the glucose costs. The presented methodology enables a simple way to identify and compare different gas supply strategies for fermentations that require more than one gaseous substrate. This includes the costs for compression of gases. Other substrate costs are easily added for overall process optimization.