Regulation of the Activity of Lactate Dehydrogenases from Four Lactic Acid Bacteria

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (P_i), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (K_act) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of P_i are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of P_i inhibits E. faecalis LDH2, whereas in the absence of FBP, P_i is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of P_i to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of P_i, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Lactic acid and animal feeds production from Sophora flavescens residues by Rhizopus oryzae fermentation

In this study, the feasibility of producing lactic acid and animal feeds from Sophora flavescens residues (SFR) by Rhizopus oryzae was explored. Results showed that the simultaneous saccharification and fermentation (SSF) is the optimal fermentation mode, which was simple and high-efficiency. When the inoculation volume of R. oryzae was 10 % and the pH value was adjusted by adding CaCO₃ in stages during SSF, the maximum concentration of lactic acid was 46.78 g/L, and the maximum lactic acid productivity reached 0.97 g/L/h. Results also showed that the protein content of the solid residues after fermentation of R. oryzae reached 12.15 %. This content was 46 times higher than that by the original SFRs and nearly 4.3 times the protein content of the solid residues after fermentation by Enterococcus faecium. In addition, the solid residues after fermentation rich in Fe and Zn could be used as animal feeds or feed additives. Thus, it is expected that this study may provide a novel approach for Chinese medicine residues treatment towards full resource recovery.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing fumR gene on cell growth and fumaric acid biosynthesis from glucose

Fumaric acid is a dicarboxylic acid used extensively in synthetic resins, food acidulants, and other applications, including oil field fluids and esters. The filamentous fungus Rhizopus oryzae is known for its ability to produce and accumulate high levels of fumaric acid under aerobic conditions. In this work, the overexpression of native fumarase encoded by fumR and its effect on fumaric acid production in R. oryzae were investigated. Three plasmids containing the endogenous fumR gene were constructed and used to transform R. oryzae, and all transformants showed significantly increased fumarase activity during both the seed culture (growth) and fermentation (fumaric acid production) stages. However, fumarase overexpression in R. oryzae yielded more malic acid, instead of fumaric acid, in the fermentation because the overexpressed fumarase also catalyzed the hydration of fumaric acid to malic acid. The results suggested that the overexpressed fumarase, encoded by fumR, by itself was not responsible for the over-production of fumaric acid in R. oryzae.     

Molecular characterization of β-fructofuranosidases from Rhizopus delemar and Amylomyces rouxii

Of the 19 strains of Rhizopus delemar deposited as Rhizopus oryzae, seven of them, NBRC 4726, NBRC 4734, NBRC 4746, NBRC 4754, NBRC 4773, NBRC 4775, and NBRC 4801, completely hydrolyzed exogenous sucrose and fructooligosaccharides. The sucrose-hydrolyzing enzyme was purified from the culture filtrate of R. delemar NBRC 4754 and classified to β-fructofuranosidase, similar to that of Amylomyces rouxii CBS 438.76. Fragments including β-fructofuranosidase genes (sucA) of seven strains of R. delemar and A. rouxii CBS 438.76 were amplified and sequenced by PCR with degenerated primers synthesized on the basis of the internal amino acid sequences of purified enzymes and successive inverse PCR. Nucleotide sequences of the obtained fragments revealed that open reading frames of 1,569 bp have no intron and encode 522 amino acids. The presumed proteins contained the typical domain of the glycoside hydrolase 32 family, including β-fructofuranosidase, inulinase, levanase, and fructosyltransferases. Amino acid sequences of SucA proteins from the seven strains of R. delemar were identical and showed 90.0 % identity with those of A. rouxii CBS 438.76. A dendrogram constructed from these amino acid sequences showed that SucA proteins are more closely related to yeast β-fructofuranosidases than to other fungal enzymes.     

The enzymatic acyl exchange of phospholipids with lipases

The acyl exchange of phospholipids with lipases was investigated. The lipase from Rhizopus delemar catalyzed the acyl exchange reaction between various phospholipids and fatty acids. When we incubated 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and oleic acid with lipase from R. delemar, the yield of diacyl phosphatidyl choline (PC) was 25% and the fatty acid composition of the converted PC was an oleic acid content of 25% and a palmitic acid content of 75%. This reaction exhibited 1-positional specificity. Three industrial lipases from Rhizopus sp., Mucor javanicus, and Candida cylindracea had the activity of the acyl exchange of phosphatidyl choline. The lipase from R. sp. gave the best result.     

Lactate formation in Caldicellulosiruptor saccharolyticus is regulated by the energy carriers pyrophosphate and ATP

Caldicellulosiruptor saccharolyticus displays superior H₂ yields on a wide range of carbon sources provided that lactate formation is avoided. Nevertheless, a low lactate flux is initiated as the growth rate declined in the transition to the stationary phase, which coincides with a drastic decrease in the glucose consumption and acetate production fluxes. In addition, the decrease in growth rate was accompanied by a sudden increase and then decrease in NADH levels. The V′_MAX of the lactate dehydrogenase (LDH) doubled when the cells entered the stationary phase. Kinetic analysis revealed that at the metabolic level LDH activity is regulated through (i) competitive inhibition by pyrophosphate (PPi, k_i=1.7 mM) and NAD (k_i=0.43 mM) and (ii) allosteric activation by FBP (300%), ATP (160%) and ADP (140%). From these data a MWC-based model was derived. Simulations with this model could explain the observed lactate shift by displaying how the sensitivity of LDH activity to NADH/NAD ratio varied with different PP_i concentrations. Moreover, the activation of LDH by ATP indicates that C. saccharolyticus uses LDH as a means to adjusts its flux of ATP and NADH production. To our knowledge, this is the first time PPi is observed as an effector of LDH.     

Stability of immobilized R. oryzae in repetitive batch productions of L(+)-lactic acid: effect of inorganic salts

Rhizopus oryzae was immobilized in polyurethane foam cubes by a natural attachment method. The effect of inorganic salts on the stability of the immobilized mycelium in repetitive batch productions of L(+)-lactic acid was studied. The amount of the inorganic salts necessary to maintain the activity of the immobilized R. oryzae in the repetitive batch fermentations strongly depended upon the initial glucose concentrations. For example, the amount of the inorganic salts should be doubled if the initial glucose concentration was twice increased. The minimum amounts of the inorganic salts were therefore determined for effective lactic acid productions in the repetitive batch fermentations with the immobilized R. oryzae.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Lactic acid production from xylose by the fungus Rhizopus oryzae

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure l(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g⁻¹. By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l⁻¹ showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l⁻¹) and glucose (19.2 g l⁻¹) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l⁻¹ h⁻¹ and 0.5 g xylose l⁻¹ h⁻¹. This resulted mainly into the product lactic acid (6.8 g l⁻¹) and ethanol (5.7 g l⁻¹).     

Immobilization of Rhizopus oryzae in a modified polyvinyl alcohol gel for L(+)-lactic acid production

The production of L(+)-lactic acid (LA) by Rhizopus oryzae immobilized in polyvinyl alcohol (PVA) was investigated. To decrease diffusional resistance, we modified the PVA gel through the addition of sodium alginate and phosphate esterification. The production of L(+)-LA improved notably in the immobilized Rhizopus oryzae. Maximum L(+)-LA production (106.27 g/L), with a yield of 73.1 % and rate of 2.95 g/L·h, was obtained at a temperature of 38 °C, 6 % PVA, and 0.8 % sodium alginate. The immobilized R. oryzae was stable in 14 serial-batch cultures using non-growth medium. The immobilized beads also displayed good tolerance to low temperature and long-term storage at 4 °C with the preservation of biochemical properties.     

Impact of NH4+ nitrogen source on the production of Rhizopus oryzae lipase in Pichia pastoris

BackgroundPichia pastoris is a highly successful system for heterologous expression. During the induction stage, the ammonium ion released into the fermentation broth has a deep impact on cell growth and protein expression. The impact of NH₄⁺ concentration on the expression of the Rhizopus oryzae lipase proAROL in P. pastoris was investigated.ResultsThe lipase activity under the optimum NH₄⁺ concentration of 440 mmol/L reached 12,019 U/mL. Increased concentrations of NH₄⁺ in the broth prevented the protease production, resulting in higher specific lipase activity in the supernatant. Furthermore, analysis of carbon metabolism and energy regeneration pattern revealed that under the definite NH₄⁺ concentrations more carbon source (methanol) was consumed with surged AOX activity and then the higher energy and amino acid precursors demand for recombinant protein synthesis is compensated for by the TCA cycle.ConclusionsIn this study, the R. oryzae lipase activity reaches the highest level ever reported under optimized NH₄⁺ concentration and the analysis of the carbon metabolism provides useful information for future optimization of protein production by P. pastoris in a molecular level.     

Relationship between saccharifying capacity and isolation sources for strains of the Rhizopus arrhizus complex

To discover the differences and modes of saccharifying ability among Rhizopus arrhizus strains isolated from various habitats, we grew strains in glutinous rice media to monitor the production of glucose from starch using spectrophotometry at regular time intervals. The saccharifying capacity of R. arrhizus var. delemar was found to be weaker than that of var. arrhizus and var. tonkinensis. These data provide additional phenotypic support for recognization of this genetically distinct lineage (var. delemar) as a separate species. Clinical isolates were generally low in saccharifying abilities, while all strains from southwest China had an excellent diastatic power.     

Purification of the extracellular pectinolytic enzyme from the fungus Rhizopus oryzae NBRC 4707

The pectinolytic enzyme from the solid-state culture of Rhizopus oryzae NBRC 4707 was purified to homogeneity by column chromatography on CM-Toyopearl 650 M and hydroxylapatite. The molecular weight of the enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 31,000 and was reduced to 29,700 after treatment with endoglycosidase H. Maximal activity was observed near pH 4.5 at 45°C. The enzyme was shown to be endopolygalacturonase, as judged from the formation of oligogalacturonides as its reaction products. The addition of purified enzyme, as expected, enhanced the formation of lactic acid and ethanol in potato pulp grown with R. oryzae.     

Identification and Characterization of Rhizot,a Novel LTR Retrotransposon of Rhizopus oryzae and R. delemar

A novel retrotransposon Rhizot was identified in Rhizopus oryzae and R. delemar. Rhizot has a unique structure that consists of a pol ORF similar to non-LTR (long terminal repeat) retorotransposons between two LTRs. Rhizot was distributed in all Rhizopus species tested. The Rhizot pol gene was transcribed in the liquid culture, and was induced by UV and oxidative stress.     

An unusual fructose 1,6-bisphosphate-activated lactate dehydrogenase from the ascidian Pyura stolonifera

Lactate dehydrogenase (LDH) was purified from the siphon muscle of the intertidal ascidian Pyura stolonifera. The enzyme is unique among chordate LDHs but resembles some bacterial and platyhelminth LDHs in being activated by fructose 1,6-bisphosphate (FBP). Concentrations of FBP in the range 5μM to 0.5 mM increase V_max of the pyruvate reductase reaction by 130% to 210%, and decrease K_m pyruvate 5 to 11 fold and K_m NADH 2.5 to 5 fold. The enzyme is also activated by inorganic phosphate, but requires a 50 fold higher concentration to attain the maximum activation achieved by 0.5 mM FBP. Of a range of metabolites tested, including other glycolytic sugar phosphates, only FBP and inorganic phosphate activated the enzyme. FBP activation was not observed with 16 representative vertebrate LDH homotetramers, but did occur to a limited extent with LDH from an echinoderm. LDH was the only pyruvate reductase enzyme detected in P. stolonifera siphon muscle, and its activity was much greater than that of phosphorylase or phosphofructokinase. The LDH reaction is utilized by P. Stolonifera during prolonged siphon closure on exposure to air when lactate, but not succinate, accumulates in the siphon muscle. While the ascidian enzyme provides the first example of a FBP activated LDH from a chordate, it remains to be determined if this unusual property has any role in metabolic regulation.     

Effect of bongkrekic acid on growth and metabolism of filamentous fungi

The antifungal activity of bongkrekic acid against 17 tested molds was determined. Bongkrekic acid prevented spore germination and mycelial proliferation of Aspergillus niger, Rhizopus oryzae and Penicillium italicum. The action of bongkrekic acid was fungicidal. Under these conditions, the incorporation of ¹⁴C-leucine and ¹⁴C-uracil into the perchloric acid insoluble material of germinating A. niger conidia was significantly reduced by bongkrekic acid. Respiratory activity of resting spores was not affected by bongkrekic acid. Respiratory activity of germinated spores was inhibited by bongkrekic acid to the extent of 30 to 60% of controls for A. niger, R. oryzae and P. italicum. It has been concluded that operation of adenine nucleotide translocation in mitochondria of tested fungi is obligatory both for normal spore germination and fungal growth.