Cloning and screening of the putative hexokinase genes from Rhizopus oryzae and their heterologous expression in Saccharomyces cerevisiae

Molecular Biology Reports - A filamentous fungus, Rhizopus oryzae (R. oryzae) is one of the ideal candidates for ethanol and lactic acid production due to its ability to grow on renewable carbon...     

Isolation and properties of lactate dehydrogenase from germinating pea plants

Lactate dehydrogenase (LDH) was isolated from pea seedlings by means of protamine sulphate and (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a MW of ca 145 500. The kinetic properties studied were the lactate oxidation pH optimum (9·1) and the pyruvate reduction pH optimum (7·1). K_m values were determined for four natural substrates (Lactate, pyruvate, NAD⁺ and NADH) and for other acids (glycollate, α-ketoglutarate and glyoxylate). The K_i value was determined for p-chloromercuribenzoate (PCMB) which is a noncompetitive inhibitor of LDH from pea plants, and the course of irreversible inhibition of the enzyme by iodoacetamide (IA) and n-ethylmaleimide (NEMI) was studied. Preincubation of LDH with the coenzyme protects against PCMB inhibition, indicating the important role of the sulfhydryl group in the active site.     

A flax-retting endopolygalacturonase-encoding gene from Rhizopus oryzae

A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R. oryzae strain YM9901 and 96% identical to a hypothetical protein (RO3G_04731.1) in the sequenced genome of R. oryzae strain 99–880. Phylogenetic analysis revealed the presence of an unique cluster of Rhizopus polygalacturonase sequences that are separate from other fungal polygalacturonases. Conservation of 12 cysteines appears to be a special feature of this family of Rhizopus polygalacturonase sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of expression of the Rhizopus oryzae uricase and urease enzymes

The regulation of intracellular urease and uricase activities was examined in Rhizopus oryzae. Urease activity (2.4 U/mg protein) was present in R. oryzae mycelium grown in minimal medium containing NH4CI as sole nitrogen source. This activity increased threefold under nitrogen derepression conditions, but no induction by urea was detected. Control of urease activity in R. oryzae differs from that found in Neurospora crassa but resembles the situation in Aspergillus nidulans. No uricase activity was detected in R. oryzae mycelium grown in minimal medium containing NH4Cl as sole nitrogen source. Uricase activity was increased 10- to 40-fold under derepression conditions and was induced by exogenous uric acid (60- to 78-fold). Control of the R. oryzae uricase differs from that found in N. crassa and A. nidulans. This is the first analysis of the regulation of enzymes from the purine catabolic pathway in any member of the Zygomycetes.     

Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes

The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226, and eLDHB lost negatively charged Glu 14, as compared to the highly conserved residues at these positions in the corresponding amino acid sequences of other mammals. These alterations were identified in six equine species by genomic DNA analysis. A comparison of the equine and human 3D structures revealed that the substituted His 88 and 226 of the eLDHA monomer and the deleted Glu 14 of the eLDHB monomer altered the surface charge of equine LDH tetramers and that these three residues were located in important regions affecting the catalytic kinetics. Also, RT-PCR amplification of the three myosin heavy chain isoforms corroborated that the cervical muscle as postural muscle of the thoroughbred horse was composed of more oxidative myofibers than the dynamic muscle. Based on this property, the mRNA expression patterns of eLDHA, eLDHB, and eGAPDH in various tissues were analyzed by using real-time PCR. The expression levels of these three genes in the cervical muscle were not always relatively higher than in the brain or heart.     

Alcohol dehydrogenase from Rhizopus javanicus.

Alcohol dehydrogenase of Rhizopus javanicus was purified, and its physical and chemical characteristics were determined. The intact enzyme was shown to have a molecular weight of approximately 60,000. Since the smallest apparent subunit was 14,000, the enzyme was presumed to be composed of four subunits. The crude mycelial extract contained multiple forms of the enzyme, which were separated by ion-exchange chromatography.     

Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose

Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, overexpressing endogenous pyruvate carboxylase (PYC) and exogenous phosphoenolpyruvate carboxylase (PEPC) to increase the carbon flux toward oxaloacetate were investigated. Compared to the wild type, the PYC activity in the pyc transformants increased 56%-83%, whereas pepc transformants exhibited significant PEPC activity (3-6mU/mg) that was absent in the wild type. Fumaric acid production by the pepc transformant increased 26% (0.78g/g glucose vs. 0.62g/g for the wild type). However, the pyc transformants grew poorly and had low fumaric acid yields (<0.05g/g glucose) due to the formation of large cell pellets that limited oxygen supply and resulted in the accumulation of ethanol with a high yield of 0.13-0.36g/g glucose. This study is the first attempt to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae.     

米根霉富马酸酶三维结构的同源建模与分析

利用Modeller7v7软件对米根霉（Rhizopus oryzoe）富马酸酶（fumarase）进行了三级结构的同源建模并对结果的空间和能量上的合理性进行了验证,进一步对酶的结构域和催化活性位点进行了研究。结果表明：富马酸酶由三个结构域组成,中心区域为一个由五个几乎平行的α螺旋组成的独特的束型结构,其催化活性位点是由三个亚基上的氨基酸相互靠近共同组成的。为以后有针对性的进行富马酸酶的定点突变提高富马酸产量提供分子水平上的理论指导。     

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).     

Production of flavour esters by Rhizopus oryzae

Microbial production of different alipathic esters with flavour characteristic has been studied. Lyophilized whole cells of Rhizopus oryzae CBS 112-07 were found to be particularly suitable to catalyse the synthesis of different flavour esters (hexyl acetate, propionate, butyrate, caprylate; geranyl acetate, propionate, butyrate and 2- and 3-methylbutyl acetate, butyrate) in n-heptane. This strain was therefore utilized for the semipreparative production of geranyl butyrate by semicontinous and continous addition of the substrates with satisfactory yields (144 g l^–1 in 264 h and 142 g l^–1 in 48 h respectively).     

Isolation and characterization of lactate dehydrogenase from white fin muscles of the skate Raja clavata

Lactate dehydrogenase (LDH) from white driving muscle of skate Raja clavata was purified by the differential precipitation of ammonium sulphate between 52 and 55% saturation. Only one protein band with the LDH activity was obtained by nondenaturing electrophoresis. The same result was obtained by the SDS-electrophoresis. The relative molecular weight calculated by this method in the presence of DS-Na was 34 kDa; Km was 29 +/- 7 and 71 +/- 16 microM for NAD.H and pyruvate, respectively. The reaction was maximally activated by 0.8-6.0 mM pyruvate and inhibited in the regions above this level. Dilution of LDH below concentration of 1 microgram/ml reduced the enzyme activity. The pH-optimum for the LDH activity ranged 7.0-8.0.     

High expression and efficient secretion of Rhizopus oryzae glucoamylase in the yeast Saccharomyces cerevisiae

Summary The expression and secretion of Rhizopus oryzae glucoamylase were studied in the yeast Saccharomyces cerevisiae. Rhizopus oryzae glucoamylase was highly expressed and efficiently secreted into a medium to a high level (above 300 mg/l) under control of a yeast promoter and the original signal sequence. Excess expression of the secretable glucoamylase with high copy number plasmid slightly decreased growth of the transformant cells in glucose medium but not in fructose medium.     

Expression of the lactate dehydrogenase genes in Xenopus species hybrids

The lactate dehydrogenase isozyme patterns of six Xenopus species-hybrid combinations have been investigated. The genes from both parental species were equally expressed in all of them. “Hybrid” isozymes originating from aggregation of subunits from both parents could be visualized and demonstrated random aggregation of enzyme subunits. Reciprocal crosses displayed the same pattern demonstrating also random aggregation of subunits.