A lipase preparation with dual pH optima,wide temperature optima and broad substrate specificity for multiple applications

Summary Optimised procedures for the production and recovery of lipase at 25 litre scale by submerged fermentation using aRhizopus species isolated from petroleum-soaked soil are developed. These lipase preparations are characterized by dual pH optima (4.0 and 8.0), wide temperature optima (over 80% activity between 40–57°C), broad specificity for substrates, high degree of tolerance for common salts and excellent stability during usage as well as storage.     

Lipases from the genus Rhizopus: Characteristics,expression, protein engineering and application

Lipases are versatile catalysts that hydrolyze ester bonds of water-insoluble glycerides or carry out reversible reactions at the water/lipid interface. The remarkable characteristics of lipases from the genus Rhizopus are their high sn-1,3-positional specificity, enantioselectivity and activity in nonaqueous media, which make them one of the most desirable enzymes for many applications, including lipid modification and biodiesel and chiral organic compound synthesis. sn-1,3-Position-specific Rhizopus lipases are particularly useful for the production of structured triacylglycerols. Significant progress has been made regarding lipases from the genus Rhizopus, including gene sequencing, elucidation of the protein structure and catalytic function, heterologous expression and redesigning Rhizopus lipases for valuable properties, which is receiving increasing academic and industrial attention. In this review, we present a comprehensive overview of Rhizopus lipases, focusing on (a) the characteristics of Rhizopus lipases, (b) Rhizopus lipase genes and structural features, (c) strategies for heterologous expression of Rhizopus lipase genes in yeast system, (d) progress in protein engineering for the improvement of the properties of Rhizopus lipases, and (e) development of biotechnological applications.     

Sucrose syrup from carob pod

Summary The ripe carob pod (pericarp) is rich in water-soluble sugars, mainly sucrose (63% on total sugars). Sucrose crystallization from aqueous carob extract is prevented by its reducing sugar content. The selective consumption of these sugars by mixed culture ofRhizopus oligosporus andSaccharomyces rouxii gives a sucrose syrup suitable for several uses e.g. sucrose extraction.     

Immunological relationship of extra-cellular polysaccharide antigens produced by different mould species

Moulds are able to produce extracellular polysaccharide antigens which are heat-stable and almost genus specific. Of 44 different strains of Penicillium 41 (93%) and all 12 strains of Aspergillus tested produced detectable quantities of an immunologically related antigen. Additionally 10 of these 56 strains produced an antigen immunologically related to the antigen produced by the genera Mucor and Fusarium. Immunologically different, but genus-specific antigens were produced by each of the species belonging to the genus Geotrichum, Fusarium, Cladosporium, Mucor and Rhizopus. The antigens produced by Mucor and Rhizopus, however, were immunologically related.     

Taxonomic Characters and Culture Conditions of a Bacterium which Produces a Lytic Enzyme on Rhizopus Cell Wall

A bacterium R–4 which produces a novel type of lytic enzyme which lyses fungal and yeast cell walls was isolated from the air and was identified to belong to the genus Bacillus.

Production of the enzyme appeared to require a high concentration of nitrogen source in medium. No inducing substance was needed for the enzyme production.

A crude preparation of the enzyme was used to characterize the lytic activity. From the lytic spectrum, the enzyme seemed to have the highest activity toward the cell walls of species in the genus Rhizopus among various fungi and yeasts tested, A proteolytic activity was shown to be parallel with the lytic activity. The lytic activity was also accompanied with the liberation of reducing sugars from Rhizopus cell wall, but no activity on some known carbohydrates tested was detected in the preparation.     

Improvement of growth ofRhizopus oligosporus on a model solid substrate

Summary Metal ions and cassava extracts stimulated growth ofRhizopus oligosporus on a model solid substrate. A large improvement in protein content was obtained by simultaneously increasing the nitrogen content and decreasing the particle size of the substrate. No improvement occurred when these conditions were applied to cassava, however, because of the sticky consistency of the cassava after gelatinization.     

蛋白和酯酶电泳在根霉鉴定上的应用

对根霉(Rhizopus)属的十个种或变种共二十四株菌的菌体可溶性蛋白和酯酶同工酶进行了电泳的研究得到对这个属分类上更多的依据.在严格控制培养,提取和电泳条件的情况下,同一株菌不同批次所得菌体蛋白电泳图谱有较好的重复性.在相同的条件下,每个种的根霉有各自特征性蛋白图谱,种内不同菌株的蛋白图谱和酯酶酶谱基本相同.特别是形态特征明显、分类地位明确的种,种内各株的图谱也较一致,如R. stolonifer;与R.circinans.在确定新变种R. delemar var. latoapicalis时,电泳图谱与R.delemar var.delema:有明显不同,起到了佐证作用.因此认为,蛋白图谱与酯酶酶谱相辅相成,在根霉种的分类中是一有效的辅助手段.     

Development of a fermentation procedure to produce a tempe-related food using common beans as substrate

Summary A basic procedure was developed to produce a tempe-like product using the mouldRhizopus oligosporus and black common beans (Phaseolus vulgaris) as substrate. The initial pH of the substrate was 5.8, and fermentation was conducted at 37°C with a relative humidity of 70% for 72 hrs. Levels of soluble solids and soluble protein increased dramatically as a result of fermentation. Some changes were as well observed in fatty acid contents of fermented samples. It was concluded that the common bean used was an acceptable substrate for preparing this product.     

A model substrate for solid-state fermentation

Summary A model substrate consisting of cassava starch embedded in kappa-carrageenan was used to mimic the growth ofRhizopus oligosporus on cassava tubers. Growth on the model substrate was similar to that during solid-state fermentation of the actual cassava. However, protein production and starch utilization were slower on the model substrate.     

Evaporative temperature and moisture control in solid substrate fermentation

Summary An integrated computerized control system, consisting of temperature and moisture sensors, a variable relative humidity air stream and water sprayers was used to control temperature and moisture in the solid substrate fermentation of extruded corn withRhizopus oligosporus in a 15 l rotary drum fermenter. Dry air was blown through the substrate to force evaporative cooling, balanced by spraying of cold water to maintain constant moisture.     

Nutritional factors affecting lipase production byRhizopus delemar CDBB H313

Summary Some nutritional factors that affect lipase yields byRhizopus delemar were studied. Dextrin proved to be the best carbon source when used at 1% level. Yeast extract was the best nitrogen source for lipase production. The presence of a lipidic source in the growth medium, at a level not higher than 2% resulted in higher enzyme production. Tween 80 exerted a positive effect on enzyme production, used in a range that goes from 0.02% to 2.00%.     

Protoplast culture of several members of the genus Physalis

High yields of protoplasts were obtained by enzymic treatment of mesophyll from five different species of the genus Physalis. Subsequent divisions and colony formation were achieved in all the species. However, numerous combinations of phytohormones failed to induce regeneration of shoots from callus tissue developed from protoplasts.     

Factors affecting the use of chloramphenicol acetyltransferase as a marker for Brassica genetic transformation

The CAT gene which codes for the enzyme chloramphenicol acetyltransferase was found to be ineffective as a reporter gene in cells and tissues of Brassica species. High levels of endogenous CAT activity were found to be widespread among this genus and did not appear to be distributed in a tissue- or cell-specific manner. Moreover, the presence of an inhibitor of CAT activity was discovered in Brassica napus and Brassica juncea. This inhibitor appeared to act selectively on bacterial CAT in transgenic plants. These findings provided an explanation for difficulties experienced in the detection of transgenic CAT activity in B. napus.     

An original habitat of tempeh molds

Tempeh is a traditional Indonesian food made from soybeans fermented with Rhizopus species. Some researchers believe the original habitat of the tempeh molds may be closely related to fresh leaves of Hibiscus species because these leaves artificially infected with the tempeh molds are used to start tempeh fermentation in cottage-scale factories. To verify this hypothesis, we investigated the occurrence of Rhizopus species in Hibiscus leaves and identified the isolated Rhizopus strains precisely. Rhizopus oryzae, one of the tempeh molds, occurred in sample leaves of some Hibiscus species with considerable frequency. This result implies that tempeh molds that lived in Hibiscus leaves might have fermented soybeans accidentally when used to wrap the cooked soybeans. The original habitat of the tempeh mold could be fresh leaves of Hibiscus species.     

Use of antagonistic bacilli for biocontrol of fungi degrading fresh wood

The species composition of micromycete complexes colonizing aspen, birch, and pine wood was studied. Calculation of the Sorens-Tchekanovsky similarity coefficients showed that these complexes shared a high degree of similarity. They were dominated by representatives of the genera Penicillium, Paecilomyces, Trichoderma, and Rhizopus. Some antagonistic bacilli inhibited growth and development of wood-decay fungi in vitro and led to the formation of spheroplasts on growing hyphae. A study of possible involvement of bacillary mycolytic enzymes in the inhibition of wood-decay fungi demonstrated selectivity of their lytic effect, which was determined by the genus and species of micromycetes and did not correlate with their relative resistance to antagonists.     

Purification and properties of an endopolygalacturonase produced byRhizopus stolonifer

Summary A polygalacturonase from culture filtrates of a strain ofRhizopus stolonifer was purified about 80 fold by ethanol precipitation, followed by ion exchange chromatography (CM-Sepharose 6B) and gel filtration (Sephadex G-100). The purified preparation was homogeneous when examined by PAGE. The enzyme is an endopolygalacturonase with an optimum catalytic activity at pH 5.0 and 45°C, and a molecular weight of 57,000±500 daltons. The activity was stimulated by Fe⁺⁺⁺, Mg⁺⁺, Co⁺⁺, and inhibited by Mn⁺⁺ and Zn⁺⁺. The enzyme was stable in the pH range of 3.0 to 5.0. The purified enzyme was specific for nonmethoxylate polygalacturonic acid, with K_m and V_max values respectively of 0.19 mg/ml and 1.3 mol/g/min. In addition, this enzymatic preparation degraded pectic substances in organge peel.     

Production of lactic acid from renewable materials by Rhizopus fungi

Lactic acid is a commonly occurring organic acid, which is valuable due to its wide use in food and food-related industries, and its potential for the production of biodegradable and biocompatible polylactate polymers. Lactic acid can be produced from renewable materials using various fungal species of the Rhizopus genus, which have advantages compared to the bacteria, including their amylolytic characteristics, low nutrient requirements and valuable fermentation by-product—fungal biomass. This paper reviews recent research in process engineering, metabolic and enzymetic mechanisms, and molecular biotechnology associated with lactic acid production by the Rhizopus fungi to get a better understanding of biochemical activities. The major process components: renewable materials, bioreactor systems and process modeling are reviewed. The role of key bioprocess parameters, such as nutrient composition, pH and growth morphology, involved in the production of lactic acid is discussed in detail. In addition, recent advances in simultaneous saccharification and fermentation, molecular genetic approaches, and enzymetic and metabolic pathways involved in the production of lactic acid by fungal strains are discussed.     

Production of a Gaseous Saturated-hydrocarbon Mixture by Rhizopus japonicus under Aerobic Conditions

The microbial production, by the genus Rhizopus, of a gaseous saturated-hydrocarbon mixture was studied under aerobic conditions. Rhizopus strains, comprising 13 strains of 9 species, were tested as to their ability to produce a gaseous hydrocarbon mixture. Except for one strain, all the strains tested produced more than two kinds of gaseous hydrocarbons simultaneously when grown in nutrient broth containing glucose. Rhizopus japonicus IFO 4758 was selected as being typical of these producers of mixed gaseous hydrocarbons. When this organism was cultivated in a synthetic medium supplemented with l-cysteine under aerobic conditions, the maximum production of the total gaseous hydrocarbon mixture reached ca. 10 nl/ml culture broth/hr. The gaseous hydrocarbon mixture produced was composed mainly of paraffin hydrocarbons, i.e., ca. 74% pentane, ca. 16% propane and a trace amount of methane. The ratios of saturated to unsaturated, and even to odd number hydrocarbons produced by this fungus were 95 : 5 and 90: 10, respectively. The biosynthetic pathways for the production of these gases are discussed in comparison with the biosynthetic pathways for ethylene and isobutene in microorganisms.     

Isolation and characterization of a novel bacterium, <Emphasis Type="Italic">Sphingomonas bisphenolicum</Emphasis> strain AO1, that degrades bisphenol A

Bisphenol A (2,2-bis(4-hydroxyphenyl) propane, BPA), which is used as a synthetic resin material or a plasticizer, is a pollutant that␣possesses endocrine-disrupting activity. Bioremediation of BPA is used to decrease its polluting effects, and here we report a novel bacterial strain AO1, which is able to degrade BPA. This strain was isolated using enrichment cultivation from a soil sample from a vegetable-growing field; the sample was one of 500 soil samples collected across Japan. Strain AO1 degraded 100 mg/l BPA to an undetectable level within 6 h in MYPG medium (containing malt extract, yeast extract, peptone, and glucose) and within 48 h in minimum medium containing 1% glucose at 30°C. Strain AO1 can utilize BPA as a sole source of carbon and as an energy source under aerobic conditions. The estrogenic activity of BPA in MYPG medium was ultimately reduced by strain AO1, although the activity initially increased. Taxonomical analysis showed that strain␣AO1 is closely related to Sphingomonas chlorophenolicum and S. herbicidovorans, neither of which have a capacity for BPA degradation. DNA–DNA hybridization showed that strain AO1 is a novel species of the Sphingomonas genus, and we designated AO1 as S. bisphenolicum.