R. oryzae

Rhizopus-soft rot is a threat in all postharvest situations including storage, marketing, and transport of crops. It causes soft rot of avocados, cassava, crucifers, pulses, yams, and sweet potatoes. This spoilage type is mainly caused by Rhizopus stolonifer and to a lesser extent by R. oryzae, Mucor piriformis, and Gilbertella persicaria (Dijksterhuis and Samson 2006). 

Li et al. (2007) reported the use of dry biomass, Rhizopus oryzae (R. oryzae) IF04697, whole cell-catalyzed methanolysis of soybean oil for biodiesel (methyl ester) in rm-butanol system. Changing one separate factor at a time (COST), live-level-four-factor Central Composite Design (CCD) were used to evaluate the effects of synthesis conditions, such as tert-butanol to oil volume ratio, methanol to oil molar ratio, water content, and dry biomass amount. Biodiesel yields of 72% were obtained under the optimal conditions using the proposed model for prediction. 

Optimization of the biotransformation showed that the best conditions for achieving high rates and molar conversions included the use of n-hexane and -heptane as solvents, a temperature of 50 °C, an equimolar concentration of the substrates around 50 mM, and a concentration of the lyophilized biocatalyst around 25-30 g/1. As a matter of fact, Table 6.1 shows how the carbon source affects the expressed activity of A. oryzae MIM and R. oryzae CBS 112.07. 

Different strains of R. oryzae and A. oryzae were able to perform efficient esterification under optimized conditions [12]. 

The resolution of the racemic mixture of 2-octanol and of other secondary alcohols (2-butanol, 2-pentanol, 2-hexanol, and 2-heptanol) by direct esterification in organic solvent was studied by using lyophiUzed mycelia of R. oryzae CBS112.07 as catalysts [18]. The profile of the resolution of (R,S)-2-octanol under optimized conditions (lg/1 of alcohol and equimolar butanoic acid as acylating agent in n-heptane, 30g/l of dry biocatalyst, and 30 °C) is shown in Figure 6.1. 

A very different situation was observed when the resolution of racemic 1,2-0-isopropyhdeneglycerol (IPG or solketal) was studied [19]. In this case, the kinetics of the esterification with butyric acid catalyzed by A. oryzae MIM and R. oryzae CBS 112.07 were investigated by carrying out independent batch tests on the commercially available R- and S-IPG (Table 6.4). 

Figure 6.1 Time course of (R)-2-octylbutanoate production catalyzed by lyophilized cells (30g/l) of R. oryzae CBS 112.07 in n-heptane at 30°C. Alcohol concentration = l. Og/l, with an equimolar concentration of butanoic acid as the acylating agent.

Table 6.4 Kinetic parameters of R- and S-IPG esterification with butyric acid catalyzed by R. oryzae CBS 112.07 and A. oryzae MIM.

Asper llus oryzae MIM was more enantioselective than R. oryzae CBS 112.07 therefore, it was used for studying the time course of the esterification with butyric acid of (R,S)-IPG under ophmal condihons (Figure 6.2). 

The enantioselective esterification of (R,S)-2-phenylpropanoic acid in organic solvent was studied by using dry myceUa of A. oryzae MIM and R. oryzae CBS... 

The two micro-organisms were enantiocomplementary since A. oryzae MIM showed preference for the formation of the S-ester, while R. oryzae CBS... 

The high molar conversion obtainable in esterifications catalyzed by dry mycelia encouraged us to investigate the partition of water in these heterogeneous systems further. The objechve was to verify whether mycelia were able to affect the thermodynamic equilibrium of the reactions by modifying the parhtioning of the water formed during the esterificahon. The synthesis of two esters by lyophilized mycelia of R. oryzae CBS 112.07 was studied and the results were compared with those obtained with a commercial immobilized CALB (Novozym 435). 

In all cases the conversions achieved by using R. oryzae CBS 112.07 were slightly higher, although both systems led to >90% conversions. 

Solvent Temperature (°C) Molar conversion at equilibrium (%) R. oryzae CBS 112.07 Novozym 435 ... 

Rhizopus oryzae is another important fungus involved in the production of organic acids in industrial fermentation. It is widely used to produce L-lactic acid as well as other organic acids. R. oryzae, produces only one stereospecific product (L-lactic acid), and not a racemic mixture (Wang et al., 2005). Much research has been done on the mechanism of lactic acid... 

Rhizopus oryzae is an indispensable microorganism in industrial fermentation, as it is widely employed to produce L-lactic acid as well as other organic acids. This organism is able to produce only one stereospecific product (L-lactic acid), rather than a racemic mixture and can, therefore, fulfill the need for producing a food additive to be used as both acidulant and preservative. During L-lactic acid fermentation many other metabolites can be produced as by-products. These include fumaric acid, malic acid, ethanol, and the like. However, these metabolites can greatly influence the downstream process and the quality of the L(+)-lactic acid produced. Fumaric acid is the main by-product, as a result of a special metabolic pathway in L-lactic acid production by R. oryzae (Wang et al., 2005). 

In addition to lactic acid producing bacteria, a few mycelial molds belonging to Rhizopus are good lactic acid producers. The ability of Rhizopus to produce only L-(-I-)-lactic acid aerobically under nitrogen-limited environments has been studied [25-28]. Compared to bacterial fermentation, Rhizopus requires only inorganic salts. In addition, Rhizopus cultures are more tolerant to a low pH environment. Consequently, pH maintenance is not as stringent as bacterial culture during lactic acid fermentation. Furthermore, Rhizopus molds are amy-lolytic that can produce lactic acid from starchy materials directly. For example, R. oryzae NRRL 395 was used to ferment starch derived from barley, cassava, corn, oat, and rice to L-lactic acid [25]. 

R. oryzae NRRL 395 is able to utilize xylose to produce lactic acid, but the production rate is much slower than with glucose as the substrate. Figure 3 shows the kinetics of lactic acid production from glucose and xylose as reported by Yang et al. [26]. 

Fig. 3. Kinetics of L-lactic acid production from glucose and xylose by R. oryzae NRRL 395 [26]...

Soccol et al. [34] used R. oryzae NRRL 395 to produce lactic acid from glucose-impregnated sugar cane bagasse. In the presence of CaCOj, 137 g/1 L-lactic acid was produced from 180 g/1 glucose with a productivity of 1.43 g/l/h. 

Water is one of the products produced by esterification of FA and glycerol. Hence, removal of this by-product shifts the equilibrium to esterification, and a high degree of esterification is achieved. Actually, in the esterification with P. camembertii and R. oryzae lipases (Section 13.3.3.1), the reaction period was shortened largely by removal of water with a vacuum pump during the reaction (Watanabe et al. 2004). 

In acidolysis of TAG with FA using immobilized R. oryzae lipase, pretreatment of the lipase in a substrate mixture containing small amounts of water was necessary... 

FIGURE 13.10 Production of MLM-type TAGs containing PUFAs using 2-MAGs as intermediates. Immobilized C. antarctica lipase is useful for the first-step ethanolysis, and immobilized R. miehei (R. oryzae) lipase is available for the second-step acylation. 

So far, the regiospecihc analysis has been conducted by Grignard degradation (Becker et al. 1993) or by hydrolysis with a 1,3-position-specihc lipase, such as lipases from pancreas, R. oryzae, and R. miehei (Luddy et al. 1964). After the reactions, FA compositions at the 2- and 1,3-positions can be determined based on the contents of FAs in 2-MAGs that are isolated from the reaction products. 

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