Rhizopus Chinensis Research Articles

Rheology, Microstructure, and Baking Characteristics of Frozen Dough Containing Rhizopus chinensis Lipase and Transglutaminase

ABSTRACTThe beneficial effects of a new recombinant lipase (Rhizopus chinensis lipase [RCL]) and transglutaminase (TG) were investigated on frozen dough systems and their breadmaking quality. Rheological properties and microstructure of doughs were measured using a dynamic rheometer, rheofermentometer F3, and scanning electron microscopy (SEM). Measurements of viscoelastic properties showed that both G′ and G″ of dough containing RCL and TG were greater than those of the control after 35 days of frozen storage. The SEM micrographs showed that dough containing RCL and TG had the most starch granules embedded in or attached to the gluten network, and the gluten seemed more powerful and resilient than for the control dough after 35 days of frozen storage. Results of the gas production and dough development tests indicated that RCL and TG improved the rheofermentative characteristics of frozen dough. RCL and TG could improve water‐holding capacity and significantly increase the glycerol content of the control dough. Image analyses showed that bread crumbs containing RCL and TG had a more open network and uniform crumb structure, which resulted in higher specific volume. This combination also yielded a product with higher sensory scores for test breads.

イネのRhizopus chinensis菌による立枯病苗およびPythium属菌による萎凋性立枯病苗の移植後の生育といもち病, 紋枯病の発生

イネのRhizopus chinensis菌による立枯病苗およびPythium属菌による萎凋性立枯病苗の移植後の生育といもち病, 紋枯病の発生

High yield Rhizopus chinenisis prolipase production in Pichia pastoris: Impact of methanol concentration

Rhizopus lipases have been successfully expressed in Pichia pastors and different fermentation strategies have been investigated. However, there is no sufficient study on the effects of methanol concentration on the production of Rhizopus lipases in P. pastors. In this study, the lipase from Rhizopus chinensis CCTCC M20102 was expressed under different fed-batch fermentation conditions at methanol concentrations ranging from 0.5 to 3.5 g/L. The lipase activity, stability, and productivities were analyzed. The optimum methanol concentration was 1 g/L, with the highest lipase activity of 2,130 U/mL, without degradation. Additional information was obtained from the analysis of methanol consumption and production rates. The results also suggested that the cell concentration at the end of the glycerol fed-batch phase was very important for cell viability and protease activity.

Microbial transformation of curcumin by Rhizopus chinensis

Curcumin (1) is a potent antioxidant and antitumor natural product. In spite of its efficacy and safety, its clinical use is hindered mainly by poor water solubility and bioavailability. Structural modification to introduce hydrophilic functions is a promising approach to resolve this problem. In the present study we first found that curcumin could be efficiently converted into glucosides by filamentous fungi including Rhizopus chinensis IFFI 03043, Absidia coerulea AS 3.3389 and Cunninghamella elegans AS 3.1207. Curcumin 4′-O-β-d-glucoside (2), together with hexahydrocurcumin (3), was isolated from a preparative-scale biotransformation with R. chinensis IFFI 03043 and characterized fully by NMR and MS. A time-course study revealed that curcumin could be efficiently converted into curcumin 4′-O-β-d-glucoside within 8 h when administered at 0.05 mmol L−1 and the productivity was 57%. Additionally, the biotransformation products of curcumin by different fungal strains were analyzed by LC/MS. At least 15 metabolites were detected, and the predominant biotransformation reaction was glucosylation. This study provides a simple, efficient and less expensive approach for the preparation of curcumin glucosides. The introduction of the glucosyl function might be able to enhance the bioavailability of curcumin.

Enhancement of activity of Rhizopus chinensis lipase by directed evolution

Enhancement of activity of Rhizopus chinensis lipase by directed evolution

Enzymic Synthesis of Gastrodin through Microbial Transformation and Purification of Gastrodin Biosynthesis Enzyme

Gastrodin, a major bioactive component of a famous Chinese herb Gastrodia elata B1., has diverse pharmaceutical functions. It is usually obtained by extraction from a plant or through chemical synthesis. However, traditional extraction from Gastrodia elata B1. is time and money consuming, while chemical synthesis is a complicated procedure and always leads to very serious environmental pollution. Thus it is urgent to explore a new gastrodin source which is more economical and environmental. The present study reports a novel approach to the production of gastrodin through biosynthesis and microbial transformation. Rhizopus chinensis SAITO AS3.1165 was screened from about 50 fungal and bacterial strains and found capable of biotransforming p-hydroxybenzaldehyde into gastrodin for use in gastrodin production. A series of purification steps including (NH(4))(2)SO(4) precipitation, ion exchange chromatography and gel filtration column chromatography was successfully used for purification of the gastrodin biosynthesis enzyme (GBE). The purity of GBE was above 95% and its molecular weight was about 63.2 kDa. We further characterized GBE's function condition, and found that the optimal temperature was 50 °C and the optimum pH 6.0. The enzyme was stable at a temperature lower than 50 °C and a pH between 6.0 and 9.0. The result indicated that gastrodin could be successfully synthesized by microbial transformation, providing a new approach for gastrodin production.

Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike

The tailed bacteriophage phi29 has 12 "appendages" (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an "autochaperone" that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function to attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is performed by phi29 gp12 for Bacillus subtilis.

Regulation of environmental factors on the expression of a solid-state specific lipase (Lip1) with Rhizopus chinensis by western blot and indirect Elisa

To identify which solid-state typical environmental factors are involved in the induction of a solid-state special lipase (Lip1), western blot and Elisa based on Lip1 antibody were used. A low water activity played a significant role in the induction of Lip1, as evidenced by the increased expression level (20–46 μg/g dry cell) along with the decrease of water activity (0.927–0.969). Physical barrier against hyphal extension was found to be another required factor, since the expression of Lip1 was significantly enhanced by 3-fold using a membrane with smaller pore size (0.45 and 0.22 μm) covered on top of surface culture.

Novel minor lipase from Rhizopus chinensis during solid-state fermentation: Biochemical characterization and its esterification potential for ester synthesis

Rhizopus chinensis produces two lipases that catalyze ester synthesis when cultured under solid-state fermentation. The Lip2 was purified to homogeneity by ammonium sulphate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. It has an apparent molecular weight of 33 kDa estimated from SDS–PAGE and 32 kDa calculated from analytical gel permeation, with synthetic activity and purification fold of 96.8 U/mg and 138.3, respectively. Maximum hydrolytic activity was obtained at pH 8.0–8.5 and 40 °C using pNPP as substrate. Slight activation of the enzyme was observed when Mn 2+ is present. The enzyme was most active on p-nitrophenyl laurate (C12). The purified lipase exhibited maximum synthetic activity at pH memory of 6.0 and 30 oC. Most of ethyl esters synthesized by lyophilized enzyme achieved good yields (>90%), and caprylic acid served as the best acyl donor. The enzyme presented a particular affinity for ethanol, n-propanol and n-hexanol, with conversion of 92%, 93% and 92%, respectively, after 20 h incubation.

Production and regulation of different lipase activities from Rhizopus chinensis in submerged fermentation by lipids

The fungal Rhizopus chinensis could produce several types of lipase, which were mainly intracellular. During the whole-cell lipase production by this strain in submerged fermentation, it was observed that two catalytic characteristics (hydrolytic and synthetic activity) of lipases were different with addition of lipids. The hydrolytic activity of the lipase was not induced by lipids efficaciously and could be detected regardless of whether substrate-related compounds were present. However, it was found that the induction of lipids for the synthetic activity lipase was significant, and that nearly no synthetic activity was detected while the medium contained no lipids. When only a little lipid (1 g/L) was added to medium, the synthetic activity increased sharply in the initial process of fermentation. Analysis of crude membrane-bound lipase by SDS-PAGE confirmed this induction. De novo biosynthesis of lipases, especially the lipase with synthetic activity occurred only when lipids existed. Cell growth and maltose repress the lipase production with synthetic activity, but have little influence on the lipase production with hydrolytic activity. Since the production process of mycelium-bound lipase with hydrolytic activity was different, it was reasonable to consider hydrolytic activity and synthetic activity for different application purposes. Whole-cell lipase obtained from fermentation process with high synthetic activity showed excellent catalytic ability in solvent free system on synthesis of ethylcaprylate and ethyloleate, the conversion could reach more than 90% in 5 h.

Rhizopus chinensis lipase: Gene cloning, expression in Pichia pastoris and properties

Lipases are the most attractive enzymes for use in organic chemical processes. In our previous studies, a lipase from Rhizopus chinensis CCTCC M20102 was found to have very high ability of esterification of short-chain fatty acids with ethanol. In this study, we reported the cloning and expression of the lipase gene from R. chinensis in Pichia pastoris and characterization of the recombinant lipase. The lipase gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOX1 promoter. In the induction phase, two bands of 37 kDa and 30 kDa proteins could be observed. The amino-terminal analysis showed that the 37-kDa protein was the mature lipase (30 kDa) attached with 27 amino acid of the carboxy-terminal part of the prosequence (r27RCL). The pH and temperature optimum of r27RCL and mRCL were pH 8.5 and 40 °C, and pH 8 and 35 °C, respectively. The stability, reaction kinetics and effects of metal ions and other reagents were also determined. The chain length specificity of r27RCL and mRCL showed highest activity toward p-nitrophenyl hexanoate or glyceryl tricaproate (C6) and p-nitrophenyl acetate or glyceryl triacetate (C2), respectively. This property is quite rare among lipases and gives this new lipase great potential for use in the field of biocatalysis.

Purification and biochemical characterization of an intracellular lipase byRhizopus chinensisunder solid-state fermentation and its potential application in the production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)

BACKGROUND: Purification and characterization of an intracellular lipase produced by Rhizopus chinenesis cultured in solid-state fermentation was investigated. The potential application in concentrating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil by the pure enzyme was also studied. RESULTS: Through four successive purification steps, the enzyme was purified to homogeneity with an apparent molecular mass of 36 kDa. The lipase was active for pH between 7.0 and 9.0 and temperatures 20–45 °C. Lipase activity was slightly increased in the presence of Ca2+ and Mg2+, but strongly inhibited by Hg2+ and SDS. The pure enzyme was most active on medium chain p-nitrophenol esters, with the highest activity towards pNP-caprylate (C8). The enzyme is a non-specific lipase, because it cleaved not only the 1,3-positioned ester bonds but also the 2-positioned bond in triolein. High EPA (17.6%) and DHA (32.9%) contents were achieved using the pure lipase (100 U) within 10 h. CONCLUSION: The enzymatic activity of the lipase on a wide variety of substrates and its stability in the presence of some organic solvents suggest that the lipase should be investigated for a range of commercial applications. The pure lipase was proved to possess potential ability for the production and concentration of EPA and DHA from fish oil. Copyright © 2008 Society of Chemical Industry

High-level expression and characterization of Rhizopus chinensis mature lipase in Pichia pastoris

High-level expression and characterization of Rhizopus chinensis mature lipase in Pichia pastoris

Membrane-bound ‘synthetic lipase’ specifically cultured under solid-state fermentation and submerged fermentation by Rhizopus chinensis: A comparative investigation

Rhizopus chinensis was able to produce synthetic lipases under both solid-state and submerged fermentations. These lipases were extracted from cell membrane using Triton X-100, and purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. Judging from SDS–PAGE, the specific synthetic lipases associated with SSF (named as SSL) and SmF (named as SML) were different in the apparent molecular mass (62 and 40 kDa). In term of hydrolytic activity, both enzymes exhibited maximum values at pH 8.0 and 40 °C; SSL appeared to be more pH tolerant and thermostable than SML. PMSF negligibly affected SSL but strongly reduced the activity of SML. Both enzymes showed clear preference for long-chained p-nitrophenyl esters, yielding maximum activity towards p-nitrophenyl palmitate (with SSL) and p-nitrophenyl laurate (with SML). In term of synthetic activity, lyophilized enzymes gave the highest values both at 30 °C, but at different pH memories (7.5 for SSL and 6.5 for SML). Most of ethyl esters synthesized by the two enzymes achieved good yields (>90%), and tetradecanoic acid and laurate acid separately served as the best acyl donors.

Production and Biochemical Characterization of an Extracellular Lipase from Rhizopus chinensis CCTCC M201021

Production and Biochemical Characterization of an Extracellular Lipase from Rhizopus chinensis CCTCC M201021

Changes in morphology of Rhizopus chinensis in submerged fermentation and their effect on production of mycelium-bound lipase

In order to control suitable mycelium morphology to obtain high lipase productivity by Rhizopus chinensis in submerged fermentation, the effects of fungal morphology on the lipase production by this strain both in shake flask and fermentor were investigated. Different inoculum level and shear stress were used to develop distinctive morphologies. Analyses and investigations both on micromorphology and macromorphology were performed. Study of micromorphology reveals that micromorphologies for dispersed mycelia and aggregated mycelia are different in cell shape, biosynthetic activity. Macromorphology and broth rheology study in fermentor indicate that pellet formation results in low broth viscosity. Under this condition, the oil can disperse sufficiently in broth which is very important for lipase production. These results indicate that morphology changes affected the lipase production significantly for R. chinensis and the aggregated mycelia were suggested to achieve high lipase production.

Effects of oils and oil-related substrates on the synthetic activity of membrane-bound lipase from Rhizopus chinensis and optimization of the lipase fermentation media

The lipase from filamentous fungi Rhizopus chinensis, as a membrane-bound enzyme, possesses the excellent catalysis ability for esterification and transesterification reactions, and has a good potential in many industrial applications. In order to improve the synthetic activity of the lipase, the effects of oils and oil-related substrates on its production and the fermentation media optimization were investigated. Based on the results, it was suggested that oleic acid could be the important substrate for the lipase production. Among various oils and oil-related substrates, olive oil containing high content of oleic acid was the optimal one for the lipase production. Using orthogonal test and response surface methodology (RSM), the composition of fermentation media was further optimized. The optimized media for lipase synthetic activity and activity yield was composed of peptone 57.94 and 55.58 g L −1, olive oil 21.94 and 22.99 g L −1, maltose 12.91 and 14.34 g L −1, respectively, with K 2HPO 4 3 g L −1, MgSO 4·7H 2O 5 g L −1 and initial pH 6.0. Under the optimal conditions, the lipase activity and the activity yield were improved 61.5 and 93.4% comparing the results before optimization, respectively. The adequate models obtained had predicted the lipase production successfully.

Biodiesel Production Catalyzed by Whole-Cell Lipase from Rhizopus chinensis

Abstract Compared with five commercial lipases, the whole-cell lipase of Rhizopus chinensis CCTCC M201021 (RCL) showed high catalytic ability in the transesterification of soybean oil to fatty acid methyl ester (biodiesel) in the solvent-free system. The effect of reaction parameters including methanol content, water content, lipase amount, and reaction temperature on transesterification was investigated. The biodiesel yield was more than 86.0% in the solvent-free system under the optimal reaction conditions. The effect of different organic solvents on the transesterification indicated that the best one was n -heptane with higher log P values (4.0–4.5). The whole-cell lipase also proved to be an efficient and potential biocatalyst for biodiesel production from oleic acid and simulant high acid value oil in the solvent-free system.

Solid-state fermentation for ‘whole-cell synthetic lipase’ production from Rhizopus chinensis and identification of the functional enzyme

Major process parameters affecting the production of whole-cell synthetic lipase (WCSL) by Rhizopus chinensis in solid-state fermentation were optimized based on synthetic activity. A combined substrate of wheat flour with wheat bran (3/2, w/w) supported both good biomass growth and enzyme production. The maximum synthetic activity of 24,447 U/kg substrate was reached by selecting a moisture content of 70%, initial pH of 6.5, supplementation of peptone (2%, w/w) as additional nitrogen source and olive oil (2%, v/w) as inducer, after 72 h of incubation. This production represented a 15.27-fold increase of lipase synthetic activity compared to the initial medium. The synthetic lipase was purified by ammonium sulfate precipitation, hydrophobic interaction and gel filtration to homogeneity, with yield and purification fold of 2.6% and 55.2, respectively, and the molecular weight was estimated to be 60 kDa by SDS-PAGE. Validation of functional enzyme was carried out by catalyzing the synthesis of ethyl flavor esters, and the result showed that conversion of ethyl octanoate and ethyl caprate reached 95%.

Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method

Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions.