The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Zhen-ming Chi - One of the best experts on this subject based on the ideXlab platform.
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Co-expression of Exo-inulinase and Endo-inulinase Genes in the Oleaginous Yeast Yarrowia lipolytica for Efficient Single Cell Oil Production from Inulin.
Applied biochemistry and biotechnology, 2017Co-Authors: Nianci Shi, Zhen-ming Chi, Zhe Chi, Weian Mao, Guang-lei LiuAbstract:Yarrowia lipolytica is a promising platform for the single cell oil (SCO) production. In this study, a transformant X+N8 in which exo- and endo-inulinase genes were co-expressed could produce an inulinase activity of 124.33 U/mL within 72 h. However, the inulinase activity of a transformant X2 carrying a single exo-inulinase gene was only 47.33 U/mL within 72 h. Moreover, the transformant X+N8 could accumulate 48.13% (w/w) SCO from inulin and the cell dry weight reached 13.63 g/L within 78 h, which were significantly higher than those of the transformant X2 (41.87% (w/w) and 11.23 g/L) under the same conditions. In addition, inulin hydrolysis and utilization of the transformant X+N8 were also more efficient than those of the transformant X2 during the fermentation process. These results demonstrated that the co-expression of the exo- and endo-inulinase genes significantly enhanced the SCO production from inulin due to the improvement of the inulinase activity and the synergistic action of exo- and endo-inulinase. Besides, over 95.01% of the fatty acids from the transformant X+N8 were C16-C18, especially C18:1 (53.10%), suggesting that the fatty acids could be used as feedstock for biodiesel production.
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Inulinase production by the yeast Kluyveromyces marxianus with the disrupted MIG1 gene and the over-expressed inulinase gene
Process Biochemistry, 2014Co-Authors: Hai-xiang Zhou, Guang-lei Liu, Zhe Chi, Fang-hua Xin, Zhen-ming ChiAbstract:Kluyveromyces marxianus has been widely used in food industries and inulinase produced by the yeast has many applications. In order to greatly enhance inulinase production by the yeast, it is very important to both derepress glucose repression and overexpress the native inulinase gene. The MIG1 gene in the yeast K. marxianus KM-0 was disrupted. It was found that a glucose-derepressed mutant (KM-69) of K. marxianus KM-0 could produce 94.6 units of inulinase activity per ml in the inulin medium within 36 h. In order to further increase inulinase production, the native inulinase gene was over-expressed in the glucose-derepressed mutant. During the 10-l fermentation, the recombinant strain KM-526 yielded 133.5 units of inulinase activity per ml within very short time (24 h). The results also showed that inulinase produced by the recombinant strain KM-526 could more effectively convert inulin into monosaccharides than that produced by K. marxianus KM-0. The results indicate that it is very important to delete the MIG1 gene in K. marxianus KM-0 and over-express the native INU1 gene in order to further enhance inulinase yield within the short time by this yeast and for efficient hydrolysis of inulin. The recombinant strain KM-526 and its inulinase have highly potential applications in food industries.
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ethanol production from inulin and unsterilized meal of jerusalem artichoke tubers by saccharomyces sp w0 expressing the endo inulinase gene from arthrobacter sp
Bioresource Technology, 2013Co-Authors: Guang-lei Liu, Zhen-ming ChiAbstract:Abstract After the endo-inulinase gene from Arthrobacter sp. was ligated the expression vectors pMIDSC31 and pMIRSC31, the endo-inulinase gene was inserted into the chromosomal DNA of Saccharomyces sp. W0. It was found that the inulinase activity of the recombinant yeast D5 in which the endo-inulinase gene was inserted into the delta sequence was higher than that of the recombinant yeast R1 in which the endo-inulinase gene was inserted into 18S rDNA sequence. More ethanol from inulin was produced by the recombinant yeast D5 than by the recombinant yeast R1. But Saccharomyces sp. W0 produced the lowest inulinase activity and concentration of ethanol. During the 3-l fermentation, the recombinant yeast D5 could produce 13.6 ml of ethanol per 100 ml of the fermented medium from 30% inulin. The recombinant yeast D5 could actively convert the unsterilized meal of Jerusalem artichoke tubers, yielding 10.1 ml of ethanol per 100 ml of the fermented medium.
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Molecular characterization and expression of microbial inulinase genes
Critical reviews in microbiology, 2012Co-Authors: Guang-lei Liu, Zhe Chi, Zhen-ming ChiAbstract:Many genes encoding exo- and endo-inulinases from bacteria, yeasts and filamentous fungi have been cloned and characterized. All the inulinases have several conserved motifs, such as WMND(E)PNGL, RDP, EC(V)P, SVEVF, Q and FS(T), which play an important role in inulinase catalysis and substrate binding. However, the exo-inulinases produced by yeasts has no conserved motif SVEVF and the yeasts do not produce any endo-inulinase. Exo- and endo-inulinases found in different microorganisms cluster separately at distant positions from each other. Most of the cloned inulinase genes have been expressed in Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Klyuveromyces lactis and Escherichia coli, respectively. The recombinant inulinases produced and the engineered hosts using the cloned inulinase genes have many potential applications. Expression of most of the inulinase genes is repressed by glucose and fructose and induced by inulin and sucrose. However, the detailed mechanisms of the repression an...
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enhanced inulinase production in solid state fermentation by a mutant of the marine yeast pichia guilliermondii using surface response methodology and inulin hydrolysis
Journal of Industrial Microbiology & Biotechnology, 2009Co-Authors: Ning Guo, Zhen-ming Chi, Fang Gong, Jun ShengAbstract:In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl2. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.
Ashok Pandey - One of the best experts on this subject based on the ideXlab platform.
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statistical optimization of solid state fermentation for the production of fungal inulinase from apple pomace
Bioresource Technology Reports, 2020Co-Authors: Ram Sarup Singh, Kanika Chauhan, Karminder Kaur, Ashok PandeyAbstract:Abstract The present work was designed to determine the suitability of apple pomace for inulinase production from Mucor circinelloides by solid-state fermentation (SSF). Central composite rotatable deign (CCRD) matrix (15 runs) of RSM was experimentally executed to investigate the influence of moisture, pH and fermentation time on fungal inulinase production from apple pomace by SSF. SSFs were performed at flask-level at 30 °C. Under optimized conditions viz., moisture 83.5%, pH 6.4 and fermentation time 5.8 days, maximum inulinase production was 411.3 IU/gds. Coefficient of variation (CV%) 0.17 indicates the lowest deviations between the experimental and predicted values. Multiple correlation coefficient (R2) was calculated to be 1.00 for inulinase production, which signifies the good correlation between the experimental and predicted results. Model's higher F-value 94,051.9 and “Lack-of-Fit” F-value 0.43 also verifies the authenticity of the quadratic model. The present article highlights the applicability of apple pomace as potent substrate for inulinase production by M. circinelloides.
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influence of aeration agitation and process duration on fungal inulinase production from paneer whey in a stirred tank reactor
Bioresource Technology Reports, 2019Co-Authors: Ram Sarup Singh, Kanika Chauhan, Ashok PandeyAbstract:Abstract In present study, inulinase production was carried out from paneer whey using Penicillium oxalicum BGPUP-4 in a stirred tank bioreactor. In preliminary experimentation in shake-flask fermentations, maximum (38.58 IU/mL) inulinase production was obtained from clarified paneer whey. Therefore, it was selected for inulinase production at bioreactor level. Probabilistic tool was used to optimise process variables for inulinase production in bioreactor. At optimal combination of process variables (agitation 180 rpm, aeration 0.98 vvm and fermentation time 5 days), maximum inulinase production obtained in stirred tank reactor was 80.2 IU/mL. Approximately 2-fold increase in inulinase production was obtained in comparison to shake-flask fermentations. A statistical evaluation of designed model predicted it significant. The findings of the present study show paneer whey as cost-effective substrate for the production of inulinase by P. oxalicum BGPUP-4 in a stirred tank reactor. Literature survey reveals, this is first report on inulinase production from paneer whey based medium.
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solid state fermentation of carrot pomace for the production of inulinase by penicillium oxalicum bgpup 4
Food Technology and Biotechnology, 2018Co-Authors: Ram Sarup Singh, Kanika Chauhan, Ashok Pandey, Jagroop Singh, Christian LarrocheAbstract:Inulinases are an important class of industrial enzymes which are used for the production of high-fructose syrup and fructooligosaccharides. Inulin, a polyfructan, is generally employed for the production of inulinase, which is a very expensive substrate. A number of agroindustrial residues have been used for cost-effective production of inulinases. In the present study, carrot pomace was selected as a substrate for the production of inulinase by Penicillium oxalicum BGPUP-4 in solid-state fermentation. Carrot pomace is one of the good substrates for bioprocesses, because it is rich in soluble and insoluble carbohydrates. A central composite rotatable design (CCRD) used in response surface methodology was employed for the optimal production of inulinase from carrot pomace. Using CCRD, 15 runs were practiced to optimize the range of three independent variables: moisture content (70-90%), incubation time (4-6 days) and pH (5.0-7.0) for inulinase production. Carrot pomace supplemented with 0.5% inulin as an inducer, 0.2% NH4H2PO4, 0.2% NaNO3, 0.2% KH2PO4, 0.05% MgSO4·7H2O and 0.001% FeSO4·7H2O was used for the production of inulinase in solid-state fermentation at 30 °C. Inulinase production (322.10 IU per g of dry substrate) was obtained under the optimized conditions, i.e. moisture content of 90%, incubation time 4 days and pH=7.0. The corresponding inulinase/invertase (I/S) ratio (3.38) was also high, which indicates the inulolytic nature of the enzyme. Multiple correlation coefficients R for inulinase production and I/S ratio were 0.9995 and 0.9947, respectively. The R value very close to one indicates an excellent correlation between experimental and predicted results.
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comparative studies on inulinase synthesis by staphylococcus sp and kluyveromyces marxianus in submerged culture
Bioresource Technology, 1999Co-Authors: P Selvakumar, Ashok PandeyAbstract:Staphylococcus sp. RRL1 and Kluyveromyces marxianus ATCC 52466 produced extra-cellular inulinase when grown in liquid medium containing inulin as the sole carbon source and the former produced about 30% more enzyme activity. The highest inulinase activity (618 U/liter) from Staphylococcus sp. was obtained in a medium with an initial pH, temperature, agitation and the size of inoculum of 6.5, 37°C, 150 rpm and 4%, respectively. Kluyveromyces marxianus ATCC 52466 gave highest inulinase activity (470.4 U/liter) in a medium with an initial pH, temperature, agitation and size of inoculum as 6.0, 30°C, 150 rpm and 4%, respectively. The Staphylococcus sp. seems to be a new and useful source of inulinase.
Fang Gong - One of the best experts on this subject based on the ideXlab platform.
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enhanced inulinase production in solid state fermentation by a mutant of the marine yeast pichia guilliermondii using surface response methodology and inulin hydrolysis
Journal of Industrial Microbiology & Biotechnology, 2009Co-Authors: Ning Guo, Zhen-ming Chi, Fang Gong, Jun ShengAbstract:In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl2. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.
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use of response surface methodology for optimizing process parameters for high inulinase production by the marine yeast cryptococcus aureus g7a in solid state fermentation and hydrolysis of inulin
Bioprocess and Biosystems Engineering, 2009Co-Authors: Jun Sheng, Zhen-ming Chi, Xianghong Wang, Kuirang Yan, Fang GongAbstract:The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies (RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD. Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of wheat bran to rice husk 0.42, temperature 29 °C, pH 5.5. Under the optimized conditions, 420.9 U g−1 of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas the predicted maximum inulinase activity of 436.2 U g−1 of inulinase activity of 436.2 U g−1 of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.
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inulinase overproduction by a mutant of the marine yeast pichia guilliermondii using surface response methodology and inulin hydrolysis
Biochemical Engineering Journal, 2009Co-Authors: Ning Guo, Fang Gong, Zhen-ming Chi, Jun Sheng, Zhe ChiAbstract:Abstract In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.
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Optimization for high-level expression of the Pichia guilliermondii recombinant inulinase in Pichia pastoris and characterization of the recombinant inulinase
Process Biochemistry, 2009Co-Authors: Tong Zhang, Fang Gong, Ying Peng, Zhen-ming ChiAbstract:Abstract The inulinase gene cloned from the marine-derived yeast Pichia guilliermondii strain 1 was expressed in Pichia pastoris X-33 and the conditions for overexpression of the inulinase were optimized. After the optimization of the conditions for production of the recombinant inulinase, 286.8 ± 5.4 U/ml and 8873 ± 55.3 U/mg of the recombinanat inulinase in the supernatant of the culture of 2-l fermentor were attained at 120 h of the fermentation and fermentation efficiency was 13.04 μg ± 0.4 of protein/ml/d. The recombinant inulinase was purified and characterized. The molecular weight of the purified recombinant inulinase was 57.6 kDa, which was higher than that of the native iunlinase. The optimal pH and temperature of the purified recombinant inulinase were 6.0 and 60 °C, respectively. Other biochemical characteristics of the purified recombinant inulinase were the same as those of the native inulinase produced by the marine-derived P. guilliermondii strain 1. The purified recombinant inulinase also had high exoinulinase activity. Therefore, the recombinant inulinase may have highly potential applications in food and pharmaceutical industies.
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cloning and characterization of the inulinase gene from a marine yeast pichia guilliermondii and its expression in pichia pastoris
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2009Co-Authors: Tong Zhang, Guang-lei Liu, Zhe Chi, Zhen-ming Chi, Fang Gong, Jun Sheng, Xianghong WangAbstract:The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.
Jun Sheng - One of the best experts on this subject based on the ideXlab platform.
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enhanced inulinase production in solid state fermentation by a mutant of the marine yeast pichia guilliermondii using surface response methodology and inulin hydrolysis
Journal of Industrial Microbiology & Biotechnology, 2009Co-Authors: Ning Guo, Zhen-ming Chi, Fang Gong, Jun ShengAbstract:In order to isolate inulinase overproducers of the marine yeast Pichia guilliermondii, strain 1, cells were mutated by using UV light and LiCl2. One mutant (M-30) with enhanced inulinase production was obtained. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant in solid-state fermentation. The initial moisture, inoculum, the amount ratio of wheat bran to rice bran, temperature, pH for the maximum inulinase production by the mutant M-30 were found to be 60.5%, 2.5%, 0.42, 30°C and 6.50, respectively. Under the optimized conditions, 455.9 U/grams of dry substrate (gds) of inulinase activity was reached in the solid state fermentation culture of the mutant M-30 whereas the predicted maximum inulinase activity of 459.2 U/gds was derived from RSM regression. Under the same conditions, its parent strain only produced 291.0 U/gds of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far.
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use of response surface methodology for optimizing process parameters for high inulinase production by the marine yeast cryptococcus aureus g7a in solid state fermentation and hydrolysis of inulin
Bioprocess and Biosystems Engineering, 2009Co-Authors: Jun Sheng, Zhen-ming Chi, Xianghong Wang, Kuirang Yan, Fang GongAbstract:The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies (RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD. Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of wheat bran to rice husk 0.42, temperature 29 °C, pH 5.5. Under the optimized conditions, 420.9 U g−1 of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas the predicted maximum inulinase activity of 436.2 U g−1 of inulinase activity of 436.2 U g−1 of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.
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inulinase overproduction by a mutant of the marine yeast pichia guilliermondii using surface response methodology and inulin hydrolysis
Biochemical Engineering Journal, 2009Co-Authors: Ning Guo, Fang Gong, Zhen-ming Chi, Jun Sheng, Zhe ChiAbstract:Abstract In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.
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cloning and characterization of the inulinase gene from a marine yeast pichia guilliermondii and its expression in pichia pastoris
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2009Co-Authors: Tong Zhang, Guang-lei Liu, Zhe Chi, Zhen-ming Chi, Fang Gong, Jun Sheng, Xianghong WangAbstract:The extracellular inulinase structural gene was isolated from the genomic DNA of the marine yeast Pichia guilliermondii strain 1 by PCR. The gene had an open reading frame of 1,542 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 514 amino acid residues of a protein with a putative signal peptide of 18 amino acids and the calculated molecular mass of 58.04 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNXPNGL) and (RDPKVF). It also had ten conserved putative N-glycosylation sites. The inulinase from P. guilliermondii strain 1 was found to be closely related to that from Kluyveromyces marxianus. The inulinase gene without the signal sequence was subcloned into pPICZαA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum activity of 58.7 ± 0.12 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. A large amount of monosaccharides, disaccharides and oligosaccharides were detected after the hydrolysis of inulin with the crude recombinant inulinase.
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Inulinase-producing Marine Yeasts: Evaluation of their Diversity and Inulin Hydrolysis by Their Crude Enzymes
Microbial ecology, 2007Co-Authors: Lingmei Gao, Zhen-ming Chi, Jun Sheng, Lin Wang, Fang GongAbstract:Total 427 yeast strains from seawater, sediments, mud of salterns, guts of the marine fish, and marine algae were obtained. After inulinase activity of the yeast cultures was estimated, we found that four strains (OUC1, G7a, OUC2, and G7a1) of the marine yeasts grown in the medium with inulin could secrete a large amount of inulinase into the medium. The results of routine identification and molecular methods show that they belong to Pichia guilliermondii OUC1, Cryptococcus aureus G7a, Yarrowia lipolytica OUC2, and Debaryomyces hansenii G7a1, respectively. The optimal pHs of inulinase activity produced by them were 6.0, 5.0, 5.0, and 5.0, respectively, while the optimal temperatures of inulinase activity produced by them were 60°, 50°, 60°, and 50°C, respectively. A large amount of monosaccharides and a trace amount of oligosaccharides were detected after the hydrolysis by the crude inulinase produced by P. guilliermondii OUC1, indicating that the crude inulinase had a high exoinulinase activity while a large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase produced both by C. aureus G7a and D. hansenii G7a1. However, no monosaccharides and disaccharides were detected after inulin hydrolysis by the crude inulinase produced by Y. lipolytica OUC2, suggesting that the crude inulinase had no exoinulinase activity.
John Lenard - One of the best experts on this subject based on the ideXlab platform.
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Characterization and partial purification of an Insulinase from Neurospora crassa.
Archives of biochemistry and biophysics, 1992Co-Authors: Hemanta K. Kole, Deborah Smith, John LenardAbstract:An insulin-binding metal- and thiol-dependent proteinase has been purified 1491-fold from high speed cytosolic fractions of the fungus Neurospora crassa. This enzyme resembles insulin-degrading enzymes (Insulinases) present in mammalian cells and in Drosophila melanogaster in the following ways: (i) it degrades radiolabeled insulin with a specificity similar to that of rat muscle Insulinase, as demonstrated by HPLC analysis of the degradation products; (ii) it is inhibited by bacitracin, EDTA, 1,10-phenanthroline, and the sulfhydryl-reactive compounds N-ethylmaleimide and p-chloromercuribenzoate, but not by inhibitors of serine proteases or by lysosomal protease inhibitors. Cross-linking with 125I-insulin labels a band of ca. 120 kDa, and several smaller bands which may represent degradation products. The N. crassa Insulinase is stimulated by Mn2+ and strongly inhibited by Zn2+; Mn2+ can also reactivate the enzyme after inhibition by EDTA, but Zn2+ is ineffective. The N. crassa protein differs in this regard from mammalian and insect Insulinases which are generally activated by both Mn2+ and Zn2+. This finding extends the apparent evolutionary conservation of these metal- and thiol-dependent proteases into the microbial realm.