Kluyveromyces fragilis KCTC 7260 and Saccharomyces cerevisiae KCTC 7904, which both grew well in pear marc extract, were selected and their growth profiles and physiological functionalities were determined. Both of the selected yeasts established maximal growth by 20 hr of cultivation at 30degrees C in pear marc extract. The cell-free extracts showed high antihypertensive angiotensin I-converting enzyme inhibitory activity of 68.9% and 52.1%, respectively. The extracts also displayed 9.2 U/mL and 12.0 U/mL of protease activity, respectively.
Kluyveromyces ; Peptidyl-Dipeptidase A ; Pyrus ; Saccharomyces cerevisiae ; Yeasts

Consolidated bioprocessing technology can be used for Kluyveromyces marxianus YX01 to produce ethanol from Jerusalem artichoke, which is one of the potential processes to produce biofuel from non-cereal crops. In this study, we combined the aeration rate with the substrate concentration to conduct cross-over experiments for K. marxinaus YX01, and studied ethanol fermentation and the influence of inulin enzyme activity. The substrate concentration had a little repressive effect on ethanol productivity. When substrate concentration reached 250 g/L under anaerobic conditions, ethanol concentration was 84.8 g/L, and ethanol yield was reduced from 86.4% (50 g/L substrate concentration) to 84.7% of the theoretical value. Aeration rate could accelerate K. marxinaus YX01 ethanol fermentation, but reduced ethanol yield. When substrate concentration reached 250 g/L under aeration at 1.0 vvm, ethanol yield was reduced from 84.7% under anaerobic conditions to 73.3% of the theoretical value. With increased concentration of the carbon source and reduced aeration rate, the inulinase of K. marxinaus YX01 reduced and the concentration of glycerol increased, however, the acetic acid increased with the increased concentration of the carbon source and aeration rate. When substrate concentration reached 250 g/L under anaerobic conditions, inulinase activity was only 6.59 U/mL; when substrate concentration reached 50 g/L under aeration at 1.0 vvm, inulinase activity was 21.54 U/mL.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; metabolism ; Helianthus ; metabolism ; Inulin ; metabolism ; Kluyveromyces ; classification ; metabolism ; Substrate Specificity

Jerusalem artichoke tubers with inulin as major component are potential feedstock for fuel ethanol production, and Kluyveromyces cicerisporus Y179 expressing high level of inulinase is suitable for ethanol production with this feedstock by simultaneous saccharification and fermentation approach. In this article, the impact of inoculum, aeration and temperature on ethanol production by the yeast was studied. The experimental results illustrated that inoculum with different levels and seed collected at different cultivation times had negligible effect, while anaerobic conditions enhanced ethanol production, and more ethanol was produced by the yeast at 30 degrees C than at 37 degrees C or 42 degrees C. The medium using Jerusalem artichoke tuber meal as sole component with 22% (W/V) total sugars was inoculated with 36 h-precultured seed at 10% (V/V), and the batch fermentation was conducted in a 5 L fermentor at 30 degrees C with a stirring speed of 300 r/min under anaerobic conditions. After 144 h, 12.3% (V/V) ethanol was produced and the yield of ethanol from sugars was 86.9% of its theoretical one, with 93.6% sugars consumed. These results indicate that K. cicerisporus Y179 is a promising candidate for industrial ethanol production using Jerusalem artichoke tuber feedstock.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; metabolism ; Helianthus ; chemistry ; Industrial Microbiology ; methods ; Kluyveromyces ; metabolism

A unique one-step ethanol fermentation process was developed with the inulinase-producing strain Kluyveromyces marxianus YX01. Firstly, the impact of temperature on ethanol fermentation was investigated through flask fermentation, and the temperature of 35 degrees C was observed to be the optimum to coordinate inulinase production, inulin saccharification and ethanol fermentation. And then, the impact of aeration and substrate concentration was studied through batch fermentation in the 2.5 L fermentor, and the experimental data indicated that the average ethanol fermentation time was decreased at the aeration rates of 50 mL/min and 100 mL/min, but higher ethanol yield was obtained under non-aeration conditions with more substrate directed to ethanol production. The ethanol concentration of 92.2 g/L was achieved with the substrate containing 235 g/L inulin, and the ethanol yield was calculated to be 0.436, equivalent to 85.5% of its theoretical value. Finally, Jerusalem artichoke grown in salina and irrigated with seawater was fermented without sterilization treatment, 84.0 g/L ethanol was obtained with the substrate containing 280 g/L dry Jerusalem artichoke meal, and the ethanol yield was calculated to be 0.405, indicating the Jerusalem artichoke could be an alternative feedstock for grain-based fuel ethanol production.
Bioreactors ; microbiology ; Ethanol ; metabolism ; Fermentation ; Helianthus ; metabolism ; Kluyveromyces ; metabolism ; Seawater ; Temperature

To improve the inulinase application in biotechnology, the characteristic of inulinase from Kluyveromyces marxianus YX01 was investigated. The inu gene of K. marxianus YX01 was transformed into Pichiapastoris GS115 host cells with molecular biology techniques. Then we achieved the heterologous expression of exo-inulinase whose molecular mass was about 86.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Furthermore, six His-tag was added to the inulinase and a two-step method was applied in the purification of inulinase, including concentration via dialysis by polyethylene glycol 20 000 and metal Ni-NTA Agarose affinity adsorption. The purification factor of purified protein was 3.6 and the recovery rate of enzyme activity was 33.1%. We characterized the purified inulinase. The optimum temperature was 60 degrees C and pH was 4.62. When inulin and sucrose were used as substrates, the K(m) and V(max) values were 80.53 g/L vs 4.49 g/(L x min) and 183.10 g/L vs 20.20 g/(L x min), respectively. In addition, metal ions including Mn2+, Ca2+, Cu2+, Zn2+ and Fe2+ exhibited different degrees of inhibition on the enzyme activity, and Cu2+, Zn2+ and Fe2+ exhibited the most significant inhibition. Our findings might lay a good foundation for industrial application of inulinase.
Glycoside Hydrolases ; chemistry ; genetics ; Industrial Microbiology ; Inulin ; Kluyveromyces ; enzymology ; genetics ; Pichia ; Sucrose ; Temperature

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.
Animals ; Cattle ; Chymosin ; biosynthesis ; genetics ; Enzyme Precursors ; biosynthesis ; genetics ; Gene Expression Regulation, Fungal ; genetics ; Genetic Vectors ; genetics ; Kluyveromyces ; genetics ; growth & development ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics

Two-hundred two yeast strains were isolated from rhizosphere (87 strains) and nonrhizosphere (115 strains) areas of potato, maize, vegetable marrow, and cabbage plants. On the basis of 26 morphological and physiological properties, the isolated yeast strains were assigned to 9 genera and 15 species. Trichosporon beigelii, Kluyveromyces marxianus and Torulaspora delbrueckii were the dominant species. Cryptococcus humicolus and Candida tropicalis were represented by considerable numbers of strains. Of low occurrence were Saccharomyces cerevisiae and Candida blankii. Other yeast species were represented by single or two strains. Total counts of yeast cells per gram dry soil ranged from 1.1x10(3) to 6.6x10(3) in soil samples of rhizosphere areas and from 6.5x10(2) to 5.6x10(3) in soil samples of nonrhizosphere areas. Types of the tested plants affected not only the total counts of yeast cells but also spectra of yeast species. Relationships of age of potato plant, moisture contents of soil samples, and its pH values and total counts of yeast cells were discussed.
Bone Marrow ; Brassica ; Candida ; Candida tropicalis ; Cryptococcus ; Egypt* ; Hydrogen-Ion Concentration ; Kluyveromyces ; Plants ; Rhizosphere ; Saccharomyces cerevisiae ; Soil* ; Solanum tuberosum ; Torulaspora ; Trichosporon ; Vegetables ; Yeasts* ; Zea mays

Ethanol fermentation from Jerusalem artichoke tubers by recombinant Saccharomyces cerevisiae strains expressing the inulinase gene (inu) from Kluyveromyces marxianus was investigated. The inu native and pgk promoters were used to drive the expression of the inu gene, and the inulinase was expressed as an extracellular enzyme. All positive clones (confirmed by PCR) were able to express inulinase as measured by enzyme activity in the culture supernatant, among which two clones HI6/6 and HPI6/3 were selected, and their inulinase activity and ethanol fermentation performance were compared with their wild type. The inulinase activities of 86 and 23.8 U/mL were achieved, which were 4.6-fold and 1.5-fold higher than that of the wild type. Furthermore, ethanol fermentation was carried out with the recombinants and medium containing 200 g/L raw Jerusalem artichoke meal, and ethanol concentrations of 55 g/L and 52 g/L were obtained, with ethanol yields of 0.495 and 0.453, respectively, equivalent to 96.9% and 88.6% of the theoretical value.
Ethanol ; metabolism ; Fermentation ; Glycoside Hydrolases ; genetics ; secretion ; Helianthus ; metabolism ; Kluyveromyces ; genetics ; Metabolic Engineering ; methods ; Plant Tubers ; metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae ; enzymology ; genetics

Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-gamma in spleen CD4+ and CD8+ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-gamma secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future.
Administration, Oral ; Animals ; Antibodies, Viral/*immunology ; B-Lymphocytes/immunology/virology ; Enzyme-Linked Immunosorbent Assay ; *Immunity, Cellular ; *Immunity, Mucosal ; Injections, Subcutaneous ; Kluyveromyces/genetics ; Mice ; Mice, Inbred BALB C ; Porcine respiratory and reproductive syndrome virus/*immunology ; Recombinant Proteins/genetics/immunology ; T-Lymphocytes/immunology/virology ; Viral Envelope Proteins/*genetics/*immunology ; Viral Vaccines/administration & dosage/*pharmacology