• Microbiology and Biotechnology Letters
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• v.19 no.5
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• pp.536-541
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• 1991

To enhance the productivity of fruit flavor compounds from whey by the lactose fermenting yeast, Kluyveromyces lactzs ATCC 8585 was treated with N-methyI-N'-nitro-N-nitrosoguanidine (NTG). After the NTG treatments, a mutant showing resistance to antifungal activity of geraniol, and strong fruity but low yeasty flavor was selected and named as K. lactis 450 K. Flavor compounds from 3-day culture broth were extracted with pentane-dichloromethane (2:l) and the concentrated oleoresins were analyzed by gas chromatography. The mutant strain produced more classes and larger amount of flavor compounds than the parent stlain. Tentatively identified volatile compounds from the culture of the mutant were: terpenes such as myrcenol; alcohols such as cis-3-hexenol, n-hexanol; esters such as ethyl isovalerate, cis- 3-hexenyl n-butyrate, n-amyl-n-hexanoate, phenyl ethyl n-propioate; ketones such as methyl vinyl ketones; other compounds such as vanillin, 3-methylcoumarin.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1471-1480
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• 2010

An extremely thermostable xylanase gene, xynB, from the hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. The response surface methodology (RSM) was also applied to optimize the medium components for the production of XynB secreted by the recombinant K. lactis. The secretion level (102 mg/l) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/l, 16 U/ml) in the original medium (yeast extract, lactose, and peptone; YLP). The secretory efficiency of mature XynB was also improved when using the YLU medium. When the mRNA levels of 13 characterized secretion-related genes in the K. lactis cultured in YLP and YLU were detected using a semiquantitative RT-PCR method, the unfolded protein response (UPR)-related genes, including ero1, hac1, and kar2, were found to be up-regulated in the K. lactis cultured in YLU. Therefore, the nutrient ingredients, especially the nitrogen source, were shown to have a significant influence on the XynB secretory efficiency of the host K. lactis.

• Food Science and Biotechnology
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• v.18 no.6
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• pp.1342-1350
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• 2009

This paper investigates the production and optimization of $\beta$-galactosidase enzyme using synthetic medium by Kluyveromyces lactis NRRL Y-8279 in stirred tank bioreactor. Response surface methodology was used to investigate the effects of fermentation parameters on $\beta$-galactosidase enzyme production. Maximum specific enzyme activity of 4,622.7 U/g was obtained at the optimum levels of process variables (aeration rate 2.21 vvm, agitation speed 173.4 rpm, initial sugar concentration 33.8 g/L, incubation time 24.0 hr). The optimum temperature and pH of the $\beta$-galactosidase enzyme produced under optimized conditions were $37^{\circ}C$ and pH 7.0, respectively. The enzyme was stable over a pH range of 6.0-7.5 and a temperature range of $25-37^{\circ}C$. The $K_m$ and $V_{max}$ values for O-nitrophenol-$\beta$-D-galactopyranoside (ONPG) were 1.20 mM and $1,000\;{\mu}mol/min{\cdot}mg$ protein, respectively. The response surface methodology was found to be useful in optimizing and determining the interactions among process variables in $\beta$-galactosidase enzyme production. Hence, this study fulfills the lack of using mathematical and statistical techniques in optimizing the $\beta$-galactosidase enzyme production in stirred tank bioreactor.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.1977-1988
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• 2015

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

• Biodesign
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• v.7 no.1
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• pp.24-27
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• 2019

Pnc1 converts nicotinamide to nicotinic acid to generate NAD⁺ through the Preiss-Handler pathway that is one of the NAD⁺-salvage pathway. By reducing levels of nicotinamide, an inhibitor of the NAD⁺-dependent histone deacetylase Sir2, yeast Pnc1 contributes gene silencing. In this study, to understand the structural features and molecular mechanism of nicotinamidase Pnc1, we overexpressed, purified, and crystallized the N-terminally His₆-tagged Pnc1 protein from Kluyveromyces lactis and obtained X-ray diffraction data at a resolution of 2.2 Å. The crystals of the K. lactis Pnc1 (KlPnc1) belonged to space group P2₁2₁2₁ with unit cell parameters a=38.5, b=77.3, c=83.3, and α=β=γ= 90°. There is one molecule in the asymmetric unit.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.5
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• pp.337-340
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• 2001

A galactooligosaccharide(GalOS)-producing yeast, OE-20 was selected from forty seven strains of yeast growing in Korean traditional Meju (cooked soybean) and the yeast was tentatively identified as Kluyveromyces maxianus var lactis by its morphology and fermentation profile. A maximum yield of 25.1%(w/w) GalOS, which corresponds to 25.1 g of GalOS per liter, was obtained from the reaction of 100 g per liter of lactose solution at 3$0^{\circ}C$, pH 7.0 for 18 h with an intracellular crude $\beta$-galactosidase. Glucose and galactosidase were found to inhibit GalOS formation. The GalOS that were purified by active carbon and celite 545 column chromatography were supplemented in MRS media and a stimulated growth was observed of some intestinal bacteria. In particular the growth rate of Bifidobacterium infantis in the GalOS containing MRS broth increased up to 12.5% compared to that of the MRS-glucose broth during a 48h incubation period.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.161-167
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• 1998

Whey is by-product from natural cheese manufacturing process. For alcoholic fermentation, the initial lactose content and pH were adjusted to 4.5% and 4.2, respectively. Two strains of yeasts (Kluyveromyces marxianus, Saccharomyces cerevisiae) and seven strains of lactic acid bacteria (Lactobacillus brevis, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus lactis, Leuconostoc cremoris, Lactococcus lactis and Streptococcus thermophilus) were examined for their alcohol production and sensory acceptability. Ethanol content in the whey fermented by lactose-fermenting K. marxianus was 2.8% at 4th day of incubation and that fermented by nonlactose fermenting S. cerevisiae was 0.2%. In case of mixed fermentation with yeasts and tactic acid bacteria (LAB being inoculated at 0 hr), the maximum ethanol production was obtained in the sample inoculated at 16 hr by s. cerevisiae, and in the sample inoculated at 24 hr by K. marxianus. The optimum temperature was $37^{\circ}C$ for alcohol production under static condition. The production of $CO_2$ gas was higher in the whey fermented by K. marxianus (1.88%) than by S. cerevisiae (0.04%). The titratable acidity of the whey gradually increased with fermentation time and its content was 0.39% at 4th day of fermentation by K. marxianus and 0.52% by S. cerevisiae. Among seven strain of latic acid bacteria tested, Lactococcus lactis exerted synergistic effect for acid production with K. marxianus. Therefore, overall results suggestd that the combination of Lactococcus lactis and K. marxianus was best choice in fermenting cheese whey for edible purpose.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2004.06a
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• pp.328-333
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• 2004

• Korean Journal of Food Science and Technology
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• v.29 no.3
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• pp.539-545
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• 1997

Lactobacillus bulgaricus and Kluyveromyces lactis were inoculated to Jangyeob and Jinpum soy milks together after the addition of different amounts of lactose to increase the contents of oligosaccharides, which were compared with single cultured samples. The contents of stachyose, raffinose, sucrose, and glucose of samples without lactose decreased by single culture method, but the oligosaccharides decreased less than in single cultured samples containing of lactose. The oligosaccharides of single cultured samples were equal or decreased compared with soy milks. While those of mixed cultured Jangyeob and Jinpum samples containing 2% lactose for 24 hr incubation increased 125.0% and 118.1%, respectively and those of samples for 36 hr incubation increased 127.0% and 141.0%, respectively, those of mixed cultured samples containing 4% lactose for 24 hr incubation increased 112.5% and 123.0%, respectively and those of samples for 36 hr incubation increased 120% and 135.9%, respectively. Therefore, the oligosaccharides in samples containing 2% lactose were slightly more than in samples containing 4% lactose. Among the cultured methods, oligosaccharides were produced in the largest amounts by the mixed culture for 36 hr. The addition of lactose in soy milks for soy yogurts was effective in the formation of oligosaccharides since the galactose, produced by the hydrolysis of lactose, was thought to be combined with sucrose by the action of ${\beta}-galactosidase$ in yeast.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.979-982
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• 2006

Two knockout vectors, in which the truncated Kluyveromyces lactis URAS gene is flanked by a direct repeat, were developed for Saccharomyces cerevisiae. Each vector was designed to harbor 5'- and 3'-end homology regions for integration. Two knockout fragments were devised to integrate into the correct locus in a complementary manner to disrupt a gene of interest and. concomitantly to make functional Kl URA3 for transfomant selection. The use of dual complementary knockout cassettes was expected to dramatically reduce integration into unwanted loci in the genome. The knockout system developed in this study was successfully used for disruption of the GAL1 gene in S. cerevisiae.