Mutant cells of Pichia

Details Mutant cells of Pichia Mutant cells of Pichia

1999-08-26

2001-11-13

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Fungi

C435S158000, C435S938000

Reexamination Certificate

active

06316245

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a fermentation process to have a high productivity with a novel mutant of Pichia, more specifically, for preparing erythritol under optimal fermentation conditions for maximum erythritol production by optimizing the environmental conditions of culture such as pH, temperature and by controlling osmotic pressure using a two stage fermentation, in which osmotic pressure is adjusted to a low level during growth phase and to a relatively high level during production phase by adding continuously glucose and NaCl or KCl.
2. Description of Prior Art
Erythritol, a four carbon sugar alcohol, is a naturally occurring substance and is widely distributed in nature. Like most of the other polyols, it is a metabolite or storage compound for seaweeds and mushrooms. Fruits like melons, grapes and pears also contain erythritol. As it is often produced by bacteria, fungi, and yeasts, erythritol also occurs frequently in fermented food systems like wines or beers, and processed vegetables such as soy sauce or the oriental miso bean paste.
Erythritol is a moderately sweet bulking agent with 60 to 70 percent of the sweetness of sucrose in a ten percent solution. Its high negative heat of solution provides the crystalline material with a strong cooling effect. As it has a taste which is very close to sucrose and with no bitter aftertaste, it is ideal to improve the taste of a combination with intense sweeteners like aspartame.
Being a small molecule, erythritol has strong colligative properties, i.e. a strong freezing point depression and boiling point elevation effect as well as a high osmotic pressure. In combination with its low hygroscopicity and viscosity in solution, it is very useful to reduce and control the water activity of foodstuffs.
Erythritol production from its natural sources such as fruits and vegetables is impractical due to the relative small amounts. Erythritol can be chemically produced by reduction of meso-tartarate, oxidation and reduction of 4,6-o-ethylidene-D-glucose, hydrolysis of dealdehyde starch, or addition of hydrogen. Since erythritol production by the chemical methods has been found to be expensive, it is worthwhile to explore an alternative method for the effective production of erythritol using microorganisms.
Erythritol can be produced by microbial methods with the osmophilic yeasts, especially species of the genus Torulopsis, such as
T. magnoliae, T. veratilis,
and
T. candida; Endomzycopsis chodati; Hansenula supelliculsa; Pichia miso; Monilliella tomentosa
var.
pollinis; Trigonopsis variabilis;
Trichosporonoides;
Candida zeylanoides;
and Aureobasidium. Some bacteria such as
Leuconostoc oenos
can also produce erythritol.
Monilliella tomentosa
var.
pollinis
produced erythritol on a medium containing 35.7% glucose with 45.6% yield. Erythritol production using this strain did not apply to industrial scale due to by-products such as glycerol and ribitol. Industrial production of erythritol has been performed by a mutant of Aureobasidium. The mutant was isolated and developed by cooperative study of Nikken Chemical and National Research Institute of Japan. The mutant produced erythritol with 47.6% yield on a medium containing 22.5% glucose and 2 g/L.h volumetric productivity.
It was found that the most polyols producing strains can grow on the conditions of high osmotic pressure such as the high concentration of sugars and salts. This fact suggests that polyols production has the relation to osmotic pressure. Reed et al. reported that glycerol productivity was improved by culturing a glycerol producing strain under the conditions of high osmotic pressure. However, erythritol production by controlling osmotic pressure has not been reported.
Therefore, in this invention, a wild strain of Pichia sp., an isolated strain from the air in 40% sucrose solution at Woosuk University, Chonbuk, Korea was selected to produce erythritol. The wild strain was mutated with NTG (N-methyl-N′-nitro-N-nitroguanidine) treatment. One of mutants has superior properties to the wild strain in erythritol yield from glucose, volumetric productivity, and sugar tolerance. By using the mutant of Pichia sp., the effect of osmotic pressure on erythritol production was investigated and two-stage fermentation, in which osmotic pressure was adjusted to a low level during growth phase and to a relatively high level during production phase, was performed by adding continuously glucose and NaCl or KCl.
SUMMARY OF THE INVENTION
The object of the present invention is to provide novel mutants cells of Pichia sp., which were deposited to Korean Culture Center of Microorganism Department of Feed Engineering, College of Engineering, Yonsei University, Sodaemun-gu, Seoul 120-749, Korea, with accession number KCCM-10129 on Jun. 12, 1998 under Budapest treaty, for preparing erythritol with high productivity.
The other object of the present invention is to provide the optimal fermentation conditions for maximum production of erythritol using mutant cells by controlling following conditions;
i) fermenting glucose medium with mutant cells wherein
a) composition of medium for maximum production of erythritol consists of 10~50 (w/v)% of glucose, 0.5~4.0 (w/v)% of yeast extract, 0.2~1.0 (w/v)% of KH
2
PO
4
, 0.01~0.04 (w/v)% of MgSO
4
.7H
2
O, 0~5 (w/v)% of NaCl, 0~5 (w/v)% of KCl.
b) pH of culture medium is 4.5~5.5.
c) temperature of cultivation is 27~33° C.
d) aeration rate of the medium is 0.5~2.0 volume of air per volume of medium per minute; and
e) agitation speed of the medium is 300~1200 rpm;
ii) removing the mutant cells and other residue from the fermentation medium; and
iii) separating and recovering erythritol from the fermentation medium of step (ii).
The further object of the present invention is to provide a fermentation process, wherein the mutant cells used for fermentation are prepared by cultivating Pichia sp. KCCM-10129 in YM medium containing 0.8~1.2 (w/v)% of glucose, 0.4~0.6 (w/v)% of peptone, 0.2~0.4 (w/v)% of yeast extract, and 0.2~0.4 (w/v)% of malt extract at 27~33° C. for 20~28 h.
The further object of the present invention is to provide a fermentation process, wherein the osmotic pressure is controlled by
i) the osmotic pressure is adjusted to 0.2~0.7 Osm/kg during growth phase and 0.8~1.2 Osm/kg during erythritol production phase; and
ii) feeding solution containing glucose, NaCl or KCl are continuously or intermittently fed into the culture broth during erythritol production phase to be 10~20% of glucose, 0~5% of NaCl or 0~5% of KCl respectively, for the effective production of erythritol.
The further object of the present invention is to provide a method of isolating Pichia sp. mutants comprising the steps of:
i) spreading and culturing a wild type Pichia sp. on yeast-malt (YM) medium containing 0.01% NTG (N-methyl-N′-nitro-N-nitroguanidine);
ii) isolating the produced colonies at least three times on YM medium;
iii) spreading and culturing the colonies of step (ii) on YM medium under UV illumination of 250~270 nm; and
iv) isolating the growing colonies.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns a method of obtaining erythritol with a high yield and a high volumetric productivity in Pichia sp. mutant by controlling osmotic pressure.
The mutant cells used for the present invention are isolated by following method.
Pichia sp. was incubated at 28~32° C. for 24 h on the fermentation agar plate containing 18~22% glucose. A single colony was incubated in a 250-mL flask containing 50 mL of YM broth. It was incubated at 28~32° C. and 220~260 rpm until the optical density of culture broth at 600 nm reached at 1.0. The grown cells were collected by centrifugation at 3000 g for 20 mn and washed with 0.1 M citrate buffer pH 5.5. The collected cells were resuspended in the buffer solution containing 0.01% NTG and incubated at 28~32° C. for 25~35 min. After NTG treatment, the cells were incubated at 28~32° C. for 8~12 h in YM broth and plated on the agar plate conta

Affiliated with

Also associated with

Rating

Mutant cells of Pichia does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Mutant cells of Pichia, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Mutant cells of Pichia will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2583409