Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Isomerase
Reexamination Certificate
2001-09-24
2004-10-05
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Isomerase
C435S100000, C435S320100, C435S252900, C435S252330, C536S023200
Reexamination Certificate
active
06800474
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a trehalose-producing microorganism and a process for producing trehalose. It also relates to a novel trehalose synthase protein, a trehalose synthase gene, recombinant plasmids carrying said trehalose synthase gene, and transformed microorganisms with said recombinant plasmids.
2. Description of the Prior Art
Trehalose is a non-reducing disaccharide, two saccharides of which are linked by &agr;-1,1 bond: &agr;-D-glucopyranosyl-&agr;-D-glucopyranoside. It has wide application in medicines, foods, and cosmetics. However, its utilization has been greatly restricted because its production to date has been inefficient and expensive.
Japanese Laid-open Patent Nos. Hei5-91890 and Hei6-145186 disclose methods for extracting trehalose from yeasts. There are several methods for isolating trehalose from fermented microorganism cultures, such as Arthrobacter (T. Suzuki, Agric. Biol. Chem., 33(2), 1969), Nocardia (Japanese Laid-open Patent No. Sho 50-154485), Micrococcus (Japanese Laid-open Patent No. Hei6-319578), amino acid-fermenting yeast, Brevibacterium (Japanese Laid-open Patent No. Hei5-211882), and yeast (Yoshikwa, etc., Biosci. Biotech. Biochem., 1994, 58, 1226-12300). Additionally, a method for producing trehalose by using recombinant plants including bacterial genes capable of converting glucose into trehalose is described in M. Scher, Food Processing, April, 95-96, 1993. Japanese Laid-open Patent No. 83-216695 discloses a method for converting maltose into trehalose by using maltose phosphorylase and trehalose phosphorylase. However, these methods are not effective, because their procedures are complicated and their yields are low.
Several enzymatic methods have been published recently. Japanese Laid-open Patent No. Hei7-143876 and EPO 628630 A2 discloses a two-step enzymatic conversion method in which starch is converted into trehalose by maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase. Japanses Laid-open Patent No. Hei7-170977 and Korean Laid-open Patent No.95-3444 disclose one-step enzymatic conversion methods in which maltose is directly converted into trehalose by trehalose synthase. However, there is still a need to increase the titer of the trehalose synthase enzyme so that production of trehalose from maltose becomes more efficient in yield and cost.
We have invested much effort over the last several years in isolating microorganisms able to convert maltose into trehalose from soil. We have successfully screened a novel strain which highly expresses trehalose and, unexpectedly, generates no byproducts, unlike all known microorganisms. Its morphological and physiological characteristics identify it as a novel
Pseudomonas stutzeri
strain. This strain has been designated as
Pseudomonas stutzeri
CJ38.
We isolated a trehalose synthase gene from chromosomes of
Pseudomonas stutzeri
CJ38 and determined its nucleotide sequence by cloning it into known vector pUC 18 with restriction enzyme Sau3AI. In addition, we isolated a trehalose synthase protein from
Pseudomonas stutzeri
CJ38 and determined its amino acid sequence using standard methods. It was found that these sequences are apparently different from the sequences of the trehalose synthase gene and all proteins known hitherto. This invention was achieved by constructing recombinant plasmnids carrying the trehalose synthase gene so that the trehalose synthase enzyme encoded in said gene can be expressed in large amounts.
SUMMARY OF THE INVENTION
The present invention provides a novel microorganism,
Pseudomonas stutzeri
CJ38, that produces trehalose from maltose. This strain was deposited at the Korea Culture Center of Microorganisms, Seoul, Korea, as the accession number KCCM 10150 on Feb. 12, 1999 under the Budapest Treaty. This strain is very valuable as it does not generate byproducts such as glucose when converts maltose into trehalose.
The present invention also provides SEQ ID NO: 2, which is a novel trehalose synthase protein with the following amino acid sequence:
Met Ser Ile Pro Asp Asn Thr Tyr Ile Glu Trp Leu Val Ser Gln
5 10 15
Ser Met Leu His Ala Ala Arg Glu Arg Ser Arg His Tyr Ala Gly
20 25 30
Gln Ala Arg Leu Trp Gln Arg Pro Try Ala Gln Ala Arg Pro Arg
35 40 45
Asp Ala Ser Ala Ile Ala Ser Val Trp Phe Thr Ala Tyr Pro Ala
50 55 60
Ala Ile Ile Thr Pro Glu Gly Gly Thr Val Leu Glu Ala Leu Gly
65 70 75
Asp Asp Arg Leu Trp Ser Ala Leu Ser Glu Leu Gly Val Gln Gly
80 85 90
Ile His Asn Gly Pro Met Lys Arg Ser Gly Gly Leu Arg Gly Arg
&ems
Chung Sung Oh
Jeon Yeong Joong
Kim Chang Gyeom
Kwon Sang Ho
Lee Jin Ho
Birch & Stewart Kolasch & Birch, LLP
Cheil Jedang Corporation
Patterson Jr. Charles L.
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