Hokkaido University Laboratory of Microbial Physiology
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>> Improving the efficiency of production of valuable substances by fermentation.
>> Searching enzymes working on amino acid analogs and analyzing their function.
>> General Research about the interaction of bacterial flora in intestine and bile acids.
>> Developping a system of genetic engineering of bifidobacterium.

Improving the efficiency of production of valuable substances by fermentation.

In order to improve the efficiency of production of valuable substances by microbial fermentation efficiently and/or to establish new processes by enzyme reaction, we work on the metablism control of microorganism and find out the novel function of microorganism. As the compounds produced by bioprocess, we target on the comparatively high value-added compounds which are amino acids, organic acids, optically-active compounds and so on.

Searching enzymes working on amino acid analogs and analyzing their function.

Amino acid converting enzyme (racemase, aminotranseferase etc...) exists in every living thing including from archaea to higher animals. Though it seems that they have been reserached well enough, we keep searching for the enzymes which convert some special amino acid. For example, we are searching for the enzyme which converts 3-hydroxy-aspartate, an analog of aspartate, and azetidine-2-carboxylic acid, an analog of proline, by a classical method of screening from soil samples to find out a new seed for research.

3-hydroxy aspartic acid which has 2 chiral carbons in a molcule is known as a competitive inhibitor of excitatory glutamic acid transporter of mammal neuron cells. Especially its L-threo form gives storng effect. Finding the enzyme which degrades only its D-threo form leads applications of enzymatic optical separation that only D-threo form is to be eliminated from DL-threo compounds by organic synthesis. (Fig. 1)

We found 2 new enzymes which are L-threo-3-hydroxyaspartate dehydratase(Psudomonas sp. T62 origin) and D-threo-3-hydroxyaspartate dehydratase (Delftia sp. HT23 origin). Nevertheless the substrates of these enzymes are just different in their optical structures, these enzymes did not have any similarities on primary structure (Fig. 2). This insight is highly interesting as a basic finding of enzyme chemistry.

General research about the interaction of bacterial flora in intestine and bile acids.

 In recent years, lactic bacteria and bifidobacteria are regarded to have a great importance about probiotics. To enjoy all of their probiotics effect, they have to be resistant to gastric acid and bile acid which can be a source of the stress in digestive tract. Also they are required to be able to reach intestinal tract alive and grow there. Bile acid is a component of the bile duodenum secretes. It is a surfactant so that it emulsifies lipids and help their digestion and absorption. In addtion to that, because of its strong character of surfactant, it is well-known that it has a strong toxicity. Our lab focuses on this bile acid and research about the interaction between the bacteria in interstine including non-probiotics bacteria and bile acids. It is very exciting fileld that microbiology can do about human health.

* Clarify the system of bile acid resistance of lactic bacteria and bifidobacteria.
We research to get basic knowledge about how lactic bacteria and bifidobacteria grow in intestine resisting against bile acid. We are trying to clarify the phosiological changes of the cell which occur when some lactic bacteria expose to bile acids by phisiolosical biochemical method or genome-based analyze (DNA-array, proteome). The achivement from this research will promote the development of probiotics works better.

* Developping the quick way of analyzing bacterial flora in intestine, FISH-FCM.
Bacteria in human and animal intestine are anaerobic bacteria which dislike oxygen, and it is difficult to culture many of them in vitro. So far, to analyze those bacteria, it was a popular way to culture them but for the reason stated, it is hard to say that the result does not really reflect the truth. We have established the FISH-FCM method which is a combination of the fluorescent in situ hybridization which enable us to detect specific strain and flow cytometry which enable us to count bacteria quickly. Using this method, we research the effect of prebiotics and probiotics on bacteria in intestine.

* Searching for microorganisms which produce secondary bile acid.
Primary bile acid which works on the lipid absorption in your body is transfered to a secondary bile acid by bacterial flora in the large intestine. Some of secondary bile acid are active in carcinogenesis promoter, but others are used as a medicine for bilestones or liver disorders. To clearfy the mechanisms of production of these various secondary bile acid, we isolate an intestine bacterium and research its conversion function.Dealing with obligatory anaerobic bacterium requires high level of skill so that it is tough work to clarify the bile acid metabolism, nevertheless its originality attracts us so much.

Developping a system of genetic engineering of Bifidobacterium.

Bifidobacterium is known as a good bacterium. Though it has various good functions which are nomalization of enteral environment and immunostimulation and more, the mechanism in detail at molcular level is not revealed yet. One of the reason of this is that the methods of gene engineering, construction of a gene defective strain and mutagenesis, are not established yet. We are developping a system of genetic engineering of bifidobacterium and finding out the gene which has a useful function.

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