Since 1970, several novel microbial enzymes producing specific oligosaccharides have been discovered. Using these new enzymes, it is now possible to produce on an industrial scale various oligosaccharides such as glycosylsucrose, fructooligosaccharides, maltooligosaccharides, isomaltooligosaccharides (branched-oligosaccharides), galactooligosaccharides, xylooligosaccharides, palatinose (isomaltulose), lactosucrose and so on. Recent developments in industrial enzymology have made possible a series of new starch oligosaccharides such as β-1, 6 linked gentiooligosaccharides, α, α-1, 1 linked trehalose, α-1, 3 linked nigerooligosaccharides and branched-cyclodextrins. Some brand-new sweeteners including trehalose and nigerooligosaccharides are being developed as food ingredients with physiologically unique functions such as superoxide dismutase-like activity and immunological activity. Also, soybean oligosaccharides containing raffinose, stachyose and other oligosaccharides mentioned above are now used in beverages, confectionery, bakery products, yogurts, daily products and infant milk. At present, the market for these oligosaccharides in Japan is expected to be more than 20 billion yen/year.
Oligosaccharides on the cell surface that play important roles in the biochemical recognition processes are potential pharmaceuticals. However it has been very difficult to synthesize these oligosaccharide structures. Recent identification of the genes for glycosyltransferases in bacteria and expression of the genes in Escherichia coli enabled the mass production of glycosyltransferases for enzymatic synthesis of oligosaccharides. Together with the improvements in the supply of sugar nucleotides, substrates of glycosyltransferases, using bacterial functions in genetically engineered bacteria, it became possible to produce a large amount of oligosaccharides.
A new functional food ingredient, phosphoryl oligosaccharides (POs), was developed from potato starch hydrolysate. POs was composed of two fractions, PO-1 and PO-2. Fraction PO-1 was the main fraction, and it was composed of maltotriose, maltotetraose, and maltopentaose to which one phosphoryl group was attached. Fraction PO-2 was predominantly composed of maltopentaose and maltohexaose to which at least two phosphoryl groups were attached. POs had the ability to form a soluble complex with calcium and had an inhibitory effect on the formation of calcium-phosphate precipitate. The effect of fraction PO-2 was stronger than that of fraction PO-1. Based on the function of the POs, described above, we applied the POs as a food ingredient. Phosphoryl oligosaccharides of calcium (POs-Ca) were an advantageous food ingredient as a soluble calcium source. From the point of view of preventing dental caries, POs can not be fermented by cariogenic microorganisms, mutans streptococci, and reduce the plaque pH fall in vitro. Moreover, POs-Ca effectively enhanced remineralization of enamel and dentin lesion.
We searched for a substance, which can make an inclusion complex with the hydrophobic low molecule that cannot form a complex with cyclodextrin. We discovered the cyclic sugar of a cyclodextran generated from dextran. There are three kinds of cyclodextrans that had been identified so far. These are cycloisomalto-heptaose, -octaose, and -nonaose. They are produced from dextran by the action of cyclodextran synthetase. Cyclodextran synthetase and byproduct degradation enzyme are required for the production of cyclodextran. We could achieve the mass production of these enzymes by use of gene modification technology. Moreover, we are looking further into increasing the production efficiency of cyclodextran. On the other hand, we also checked the usefulness of this substance, and confirmed that cyclodextran had the following functions. Cyclodextran and its derivatives have functions, such as a cariostatic action, a hydrophobic substance solubilized action, an anti-HIV action, and an anti-ulcer action. Therefore, cyclodextran is a multi functional cyclic oligosaccharide like cyclodextrin, and we particularly anticipate the use of its cariostatic action.
We have developed large-scale production of alkaline cellulases, alkaline proteases, and alkaline α-amylases, and the enzymes have been incorporated into heavy-duty compact detergents and/or bleaches. The problem with traditional detergent enzymes is that they are seriously inactivated by chemical oxidants and chelating reagents, and these enzymes are thermally unstable, especially when they are used in automatic dishwashers. We have found an alkaline liquefying α-amylase AmyK (formerly designated LAMY) from alkaliphilic Bacillus sp. strain KSM-1378. AmyK is highly active at alkaline pH, compared with other industrial α-amylases reported so far, and resistant to various surfactants. However, AmyK is less thermostable than the Bacillus licheniformis α-amylase (BLA), therefore, improvement in the thermostability of AmyK is desirable for use at high temperatures under alkaline conditions in automatic dishwashers. Moreover, AmyK and other Bacillus α-amylases are inactivated by chemical oxidants. We tried to improve the oxidative stability of AmyK by replacing a Met residue with non-oxidizable amino acids as in the case of alkaline proteases that acquired oxidative stability by site-directed mutagenesis. In this article, we describe the properties and deduced amino acid sequence of AmyK, and improvement in thermostability and oxidative stability of the enzyme by site-directed mutagenesis.
Modifying the properties of enzymes and proteins has become a relatively routine practice in both the academic and the bioindustrial sectors since the first introduction of the concept in the late 70's to early 80s. The original tools of site-specific modification and three dimensional structural analysis based design have expanded to include regio-targeted mutagenesis, homology based recruitment of amino acid residues and in vivo and in vitro recombination of genes. The advent of PCR based methodologies for gene cloning and manipulation have greatly increased the ability to design altered proteins yet at the same time have made the process less time-consuming as well. Add to this the ability to introduce thousands, if not millions, of variations into existing proteins, the rapid development of robotic based handling and screening systems and one has the basis for a revolutionary excursion into protein design. Yet in spite of all this capability, the successful outcome of a commercial protein engineering project remains based in three quite simple fundamental principles. First, one must have a clear understanding of the real world application conditions under which an enzyme is used and how these conditions can define what actually constitutes an improvement in the performance of the enzyme. Second, one must be able to quantitate what the improved property can deliver in terms of process value to the customer. And third, but clearly not the least, one must understand the key commercial features which can expedite or hinder the actual commercialization of a new product. These three questions can be restated in non-scientific terms as: what does the customer need?; what value will this bring to the customer?; and how can the product be delivered to the customer so that value is recognized by all? These three questions will form the basis of the following discussion on engineering improved amylolytic enzymes to the starch industry.