Pectin is a complex polysaccharide which is present primarily in the intercellular layers of plants, and which functions as an adhesive substance among cells. Pectin-degrading enzymes are generally referred to as pectinases.
Pectinases are used not only in food processing such as refining of fruit juices, but they are also used in fields such as the refining of fibers (e.g., Patent Reference 1), and in addition, attempts have been made to use them in pharmaceuticals (e.g., Patent Reference 2). With this diversification in demand, novel pectinases have been sought which have a variety of optimal pH and temperature characteristics, depending on the purpose for their use.    Patent Reference 1: Japanese Patent Application Kokai Publication No. 2009-35853    Patent Reference 2: Japanese Patent Application Kokai Publication No. H09-315999
Among the pectinases, some types of enzymes break down plant fibers by reducing the molecular weight, fragmenting, or dissolving insoluble pectin (protopectin) present in the intercellular layers of plants, and also exhibit activity which frees single cells from each other (known as maceration or protopectinase activity, but referred to in this Specification as “maceration activity”). On the other hand, there are also enzymes which possess pectin-degrading activity but which do not possess maceration activity, and even if they exhibit both types of activity, the enzymes may have a different optimal pH for each respective activity.
Enzymes possessing maceration activity are necessary, along with cellulase, for isolating protoplasts from plants and indispensible for fundamental botanical research. It has been reported that enzymes such as polygalacturonase and polymethylgalacturonase which hydrolyze the α-1,4 bond of polygalacturonic acid, and lyases such as pectin lyase and pectate lyase which cleave these bonds with a β-elimination reaction, also possess some type of maceration activity.
Soft plant rot is known to occur as a softening plant disease in which plant tissues are degraded by these maceration enzymes which are produced by microorganisms. Among these, pectate lyase, derived from the genus Erwinia, is well known (Florence Tardy, William Nasser, Janine Robert-Baudouy, and Nicole Hugouvieux-Cotte-Pattat, J. Bacteriol., 179, 2503-2511, April 1997).
Detailed research has been conducted regarding Erwinia-derived pectate lyases and on the relationship between their maceration activity and pectinase activity. For example, Pectate lyase C (PelC), derived from Erwinia  chrysanthemi, is a pectinase which has pectate lyase activity and maceration activity. Comparing experiments on single or double amino acid changes of PelC in various PelC produced using a method of site-specific elicitation of mutations described later have shown that these enzymes possess their respective intrinsic pectate lyase activity and maceration activity which is proportional to the pectate lyase activity (Nobuhiro Kita, Carol M. Boyd, Michael R. Garrett, Frances Jurnak, and Noel T. Keen, J. Biol. Chem., 271, 26529-26535, Oct. 25, 1996). It should be noted that all of the pectate lyases appearing in the article had an optimal pH on the alkaline side, regardless of the microorganism from which they were derived.
Maceration enzyme preparations for the industrial use are primarily derived from microorganisms such as filamentous fungi belonging to the genus Aspergillus and Rhizopus, and microorganisms in the genus Bacillus. For example, Pectolyase Y-23 (Registered trademark of Kyowa Hakko Co., Ltd.) is an enzyme preparation used in research which contains, as primary enzymes, pectin lyases and polygalacturonases derived from Aspergillus japonicum, and is well known for its potent maceration activity. The optimal pH of Pectolyase Y-23 is 5.5. Macerozyme R10 (Registered trademark of Yakult Pharmaceutical Co.) is also widely used. Macerozyme R10 is a pectinase preparation which has polygalacturonase activity and an optimal pH of 5.0. In these fields of basic research, there is a desire for novel maceration enzymes with optimal pH values which are tailored to the characteristics of plants.
These maceration enzymes are used not only in basic research on plants, but efforts have also been made to use them in the manufacture of food products by single cell formation of plants (e.g., Patent Reference 3). In comparison with extraction using alcohols and hot-water extraction in manufacturing processes for botanical functional raw materials, single cell formation of plants using maceration enzymes is a milder process which is implemented at normal temperatures and pressures. It therefore does little harm to the environment, and has the advantages that the natural plant components are not readily modified and the taste of the resulting food is enhanced, because of a masking effect on intracellular components by the cell wall and the cell membrane.    Patent Reference 3: Japanese Patent Application Kokai Publication No. H09-75026
In the conventional mass production of food products by single cell formation of plants using maceration enzymes, there was the problem of low yields from plant raw materials, because large quantities of undissociated tissue remained after enzymatic reactions. It was thus necessary to carry out prolonged enzymatic treatment or to use physical treatment such as stirring or beating, in order to raise yields.
However, when prolonged enzymatic treatment was carried out for 1-2 days under mild conditions such as normal temperature, for example, harmful microorganisms remaining in the raw materials grew during the enzymatic reaction, and it was difficult to separate the plant cell product from the harmful microorganisms with a filter, so it was necessary to sterilize by heating the enzymatic reaction solution to about 100° C. If enzymatic treatment is carried out at about 40° C. over a short period of 3-5 hours, some degree of vigorous stirring or beating was needed (for example, in Patent Reference 4, in which is described a mechanism provided with a spiral stirring member held rotatable around an axis of rotation in a tube forming an enzymatic treatment unit). These treatments significantly destroy or damage cell membranes formed from lipid double membranes, making it difficult to retain the natural components, in particular the water-soluble components, within the cell.    Patent Reference 4: Japanese Patent No. 3,986,541
In view of the current state and problems of the prior art, the present invention has as its object to provide a novel pectin lyase, a pectin lyase gene, an enzyme preparation containing the pectin lyase, and a method for producing single cells of plant tissue, which is capable of improving yields from plant raw materials when obtaining single cells from plant tissue by using a maceration enzyme, and when producing food by single cell formation of plants, without needing heat treatment for sterilization which was necessary when carrying out prolonged enzymatic treatment, or physical treatment such as stirring or beating.