Zymolyase (lyticase) is a very important enzyme in microbiology, isolated from Arthrobacter luteus
. Zymolyase actively degrades yeast cell walls [1
]. The principal ingredient in zymolyase is β-1,3-glucan laminaripentaohydrolase, which hydrolyzes glucose polymers linked by β-1,3-bonds, producing laminaripentaose [1
]. Zymolyase also includes protease and mannase. These enzymes strongly degrade yeast cell walls. By changing pH or temperature, cell wall lysis and dehydration of glucan are easily achievable. For this reason, zymolyase is frequently used for the preparation of nucleic acids or soluble proteins from yeast cells, and many commercial kits involving such enzyme treatment have been released. However, digestion must be performed by incubating at moderate temperature for a given length of time, thereby having potential to change the yeast reaction. Forsburg [4
] introduced the RNA preparation method, involving 6 U/ml of enzyme at 37 °C for 30 min, and Klassen [5
] introduced 8 U/ml of enzyme over 1 h. These conditions may be considered moderate, and many commercial kits use higher concentrations (10–500 U/ml) and short-term incubation (15–30 min) at 37 °C. This is a strategy to avoid cell response resulting from enzymatic treatment.
Recently, there have been a number of studies on the influence of zymolyase. De Groot [6
], for example, measured cell density under 0.1 U/ml concentration at 37 °C for 1–4 h, and also reported the induction of cell wall genes and mitogen-activated protein kinase (MAPK)-related genes under 0.26 U/ml of zymolyase at 28 °C for 1–4 h, or 0.8 U/ml at 24 °C for 3 h, or 5 U/ml 24 °C for 2 h [7
]. Bermejo [10
] also investigated the influence of mutation on the MAPK pathway receptor under 0.8 U/ml at 24 °C for 3 h. Since these studies utilize long-term incubation, from 1 to 4 h, at low concentrations of digestion enzyme, gene expression data show significant changes. However, the reaction times of digestion reported by these studies are relatively longer than under RNA preparation conditions. If gene expression changes also occur during short-term reaction times such as those used for RNA preparation, then RNA preparation using the digestion method is confronted with a critical problem. Because the concentrations of digestive enzyme in RNA preparations are generally higher than in enzymatic studies, it is thought that stress is generated.
Moreover, Bermejo [11
] reported that SLT2
, a protein kinase of the cell integrity pathway on the MAPK cascade, was induced by 0.4 U/ml of zymolyase treatment at 24 °C for 15 min, and induction was maximized for 2 h. Generally, the incubation time for zymolyase treatment is 15–30 min; therefore, this report suggests that unintended gene expressions will be caused by zymolyase treatment for RNA preparation. What then is the influence of such treatment? Many recent protocols do not use enzyme treatment because it is known to cause complications. However, the concentrations and incubation times used in previous research do not correspond to each other, and therefore information regarding the influence of the enzymatic process in RNA preparation is insufficient and complicates our understanding of this method. To address this problem, we investigated the true influence of the enzyme method on RNA preparation.
There are several methods of yeast RNA preparation. The hot phenol method, the glass beads method, and the enzyme digestion method are generally used. Since the treatment time is the shortest in the glass bead method, the possibility of changing the gene expression is thought to be very low. Phenol denatures cell proteins and enzymes, so the hot phenol method is expected to suspend cell activity promptly. We used a commercial kit able to exploit the advantages of both methods. The influence of enzyme treatment was detected as gene expression changes using a DNA microarray.