Study on the effects of environmental factors on enzyme activities during growth of Hypsizygus marmoreus

The sensitivity of Hypsizygus marmoreus to environmental factors such as temperature, humidity, illumination and CO2 concentration varies greatly in different growth stages. In this paper, the effects of various environmental factors on the growth and development of H. marmoreus were investigated by measuring the enzyme activities of H. marmoreus at different growth stages under different microenvironment conditions in the mushroom room, so as to confirm the influence mechanism of environmental factors on the growth of H. marmoreus. The results showed that at budding stage xylanase and laccase were found significantly positively correlated with CO2 concentration and light intensity, and dramatically negatively correlated with humidity while carboxymethyl cellulose and manganese peroxidase were markedly positively correlated with humidity, and significantly negatively correlated with CO2 concentration and light intensity. On the other hand, in mature fruit bodies xylanase activity was found significantly positively correlated with CO2 concentration and light intensity, and dramatically negatively correlated with humidity while manganese peroxidase activities were found significantly positively correlated with humidity, and dramatically negatively correlated with light intensity. The activity of β-glucosidase in budding and mature fruiting bodies was markedly negatively correlated with CO2 concentration and significantly positively correlated with humidity.


Introduction
Hypsizygus marmoreus (Peck) H.E. Bigelow belongs to Basidiomycotina, Hymenomycetes, Agaricales, Tricholomatacese, Hypsizigus [1]. Since commercial cultivation of H. marmoreus began in Japan in the 1970s, it has become one of the most important edible fungi varieties in East Asia [2]. Humidity, CO 2 concentration and light time requirements varies with different growth stages in H. marmoreus. Its cultivation environment is a complex non-linear and timevarying system. The effects of various environmental factors on the qualities and yield of
Materials. The cultivation materials of H. marmoreus consist of cottonseed hull, corn flour, corncob, wheat bran, rice bran and sawdust. The fruiting body samples at bud stage and mature stage shown in Fig 1 were cultivated in the mushroom house of Tianjin Hong bin He sheng Agricultural Technology Development Co., Ltd.
Sampling method. Bottle cultivation method was used with a batch size of 60000 bottles. Each bottle of 1100 ml volume consisted of 230±2g and 660±5g dry and wet raw material, respectively. Stepped high pressure sterilization method was used to sterilize, and kept at 121˚C for 90 min. The cultivation period was 121-123 days. A mushroom house was randomly selected to measure environmental parameters and collect samples (Fig 2). There are six tiers in the mushroom house of size (18mx13mx4.8m) at a gap to 55 cm between tiers. Samples of cultivation bottles were collected from 1 st , 3 rd and 6 th tier. Three positions were selected in each tier (Left, right and middle positions) (Fig 3). The first tier is the bottom floor and the sixth tier is the highest tier. Samples were collected from each sampling point and mixed for enzyme activity determination.
Measurement of humidity, CO 2 and light intensity. Humidity was measured by hygrometer at different places of different tiers (Youlide UNI-T), CO 2 was measured by BW GasAlert Micro 5IR, and light intensity was measured by portable light intensity tester (Asensetek ALP-01). The air supply system is designed to supply fresh air through the roof of the mushroom house and extract air from the ground layer (open and close at regular intervals). After the mycelium recovery period, light stimulation was given for 20 days, and the light duration and frequency were gradually extended according to the procedure (Table 1). Only the light intensity was measured in this study, not the illumination time and time interval. The CO 2 concentration in the mushroom house was regulated by a ventilator 35 cm above the ground. The humidity control in different growth stage is controlled by the humidifier, which is turned on at a certain interval and turned off after humidification for a certain period of time. Humidity controlled at 90%±5%.
Enzyme activity determination. Preparation of crude enzyme solutions. Samples collected were weighed 10 g and placed in a mortar. After adding 30 mL ice-water mixture and 3 g quartz sand, the samples were ground into homogenate and transferred into 10 mL centrifugal tube. The samples were centrifuged for 10 min in a cryogenic centrifuge at 5366.4×g, and the supernatant was refrigerated at 4˚C.
Determination of laccase activity. The changes of absorbance in 3 minutes at 420 nm were determined by adding 50μL crude enzyme solution, 150μL acetic acid-sodium acetate buffer  (pH 5.0, 50mmol�L -1 ), 100μL 1mmol�L -1 ABTS into 96-well plate. Three replicates were measured for each sample. The enzyme activity was defined as the amount of enzyme required to oxidize 1 micromol ABTS/min. Activity of laccase was calculated by pressing down formula [9][10][11].
ΔOD is the difference of absorbance between the start and end. V 1 is the total volume of enzymatic reaction mixture. V 2 is the amount of enzymatic solution added. Molar extinction coefficient (ε) of ABTS is 36000 M -1 �cm -1 (420nm in acetic acid-sodium acetate buffer). Δt is the reaction time (3 min).
Determination of carboxymethyl cellulose activity. Citric acid buffer (pH 5.0, final concentration 50mmol�L -1 ) containing 1.5 mL of 1% CMC-Na as reaction substrate and 0.5 mL crude enzyme solution was placed in 25 mL plugged test tube. 3 mL DNS (3, 5-dinitrosalicylic acid) was added immediately after 30 minutes incubation at 50˚C in water bath, boiled for 10 minutes, and cooled under running water to room temperature. The volume is fixed to 25 mL by distilled water. After shaking, 200 μL was taken out in 96-well plate. The absorbance at 540 nm was measured by microplate reader (PerkinElmer Enspire). The crude enzyme solution boiled for 10 minutes was used as the control group. Enzyme activity was defined as the amount of enzyme required to catalyze the substrate to produce 1 μmol glucose per minute as a unit of enzyme activity [10,12,13]. The difference between the measured absorbance and the control was substituted into the following formula to obtain the enzyme activity.
Among them, ΔOD value is the difference between the absorbance value measured in treatment and that measured in control group, which is substituted into the standard curve equation to obtain the concentration of glucose. V 0 is the volume after final dilution (25mL). V 1 is the total volume of the enzyme reaction mixture (the total volume of the enzyme solution and the reaction substrate). M glucose is the molar mass of glucose used in the experiment, Δt is the time of enzymatic reaction (30min), n is the intercept, m is the slope and V 2 is the volume of enzymatic liquids added. V 1 divided by V 2 is the dilution multiple.
Determination of xylanase activity. A volume of 0.1 mL crude enzyme solution was added into 25 mL plugged test tube with 0.9 mL of 1% birchwood xylan (0.05mol�L -1 citric acid buffer, pH 5.0) as reaction substrate. The reaction system was incubated at 50˚C for 30 minutes in a water bath, then removed and immediately added 3 mL DNS reagent, and boiled for 10 minutes. After cooling to room temperature under water, the volume is fixed to 25 mL by distilled water. The absorbance value at 540 nm was determined by microplate reader (PerkinElmer Enspire), using 200 μL in a 96-well enzyme-labeling plate. The inactivated crude enzyme solution boiled for 10 minutes beforehand was used as control group. The unit of enzymatic activity is defined as the amount of enzymes required to decompose 1 μmol xylan per minute [13,14].
M xylose is the relative molecular weight of xylose used in the formula, and the other items are the same as the formula for calculating the activity of CMCase.
Determination of manganese peroxidase activity. A volume of 2 mL of 50 mmol�L -1 lactic acid-sodium lactate buffer solution (pH 4.5), 0.1 mL of 1.6 mmol�L -1 MnSO 4 solution and 0.8 mL crude enzyme solution were added to 10 mL centrifugal tube. The reaction was initiated by adding 0.1 ml 1.6 mmol�L -1 H 2 O 2 at room temperature. After 3 minutes, the tube solution was quickly put into ice water. 200 μL reaction solutions were added into 96-well plate and the absorbance at 240 nm was measured by microplate reader (PerkinElmer Enspire). Enzyme activity was defined as the amount of enzyme required to oxidize 1μmol Mn 2+ per minute. The enzymatic activity of MnP was calculated according to the following formula. The meanings of each item in the formula were the same as those in the laccase formula. Molar extinction coefficient of Mn 3+ -lactate is 7800M -1 �cm -1 at 240nm [14,15].
Determination of β-glucosidase. A volume of 1.5 mL of 3% salicin solution in 50mmol�L -1 citric acid buffer (pH 5.0) as reaction substrate and 1 mL crude enzyme solution were added to 25 mL plugged test tube, bathed in water at 50˚C for 30 minutes. The above samples were added with 3 mL DNS, boiled for 10 minutes, cooled to room temperature in flowing water, added distilled water to the scale line, shaken well, and then added 200 μL sample into 96-well plate to determine its absorbance at 540 nm by microplate reader (PerkinElmer Enspire). The crude enzyme solution boiled and inactivated for 10 minutes served as control [16]. The enzymatic activity of β-glucosidase was calculated according to the following formula. The meanings of each item in the formula were the same as those in the CMCase.
Preparation of standard curves. (1) Standard curve preparation of xylose The standard xylose of 0.1 g was dissolved in 50 mL citric acid-sodium citrate buffer solution. After stirring and dissolving, it was transferred into 100 mL volumetric bottle. The standard xylose solution of 1 mg�mL -1 was prepared by adding citric acid-sodium citrate buffer. Ten clean plugged test tubes were added with 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL and 0.8 mL standard solution respectively. Distilled water was added to make volume of 1 mL, followed by addition of 3 mL DNS reagent, and boiled for 10 minutes. Distilled water was added to make volume 25 mL after cooling to room temperature. 200 uL was added to 96-well enzyme plate to determine its absorbance at 540 nm by microplate reader (PerkinElmer Enspire). Standard curve was made with absorbance value as ordinate and xylose concentration as abscissa.
Standard curve of xylose linear regression was performed with absorbance (y) as ordinate and xylose content (x) as abscissa. The regression equation was obtained as follows: y = 0.2242x + 0.0135 (R 2 = 0.9988, n = 8). The linear relationship between xylose and absorbance was good in the range of 0.1 mg�mL -1~0 .8 mg�mL -1 .
(2) Standard curve preparation of glucose: The standard curve of glucose was prepared in the same way as that of the above steps. Xylose was replaced by glucose.
Standard curve of glucose linear regression was performed with absorbance (y) as ordinate and glucose content (x) as abscissa. The regression equation was obtained as follows: y = 0.305x-0.0064 (R 2 = 0.9991, n = 7). The linear relationship between glucose and absorbance was good in the range of 0.2 mg�mL -1~1 .2 mg�mL -1

Statistical analysis
SPSS Statistical 17.0 statistical software was used for data processing. Variance analysis was used for comparison between groups at P < 0.05. Bivariate correlation was determined for analysis.

Changes of environmental factors
It can be seen from Fig 4A that from 1 st to 6 th tiers, the concentration of CO 2 was on the rise, and 6 th tier was the strongest. The reason for the rising trend of CO 2 concentration is may be due to the ventilator of the mushroom house on 1 st layer of the mushroom house. Humidity decreased gradually with the increase of tiers, and 6 th tier was slightly lower than 1 st and 3 rd tier (see Fig 4B). The intensity of light in 3 rd layer was the strongest, while that in 6 th layer was slightly higher than that in 1 st tier (see Fig 4C). Statistical analysis showed that there were significant differences in CO 2 concentration and light intensity between tiers 1 st , 3 rd and 6 th (P < 0.01), and there were significant differences in humidity between tiers1 st , 3 rd and 6 th (P < 0.05).

Xylanase activity assay
The data showed that the xylanase activity in fruiting bodies of H. marmoreus increased with the increase of sampling tiers. The highest xylanase activity was found in fruiting bodies of tier 6 i.e. in mature fruiting bodies 314.0 U/L and in buds 281.9 U/L (see Fig 5). Statistical analysis showed that there were significant differences in xylanase activity between 1 st , 3 rd and 6 th tier in budding and mature fruiting bodies (P<0.01).

Carboxymethyl cellulase assay
The enzyme activity in budding fruiting bodies of 1 st tier was the highest (14.3 U/L), while that of 6 th tier was the lowest (6.4 U/L) (see Fig 6A), yet, that in the mature fruiting bodies of 6 th tier (9.8U/L) was highest (see Fig 6B). Significant differences in CMCase activity between 1 st , 3 rd and 6 th tier in the budded and mature fruiting bodies could be observed (P<0.01).

Laccase assay
Laccase activity in budding fruiting bodies of H. marmoreus of 6 th tier was the highest (40.4 U/ L) (see Fig 7A), while that of 1 st tier was the lowest (19.2 U/L) yet, that in the mature fruiting bodies of 6 th tier (77.7U/L) was lowest (see Fig 7B). Laccase activity showed significant variation in budding and mature fruiting bodies of 1 st , 3 rd and 6 th tier (P<0.01).

Manganese peroxidase assay
The maximum MnP activity (66.2 U/L) was observed in budding fruiting bodies of 1 st tier (see Fig 8A), while that of 6 th tier was the lowest (54.1 U/L). In matured fruiting bodies, the MnP activity was observed to be the highest in 1 st tier (6.3U/L) (see Fig 8B). It was also observed that the MnP activity in the mature fruiting body of 1 st tier significantly differed from that in 3 rd tier and 6 th tier (P < 0.01) while no significant difference was observed between 3 rd and 6 th tier. Moreover, the MnP activity of the mature fruiting bodies from H. marmoreus was very low with respect to budding fruiting bodies. and 9B). The enzyme activity differed significantly among 1 st , 3 rd and 6 th tiers in the budded fruiting bodies (P<0.05; P<0.01) whereas significant differences were observed in mature fruiting bodies of 1 st and 6 th tier, and between 3 rd tier and 6 th tier (P<0.01). There was no significant difference in the activity of β-glucosidase between mature fruiting bodies of 1 st and 3 rd tier.

Correlation analysis of CO 2 and five enzyme activities
Correlation analysis showed that there was a significant positive correlation between xylanase activity and CO 2 concentration in the budding and mature fruiting bodies (P<0.01). CMCase activity and MnP activity in budding fruiting bodies were significantly negatively correlated with CO 2 concentration (P<0.01). Laccase activity was markedly positively correlated with CO 2 concentration in the budding fruiting bodies (P<0.01). The activity of β-glucosidase in the budding and in the mature fruiting bodies was significantly negatively correlated with CO 2 concentration (P<0.01). CMCase activity of mature fruiting bodies was positively correlated with CO 2 concentration. Laccase and MnP activities of mature fruiting bodies were negatively correlated with CO 2 concentration (see Table 2).

Correlation analysis of humidity and five enzyme activities
Correlation analysis showed that there was a significant negative correlation between xylanase activity and humidity in the budding and mature fruiting bodies (P<0.01). MnP activity and β-glucosidase activity of budding and mature fruiting bodies were dramatically positively correlated with humidity (P<0.01, P<0.05). CMCase activity in the budding fruiting bodies was significantly positively correlated with humidity (P<0.01) while negatively correlated in mature fruiting bodies. Laccase activity was significantly negatively correlated with humidity in the budding fruiting bodies (P<0.01), while positively correlated in mature fruiting bodies (see Table 3).

Correlation analysis of light intensity and five enzymes activities
Significant positive correlation was observed between xylanase activity and light intensity in the budding and mature fruiting bodies (P<0.01) (see Table 4). CMCase activity in the budding fruiting bodies was markedly negatively correlated with light intensity (P<0.05), while was not markedly negatively correlated in mature fruiting bodies. Laccase activity in the budding fruiting bodies was remarkably positively correlated with light intensity (P<0.05), while positively correlated in mature fruiting bodies. The MnP activity in the budding and mature fruiting bodies was significantly negatively correlated with light intensity (P<0.01). βglucosidase activity in the budding and mature fruiting bodies was negatively correlated with light intensity (P<0.05). (see Table 4).

Discussion
The main environmental factors of mushroom growth and development include temperature, humidity, luminosity and the air composition of the surrounding matrix, such as the concentration of oxygen and carbon dioxide [4,17]. Mushroom absorbs its nutrients from the substrate (grasses, wood and agricultural residues) through its mycelium, obtaining substances necessary for its development, such as carbon, nitrogen, vitamins and minerals [18]. The utilization of different types of substrate by fungus (mushroom) will depend on its capacity to secrete enzymes such as oxidative (ligninase, laccase, MnP) and hydrolytic (cellulase, xylanase and tannase) enzymes, which are involved in utilizing lignocellulosic substrates [4,19]. In this research, it was found that the enzymatic activity of H. marmoreus in the growth process was related to the light intensity, humidity and CO 2 concentration in the growth environment. It is important to keep the CO 2 concentration in a certain range during the darkened spawn-running and fruiting phases. If the growth environment contains high concentration of CO 2 , the mushroom with thick and short stipe pileus will be produced in the growth process [6,18]. The appearance of mushroom is closely related to its growth process, and the changes of enzyme activities related to the growth and development of mushroom are the deep-seated reasons.
Xylanase plays a role in decomposing hemicellulose (xylan) in cultivation material during the growth of mushroom [20,21], helping fungal mycelium to utilize nutrients in cultivation    material and make fruiting body grow. The results showed that CO 2 and light intensity have a positive effect on xylanase activity in a certain concentration range, while relative humidity has a negative effect. It could be inferred from the present study that the xylanase activity of fruiting bodies at ripening stage was significantly higher than that at budding stage, suggesting that xylanase accumulated in fruiting bodies during the development of fruiting bodies. The result is consistent with the literature report from Flammulina velutipes [21]. This indicated that xylanase played an important role in the growth and development of mushrooms in general and H. marmoreus in particular.
CMCase converts cellulose to cellobiose and glucose. Cellobiose and other cello-oligosaccharides could be hydrolyzed by extracellular or intracellular β-glucosidase after cellobiose is transported into the cell by cellodextrin transporters [22,23]. Results obtained in this study indicated that light intensity have a negative effect on CMCase and β-glucosidase. At budding stage, carbon dioxide had a negative effect on CMCase activity while relative humidity had a positive effect. Carbon dioxide had a negative effect on β-glucosidase while relative humidity had a positive effect. Therefore, relatively low CO 2 concentration, light intensity and relatively high relative humidity may be conducive for the accumulation of CMCase in the budding stage of H. marmoreus. In addition, compared with xylanase, CMCase, laccase and MnP, the activity of β-glucosidase in the fruiting body of H. marmoreus was lower during the growth and development. It could also be inferred that the activity of β-glucosidase in H. marmoreus was lower than others mushroom [21].
In fungi, laccase is known as a ligninolytic enzyme and appears to be involved in fruiting body formation [23]. The lignin in the culture medium was hydrolyzed by laccase and MnP. In this research, the results indicated that laccase was gradually accumulated or increased in the fruiting body of H. marmoreus with the growth phases. At budding stage, Carbon dioxide and light intensity had a positive effect while relative humidity had a negative effect on laccase. Comparing the activity of MnP in mature fruiting bodies with that in budding fruiting bodies of H. marmoreus, the activity of MnP in mature fruiting bodies was too low. The reason may be that the fruiting bodies of H. marmoreus do not need MnP to provide nutrition at this physiological stage of fruiting. During the growth and development of H. marmoreus, carbon dioxide and light intensity had a negative effect on MnP while relative humidity had a positive effect. In budding stage, the CO 2 , relative humidity and light intensity had an inconsistent correlation. This reason needs to be further studied.
In this study, it can be seen that the enzyme activity differed depending on different environmental conditions tested in this study. The reason for these changes may be attributed to the influence of environmental factors on cell respiration during the growth and development, which affected the expression of these enzymes. In particular, because the ventilation fan in the mushroom house is only 35cm above the ground, the air flow, CO 2 and O 2 concentrations are  different at different heights and tiers of the mushroom rack resulting changes in enzyme activities. Therefore, it is very important to regulate and control the environmental factors CO 2 , relative humidity and light intensity in the cultivation of H. marmoreus, which will directly affect the growth and development and yield of H. marmoreus.