Current Microbiology

Enhanced Cellulase Production of the Trichoderma viride

mutated by microwave and ultraviolet

Journal: Current Microbiology

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Manuscript ID: CMB-09-01-0049

Manuscript Type: Original Manuscripts

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Date Submitted by the

18-Jan-2009
Author:
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Complete List of Authors: Li, Xing-hua; Zhejiang University, College of Animal Sciences
Yang, Huan-jun; Zhejiang University, College of Animal Sciences
Roy, B.; Zhejiang University, College of Animal Sciences
Park, E. Y.; Shizuoka University
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Jiang, Li-jun; Zhejiang University, College of Animal Sciences

Wang, Dan; Zhejiang University, College of Animal Sciences
Miao, Yun-gen; Zhejiang University, College of Animal Sciences
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Keywords: cellulase, Trichoderma viride, mutation, microwave, ultraviolet

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Page 1 of 15 Current Microbiology

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4 Enhanced Cellulase Production of the Trichoderma viride mutated
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6 by microwave and ultraviolet
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10 Xing-hua Li a, Hua-jun Yang a, B. Roya, E. Y. Parkb, Li-jun Jiang a, Dan Wang a,
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12 Yun-gen Miao a,*
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15 a
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Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of
17 Agriculture, Zhejiang University, Hangzhou 310029, PR China;
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19 Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of
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21 Agriculture, Shizuoka University, Shizuoka 422-8529, Japan

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30 Address correspondence to:

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College of Animal Sciences, Zhejiang University, Hangzhou 310029
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33 P. R. China
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35 Tel: +86 571 86971659
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37 E-mail: miaoyg@zju.edu.cn
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48 Running title: Cellulase Production of the Trichoderma viride
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50 Keywords: cellulase, Trichoderma viride, mutation, microwave, ultraviolet
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54 §
The work was supported by a key project of Zhejiang Government No. 2008C24011 and by
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56 the Hi-Tech Research and Development Program of China (No. 2008AA10Z132 and No.
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58 2006AA10A119).
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5 Abstract Cellulase-producing fungi Trichoderma viride were cultured and fermentated on the
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7 solid-state wheat bran fermentation medium. The characteristics of its carboxymethyl
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9 cellulase (CMCase) in the condition of this solid-state fermentation were evaluated, that the
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optimum culture time, optimum pH and optimum temperature for CMCase activity of T.
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13 viride fermentated in this solid state was 60 h, 5.0 and 50℃ respectively. Carboxymethyl
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15 cellulose sodium (CMC-Na) and Congo red were used to screen the strains that had stronger
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17 ability to produce enzymes. After the compound mutagenesis by microwave and ultraviolet,
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seven mutant strains (M-B1 to M-B7) were selected and their CMCase activities were assayed.
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20 Five of them (M-B1,M-B2, M-B3, M-B5 and M-B7) had significantly stronger ability to
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22 produce enzymes than normal wild type, and they were also very stable for a long period of
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24 up to 9 generations to produce cellulase.
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Keyword cellulase, Trichoderma viride, mutation, microwave, ultraviolet
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5 Introduction
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7 The industrial revolution generated an increasing need for energy that was fueled mainly by
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9 fossil fuels. With the progress of industrialization, petroleum was in great demand. As a
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11 consequence, serious environmental problems have arisen [1-3].
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Recently, products utilizing biomass as an alternative resource have been developed for
14 many markets. One of these, cellulolytic biomass, is known as a carbon-neutral material
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16 because it does not increase the amount of carbon dioxide in the air [4, 5].
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18 Cellulases catalyze the hydrolysis of cellulose which are mainly three types:
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20 endoglucanases (EC. 3.2.1.4), cellobiohydrolases (EC. 3.2.1.91) and β-glucosidases
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21 (EC.3.2.1.21)[6-8]. With the recent development of biotechnology, there has been vast interest
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23 to use cellulose digestive microorganisms to convert cellulosic biomass to glucose that can be
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25 used in different applications such as production of fuel ethanol, use in animal feed, use in
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27 waste water treatment and in brewing industry.
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Many microorganisms that produce various cellulolytic enzymes have been studied for
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30 several decades. The genus of Trichoderma has been especially famous for producing
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32 cellulolytic enzymes with relatively high enzymatic activity[9].
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34 However, these technologies have hardly been realized in practice because of their high
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36 running cost and low yields of this enzyme. Therefore, investigation on ability of microbial
37 strains to utilize inexpensive substrate and improvement of enzyme productivity is thus an
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39 important object for research.
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41 Wheat bran, the agricultural byproduct, is a cheaply available resource in China, and has
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43 potentiality as an industrial fermentation substrate.
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In the current work, wheat bran was used as substrate in order to reduce the cost of
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46 cellulase production. The aim of this study was to obtain high levels of extracellular cellulases
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48 by mutating the T. viride using physical and chemical methods, and maximize cellulase
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50 activity by optimizing the parameters, including the culture time of T. viride, pH of enzyme
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reaction and temperature of enzyme reaction for the normal and mutated T. viride in
53 solid-state wheat bran fermentation medium.
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57 Materials and Methods
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59 Fungal strain
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The T. viride strain AS 3.3711 (purchased from China General Microbiological Culture

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3 Collection Center) for this experiment is a high cellulase activity-producing fungal strain. It
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5 was cultured on potato dextrose agar slants (PDA：potato leachate, 1.0 L; glucose 20.0 g; agar,
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15.0 g; nature pH) at 28℃ for 7-10 days, and stored at 4℃ when spores were formed.
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12 Media
13 The medium used for fermentation contained 0.8 g of wheat bran and 720 µL of mineral salt
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15 solution A (MgSO4, 0.05% (m/v); K2HPO4, 0.1% (m/v), nature pH), mixed well, and then put
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17 into the test tube. The medium used for screening contained 0.5 g of carboxymethyl cellulose
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19 sodium (CMC-Na, Sigma), 1.5 g of agar, and 100 mL of mineral salt solution B ((NH4)2SO4,
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20% (m/v); MgSO4, 0.05% (m/v); K2HPO4, 0.1%(m/v); NaCl, 0.05% (m/v), nature pH).
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24 Cellulase Production
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26 The spores were washed down from the PDA agar slants by distilled water. For inoculum
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28 preparation, 20 µL of spore suspension (containing 108 conidia/ml) of T. virid was inoculated
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onto the test tube of fermentation medium, cultured at 28℃ until spores were full of the
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31 medium. Small scale experiments were carried out in the same way as the inoculum
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33 preparation, except that the spores were washed down from the last fermentation media, not
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35 the PDA agar slants, and were cultured for 60 h.
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Enzyme extraction and activity assay
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40 Cellulases were extracted by suspending the fermented wheat bran in 20 mL distilled water
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42 and mixing it for 1 h at 220 rpm. Following this, the suspended material and the fungal
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44 biomass were separated by centrifugation at 4000rpm for 10 min). The clarified supernatant
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was used as the source of enzymes.
47 Carboxymethyl cellulase (CMCase) activity was determined by measuring the amount of
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49 glucose released from CMC-Na by the DNS method with glucose as the standard. Reaction
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51 mixtures contained 2 ml of 10 g·L－1 CMC-Na in 0.1 M citrate buffer (pH 5.0) and 0.5 ml of
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53 each enzyme solution appropriately diluted with 0.1 M citrate buffer (pH 5.0), and was mixed
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55 well. Each diluted enzyme solution in controls were inactivated first in a boiling-water bath
56 for 10 min.
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58 After incubation at 50℃ for 30 min, the controls and samples were taken out of the 50
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60 ℃ bath. The control reactions were terminated by adding 2.5 ml of 3, 5-dinitrosalicylic acid
(DNS), but the sample reactions, at first, were quickly moved into the boiling-water bath for

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3 10 min, then were added 2.5 ml of DNS to terminate the enzyme-substrate reaction.
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5 The mixtures were also mixed well, then placed into a boiling-water bath for 5 min, and
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7 cooled to room temperature. The absorbance of the reaction solutions were measured at 530
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9 nm. The International unit of activity (IU) is defined as the amount of enzyme, which liberates
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l µmol of glucose per minute in a standard assay.
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14 Characteristics of carboxymethyl cellulase
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16 The optimum culture time of T. viride cultured in solid-state wheat bran fermentation medium
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was determined by measuring the carboxymethyl cellulase (CMCase) activity at the different
19 culture times (12-192 h) with CMC-Na as the substrate at 50℃ and pH 5.0.
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21 The optimum pH of enzyme was evaluated by measuring the CMCase activity at the
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23 optimum culture time with CMC-Na as the substrate at 50℃ and different pH. The following
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25 buffers were used: 0.1 M citrate buffer (pH 3.0-6.0), 0.2 M phosphate buffer (pH 7.0-8.0), 0.2
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27 M Glycine/NaOH buffer (pH 9.0- pH 10.0).
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29 The optimum temperature of the enzyme was evaluated by measuring the CMCase
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activity at the optimum culture time and pH, at the different temperature (40-90℃) with
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CMC-Na as the substrate.
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34 The thermotolerance of CMCase was determined at the optimum culture time, pH and
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36 temperature with CMC-Na as the substrate, after incubating the enzymes at different
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38 temperatures (40-90℃) for 10 min. Then, the enzymes were assayed for the remaining
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40 activity.
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43 Preparation of single spore suspension
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45 The spores which are full of the test tube of solid-state wheat bran fermentation medium were
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47 washed down with distilled water, diluted properly, and then plated onto the CMC-Na
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49 screening medium. Picked a single strain and cultured it on the olid-state wheat bran
50 fermentation medium. When spores were again full of the fermentation medium, spores
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52 washed them down with distilled water, diluted properly and then counted to 108 conidia per
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54 milliliter with the cell counting plate under the microscope.
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Mutagenesis with microwave treatment
59 Use a microwave oven (maximum power: 700 W; microwave frequency: 2450 MHz) to
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radiate the single spore suspensions for different times (15-195 S) in the ice bath in order to

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3 eliminate heating effect of the oven.
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7 Mutagenesis with ultraviolet treatment
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9 Ultraviolet treatment (power: 15 W) was given to the spore for 15min with the 15 cm of
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distance from the UV light to the screening medium plated with the single spore suspension.
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14 Screening of T. viride mutant
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16 After each mutagenesis, the mutated spore suspensions were plated onto the CMC-Na
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18 screening medium, and cultured at 28℃. Four days later, the hydrolysis halos were made
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visible by Congo red staining. The fatality rate of spores were counted.
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21 The qualitative cellulase activities were judged by the ratio of the hydrolysis halo
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23 diameter to the mutant strain colony diameter (H/C). Selected the mutant strains producing
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25 the bigger ratio (H/C > 1.5), cultured them onto the fermentation medium, and prepared the
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27 spore suspensions to be mutated in another way.
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The final screened mutant strains were cultured in fermentation medium, and their
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30 CMCase activities were analyzed in the way as the normal strains under the optimum
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32 condition of culture time, pH and temperature.
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Hereditary stability studies of mutants
37 Mutants obtained by the compound mutation of two methods above were studied for their
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39 stability for cellulase production for 9 generations. Mutants after every fermentation were
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41 inoculated on the wheat bran fermentation medium and used for inoculating next fermentation
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44 Statistical analysis
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46 Analysis of variance (ANOVA) was done with Statistics software for version 5.0. Before
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48 analysis, the assumptions of ANOVA were tested, that is data normality, variance
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50 homogeneity and factor additivity. Tukey honest significant difference (HSD) test and
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Dunnett t (2-tailed) test were used for multiple comparisons. All experiments were performed
53 in triplicate.
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57 Results
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59 Characteristics of carboxymethyl cellulase
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The ANOVA showed that there were extremely significant differences of the cellulase

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3 activities between different culture time, different pH and different temperature, respectively
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5 (ANOVA, p<0.01).
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7 The cellulase enzymes excreted by T. viride which were cultured in solid-state wheat
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9 bran fermentation medium exhibited extremely significant maximum carboxymethyl cellulase
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(CMCase) activity (Tukey honest significant difference (HSD) test, p<0.01) at 60 h of culture
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12 time (Fig. 1A), and the CMCase activity attained extremely significant highest (Tukey honest
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14 significant difference (HSD) test, p<0.01) at the environmental condition of pH 5.0 and
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16 temperature 50℃ (Fig. 1B & Fig. 1C). It was stable at pH range of 4.0 to 7.0 (Fig. 1B). The
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18 enzyme was found to retain more than 80% of it maximal activity for at least 20 min at 50-70
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20 ℃ and 10 min at 80℃, respectively. Interestingly, it retained activity approximately 50% of
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22 its maximal activity after incubating at 90℃ for 10 min (Fig. 1B).
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Figure 1
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Mutagenesis of T. viride and screening of mutant

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31 Normal wild type strain (W1), isolated by its ability to grow on CMC-Na as carbon source
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and major hydrolytic zones was subjected to successive mutagenic treatment with microwave
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and ultraviolet.
36 The normal wild T. viride strain (W1) spores were exposed to microwave (maximum
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38 power: 700 W; microwave frequency: 2450 MHz) for different times (15-195 S), using the ice
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40 bath to eliminate heating effect of the oven on the spores.
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42 The lethality rate of T. viride spores exposed to microwave responding with varying
43 times was illustrated in Fig.2. With 135 second of exposure to microwave, the lethality rate of
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45 T. viride spores attained above 85%, and after 165 second it was approximately 100%. It
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47 suggested that the T. viride spores were very sensitive to the microwave. However, the rate of
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49 positive mutation as to the survival colonies was increasing with the time increased (data not
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shown).
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54 Figure 2
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57 After the treatment of microwave, selected seven mutated strain colonies (M-A1 to
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59 M-A7) cultured on the CMC-Na screening medium, which produced bigger ratio of the
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hydrolysis halo diameter to the mutant strain colony diameter (H/C > 1.5), cultured them onto

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the fermentation medium. When a lot of spores appeared, stored at 4℃ in order to be mutated
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5 later in another way.
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7 The microwave mutated T. viride strain spores were prepared to single spore suspensions
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9 as mentioned above. These single spore suspensions were plated onto the screening medium,
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11 and exposed to ultraviolet for 15min with the 15 cm of distance from the UV light to the
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medium plates.
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14 After that, seven mutated strain colonies (M-B1 to M-B7) cultured on the screening
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16 medium were selected out in the same way, cultured on the fermentation medium, and then
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18 their CMCase activities were assayed as above.
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23 After the compound mutagenesis of microwave and ultraviolet, seven mutant strains were
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25 isolated (M-B1 to M-B7) and used to fermentate. The selection of hypercellulaseproducing

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27 mutants was based on the diameter of the clearing zone surrounding the colony on the
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plate-screening medium (H/C > 1.5). Their CMCase activities were assayed as before
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34 Table 1
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37 The results presented in Table 1 indicated the production of CMCase activity by mutants
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39 mutated by the compound effect of microwave and ultraviolet. Five of the seven mutant
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41 strains (M-B1,M-B2, M-B3, M-B5 and M-B7) secreted significantly more CMCase than the
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43 wild normal type. No difference in the growth of both wild type and mutant strain was
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observed as assessed by measurement of wet biomass (data not shown). This result indicates
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46 that the enhancement of enzyme production by the mutant strain was not because of an
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48 increase in growth but to the enhancement in production and secretion of the enzyme.
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50 Interestingly, the CMCase activity of mutant strain M-B4 and M-B6 were not statistically
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bigger than the wild strain. It suggested that the CMC-Na screening medium was not found to
53 give absolutely reliable indication of elevated cellulolytic activities.
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55 The five mutants selected in the second mutation of ultraviolet (M-B1,M-B2, M-B3,
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57 M-B5 and M-B7) were found to be stable for cellulase production for a studied period of 9
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59 generations (Table.2).
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Table 2
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7 Discussion
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9 The enzymatic degradation of waste cellulose by cellulolytic microorganisms has been
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11 suggested as a feasible alternative for the conversion of cellulosic wastes into fuel ethanol.
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13 Fungi are the main cellulase-producing microorganisms, although a few bacteria and
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15 actinomycetes have also been reported to yield cellulase activity [10-13]. Microorganisms of
16 the genera Trichoderma and Aspergillus are thought to be cellulase producers, and crude
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18 enzymes produced by these microorganisms are commercially available for agricultural use.
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20 However, Attempts to use these enzymes in the degradation of cellulosic wastes have not been
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22 successful for several reasons such as, low enzymatic yields, and low specific activities, end
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product inhibition of the enzymes.
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25 Strain improvement by mutations is an age-old and a successful method [14-18]. We

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27 used this method to isolate some mutant strains for better enzyme- excreting efficiency. It was
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29 reported that when fungi were grown with mutagens at sublethal concentrations secretory
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31 enzyme production increased. Mutant strains M-B1, M-B2, M-B3, M-B5 and M-B7 produced
32 high levels of CMCase, which were obtained by compound mutation of microwave and
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34 ultraviolet, and these, were also stable for a long period of 9 generations to produce cellulase.
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36 To analyze the reasons for enhanced ability to produce CMCase enzymes of mutant strains,
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38 we are planning to do molecular studies. Overproduction of cellulases makes these mutants

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good candidates for easy and economic cellulosic waste degradation.
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A fundamental problem in the development of a continuous process for the production of

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43 fungal enzymes, such as cellulases by Trichoderma viride, is the maintenance of conditions
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45 for maximal enzyme production. Because during the process of fermentation, the
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physiological state of the microorganism may not always be kept at its optimum for enzyme
48 production, as a result of which, the physiological balance may be set back from production to
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50 non-productive cell growth or forward to unwanted sporulation, leading in both cases to
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52 decreased enzyme productivity. The balance between these two undesirable physiological
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54 stages and the maximum productive stage on the basis of a fixed, time-dependent feeding rate
55 over along period of time is rather sensitive. Thus there are many studies about the fixed
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57 optimum fermentation condition for some popular cellulolytic microorganisms.
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Acknowledgements

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3 The work was supported by a key project of Zhejiang Government No. 2008C24011 and by
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5 the Hi-Tech Research and Development Program of China (No. 2008AA10Z132 and No.
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7 2006AA10A119).
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11 References
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1. Aristidou A, Penttila M (2000) Metabolic engineering applications to renewable resource
14 utilization. Curr Opin Biotechnol 11(2): 187-198
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16 2. Jeffries TW, Jin YS (2000) Ethanol and the rmotolerance in the bioconversion of xylose by
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18 yeasts. Adv Appl Microbiol 47: 221-268
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20 3. Zaldivar J, Nielsen J, Olsson L (2001) Fuel ethanol production from lignocellulose: a
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27 prospects. Appl Microbiol Biotechnol 69(6): 627-642
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32 6. Jagtap S, Rao M (2005) Purification and properties of a low molecular weight
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34 1,4-beta-d-glucan glucohydrolase having one active site for carboxymethyl cellulose
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36 and xylan from an alkalothermophilic Thermomonospora sp. Biochem Biophys Res
37 Commun 329(1): 111-116
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39 7. Guo R et al (2008) Molecular cloning and characterization of two novel cellulase genes
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41 from the mollusc Ampullaria crossean. J Comp Physiol [B] 178(2): 209-215
iew

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43 8. Thongekkaew J et al (2008) An acidic and thermostable carboxymethyl cellulase from the
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yeast Cryptococcus sp. S-2: purification, characterization and improvement of its
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46 recombinant enzyme production by high cell-density fermentation of Pichia pastoris.
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48 Protein Expr Purif 60(2): 140-146
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50 9. Miettinen-Oinonen A, Suominen P (2002) Enhanced production of Trichoderma reesei
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endoglucanases and use of the new cellulase preparations in producing the stonewashed
53 effect on denim fabric. Appl Environ Microbiol 68(8): 3956-3964
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55 10. Lowe SE, Theodorou MK, Trinci AP (1987) Cellulases and xylanase of an anaerobic
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57 rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan.
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59 Appl Environ Microbiol 53(6): 1216-1223
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11. Lynd LR et al (2005) Consolidated bioprocessing of cellulosic biomass: an update. Curr

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3 Opin Biotechnol 16(5): 577-583
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5 12. Penttila M et al (1986) Homology between cellulase genes of Trichoderma reesei:
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7 complete nucleotide sequence of the endoglucanase I gene. Gene 45(3): 253-263
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9 13. Tomme P et al (1988) Studies of the cellulolytic system of Trichoderma reesei QM 9414.
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Analysis of domain function in two cellobiohydrolases by limited proteolysis. Eur J
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12 Biochem 170(3): 575-581
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14 14. Kumakura M, Kaetsu I, Nisizawa K (1984) Cellulase production from immobilized
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16 growing cell composites prepared by radiation polymerization. Biotechnol Bioeng
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26(1):17-21
19 15. Chadha BS, Garcha HS (1992) Mixed cultivation of Trichoderma reesei and Aspergillus
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21 ochraceus for improved cellulase production. Acta Microbiol Hung 39(1): 61-67
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23 16. Hayward TK et al (2000) Improvements in titer, productivity, and yield using Solka-floc
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for cellulase production. Appl Biochem Biotechnol 84-86: 859-874
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17. Bailey MJ, Tahtiharju J (2003) Efficient cellulase production by Trichoderma reesei in
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28 continuous cultivation on lactose medium with a computer-controlled feeding strategy.
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32 18. Villena GK, Gutierrez-Correa M (2006) Production of cellulase by Aspergillus niger
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biofilms developed on polyester cloth. Lett Appl Microbiol 43(3): 262-268
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27 Fig.1 Characterization of carboxymethyl cellulase (CMCase) of T. viride cultured in solid wheat
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29 bran fermentation medium. (A) Optimum culture time of T. viride on the activity of CMCase.
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Culture time profile was determined by incubating the enzyme at 50 in 0.1 M citrate buffer (pH
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32 5.0) for 30 min with CMC-Na as the substrate at varying times (time 12-192 h). (B) Optimum pH
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34 on the activity of CMCase. pH profile was determined in the same way as (A) at the optimum
35 culture time and varying pHs (pH 3.0-10.0 h). (C) Optimum temperature on the activity of CMCase.
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37 Temperature profile was determined in the same way as (A) at the optimum culture time, optimum
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38 pH and varying reaction temperature (reaction temperature 40-90 ). (D) Thermotolerance of

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40 CMCase. The thermal stability of CMCase was carried out by incubating at different temperatures
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(as indicated in the Fig.1D) for 10, 20, 30, 40, 50 and 60 min before the remaining activity was
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43 assayed in the same way as (A) at the optimum culture time, optimum pH and optimum reaction
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temperature.
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19 Fig.2 The lethality rate of T. viride spores exposed to microwave for different times.
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Fo

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rP

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ee

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rR

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ev

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 Current Microbiology Page 14 of 15

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5 Table.1 The CMCase activity of mutant strains mutated by the compound effect of microwave and
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ultraviolet.
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Fo

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23 Data of CMCase activities were showed as mean±S.D. Dunnett t (2-tailed) test was used to
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rP

25 compare the CMCase activities of every mutant strain to that of normal strain. The double star
26 superscript indicated that there was greatly significant difference of CMCase activity between the
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28 mutant and normal strain.
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ee

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rR

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ev

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Page 15 of 15 Current Microbiology

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5 Table.2 The CMCase activities of mutant strains for 9 generations
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20 Data of CMCase activities were showed as mean.
Fo

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rP

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ee

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rR

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ev

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