Skip Navigation


PEDS Advance Access originally published online on October 30, 2007
Protein Engineering Design and Selection 2007 20(11):535-542; doi:10.1093/protein/gzm052
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
20/11/535    most recent
gzm052v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ni, J.
Right arrow Articles by Watanabe, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ni, J.
Right arrow Articles by Watanabe, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Random exchanges of non-conserved amino acid residues among four parental termite cellulases by family shuffling improved thermostability

Jinfeng Ni1,2, Motomi Takehara1, Mitsuhiro Miyazawa1 and Hirofumi Watanabe1,3

1 National Institute of Agrobiological Sciences, 1–2 Owashi, Tsukuba, Ibaraki 305-8634, Japan 2State Key Laboratory of Microbial Technology, Shangdong University, 27 Shandanan Road, Jinan, Shangdong 250100, People Republic of China

3 To whom correspondence should be addressed.E-mail: hinabe{at}affrc.go.jp, gca01405{at}nifty.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
There have been two major problems preventing applications of termite cellulases; one was difficulty for their hetelologous overexpression, and another is their low thermostability. We previously achieved adaptation of termite cellulase genes to an overexpression system of Escherichia coli by family shuffling of four orthologous cDNAs (Biosci. Biotechnol. Biochem., 2005; 69: 1711–1720). Using the adapted mutant cDNAs as parental genes combined with native-form cDNAs, we performed further family shuffling and obtained mutant cDNAs, which gave enzymes with improved thermostability. The best-evolved clone (PA68) was improved by 10°C in maximum stability (retaining 90% original activity for 30 min incubation) from the parental enzymes, and kept 54% of its original activity for 150 min at 50°C, whereas the most thermostable enzyme amongst the parents (A18) retained 30% of its original activity. PA68 showed 889 (µmoles of reducing sugars/min/mg of protein) in Vmax and 560 (µmoles of reducing sugars/min/mg of protein) in the specific activity against carboxymethylcellulose, which corresponds to 9.8 and 13.1 times of those of one of the ancestral enzymes rRsEG. In summary, we improved thermostability of the termite cellulase and increased the Vmax value and specific activity by combining only cDNAs encoding enzymes adapted for normal temperatures.

Keywords: endo-beta-1,4-glucanase/Escherichia coli/family shuffling/Isoptera/mutant


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Terrestrial plants annually fix 6.0–6.3x1016 g carbon as cellulose, hemicellulose and lignin (Prentice et al., 2001Go). Decomposition of these materials is carried out mainly by bacteria and fungi in the temperate zones, but also by termites (Insecta; Isoptera) in the tropical and subtropical zones (Yamada et al., 2005Go). Undoubtedly, the decomposition potential of termites is supported by their cellulolytic enzymes, and thus is expected to have industrial applications. In termites, two producers of cellulolytic enzymes are known; one is symbiotic protozoa and the other is the termites themselves (O’Brien and Slaytor, 1982Go; Breznak and Brune, 1994Go; Watanabe and Tokuda, 2001Go). The endogenous cellulases of termites belong to the glycoside-hydrolase family 9 (GH9) (Watanabe and Tokuda, 2001Go). Of the termite species from which endogenous cellulase cDNAs were isolated, all cellulases have a single domain structure with a catalytic module and consist of 448 amino acids (432 in mature forms) with more than 70% overall identity in the amino acid sequences (Watanabe and Tokuda, 2001Go; Nakashima et al., 2002Go; Li et al., 2003Go). They are active against crystalline celluloses, despite their single domain structure (Watanabe et al., 1997Go; Nakashima et al., 2002Go; Tokuda et al., 2005Go), although enzymes without cellulose-binding domains are generally considered inactive against such substrates (Bayer et al., 1998Go). The structure of the termite gut would explain the reason for the absence of the cellulose binding domains from the termite cellulases. The digestive tract of termites is a closed cavity for enzymic reactions where cellulases and substrates (masticated wood) are retained at high concentration, which prevents diffusion of the enzymes and is comparable to industrial biomass-conversion systems combining mortars, enzymes and a tank (Watanabe, 2003Go). To establish a foundation for application research of termite cellulases, recombinant production of objective enzymes is inevitable since termites cannot be cultivated like microbes as an on-demand enzyme source. Recently, we accomplished overexpression of the termite endogenous cellulase with minimum modification of its first structure by family shuffling of four orthologous cDNAs from different termite species (Ni et al., 2005Go). Other requirements for industrial applications of cellulases, e.g. thermostability and activity under alkaline conditions, are noted in particular for their applications in detergent production and enzymic ethanol production from biomass (Ito et al., 1998Go; Sheehan and Himmel, 1999Go). Termite endogenous cellulases show activity in a relatively broad pH range from pH 3 to over pH 10; however, it does not have high thermostability (Tokuda et al., 1997Go; Watanabe et al., 1997Go). Here, we report improved thermostability of a mutant cellulase made by family shuffling of four homologous cDNAs of termite cellulases.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Bacterial strains, plasmids and culture conditions

A normal expression plasmid vector pAD10{alpha}, which was derived from pGAD10 (Takara Bio, Inc. Otsu, Shiga, Japan) by detaching the GAL4 domain and inserting the {alpha}-factor signal sequence (Ni et al., 2005Go), was employed by family shuffling and following the screening process in E.coli DH5{alpha} (Toyobo, Osaka, Japan). Overexpression was conducted using pQE30 overexpression vector (Qiagen, Inc., Valencia, CA, USA) with E.coli JM109 (Toyobo). Procedures for constructing a cDNA library in pAD10{alpha}, subcloning to pQE30 employed the primer designs, and the culture conditions for screening and overexpression were previously described (Ni et al., 2005Go) except that Luria-Bertani (LB) ampicillin medium [1% tryptone, 0.5% yeast extract, 1% sodium chloride (w/v) with 100 µg/ml of ampicillin] was used for overexpression cultures [originally a fully synthetic medium was employed (Ni et al., 2005Go)].

Recovery of crude enzyme extract from E.coliand affinity purification

Transformants of E.coli JM109 were recovered from 10 ml of overnight overexpression cultures by centrifugation, and the recovered cells were stored at –30°C overnight. Crude enzyme extract was recovered from the cells using CelLytic B bacteria extracting reagent (Sigma–Aldrich Corporation, St Louis, MO, USA) according to the product manual. Recombinant enzymes were purified from the crude extracts using His-tag affinity resin (MagExtract His-tag fusion protein purification kit, Toyobo) (Ni et al., 2005Go).

Family shuffling

Seven mutant cDNAs of the second generation, which were previously screened from a shuffled cDNA library combined with four native termite cellulase cDNAs (RsEG, NtEG, CfEG and CaEG from termites Reticulitermes speratus, Nasutitermes takasagoensis, Coptotermes formosanus and C. acinaciformis, respectively) and two mutant cDNAs (EGD1 and EGD2) of the first generation (Ni et al., 2005Go), were used as parent cDNAs for family shuffling. The shuffling scheme from the four native cDNAs to the third generation of mutant cDNAs is described in Fig. 1. Parental cDNAs were digested with DNase I and recombined with PCR following the method described previously (Ni et al., 2005Go).


Figure 1
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Flowchart of family-shufflings and selections to obtain termostable mutant enzymes. For a family shuffling, a mixture of parental cDNAs was fragmented randomly by DNAseI digestion, and then denatured (single-stranded) and re-annealed (recombinated). The recombinated cDNA was amplified with a pair of primes of 5’ and 3’ regions by PCR, and thus truncated clones were neglected from the library. The mutant cDNAs in the generated library were transformed in the host cells and clones were selected in appropriate conditions. Detail experimental conditions were described in the text and Ni et al. (2005Go). {dagger}Clones were obtained by Ni et al. (2005Go).

 
Detection and measurement of cellulase activityof recombinant termite cellulases

For pre-screening colonies of E.coli JM109 expressing recombinant cellulases, the Congo Red plate assay, which detects cellulase activity as a clear zone around growing colonies against a red-stained background on agar plates containing CMC [carboxymethyl cellulose, sodium salt: average molecular weight, 250 000; the degree of carboxymethyl substitution, 0.7 (w/v); Sigma–Aldrich Corporation] as a substrate (Teather and Wood, 1982Go), was employed before screening by quantitative measurements. For quantitative measurements of cellulase activities, enzyme samples (25 µl) were incubated with 100 µl of CMC solution [1%(v/w)] in sodium acetate buffer (0.1 M, pH 5.5) at 37°C for 5 min (the standard condition for enzyme assay). Reactions were stopped by the addition of Tetrazolium Blue reagent (800 µl), and then the reaction mixtures were heated at 95°C for 5 min and the absorbance (660 nm) was measured to quantify the reducing sugars generated in the enzymic reactions (Jue and Lipke, 1985Go). Optimum temperatures of sample enzymes were measured by changing the reaction temperatures from 20 to 75°C at 5°C intervals. Thermal stability was measured by pre-incubating sample enzyme solutions at different temperatures (from 20 to 75°C at 5°C intervals) for 30 min before enzymic assay at 37°C. For measurement of kinetic constants (Km and Vmax) of sample enzymes, substrate concentrations of enzyme reactions were varied from 4 to 20 mg/ml. Lineweaver–Burk plots were carried out using Microsoft Excel 2002. Protein concentrations of sample enzyme solutions were measured using Quick Start Bradford Dye Reagent (no. 500-0205, Bio-Rad Laboratories, Inc., Hercules, CA, USA) with Bovine Serum Albumin Standard Set (no. 500-0207, Bio-Rad Laboratories) according to the product manuals.

Screening of mutant cDNA clones

For screening of thermostable mutant cellulases, the crude enzyme extracts of transformants harbouring mutant cDNAs were pre-incubated at 50°C for 30 min in a 1.5 ml disposable centrifuge tube on a heating block, and then their cellulase activity was measured under the standard condition described in the previous section. For screening of alkaline active mutant cellulases, enzymic reactions were conducted at pH 9.0 by replacing the buffer of the standard condition with Tris–HCl buffer (0.1 M, pH 9.0).

Measurement of thermal stability of mutant cellulases

The thermal stability of sample enzymes was measured by two methods. One is the thermal inactivation test with pre-incubation at a constant temperature (50°C) over various periods: enzymic samples were pre-incubated for 10, 20, 30, 60, 90, 120 and 150 min before enzymic assay at 37°C. The other is the thermostability test with pre-incubation for a constant period at various temperatures: enzymic samples were pre-incubated at various (but constant) temperatures from 20 to 65°C at 5°C intervals for 30 min before measurement of cellulase activity at 37°C.

Measurement of pH–activity relationshipof mutant cellulases

To evaluate the effect of reaction pH, the buffer of the standard condition for enzyme assay was replaced by a series of McIlvaine’s buffers (di-sodium phosphate–citric acid) of different pH conditions (pH 3–9) (McIlvaine, 1921Go).

Analysis of substrate specificity of sample enzymes

To analyse the substrate specificity of recombinant termite cellulases, CMC was replaced by a variety of substrate solutions and suspensions. The substrates and their final concentrations (w/v) were: Avicel (0.5%), micro-crystalline cellulose (Sigmacell type 20) (0.5%), lichenan (0.2%), laminarin (0.5%), birchwood xylan (0.5%), chitin (1%), barley β-glucan (0.5%), CM-curdlan (carboxymethyl-β-1,3-D-glucan, Wako Pure Chemical Industries, LTD, Osaka, Japan) (1%), HEC (hydroxyethyl cellulose: Nakarai Tesque Inc., Kyoto, Japan) (1%), insoluble cello-oligosaccharide (ICO, average degree of polymerization: 34) made by the method of Sawano et al. (1988Go) (1%, suspension), phosphoric acid-swollen cellulose (PASC) made from Avicel according to the method of Wood (1988Go) (10%). All substrates were purchased from Sigma–Aldrich unless otherwise indicated. Each 10 µl of sample enzyme solution was added to 400 µl of the substrate solutions, and incubated for 5 min at 37°C except for reactions with PASC and ICO (10 min), and those with lichenan, Avicel, Sigmacell Type 20 and xylan (60 min). Reducing sugars generated in the enzymic reactions were measured by the addition of 800 µl of Tetrazolium Blue reagent. The amount of reducing power, which was originally present in the samples, was measured by adding Tetrazolium Blue reagent to the substrate solution before addition of the enzymic samples, and the values obtained were deducted from those from enzymic assays.

SDS–PAGE and western blotting

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Laemmli, 1970Go) was performed using Ready Gel E-R520L (5–20% gradient; ATTO, Japan). Proteins on the gel were detected by staining with Coomassie Brilliant Blue R-250. For western blotting, proteins in the PAGE gel were transferred to PVDF (polyvinylidene difluoride) membrane (Sequi-Blot, no. 162-0182, Bio-Rad) using a semi-dry transfer cell (TRANS-BLOT SD, Bio-Rad) with transfer buffer [25 mM Tris, 150 mM glycine and 10% (w/v) methanol pH 8.3 (non-adjusted)] soaked in extra thick blotting paper (Bio-Rad Laboratories) at 25 V for 70 min. His x 6 tagged protein was detected by Anti-His antibody conjugated to horseradish peroxidase (Qiagen) with ECL western-blotting detection reagent (GE Healthcare UK Ltd, Amersham Place, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions.

Prediction of 3D structure by homology modelling

Homology modelling for prediction of 3D structure of the selected mutant cellulase was done by submitting its amino acid sequence to the Swiss Model server (http://swissmodel.expasy.org//SWISS-MODEL.html) using NtEG (1ks8A) as a template (Schwede et al., 2003Go).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
Screening for thermostable and alkaline activemutant cellulases

The third generation mutant cDNAs, which were reconstructed from second family shuffling of the parent mutant cDNAs, were cloned into pAD10{alpha} and transformed into E.coli JM109. The transformants were placed in Congo Red assay plates and 1150 colonies with reconstructed cellulase activity were recovered from a total of 3000 colonies. Of these, 192 and 96 colonies with remarkable halo-forming activity were cultured in 1 ml of LB ampicillin medium and their crude extracts were recovered for the quantitative cellulase assays of alkaline activity and thermostability, respectively. Top 20 most active cellulases (crude extract) from the thermostable and alkaline-active clones are listed in Fig. 2A and B, respectively. When the activities were expressed in specific activities (µmole of reducing sugars generated by the enzymatic reaction per min per mg of protein in the crude extracts), both these sets of values were almost parallel (colonies showing high activity/ml showed high specific activity) in thermostable clones (Fig. 2A and C), whereas in alkaline active clones, such a tendency was not observed (Fig. 2B and D). This result suggests that the enhanced alkaline activity was stemmed from the modification. These 40 mutant cDNA clones were subcloned into pQE30 plasmid vector and were again transformed into E.coli JM109. The transformants were cultured for overexpression and the affinity-purified recombinant enzymes were recovered from their crude extracts. The clones were screened for productivity in the pQE30 vector, cellulase activity and thermostability of the purified mutant enzymes regardless of the conditions for the former screening process (alkaline or thermostability). As a result, purified enzymes from the two transformants of mutant cDNAs (TA51 and PA68, respectively) showed high productivity of recombinant enzymes (Fig. 3) at the same time compatible with thermostability. Thus, these two mutant cDNAs were selected for the following studies. The productivity of two mutants reached to 20 mg of mutant enzyme per litter of bacterial culture in the maximum condition.


Figure 2
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Comparison of cellulase activity in the crude extracts of selected transformants. Cellulase activity of the crude extracts of the thermostable clones (A) and the alkaline active clones (C). Their specific activities were drawn in (B) and (D), respectively. One unit of cellulase activity is defined as one micromole of reducing sugars generated in 1 min of reaction.

 

Figure 3
View larger version (68K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. SDS–PAGE and western-blotting of the crude extract of wile type and mutant cellulase transformants. Details of experimental conditions were written in Materials and methods. M: molecular weight markers.

 
Thermostability of the mutant cellulases

The thermostability of mutant cellulases TA51 and PA68 was compared with part of their parent enzymes. At a constant temperature of 50°C, rRsEG (recombinant product of RsEG), EGD1 and EDG2 (mutant cellulases from the first round family shuffling) completely lost their enzymic activities within 20 min. The mutant cellulase A74, from the second-round family shuffling, retained 20% of its original activity for 20 min; however, completely lost its activity at 60 min. Whereas the other second generation mutant cellulase A18 retained 30% of its original activity after pre-incubation of 150 min. Against these parental enzymes, PA68 retained 54% of its original activity for 150 min at 50°C, while TA51 completely lost its original activity within 60 min, although the thermal stability of TA51 improved against all of the parent enzymes except A18 (Fig. 4A). In the thermostability test, PA68 showed the best stability among all the clones compared. The other third generation clone TA51 showed the second best stability among all recombinant enzymes compared except that the remaining activity at 50°C was inferior to that of A18. The complete deactivating temperatures were 5°C (TA51) and 10°C (PA68) higher than the other recombinant cellulases (Fig. 4B).


Figure 4
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Thermal stability of wild-type and mutant cellulases. Thermal inactivation experiment at 50°C (A) and thermostability test (B) were conducted. Detail experimental conditions were described in Materials and methods.

 
Optimum pH of the mutant cellulases

The mutant cellulases generally showed optimum activity between pH 6.9 and pH 7.2. There was no alkaline shift in optimum pH among the mutant cellulases compared with those of the wild-type parents (rRsEG and rNtEG). In addition, there was no notable improvement in alkaline activity in the mutant cellulases of the third generation (TA51 and PA68) compared with the second generations (A18 and A74).

Kinetic properties of the mutant cellulases

Changes in the mutant cellulases were reflected mainly on the specific activities and Vmax of the property parameters (Table I). For the specific activities (activity per mg of the sample enzyme), TA51 and PA68 showed 12–15 times higher values than those of the native-form recombinant cellulases (rRsEG and rNtEG). For the Vmax values, TA51 and PA68 showed 5–22 times higher values than those of rRsEG and rNtEG. For the Km values, all values from the native and mutant cellulases were between 5.7 and 15.0 (differences between the lowest and highest values were less than three times). Comparing Km values to the native-form recombinant enzymes, TA51 showed a slightly lower value than those of rRsEG and rNtEG, but the PA68 values were between rRsEG and rNtEG.


View this table:
[in this window]
[in a new window]

  		Table I. Enzymatic properties of parent and mutant recombinant termite cellulases

 
Substrate specificity of mutant cellulases

The substrate specificity of mutant cellulases was not qualitatively changed from the native-form recombinant cellulases. The mutant and native-form cellulases showed activity only against substrates including β-1,4-linkages of glucose units, but not against glucose polymers with only β-1,3-linkages (CM-curdlan) or β-1,4-linked polymers of other sugars (chitin and xylan) (Table II). The only major difference between the native-forms and mutants was found in their relative activities on insoluble celluloses (barley β-glucan, laminarin, lichenan, Sigmacell Type 20 and Avicel) versus soluble celluloses (CMC and HEC). Mutant cellulases generally showed lower relative activity on insoluble substrates than their parent rRsEG (Table II).


View this table:
[in this window]
[in a new window]

  		Table II. Substrate specificity of wild-type and mutant recombinant termite cellulases

 
Predicted 3D structure of PA68 mutant cellulase

The 3D structure of the mutant cellulase PA68 was predicted as described in Fig. 5. There was no mutations occurred in the higher structure, but in the {alpha}-helices and random coils located far from the catalytic cleft, except for Q202K, N245S and I408V situated just outside of the cleft (Fig. 5).


Figure 5
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. A stereo schematic view of the homology model of the mutant cellulase PA68 using the cellulase of Nasutitermes takasagoensis (NtEG, PDB entry 1KS8) as a template. The amino acids forming the catalytic cleft are described as ‘balls and sticks’, and mutated amino acids were described as red ‘ribbons’. No mutations were occurred on the amino acids described as ‘strands’. The catalytic cleft was viewed from the –2 site (non-reducing side of the substrate) toward the +2 site.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 Funding
 References
 
Although PA68 was screened by activity of the crude extract of its transformant under alkaline conditions (pH 9.0 for PA68), the purified PA68 mutant cellulase showed limited improvement in alkaline activity but remarkable improvement in thermal stability. On the other hand, for the mutant cellulase TA51, which was screened by the thermal stability test at 50°C for 30 min of pre-incubation, the improvement in thermal stability was limited compared to that of PA68. As structural factors determining optimum pH and pH stability, pKa values of acid/base catalysts are important in fungal β-1,4-galactanases (Le Nours et al., 2003Go). While in the current study, because the acid/base catalysts and all of the substrate interacting residues are conserved among the four parental genes, the mutations introduced by the family shuffling (random homologous recombination among homologues) were limited to the non-conserved regions (Fig. 6). This would explain why the mutant cellulase PA68 did not show optimum pH shifted to the alkaline side even though it was selected with alkaline activity of the crude extract. The high alkaline activity of the PA68 crude extract would be given by its enhanced production by E.coli, which causes higher specific activity of the extract. A possible interaction of the cellulase with other hydrophilic proteins produced by the host cells would also protect the activity under alkaline conditions. Addition of stabilising proteins such as bovine serum albumin and casein are known to stabilize enzymes under extreme conditions (Hess and FitzGerald, 1998Go; Marini et al., 2000Go; Santhoshkumar and Sharma, 2001Go).


Figure 6
View larger version (61K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Alignment of amino acid sequences of parent and mutant termite cellulases. White characters in reverse background indicated coincidences of amino acids in sequences of four and over. Closed and open bars above sequences indicate {alpha}-helix and sheet regions, respectively, and asterisks (*) under sequences designate predicted residues forming the catalytic cleft and interacting with substrates (Ni et al., 2005Go). The predictions are based on former crystallographic studies (Khademi et al., 2002Go; Sakon et al., 1997Go).

 
The crude extract of the mutant cellulase TA51 showed significant improvement in thermostability, while the same enzyme purified by HisTag affinity method did not show any improvement in thermostability. This result suggests that the improvement of the crude extract was probably due to other components in the crude extract other than the mutant enzyme. Small molecules as polyamines or amino acids, which prevent aggregation of proteins, can improve thermostability (Shiraki et al., 2002Go; Kudou et al., 2003Go). Such factors might be found in the crude extract of host cells.

The majority of amino acid substitutions introduced into PA68 and TA51 avoided the positions, which were assumed to interact with the substrates (Fig. 5). The substrate-interacting amino acids Asp53 (predicted nucleophile), Asp56 (predicted co-nucleophile), His123, Trp126, Phe204, Gly248, Trp252, Asp253, Trp300, Arg304, His305, His358, Arg360, Tyr407 and Glu411 were all conserved in the native and mutant cellulases. The other substrate-interacting residues Gly208, Phe250, Asn254 were conserved in all of the mutants and native enzymes except NtEG (Fig. 6). The remaining substrate-interacting residue Tyr376 was conserved amongst the mutants PA68, TA51, and the native forms CfEG and RsEG. Thus, while no mutations were introduced in the catalytic cleft of PA68 and TA51, three mutations (Q202K, N245S and I408V) were introduced just outside of the cleft. The possible roles of these three mutations in the improvement of the thermostability should be elucidated in future study and considered in molecular design of thermostable enzymes based on the termite cellulases.

rRsEG showed activity against polysaccarides with β-1,4-linkages, and the mutant cellulases inherited this substrate specificity. The unchanged structure of the catalytic cleft of the mutants would also explain the conserved substrate specificity. The change in specific activities of the mutant cellulases against the insoluble celluloses were relatively small comparing to its drastic change against CMC, so that the ratio of the activity on Sigmacell 20, Avicel and ICO against that on CMC was reduced in the mutant cellulases comparing with rRsEG (Table II). Amongst such mutant cellulases, PA68 slightly recovered the activity against these native form celluloses comparing with the second generations, although the specific activity against CMC was as high as other mutant cellulases (Table I). Combined with the improvement of the thermostability, we expect PA68 as the parent for further improvement toward hydrolysis of native form cellulose.

The current study demonstrated that non-rational design of enzymes could improve the enzymic properties with regionally directed introduction of mutations. In some experiments, DNA shufflings were conducted with single cDNA (Oh et al., 2002Go; Pedersen et al., 2002Go; Kim et al., 2003Go) to obtain mutant cDNAs encoding thermostable enzymes, while in other cases, the improvement of thermal stability by directed evolution was accomplished by error-prone PCR (Cherry et al., 1999Go; Johannes et al., 2005Go; Miyazaki et al., 2006Go; Yun et al., 2006Go). In the current study, we used family shuffling to generate a mutant cDNA library (Ni et al., 2005Go). The parental four cDNAs encoded highly homologous enzymes [sharing 317 identical amino acids (73%)] which share conserved catalytic residues and their neighbours. As the result, the variety in the library would be limited within the regions, which do not directly support catalytic function. Thus, the improved thermostability of the mutant enzymes was accomplished with minimum changes in the non-catalytic regions of the parent cDNAs, which mainly forms {alpha}-helix loops and fewer mutations occurred in the random coil structures between the loops. Our previous study accomplished increase of activity and productivity of the target enzyme by the family shuffling of the same four parent cDNAs (Ni et al., 2005Go). The current study demonstrated that the same method could be applied aiming for improvement of thermostability from the same non-thermostable parents.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary data (colour image for Fig. 5) are available at PEDS online.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
This work was supported by the Insect Technology Project of The Ministry of Agriculture, Forestry and Fisheries of Japan (April 1, 2003–March 31, 2007) and JSPS (Japan Society for the Promotion of Science) postdoctoral fellowship to J.N. (September 13, 2004–September 12, 2006).


   Footnotes
 
Edited by Stephen Withers


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Bayer E.A., Chanzy H., Lamed R., Shoham Y. Curr. Opin. Struct. Biol. (1998) 8:548–557.[CrossRef][Web of Science][Medline]

Breznak J.A., Brune A. Annu. Rev. Entomol. (1994) 39:453–487.[CrossRef][Web of Science]

Cherry J.R., Lamsa M.H., Schneider P., Vind J., Svendsen A., Jones A., Pedersen A.H. Nat. Biotechnol. (1999) 17:379–384.[CrossRef][Web of Science][Medline]

Hess J.F., FitzGerald P.G. Mol. Vis. (1998) 4:29–33.[Medline]

Ito S., Kobayashi T., Ara K., Ozaki K., Kawai S., Hatada Y. Extremophiles (1998) 2:185–190.[CrossRef][Medline]

Johannes T.W., Woodyer R.D., Zhao H. Appl. Environ. Microbiol. (2005) 71:5728–5734.[Abstract/Free Full Text]

Jue C.K., Lipke P.N. J. Biochem. Biophys. Methods (1985) 11:109–115.[CrossRef][Web of Science][Medline]

Khademi S., Guarino L.A., Watanabe H., Tokuda G., Meyer E.F. Acta Crystallogr. D Biol. Crystallogr. (2002) 58:653–659.[CrossRef][Medline]

Kim Y.W., Choi J.H., Kim J.W., Park C., Cha H., Lee S.B., Oh B.H., Moon T.W., Park K.H. Appl. Environ. Microbiol. (2003) 69:4866–4874.[Abstract/Free Full Text]

Kudou M., Shiraki K., Fujiwara S., Imanaka T., Takagi M. Eur. J. Biochem. (2003) 270:4547–4554.[Web of Science][Medline]

Laemmli U.K. Nature (1970) 227:680–685.[CrossRef][Medline]

Le Nours J., Ryttersgaard C., Lo Leggio L., Ostergaard P.R., Borchert T.V., Christensen L.L., Larsen S. Protein Sci. (2003) 12:1195–1204.[CrossRef][Web of Science][Medline]

Li L., Frohlich J., Pfeiffer P., Konig H. Eukaryot. Cell (2003) 2:1091–1098.[Abstract/Free Full Text]

Marini I., Moschini R., Corso A.D., Mura U. J. Biol. Chem. (2000) 275:32559–32565.[Abstract/Free Full Text]

McIlvaine T.C. J. Biol. Chem. (1921) 49:183–186.[Free Full Text]

Miyazaki K., Takenouchi M., Kondo H., Noro N., Suzuki M., Tsuda S. J. Biol. Chem. (2006) 281:10236–10242.[Abstract/Free Full Text]

Nakashima K., Watanabe H., Saitoh H., Tokuda G., Azuma J.I. Insect. Biochem. Mol. Biol. (2002) 32:777–784.[CrossRef][Web of Science][Medline]

Ni J., Takehara M., Watanabe H. Biosci. Biotechnol. Biochem. (2005) 69:1711–1720.[CrossRef][Medline]

O’Brien R.W., Slaytor M. Aust. J. Biol. Sci. (1982) 35:239–262.

Oh K.H., Nam S.H., Kim H.S. Biotechnol. Prog. (2002) 18:413–417.[CrossRef][Medline]

Pedersen J.S., Otzen D.E., Kristensen P. J. Mol. Biol. (2002) 323:115–123.[CrossRef][Web of Science][Medline]

Prentice I.C., Farquhar G.D., Fasham M.J.R., Goulden M.L., Heimann M., Jaramillo V.J., Kheshgi H.S., Quere C.L., Scholes R.J., Wallace D.W.R. The Carbon Cycle and Atmospheric Carbon Dioxide—Houghton J.T., Ding Y., Griggs D.J., Noguer M., van der Linden P.J., Dai x., Maskell K., Johnson C.A., eds. Cambridge: Cambridge University Press. 193–237. Climate Change 2001: The Scientific Basis.

Sakon J., Irwin D., Wilson D.B., Karplus P.A. Nat. Struct. Biol. (1997) 4:810–818.[CrossRef][Web of Science][Medline]

Santhoshkumar P., Sharma K.K. Mol. Vis. (2001) 7:172–177.[Web of Science][Medline]

Sawano M., Sakamoto R., Arai M. Methods Enzymol. (1988) 160:274–299.[CrossRef][Web of Science]

Schwede T., Kopp J., Guex N., Peitsch M.C. Nucleic Acids Res. (2003) 31:3381–3385.[Abstract/Free Full Text]

Sheehan J., Himmel M. Biotechnol. Prog. (1999) 15:817–827.[CrossRef][Medline]

Shiraki K., Kudou M., Fujiwara S., Imanaka T., Takagi M. J. Biochem. (Tokyo) (2002) 132:591–595.[Abstract/Free Full Text]

Teather R.M., Wood P.J. Appl. Environ. Microbiol. (1982) 43:777–780.[Abstract/Free Full Text]

Tokuda G., Watanabe H., Matsumoto T., Noda H. Zoolog. Sci. (1997) 14:83–93.[CrossRef][Web of Science][Medline]

Tokuda G., Lo N., Watanabe H. Physiological Entomology (2005) 30:372–380.[Web of Science]

Watanabe H., Nakamura M., Tokuda G., Yamaoka I., Scrivener A.M., Noda H. Insect Biochem. Mol. Biol. (1997) 27:305–313.[CrossRef][Web of Science][Medline]

Watanabe H., Tokuda G. Cell Mol. Life Sci. (2001) 58:1167–1178.[CrossRef][Web of Science][Medline]

Watanabe H. Farming Japan (2003) 37:15–19.

Wood T.M. Methods Enzymol. (1988) 160:19–25.[CrossRef][Web of Science]

Yamada A., Inoue T., Wiwatwitaya D., Ohkuma M., Kudo T., Abe T., Sugimoto A. Ecol. Res. (2005) 20:453–460.[CrossRef]

Yun C., Matsuda H., Kawamukai M. Biosci. Biotechnol. Biochem. (2006) 70:559–563.[CrossRef][Medline]

Received January 11, 2007; revised July 25, 2007; accepted September 14, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow All Versions of this Article:
20/11/535    most recent
gzm052v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Ni, J.
Right arrow Articles by Watanabe, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ni, J.
Right arrow Articles by Watanabe, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?