APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/00/$04.0010

Feb. 2000, p. 788–793 Vol. 66, No. 2

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Bacterial Cell Surface Display of an Enzyme Library forSelective Screening of Improved Cellulase Variants

YONG-SUNG KIM, HEUNG-CHAE JUNG, AND JAE-GU PAN*

Bioprocess Engineering Division, Korea Research Institute of Bioscience and Biotechnology,Yusong, Taejon 305-600, Korea

Received 16 June 1999/Accepted 9 September 1999

The bacterial surface display method was used to selectively screen for improved variants of carboxymethylcellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the icenucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cellsurface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates thancontrols, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formeduniform tiny colonies. These variations in growth rate were assumed to result from altered availability ofglucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicatethat large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmedby assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes inthe cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused andfree forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acidsubstitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterialsurface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growthrates. This technique will provide a useful technology platform for directed evolution and high-throughputscreening of industrial enzymes, including hydrolases.

Directed evolution is increasingly being used to improvebiocatalysts (13). As more powerful combinatorial mutagenesismethods (e.g., combinatorial cassette mutagenesis, StEP andDNA shuffling [5]) become available, designing selection orscreening strategies becomes the most critical step in the suc-cessful exploitation of generated molecular diversity (8, 13).Screening can be based on chromogenic substrates or easilyobserved colony phenotypes (16, 29), but the most direct meth-ods of screening and selection link improved enzyme activity tothe survival or growth rates of cells (12, 14, 21). Examples ofthis method include selection on plates containing increasingantibiotic concentrations (23, 28) and complementation selec-tion with auxotrophs (1, 27). Unfortunately, these selectionprocedures are designed for specific enzyme activities, makingthe generalized identification of improved enzyme variants dif-ficult (21). In addition, these methods work only if the activityof the target enzyme does not interfere with cellular metabo-lism and can be distinguished from the background of all othercell reactions (2).

An alternative selection method is to display libraries ofmutated proteins on phage or microbial cell surfaces and thento select mutant enzymes having desirable properties (4, 25).Enzymes displayed on phage, for example, may be screened forimproved affinity for desired substrates and the correspondingclones selected by panning techniques (22, 25). Recently, alibrary of surface protease OmpT was displayed, and clonesshowing improved substrate affinity were isolated by flow cy-tometry (18). In our study, we have used the ice nucleationprotein (Inp)-based bacterial surface display system (10, 11) toselectively screen enzyme libraries for improved catalytic ac-tivity. Our model enzyme is carboxymethyl cellulase (CMCase).

Because CMC (carboxymethyl cellulose) is a high-molecular-weight polymer, it is not transported into cells. Thus, cells thatdisplay CMCase on their surfaces only hydrolyze CMC in agarplates. Cell colonies that hydrolyze CMC can be easily recog-nized because they are surrounded by a clear halo after stain-ing with Congo red (11). Because Escherichia coli cells display-ing CMCases form tiny colonies on M9 minimal mediumcontaining CMC as the sole carbon source, we reasoned thataltered growth rates of transformants would be correlated withthe activities of displayed CMCase variants. Thus, by selectingrapidly growing colonies, only the cells containing improvedCMCase variants would be isolated and checked further, ob-viating laborious random plating and assay procedures. In thisreport, we describe a growth-based direct screening methodfor the identification of improved CMCases based on the dis-play of an enzyme library on the bacterial cell surface. Thistechnique may provide a useful technology platform for high-throughput selective screening of industrially important hydro-lytic enzymes.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. E. coli JM109 (recA1supE44 endA1 hsdR17 gyrA96 relA1 thi D(lac-proAB) F9 [traD36 proAB1 lacIq

lacZ DM15]) was used as a host cell for DNA manipulations and gene expression.pKK223-3 containing a tac promoter (Amersham Pharmacia Biotech, Uppsala,Sweden) was used so that the expression of the Inp fusion protein or foreignproteins could be induced with isopropyl-b-D-thiogalactoside (IPTG) for high-level gene expression in E. coli. pSSTS110 (10) was employed for surface displayof CMCase. A CMCase gene (endo-b-1,4-glucanase, EC 3.2.1.4) from B. subtilisBSE616 (GenBank accession number D01057) was originated from plasmidpUBS101 (19). Recombinant E. coli cells were grown at 37°C in Luria-Bertani(LB) medium containing yeast extract, 5 g/liter; tryptone, 10 g/liter; and NaCl, 5g/liter. When appropriated, ampicillin was added to a final concentration of 100mg/ml. Cell growth was determined by measuring optical density of the culture at600 nm (OD600) with an Ultraspec 2000 spectrometer (Amersham PharmaciaBiotech).

HPLC analysis of CMC hydrolysates. The products of CMC hydrolysis byCMCase were analyzed using high-pH anion-exchange chromatography (Dionex,Sunnyvale, Calif.). Separation of b-D-glucose (G1) and cellooligomers, such as

* Corresponding author. Mailing address: Bioprocess EngineeringDivision, Korea Research Institute of Bioscience and Biotechnology(KRIBB), P.O.B. 115, Yusong, Taejon 305-600, Korea. Phone: 82-42-860-4483. Fax: 82-42-860-4594. E-mail: jgpan@kribb4680.kribb.re.kr.

788

on July 16, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

cellopentaose (G5), cellotetraose (G4), cellotriose (G3), and cellobiose (G2),was accomplished by using a CarboPac PA1 analytical column (Dionex, 4 by 250mm) and a CarboPac PA1 guard column (4 by 50 mm) with a mobile phasecontaining a mixture of eluent 1 (deionized water), eluent 2 (200 mM NaOH),and eluent 3 (200 mM NaOH, 1 M sodium acetate) at a flow rate of 1.0 ml/min.A PAD system with a gold electrode was used for detection of carbohydrates. ADionex Advanced Computer Interface (ACI) model III was used for data ac-quisition with Dionex AI-450 software, version 3.32. For hydrolysis of cellopen-taose (Sigma, St. Louis, Mo.), a reaction mixture containing 100 ml of 10 mg ofcellopentaose per ml, 20 ml of purified CMCase (0.09 mg/ml), and 80 ml of 50mM sodium phosphate buffer (pH 5.5) was incubated for 120 min at 37°C. Toanalyze the CMC hydrolysate, a reaction mixture containing 100 ml of 10 mg ofCMC per ml, 20 ml of 3 3 108 cells displaying an evolved CMCase variant (2R52)and 80 ml of 50 mM sodium phosphate buffer (pH 5.5) were incubated for 180min at 37°C. The cells were removed by centrifugation before high-pressureliquid chromatography (HPLC) analysis.

Random mutagenesis and enzyme library display. Random mutagenesis of theCMCase gene as shown in Fig. 1 was performed by DNA shuffling as describedpreviously (23, 30). Briefly, the substrates for the shuffling reaction were 1.3-kbdouble-stranded-DNA PCR products derived from pYSK3 by using a recombi-

nant Taq DNA polymerase (TaKaRa Shuzo Co., Shiga, Japan) with two primers,YSK1 and YSK2, which are annealed to the outside of CMCase gene. PCRconditions were 30 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s. Afterdigestion of about 5 mg of the DNA substrates with DNase I (BoehringerMannheim, Dusseldorf, Germany), 50- to 200-bp fragments were recovered on a2% agarose gel and reassembled by PCR without primers by using a PCRprogram of 60 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 65 s. A 50-folddilution of PCR assembled products was used for the final production of singlePCR products of the correct size (1.3 kb) with 30 pmol of each primer and 30additional PCR cycles (94°C for 60 s, 55°C for 60 s, and 72°C for 60 s). For thisPCR amplification, two internal primers, YSK3 and YSK4, which anneal justinside of the first primer set, were used. After successful reassembly and ampli-fication, reactions were verified by 0.8% agarose gel electrophoresis, and theshuffled products were purified with a Wizard PCR Prep Kit (Promega, Madison,Wis.), digested with terminal restriction enzymes, XmaI and HindIII, and sub-cloned into pYSK3. This process produced the plasmids containing the mutatedCMCase genes that were fused to the 39 end of the Inp gene. Plasmids were usedto transform competent E. coli JM109 cells by a high-efficiency transformationmethod (9).

FIG. 1. (A) Schematic diagram of plasmid construction and DNA shuffling procedure of the CMCase gene. The cel and mcel mean the CMCase and matureCMCase gene, and X and H show XmaI and HindIII, respectively. (B) Oligonucleotide primers used for PCR reactions. See the text for detail description.

VOL. 66, 2000 SCREENING OF IMPROVED CELLULASE VARIANTS 789

on July 16, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

Construction of plasmids for surface display and intracellular expression ofCMCase. For surface display of CMCase, the corresponding gene was subclonedinto an Inp surface display vector, pSSTS110, as described below. The 1.3-kbPCR products derived from pUBS101 by using Pfu DNA polymerase (Strat-agene, La Jolla, Calif.) with two primers (CMCF1 and CMCF2) were digestedwith XmaI and HindIII and then ligated with pSSTS110 which had been digestedwith the same enzymes, generating pYSK3. This plasmid contains only the geneencoding the mature form of CMCase from amino acids 31 to 499 and lacks thesignal sequence required for secretion. To achieve intracellular expression of thefree form of CMCase, 1.3-kb DNA fragments of the CMCase gene were ob-tained by 30 cycles of PCR (94°C for 30 s, 50°C for 30 s, and 72°C for 60 s) withtwo primers, YSK7 and YSK8. For correct translation of the CMCase gene in E.coli, the ATG start codon was added as indicated in boldface in Fig. 1B, and theXmaI and HindIII sites are underlined. PCR products were purified, digestedwith XmaI/HindIII, and ligated with pKK223-3 that had been digested with thesame enzymes, resulting in a free-form expression vector, pYSK1.

Selection and screening. Transformants displaying a library of CMCase vari-ants on the surface of E. coli cells were spread on M9 minimal medium platescontaining 0.5% (wt/vol) CMC (Sigma), 1 mM of IPTG (Sigma), 100 mg ofthiamine per ml, and 50 mg of ampicillin per ml (M9-CMC plates). For the firstpositive selection, 150 larger colonies were picked up after a 72-h incubation at37°C and subsequently transferred onto an LB plate containing 100 mg of am-picillin per ml and 1 mM IPTG (LB-Amp-IPTG). Halo-forming activities of thecells were analyzed by the Congo red method (11). After growth for 15 h at 37°C,bacterial colonies were overlaid with 10 ml of sterile top agar containing 0.5%CMC and then incubated at 37°C for 6 h to allow hydrolysis of CMC. After thisincubation, the plates were flooded with 0.2% (wt/vol) Congo red. After 30 min,

the Congo red solution was poured off, and the plates were washed with 10 ml of1 M NaCl for 10 min. Colonies that hydrolyze CMC were identified by yellowhalos, where Congo red staining is absent (20). During the first round of randommutagenesis and selection, 150 colonies were identified that showed highergrowth rates and larger halos than control colonies containing the parent CMCase.

FIG. 2. HPLC chromatogram of CMC hydrolysates. (A) Standard chromato-gram of glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4), andcellopentaose (G5) at a concentration of 2 g/liter. (B) Chromatogram of cello-pentaose hydrolysate produced by treatment with the purified parent CMCase.(C) Chromatogram of CMC hydrolysate produced by treatment with E. coliwhole cells displaying Inp-CMCase fusion proteins.

FIG. 3. Colonies of E. coli JM109 displaying CMCase on their surfaces (mag-nification, 32.5). Parent CMCase (A) and the shuffled CMCase library (B) fromthe first round of mutagenesis are shown. Colonies were formed after 72 h ofgrowth on M9 minimal agar plate at 37°C containing 0.5% (wt/vol) CMC as thesole carbon source, 50 mg of ampicillin per ml, 100 mg of thiamine per ml, and1 mM IPTG. (C) Transformants of the shuffled CMCase library were spread andgrown for 24 h on M9 glucose agar plate at 37°C.

790 KIM ET AL. APPL. ENVIRON. MICROBIOL.

on July 16, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

These colonies were used for the next round of mutagenesis and selection; 1.3-kbfragments of evolved CMCases were amplified by colony PCR and then used asPCR templates for the next rounds. Colony PCR with YSK1 and YSK2 asprimers was performed as described elsewhere (6) under PCR conditions of 30cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 60 s. Three rounds of randommutagenesis and screening were carried out, and 150 to 200 clones from eachround were selected and characterized in detail.

CMCase assay. Whole-cell and free-form CMCase activities were determinedaccording to previous methods (11, 19). Enzymatic reactions were performed for30 min at 37°C with mixing. One unit of enzyme was defined as the quantity ofenzyme capable of releasing 1 mmol of glucose equivalent per min.

SDS-PAGE and Western blot analysis. The expressed enzyme variants wereanalyzed by standard sodium dodecyl sulfate (SDS)–10% polyacrylamide gelelectrophoresis (PAGE) and Western blot with rabbit anti-CMCase antibody.The soluble and insoluble fractions from total expressed CMCase were per-formed by centrifugation method. Briefly, 2.5 3 108 cells from 1 ml of culture(1.0 OD600) were pelleted by centrifugation at 12,000 rpm for 5 min. The cellswere washed twice with 0.85% saline solution and resuspended in lysis buffer (50mM Tris, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride; pH 8.0). Then thecells were lyzed by ultrasonifier 450 (Branson, Danbury, Conn.). The solublefraction was obtained from the supernatant after centrifugation at 12,000 rpm for10 min, while the insoluble fraction was collected from the pellets.

DNA sequencing. The 1.3-kb DNA fragment encoding the evolved CMCaseand its flanking regions was sequenced in both forward and reverse directions byusing a BigDye Terminator Ready-Reaction kit and an ABI Prism 377 DNASequencer (Perkin-Elmer/Applied Biosystems, Foster City, Calif.).

RESULTS AND DISCUSSION

Generation of glucose by surface-displayed CMCases andgrowth of E. coli. The B. subtilis BSE616 CMCase (19) used inthis work is similar to that of Bacillus sp. D04 (7), having bothendo- and exoglucanase activities. Our preliminary HPLCanalysis showed that hydrolysis of CMC by this enzyme pro-duced glucose, cellobiose, and oligosaccharides. The CMCasealso cleaved cellopentaose (G5) to cellotetraose (G4), cellot-riose (G3), cellobiose (G2), and glucose (G1). In order todetermine if Inp-CMCase fusion proteins have the same cat-alytic activity as free-form CMCase, the products of hydrolysiswere analyzed by using a Dionex high-pH anion-exchangeion chromatography system (Fig. 2). Treatment of CMC with

surface-displayed CMCases (Fig. 2C) and purified unfusedCMCases (Fig. 2B) generated the same products, includingglucose, cellobiose, cellotriose, and cellotetraose. The glucoseliberated by hydrolysis of CMC can be used as a carbon sourcefor cell growth. As expected, E. coli cells displaying the non-mutated parent CMCase formed uniform tiny colonies on M9-CMC minimal medium plates, reflecting their homogeneousgrowth rates (Fig. 3A).

E. coli cells displaying a CMCase library form heteroge-neous colonies on CMC plates. Following random mutagenesisof the 1.3-kb CMCase gene by DNA shuffling (Fig. 1), mutatedDNA fragments were subcloned into the Inp surface displayvector, pYSK3. This CMCase library was transformed into E.coli. Transformed cells were spread on M9-CMC minimal me-dium agar plates and incubated for 72 h at 37°C, producing 100to 300 colonies per plate. The library size was more than 1.0 3106 colonies per transformation. E. coli colonies displayingCMCase variants showed heterogeneity in colony size, reflect-ing differences in growth rates (Fig. 3B). Such differences incolony size, which are presumably due to differences in thehydrolytic activity of CMCase variants, were the basis ofscreening for putative improved CMCase variants. Colony sizedid not vary along with varying levels of expression of CMCasevariants. Total cellular level of expressed Inp-CMCase variantfusions in the cell was considered to be constant, which wasconfirmed by SDS-PAGE (data not shown). This might resultfrom the fact that the initiation of transcription and translationof them was started from the completely same genetic ele-ments, e.g., the tac promoter and Shine-Dargarno sequence,and mutated CMCase constitutes only one-third of fusion pro-teins from C-terminal end. Moreover, colonies did not vary insize directly after recovery from transformation. When thetransformants were spread on M9 glucose agar plate, colonieswere formed more homogeneously in size (Fig. 3C), indicatingthat transformation did not affect the colony size. It is notable

FIG. 4. Congo red staining of E. coli JM109 colonies displaying evolved CMCase variants on LB-Amp-IPTG agar plates topped with 0.5% (wt/vol) CMC. (A)Colonies selected from M9-CMC plates showing outgrowth. (B) Colonies randomly chosen from a library of transformants grown on LB-Amp plates. Control coloniesare shown in each photograph. The first control colony is JM109(pYSK3), the second is JM109(pEIN229), and the third is JM109(pKK223-3); all other colonies wereselected as CMCase variants.

VOL. 66, 2000 SCREENING OF IMPROVED CELLULASE VARIANTS 791

on July 16, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

that most colonies on plates containing mutant libraries weresmaller than those containing the parent CMCase, indicatingthat most mutations occurred were deleterious (Fig. 3B).

Rapidly growing colonies produce larger halos on CMCplates. The 150 to 200 colonies originally selected for rapidgrowth on M9-CMC plates were transferred to LB agar platescontaining 0.5% CMC (LBC). These colonies were assayed forCMCase activity by checking their ability to form halos de-tected by staining with Congo red. Congo red reacts with cer-tain polysaccharides, including b-D-glucans and substituted cel-lulose, to form colored dye-polysaccharide complexes, but it

does not bind with glucose or cellooligosaccharides such ascellobiose, cellotriose, or cellotetraose. Thus, after stainingwith Congo red, a clear yellow zone (halo) forms around col-onies that hydrolyze CMC (26). All rapidly growing colonieson the M9-CMC plates produced larger halos on LBC platesthan those displaying the parent CMCase, indicating improvedactivity of surface-displayed CMCases (Fig. 4A). In contrast,Congo red staining assays showed that transformants that wereselected randomly from normal LB-Amp plates produced ei-ther no halos or halos of similar size to those produced by cellsdisplaying the parent CMCase (Fig. 4B). This observationdemonstrates that isolating cells from outgrowing colonies isan essential step in the selection process.

Activities of evolved CMCases. To further improve enzymeactivity, two more rounds of random mutagenesis, display, andselection were performed. The surface enzyme activities of theevolved CMCase variants obtained through three rounds ofDNA shuffling were characterized (Fig. 5A). All the clonesobtained through these selection and screening steps showedhigher hydrolytic activities than clones containing the parentCMCase. Three clones from the first round (1R86, 1R169, and1R186) showed 1.2- to 1.8-fold increases in activity, while threeclones from the second (2R29, 2R33, and 2R59) and the thirdround (3R38, 3R139, and 3R256) showed 2.2- to 3.8-fold and3.2- to 5-fold increased activities, respectively. These resultsdemonstrate that colony outgrowth on M9-CMC plates andformation of larger halos on LBC agar plates were indeed dueto the increased activities of surface-displayed CMCases.

To determine if increased enzyme activity was affected byfusing CMCase to Inp, evolved CMCase genes were excisedfrom Inp and expressed in the cytoplasm. The activities of eachof the unfused CMCases from cell extracts were assayed. Fig-ure 5B shows that specific activities of free-form CMCase vari-ants were much higher than for the parent enzyme. For exam-ple, 2R59 from the second round and 3R256 from the thirdround showed about 1.8- and 2.2-fold higher activities, respec-tively, compared to the parent CMCase (Fig. 5B). It is proba-ble that the extent of improvement of free-form enzymes islower than the improvement of Inp-CMCase fusions becausethe CMCases were selected in the Inp-fused form during theevolution experiments. Nevertheless, the trend toward im-proved activities of evolved enzymes was well correlated inInp-CMCase fusion proteins and unfused CMCases (Fig. 5).Additionally, Western blot analysis showed that very similarlevels of the evolved and parent CMCases were produced (Fig.5C), indicating that changes in the activities of the variantswere not due to the changes in protein expression levels. Inorder to determine whether mutations affected solubility ofenzyme variants, soluble and insoluble fractionation of totalcellular proteins and subsequent Western blotting were per-formed. The results showed that more than 98% of the ex-

FIG. 5. Comparisons of CMCase activities and expression levels of improvedvariants and the parent CMCase. (A) Enzyme activities of the surface-displayedenzymes. (B) Enzyme activities of their corresponding free-form enzymes. (C)Western blot of the soluble fractions of corresponding enzyme variants in sameorder.

TABLE 1. Amino acid substitution and the number of silent mutations of selected CMCase variants

Mutant Amino acid substitutions No. of silent mutations

1R86 K45E, K55R, R83W, I191T, N223Y, T256A, Q350R, N381D 21R169 F91L, I188V, S335L, T342A, T481S 81R186 G123E, I339V, N415D 22R29 A222V, S308P, L315M, S335L, A360S, N368Y, A397T 32R33 N39I, K60R, N244S, G267D, Q357R, I370V, T382I 22R59 D317N, K347N 03R38 K109N, truncated form containing amino acids 31 to 368 53R139 K109N, G123E, S335L, Q357R 23R256 V90I, T260A, T285I, K347R, Y358H, H424Y, A430P 7

792 KIM ET AL. APPL. ENVIRON. MICROBIOL.

on July 16, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from

pressed enzyme variants were soluble (Fig. 5C). Even thoughthe insoluble form of enzymes were detected only in the case ofvariants, 1R186 and 3R256, the amount was less than 2% oftotal expressed enzymes (data not shown).

These results demonstrate that potential structural con-straints caused by fusing CMCase to a large Inp molecule didnot significantly hamper the display of a functional CMCase. Inscreening systems involving secreted enzymes, special devicesare often required to prevent the diffusion of enzymes to othercells (17). Assays of secreted enzymes require expression-nor-malization before the evaluation of changes in specific enzymeactivities (15). Expression levels did not change when enzymelibraries were displayed by fusion to the anchoring motif, Inp.

Sequence analysis. Complete DNA sequences for nine evolvedCMCase genes were obtained and their mutation sites wereidentified. Table 1 summarizes the positions of amino acidchanges in each gene and the number of silent mutations withrespect to the parent CMCase gene. Mutated proteins hadbetween 1 and 8 amino acid substitutions in the 469 aminoacids that make up the mature CMCase. Meanwhile, they alsocontained silent mutations for up to 8 base changes. All thenucleotide substitutions were randomly distributed throughoutthe CMCase gene. Exceptionally, 3R38 was a truncated formcontaining only amino acids 31 to 368 of CMCase. It is possiblethat use of the surface display method may affect the process ofdirected evolution since selections are made on fusion pro-teins. This may explain why Inp fusions of CMCase variantsshowed consistently higher activities than the correspondingfree enzymes. Although it is premature to comment on theevolved CMCase variants without structural information, itshould be interesting to determine whether directed evolutionof enzymes in their fused form generates a characteristic set ofvariants.

Conclusion. The surface display of hydrolytic industrial en-zyme libraries could provide a high-throughput screening en-vironment according to the ability of the cells to grow onnonutilizable substrates as described here. The bacterial sur-face display method would be a technology platform for selec-tion of improved enzymes produced by directed evolution, ifInp-enzyme variants, for example, were functionally displayed.In addition, enzymes such as proteases and lipases, which aretoxic to the cells when expressed in the cytosol, can be selectedfor improved characteristics by using these techniques. Whilemost directed evolution experiments have attempted to im-prove binding of non-natural substrates and/or enzyme stabilityin non-natural environments (2), we have shown here thatoverall catalytic efficiency of cellulase toward its natural sub-strate (cellulose) can be improved under physiological condi-tions.

ACKNOWLEDGMENTS

This work was supported in part by grant NB0470 from the Ministryof Science and Technology of Korea.

We thank S. H. Koh for his help with the HPLC analysis of CMChydrolysates, E. S. Choi for critical reading of the manuscript, andS. H. Park for the CMCase gene and for helpful discussion.

REFERENCES1. Asanuma, S., A. Yamagishi, N. Tanaka, and T. Oshima. 1998. Serial increase

in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillussubtilis by experimental evolution. Protein Sci. 7:698–705.

2. Arnold, F. H., and A. A. Volkov. 1999. Directed evolution of biocatalysts.Curr. Opin. Chem. Biol. 3:54–59.

3. Cowan, D. A. 1991. Industrial enzymes, p. 311–340 In V. Moses and R. E.Cape (ed.), Biotechnology: the science and the business—1991. HarwoodAcademic Publishers, Chur, Switzerland.

4. Georgiou, G., C. Stathopoulos, P. S. Daugherty, A. R. Nayak, B. L. Iverson,and R. Curtiss III. 1997. Display of heterologous proteins on the surface ofmicroorganisms: from the screening of combinatorial libraries to live recom-binant vaccines. Nat. Biotechnol. 15:29–34.

5. Giver, L., and F. H. Arnold. 1998. Combinatorial protein design by in vitrorecombination. Curr. Opin. Chem. Biol. 2:335–338.

6. Gussow, D., and T. Clackson. 1989. Direct clone characterization fromplaques and colonies by the polymerase chain reaction. Nucleic Acids Res.17:4000.

7. Han, S. J., Y. J. Yoo, and H. S. Kang. 1995. Characterization of a bifunctionalcellulase and its structural gene: the cel gene of Bacillus sp. D04 has exo- andendoglucanase activity. J. Biol. Chem. 270:26012–26019.

8. Harayama, S. 1998. Artificial evolution by DNA shuffling. Trends Biotech-nol. 16:76–82.

9. Inoue, H., H. Jojima, and H. Okayama. 1990. High efficiency transformationof Escherichia coli with plasmids. Gene 96:23–28.

10. Jung, H. C., J. M. Lebeault, and J. G. Pan. 1998. Surface display of Zy-momonas mobilis levansucrase by using the ice-nucleation protein of Pseudo-monas syringae. Nat. Biotechnol. 16:576–580.

11. Jung, H. C., J. H. Park, S. H. Park, J. M. Lebeault, and J. G. Pan. 1998.Expression of carboxymethylcellulase on the surface of Escherichia coli usingPseudomonas syringae ice nucleation protein. Enzyme Microb. Technol. 22:348–354.

12. Kast, P., and D. Hilvert. 1997. 3D structural information as a guide toprotein engineering using genetic selection. Curr. Opin. Struct. Biol. 7:470–479.

13. Kuchner, O., and F. H. Arnold. 1997. Directed evolution of enzyme catalysts.Trends Biotechnol. 15:523–530.

14. MacBeath, G., P. Kast, and D. Hilvert. 1998. Redesigning enzyme topologyby directed evolution. Science 279:1958–1961.

15. Miyota, Y., S. Komada, H. Momose, and S. Taguchi. 1998. Quantitative assaysystem for specific enzyme activity using antibody: the case of protease,subtilisin BPN9. J. Biotechnol. 66:157–163.

16. Moore, J. C., and F. H. Arnold. 1996. Directed evolution of a para-nitro-benzyl esterase for aqueous-organic solvents. Nat. Biotechnol. 14:458–467.

17. Naki, D., C. Paech, G. Ganshaw, and V. Schellenberger. 1998. Selection of asubtilisin-hyperproducing Bacillus in a highly structured environment. Appl.Microbiol. Biotechnol. 49:290–294.

18. Olsen, M., G. Georgiou, and B. Iverson. 1998. Display of enzyme libraries onE. coli: screening of combinatorial libraries by FACS. IBC’s Second Inter-national Symposium on Directed Evolution of Industrial Enzymes, San Di-ego, Calif.

19. Park, S. H., H. K. Kim, and M. Y. Pack. 1991. Characterization and structureof the cellulase gene of Bacillus subtilis BSE616. Agric. Biol. Chem. 55:441–448.

20. Park, S. H., and M. Y. Pack. 1986. Cloning and expression of a Bacilluscellulase gene in Escherichia coli. Enzyme Microb. Technol. 8:725–728.

21. Skandalis, A., L. P. Encell, and L. A. Loeb. 1997. Creating novel enzymes byapplied molecular evolution. Chem. Biol. 4:889–898.

22. Soumillion, P., L. Jespers, M. Bouchet, J. Marchand-Brynaert, P. Sartiaux,and J. Fastrez. 1994. Phage display of enzymes and in vitro selection forcatalytic activity. Appl. Biochem. Biotechnol. 47:175–189.

23. Stemmer, W. P. 1994. Rapid evolution of a protein in vitro by DNA shuffling.Nature 370:389–391.

24. Tomme, P., R. A. J. Warren, and N. R. Gilkes. 1995. Cellulose hydrolysis bybacteria and fungi. Adv. Microb. Physiol. 37:2–81.

25. Wang, C. I., Q. Yang, and C. S. Craik. 1996. Phage display of proteases andmacromolecular inhibitors. Methods Enzymol. 267:52–68.

26. Wood, P. J., J. D. Erfle, and R. M. Teather. 1988. Use of complex formationbetween Congo red and polysaccharides in detection and assay of polysac-charide hydrolases. Methods Enzymol. 60:59–74.

27. Yano, T., S. Oue, and H. Kagamiyama. 1998. Directed evolution of anaspartate aminotransferase with new substrate specificities. Proc. Natl. Acad.Sci. USA 95:5511–5515.

28. Zaccolo, M., and E. Gherardi. 1999. The effect of high-frequency randommutagenesis on in vitro protein evolution: a study on TEM-1 beta-lactamase.J. Mol. Biol. 285:775–783.

29. Zhang, J. H., G. Dawes, and W. P. Stemmer. 1997. Directed evolution of afucosidase from a galactosidase by DNA shuffling and screening. Proc. Natl.Acad. Sci. USA 94:4504–4509.

30. Zhao, H., and F. H. Arnold. 1997. Optimization of DNA shuffling for highfidelity recombination. Nucleic Acids Res. 25:1307–1308.

VOL. 66, 2000 SCREENING OF IMPROVED CELLULASE VARIANTS 793

on July 16, 2018 by guesthttp://aem

.asm.org/

Dow

nloaded from