Characterization of a chitinase gene (ch/A) from Serratia marcescens BJL200 and one-step purification of the gene product

Keywords: Serratia marcescens, Chitinase, Hydrophobic interaction chromatography

The nucleotide sequence of the chiA gene from Serratia marcescens strain BJL200 was determined. The gene was found to encode a protein of 563 amino acid residues, with a typical N-terminal signal peptide of 23 residues, that is cleaved off during export. The gene exhibited striking differences with two previously characterized chiA genes of S. marcescens in the region corresponding to amino acid residues 410-467 of the gene product. Periplasmic fractions of an Escherichia coli strain harbouring the cloned gene were used as starting material for the development of a fast, one-step purification protocol for the chitinase that is based on hydrophobic interaction chromatography. 

Introduction

The Gram-negative enterobacterium Serratia marcescens produces several chitinolytic enzymes [1] and is one of the most efficient bacteria for the biological degradation of chitin [2]. Chitinolytic enzymes are of biotechnological interest, since their substrate, chitin, is a major structural component of fungal cell walls. Thus, chitinolytic enzymes could in principle be employed as natural anti-fungal agents, for example by expressing their genes in crop plants or in bacteria used in fermentation processes prone to fungal attack [3,4]. Sundheim et al. [5] cloned two chromosomal fragments encoding chitinolytic (and antifungal) activity from S. marcescens BJL200. In the present study we have analysed one of these fragments and determined the nucleotide sequence of the chitinase gene located on it. 
Protein engineering could be used to improve the stability and activity of the naturally occurring Serratia chitinases, in order to improve their applicability. Efficient protein engineering requires simple and fast procedures for purification, that permit the rapid processing of large numbers of mutant proteins. Therefore, we have developed a fast, one-step procedure for purification of the chitinase from periplasmic fractions of E. coli harbouring the gene characterized in the present study. 

Materials and Methods


Bacterial strains and plasmids

Escherichia coli DH5a [6] was grown at 37°C in Luria Broth (LB). For plates, LB medium was solidified with 1.5% agar. The chitinase encoding plasmid pLES4 has been described before [5]. Plasmid pGEM®-7f(+)Z was obtained from Promega (Madison, WI). Escherichia coli strains containing these plasmids were grown in the presence of 50/~g ml-1 ampicillin. 

Chitinase assays

Chitinase activity was detected using chitincontaining agar plates and quantified using 4methylumbelliferyl-#-o-N,N'-diacetyl-chitobioside (4-MU-(GIcNAc) 2) as described previously [4]. 

DNA techniques

Recombinant DNA work was performed, using standard protocols [7]. Subclones for sequencing were obtained by restriction endonuclease deletions or by using the Promega Erase-a-Base system. pUC/M13 forward and reverse primers were primarily used as primers for sequencing. To cover gaps and/or some regions showing high compression, additional oligonucleotide primers were synthesized. Ambiguous sequences due to band compression were resolved by running formamide gels (40%) as described by the supplier of the sequencing chemicals (USB Comments 17(1), 1990). 

Chitinase purification

E. coli cells harbouring pMAY20-1 were grown overnight to early stationary phase and the cells were fractionated by osmotic shocking [8]. The periplasmic extract (1/10 of the original culture volume) was diluted 1.4 times and adjusted to 0.65 mM MgC12, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM EDTA, 20 mM Tris. HC1 (pH 8.0), 0.5 M ammonium sulfate. 2 ml was loaded onto a phenyl-superose HR 5/5 column (5 x 50 mm; Pharmacia-LKB, Uppsala, Sweden), which was inserted into an FPLC chromatography system (Pharmacia-LKB) and equilibrated with buffer A (20 mM Tris-HC1 (pH 8.0), 1 mM EDTA, 0.1 mM PMSF, 0.5 M ammonium sulfate). The column was washed by applying a 10-ml linear gradient from 100% buffer A to 100% buffer B (20 mM Tris. HC1, pH 8.0, 1 mM EDTA, 0.1 mM PMSF, 4% (v/v) isopropanol), followed by several ml isocratic flow at 100% buffer B to elute the chitinase. Chromatography was performed at room temperature, using a flow rate of 0.2 ml min-1. Protein concentrations were determined by the method of Bradford, using BSA as a standard. 
Purity of chitinase samples was analysed using standard SDS-PAGE and size exclusion chromatography on a Superose-12 column (Pharmacia-LKB), equilibrated in 75 mM ammonium bicarbonate (pH 8.0), using a flow rate of 0.35 ml rain -~, in an FPLC chromatography system, at room temperature. The N-terminal amino acid sequence of the purified protein was determined using an Applied Biosystems automatic sequencer, model 477A. The purified chitinase was hydrolysed and analysed on an amino acid analyser as described [9]. 

Results and Discussion


Subcloning and nucleotide sequence of the chitinase gene

Previously, plasmid pLES4 consisting of an 18-kb EcoRI chromosomal fragment from S. marcescens BJL200 in pBR325, was shown to confer chitinase activity to E. coli [5]. The region encoding the chitinolytic activity was localized to a 4-kb EcoRI-HindIII fragment, by constructing deletion clones of pLES4 and testing the ability of these clones to confer chitinase activity to E. coli (results not shown). The fragment was subcloned into the high copy number vector pGEM®-7f( + )Z, resulting in plasmid pMAY20-1, and sequenced. 
The nucleotide sequence for 2698 bp of S. marcescens DNA is shown in Fig. 1. An open reading frame (ORF) was identified between nucleotides 413 and 2141. This ORF contains a start The deduced amino acid sequence of the chitinase A, shown below the nucleotide sequence, is numbered on the right-hand side. Amino acid residues that are different in the chitinase A sequences reported in [11] and [12] are given in small types italic and small types, respectively, under the deduced amino acid sequence. The signal peptide cleavage site is indicated by a vertical arrow. The sequence of the chi.A gene has been submitted to the EMBL Data Library and has been assigned the accession number Z36294. 
TCGAGGATTATTGCGGAATTTTTTCGCTTCGGCAATGCATCGCGACGATTAACTCTTTTATGTTTATCCTCTCGGAAT~AAGGAATCAGTTATGCGC~T~CCGCTG~GCM R K ¥ N K P L L A i0 481 GCTGTTGATCGGCAGCACGCTGTGTTCCGCGGCGCAGGCCGCCGCGCCGGGCAAGCCGACCATCGCC TGGGGCAACAC CAAGTTCGCCATCGTTGAAGTTGACCAGGCGGCTACCGCTTA L L I G S T L C S A A Q AtA A P G K P T I A W G N T K ¥ A I V 8 V D Q A A T A Y 50 601 TAATAGTTTGGTGAAGGTA~TGCCGCC GATGTTTCGGTCTCCTGGAATTTATGGAATGGCGACACCGGTACGACGGCAAAAGTTTTATTAAATGGCAAAGAGGCGTGGAGCGGCCC N S L V K V K N A A D V S V S W N L W N G D T G T T A K V L L N G K 8 A W S G P 90 n a t t n a g p 721 GTCAACCGGTTCTTCCGGTACGGCGAATTTTAAAGTCAATAAAGGC GGCCGTTATCAAATGCAGGTGGCATTGTGCAATGCCGACGGC TGCAGCGCCAGCGATGCCACCGAAATTGTGGT S T G S S G T A N F K V N K G G R Y Q M Q V A L C N A D G C S A S D A T E I V V 130 t t 841 GGCC GACACC GACGGCAGCCATTTGGCGCCGTTGA~GCCGC TGCTGGAAAAGAATAAACCGTATAAACAGAACTCCGGCAAAGTGGTCGGTTCTTATTTCGTC GAGTGGGGCGTTTA A D T D G S H L A P L K E P L L E K N K P Y K Q N S G K V V G S Y F V E W G V Y 170 a P 961 CGGGCGCAATTTCACCGTCGACAAGATCCCGGCGCAGAACCTGACCCACC TGC TGTAC GGCTTTATCCCGATCTGCGGTGGCAACGGCATCAAC GACAGCCTGA~GAGATCGAAGGCAG G R N F T V D K 1 P A Q N L T H L L Y G ¥ I P I C G G N G I N D S L K E I E G S 210 1081 CTTCCAGGCGCTGCAGCGCTCCTGCC~GGCCGCGAGGAC TTCAAAGTCTCGATCCACGATCCGTTCGCCGCGCTGCAAAAAGCGCAGAAGGGCGTTACCGCCTGGGATGAC CCC TACAA ¥ Q A L Q R S C Q G R E D F K V S I H D P F A A L Q K A Q K G V T A W D D P Y K 250 1201 GGGCAAC TTCGGCCAGCTGATGGCGCTGAAACAGGCGCATCCTGACCTGAAAATTCTGCC GTCGATCGGCGGCTGGACGCTGTC CGACCCGTTCTTCTTCATGGGC GATAAGGTGAAGCG G N F G Q L M A L K Q A H P D L K I L P S I G G W T L S D P F F F M G D K V K R 290 1321 CGATCGCTTCGTCGGTTCGGTGAAAGAGTTCCTGCAGACCTGGAAGTTCTTCGATGGC GTGGATATCGACTGGGAGTTCCCGGGCGG~GGCGCCAACCC GAACC TGGGCAGCCCGCA D R F V G S V K E F L Q T W K F F D G V D I D W E F P G G K G A N P N L G S P Q 330 1441 GGAC GGGGAAAC CTATGTGCTGCTGATGAAGGAGCTGCGGGC GATGCTGGATCAGCTGTCGGCGGAAACCGGCCGCAAATATGAACTGAC CTC TGCCATCAGC GC CGGCAAGGACAAGAT D G E T Y V L L M K 8 L R A M L D Q L S A 8 T G R K Y E L T S A I S A G K D K I 370 1561 CGACAAGGTGGCTTACAACGTTGCGCAGAACTCGATGGATCACATCTTCCTGATGAGCTAC GACTTCTATGGCGCC TTC GATCTGAAGAACCTGGGGCATCAGACCGCGCTGAATGCGCC D K V A Y N V A Q N S M D H I F L M S Y D F Y G A F D L K N L G H Q T A L N A P 410 r r 1681 GGCCTG~GCCG~CACCGC'P~A~.CCACGGTG~"CGGCGTC~TGCGCTGCTGGC~GGGCGTC~.GCCGGGCAA~TCGTGGTCGG~.CCGCCATGTATGGCCGcGGC TG~C CGG A W K P D T A Y T T V N G V N A L L A Q G V K P G K I V V G T A M Y G R G W T G 450 p g s r h r 1 h h g • r r q c a a - g p g s r h r 1 h h g e r r e c a a - g 1801 GGTG~.C GG C TAC CAG~t~.ACA'PI~C C G'PI~C~CTGG TAC ~C P~.C C G GGC C ~ G'I~i~GGCAC C TGGGA~,.AC GGCAT C GTG~C TAC C GC C/~ATC G C CGGC CAG TT~T~u~GC GG C Gd~- V N G Y Q N N I P F T G T A T G P V K G T W E N G I V D Y R Q I A G Q F M S G E 490 a t g p k g h r a - e s 1921 GTGGCAGTATACCTACGACGCCACGGCGGAAGCGCCTTACGTGTTCAAACCTTC CAC CGGCGATCTGATCAC CTTCGACGATGC CCGCTCGGTGCAGGC CAAAGGCAAGTACGTGCTGGA W Q Y T Y D A T A 8 A P Y V F K P S T G D L I T F D D A R S V Q A K G K Y V L D 530 2041 TAAGCAGCTGGGCGGCCTGTTCTCCTGGGAGATCGACGCGGATAACGGCGATATTCTCAACAGCATGAAC GCCAGCCTGGGCAACAGC GC CGGCGTTCAATAATCGGTTGCAGTGCGTTG --563 K Q L G G L F S W E I D A D N G D I L N S M N A S L G N S A G V Q * 2161 CCGGGGGATATCC~TCGCCCCCGGCT~TTTCGCCGC~TTTTTTACGCCGCACAGATTGTGGCTCTGCCCCGAGCA*~`ACGCGCTCA~CGGACTCACCCTTTTGGGTAATCC~
codon (ATG) at nucleotide position 452 which is preceded by a putative Shine-Dalgarno sequence (AAGGA). The ORF could thus encode a protein of 563 amino acids with a molecular mass of 60 943 Da. The N-terminal 23 amino acids of the chitinase have the typical features of a signal peptide [10]. N-terminal amino acid sequencing of the purified chitinase gave a sequence corresponding to residues 24-31 of the protein encoded by the identified ORF, which confirms that this ORF encodes the chitinase and that the enzyme is processed between residues 23 and 24 during translocation. The deduced amino acid sequence of the chitinase exhibited 95 and 96% identity with chitinases encoded by the chiA genes of two other S. marcescens strains [11,12]. Comparison of the coding regions of the three chiA genes showed that the chitinase gene described here contains three nucleotide insertions (positions 1680, 1693 and 1738) compared to both chiA genes [11,12], and another three nucleotide insertions (positions 1841, 1842 and 1852) compared to the chiA sequence reported in [11] only (Fig. 1). These insertions all concern G or C and occur in parts of the sequence with a high G + C content. As a consequence of the insertions, the chitinase described here contains two additional amino acids and two stretches of amino acid residue changes (residues 410-429 and 464-467), compared to the chitinase reported in [11] (Fig. 1). The chitinase sequences deduced from the previously published genes contain a high number of arginines and histidines in the region between amino acid residue 410 and 423. The chitinase deduced from the gene described here does not contain this remarkable cluster of positively charged residues, which was confirmed by amino acid analysis of the purified protein (results not shown). In addition to the nucleotide insertions, the coding region of the present gene differs at 52 and 42 positions from the coding regions of the chiA genes reported in references [11] and [12], respectively (not shown). Of these nucleotide substitutions, 7 and 4 result in amino acid residue differences between the chitinases (Fig. 1). 

Fast purification of the chitinase produced by E. coli (pMA Y20-1)

Early stationary cultures of E. coli(pMAY20-1) contained approximately equal amounts of chitinase in the supernatant and in the periplasmic fraction obtained by osmotic shocking of the cells. Pilot experiments showed that ammonium sulfate-precipitated culture supernatant (20 times concentrated) was not optimal as starting material for the purification: large amounts of (partly non-proteinaceous) material absorbing at 280 nm remained associated with the chitinase in various purification steps (results not shown). 
The use of periplasmic extract as starting material permitted the purification of the chitinase by one, fast hydrophobic interaction chromatography step (Fig. 2). The chromatography procedure was optimized by using a high resolution column, minimizing the ammonium sulfate concentration in the starting buffer, and identifying the optimal concentration of isopropanol in buffer B. A typical chromatographic run, with a sample size corresponding to 14 ml of the original culture volume, yielded 150-200/xg pure protein, with a recovery of 70-90% (Table 1). SDS-PAGE showed that the chitinase peak contained a highly pure protein of approximately 62 kDa (Fig. 3). The material eluted from the phenyl-superose column gave one symmetrical peak on a flat baseline upon size-exclusion chromatography, confirming the homogeneity of the material (results coli(pMAY20-1). Chromatography of periplasmic extract of E. cofi(pMAY20-1) on a 1-ml phenyl-superose column was performed as described in Materials and Methods. At the point indicated by the arrow the sensitivity of the UV detector was increased by a factor two. All fractions eluted from the column were tested for chitinase activity, which was only found in the sharp peak eluting at 18 ml. not shown). Similar results were obtained when the standard buffers A and B in the hydrophobic interaction chromatography step were replaced by 25 mM ammonium bicarbonate, 0.5 ammonium sulfate, and 25 mM ammonium bicarbonate, 4% (v/v) isopropanol, respectively (chro-1 2 3 4 5 
403 matograms not shown). The use of this buffer system permits direct freeze-drying of the purified enzyme (Fig. 3; lanes 4 and 5). The purification protocol described here consists of only one, fast chromatography step and yields highly pure protein that can easily be concentrated by freeze-drying. The method is complementary to a more complex, large-scale protocol that was used to purify chitinase A from ammonium sulfate-precipitated culture supernatant of an E. coli strain harbouring chiA from S. marcescens QMB 1466 [11,13]. The three-dimensional structure of the latter enzyme is currently being determined [13]. The availability of chitinase A-producing E. coli strains, the crystal structure of a chitinase A, a large-scale purification protocol, and the fast high-performance purification method described above forms an excellent starting point for protein engineering studies. 



  TGCGAAGGCATGC TC TTTC C TTAAGTGTTGTTCACAGTAC CAACACAGTCAGGG TGTTG TCAAT~ATGACAACAC C C 121 TGAC TGCAGAGTGTGGTACAATCC TGATAAATATTTATCTTTC C TTAATAAAAAATTCAC TATC CATATTTGTC GTGCTTTC TTTTATTTATATTAAAATAAATTCAC GCTTGCTGAATA 241 AAAC C CAGTTGATAGC GCTCTTGT~CAC GC C TT~TATTTATAGTC TGAATGTACGC GGTGG C~AATGATTATTTC GCCAC GTGGAAAGAC GC TGTTGC TA~A~TCT 361 


Fig. 1 .

 1 Fig.1. Nucleotide sequence of the chitinase encoding region in pLES4. The sequence is numbered on the left-hand side. A potential ribosome-binding site is double underlined. A putative promoter region, outlined on the basis of transcription initiation analysis[11], is underlined. A putative transcription terminator (ziG = -25,4 kcal mol-1) 18 bases downstream of the TAA stop codon is marked with inverted arrows. Nucleotide insertions (as compared to other chiA genes; see text) are marked with solid triangles. The deduced amino acid sequence of the chitinase A, shown below the nucleotide sequence, is numbered on the right-hand side. Amino acid residues that are different in the chitinase A sequences reported in[11] and[12] are given in small types italic and small types, respectively, under the deduced amino acid sequence. The signal peptide cleavage site is indicated by a vertical arrow. The sequence of the chi.A gene has been submitted to the EMBL Data Library and has been assigned the accession number Z36294. 


Table 1

 1 Hydrophobic interaction chromatography of S. marcescens chitinase on a phenyl-superose column 


		Volume	Total protein	Total activity a	Recovery of	Specific activity
		(/zl)	(/zg)	(/xmol min-l)	activity (%)	(/zmol min-1 mg i)
	Diluted periplasmic extract	2000	1168	2.8	100	2.4
	Peak fractions	880	166	2.2	79	13.3

a Chitinase activity is expressed in amount of 4-MU released per minute. 
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Acknowledgements

We thank Leif Sundheim for providing us with pLES4 and Knut Sletten for amino acid analysis and sequencing of the purified chitinase. This work was supported by a grant from the Norwegian Research Council. 


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Fig. 3. SDS-PAGE analysis of the purification of the chitinase produced by E. coli(pMAY20-1). Lane 1, undiluted periplasmic extract of E. coli(pMAY20-1); lanes 2 and 3, fractions taken from the peak at 18 ml in Fig, 2; lanes 4 and 5, fractions corresponding to those in lanes 2 and 3, but obtained after hydrophobic interaction chromatography with ammonium bicarbonate buffers (see text) followed by concentration via freeze-drying. The positions and molecular masses (in kDa) of protein size markers are indicated on the left.
May B Brurberg, Vincent G H Eijsink, Ingolf F Nes
Characterization of a chitinase gene (ch/A) from Serratia marcescens BJL200 and one-step purification of the gene product