Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE11-1 and its effect on the production of keratinase in Bacillus subtilis

Accepted Manuscript Title: Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE11-1 and its effect on the production of keratinase in Bacillus subtilis Author: Baihong Liu Juan Zhang Zhen Fang Guocheng Du Jian Chen Xiangru Liao PII: DOI: Reference:

S1359-5113(14)00269-4 http://dx.doi.org/doi:10.1016/j.procbio.2014.04.021 PRBI 10132

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

18-12-2013 4-4-2014 30-4-2014

Please cite this article as: Liu B, Zhang J, Fang Z, Du G, Chen J, Liao X, Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE111 and its effect on the production of keratinase in Bacillus subtilis, Process Biochemistry (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highlights

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Optimizing the C-terminus of propeptide will affect the cleavage efficiency of propeptide.

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Inserting linkers and deleting residues at P2 position decreases the mature keratinase production.

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The primary structure of C-terminus propeptide is crucial for the mature keratinase production.

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Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE11-1

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and its effect on the production of keratinase in Bacillus subtilis

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Baihong Liua, e, Juan Zhangb, e*, Zhen Fanga, e, Guocheng Dud, e, Jian Chenc, e, Xiangru Liaob, e*

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a Synergetic Innovation Center of Food Safety and Nutrition, Wuxi 214122, China

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b Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

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c National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi

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214122, China

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d The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan

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University, Wuxi 214122, China

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e School of Biotechnology, Jiangnan University, Wuxi 214122, China

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* Corresponding authors: Xiangru Liao, Tel.: +86-510-85913661, Fax: +86-510-85910799, E-mail:

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[email protected]. Juan Zhang, E-mail: [email protected];

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Abstract

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The keratinase from Bacillus licheniformis BBE11-1 is a serine protease and expressed as a

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pre-pro-precursor. To produce a mature and active keratinase, the propeptide must be cleaved on the

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C-terminal via cis or trans. In this study, to enhance the production of keratinase in Bacillus subtilis,

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single amino acid substitutions, single residue deletions and linkers were introduced at the C-terminus

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of the propeptide. The results showed that optimize the residue of cleavage site of propeptide will affect

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the cleavage efficiency of propeptide, and the mature enzyme yield of Leu(P1)Ala mutant increases

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50% compared with the wild-type. In addition, inserting linkers and deleting individual residues at the

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C-terminal of the propeptide decreases the mature keratinase production. Our results indicated that the

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primary structure of the C-terminus of propeptide is crucial for the mature keratinase production.

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Propeptide engineering at C-terminus may be an effective approach to increase the yield of keratinase.

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Keywords: Keratinase, Bacillus licheniformis, Propeptide, Cleavage efficiency

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1. Introduction

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Keratinases are serine and metalloproteases enzymes hydrolyze insoluble keratins [1]. Many

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keratinases can degrade feathers into amino acids and soluble peptides, which is a potential application

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instead of expensive food crops in the field of animal feed production [2]. In the textile processing

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industry, keratinases can be used to replace conventional pollution-generating physicochemical

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methods, impart shrink-resistance, and efficiently improve the handling properties [3]. Furthermore,

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keratinases are used to manufacture detergents [4], medicines [5], cosmetics [6], and leather. Their

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novel applications such as decontaminating materials by degrading prion are also reported [7].

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Keratinase from Bacillus licheniformis BBE11-1 (GenBank accession nos. JX504681) is a serine

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protease which homology with subtilisin E, subtilisin BPN’ and the kerA [8]. Like subtilisins, it

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displays in a pre-pro-form, with a propeptide before N-terminal region of the mature protein domain.

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The propeptide is essential as an intermolecular chaperone that guides correct folding of the mature

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domain in three steps of the activation process [9-10]: (1) Assistance in the correct folding of the

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mature enzyme domain as an intramolecular chaperone, (2) Autoprocessing and changing the structure

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of the mature enzyme domain, and (3) Temporary binding to the mature enzyme domain before degradation via cis [11] (the mature enzyme cleaves its own propeptide) or trans [12] (the mature enzyme cleaves the propeptide of another molecule). To form a mature and active protease, degradation is required, because the propeptide can inhibit the

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active site of mature enzyme and form a stable and inactive propeptide-enzyme complex [13].

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Therefore, propeptide release is an important rate-determining step for mature keratinase production

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[13-14]. Before degradation, the propeptide must be cleaved on the C-terminal side of residue at P1

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position. Thus, the C-terminal of propeptide may plays an important role for mature enzyme

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production. In this study, to enhance the production of keratinase in Bacillus subtilis, and investigate the effect of

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C-terminal structure of propeptide on the mature enzyme secretion, the residue at P1 position is

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replaced with different types of residues to investigate the function of Leu(P1) on effective cleavage of

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the propeptide and its effect on the yield of mature keratinase. In addition, inserting linkers of different

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lengths and deleting individual residues at the C-terminal of the propeptide are performed to analyze

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the effect on the mature keratinase production caused by changes of primary structure at the C-terminus

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of propeptide.

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2. Materials and methods

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2.1. Bacterial strains, plasmids and culture conditions

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The Escherichia coli strain JM109 was purchased from TaKaRa (TaKaRa, China). E. coli cells with

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plasmids were cultured aerobically at 37 °C in Luria-Bertani medium containing 100 μg/mL ampicillin.

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Bacillus licheniformis BBE11-1 (GenBank accession no. JQ894491) was used as the source of genomic

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DNA. The strain Bacillus subtilis WB600 and the vector pMA5 [15] were used for the expression of

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keratinase. The pMD19-T vector (TaKaRa, China) was used for ker (keratinase) gene clone. 2.2. Site-directed mutagenesis

Site-directed mutagenesis was performed using the MutanBEST kit (TaKaRa, China). A one-step PCR method was carried out using the PrimeSTAR HS DNA polymerase (TaKaRa, China), the plasmid

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ker/pMD19-T as a DNA template DNA, and oligonucleotide primers. To ensure propeptide cleavage

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under the same conditions, different linkers (GGG, GGGG, GGGGS, PTPPTTPT, namely, ins3G, ins4G,

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ins4GS and insPT, respectively) and three deletion-mutations (deletion of residues at the P2 to P4

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position; named del-A, del-AH and del-AHA, respectively) were constructed at the Ala(P2) position of

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the propeptide. The sequences of the mutagenic primers are shown in Table 1. The PCR products were

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treated with a blunting kination enzyme and Ligation Solution I (TaKaRa, China), ligated into circular

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plasmids, and then transformed into E. coli JM109. The successful introduction of the desired

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mutations was confirmed by DNA sequencing (Sangon, China).

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2.3. Construction of expression vector

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Ker was amplified from the pMD 19-T-keratinase vector, and the Nde I and Bam HI cloning sites were

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introduced into the primers to permit ligation into the pMA5 vector. The 5’ primer was 5’-

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GGAATTCCATATGATGAGGAAAAAGAGTTTTTGG, and the 3’ primer was 3’-

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CGCGGATCCTTATTGAGCGGCAGCTTCG. The ligation mixture was used to chemically transform

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competent Escherichia coli JM109. The plasmids isolated from these transformants were confirmed by

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DNA sequencing, and the plasmid with the correct sequences was designated pMA5-ker. B. subtilis

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WB600 was chosen as the host and transformed by pMA5-ker plasmid. After 12 h of incubation at

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37°C on LB agar containing 20 μg/ml kanamycin, transformants were confirmed by colony PCR and

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DNA sequencing, and were used for further expression and purification.

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2.4. Expression, purification of keratinase To confirm the expression of keratinase, wild-type (WT) and different mutated transformants with pMA5-ker plasmids were cultured in a 250 mL shake flask containing fermentation medium (yeast extract, 5 g/l; peptone, 10 g/l; NaCl, 10 g/l; glucose, 10 g/l; MgSO4, 0.1 g/l) at 37°C for 24 h. And then,

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the culture broth was centrifuged at 8000 g for 10 min, and the keratinase was concentrated by

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ultrafiltration using a 10 kDa membrane (Pellicon® XL filter; Millipore corporation, USA).The enzyme

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solution was then injected into an AKTA purifier (GE Healthcare, USA) through Phenyl Sepharose Fast

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Flow columns (5 ml) for hydrophobic interaction chromatography (HIC) (GE Healthcare, USA). After

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eluting the unbound proteins, a gradient elution was performed using ammonium sulfate (from 1 to 0

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M), and the fractions were collected for the specific activity assay and SDS-PAGE analysis. Total protein concentration was determined by a BCA assay kit (Tiangen, China) using bovine serum

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albumin as a standard. Proteins were analyzed on 12% polyacrylamide gels under denaturing

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conditions.

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2.5. Determination of keratinase activity

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The keratinase activity was determined according to the modified method of Yamamura et al [16]. A

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portion (0.5 ml) of the enzyme solution was incubated with 1.5 ml of 10 g/l keratin (J&K, China) in 50

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mM Gly/NaOH buffer (pH 10.5) at 40°C for 15 min. The reaction was terminated with 2 ml of 60 g/l

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trichloroacetic acid (TCA) and then allowed to stand for 10 min. After centrifugation (Tomy MRX-152,

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Japan) (15,000 g; 10 min), the supernatant (0.5 ml) was mixed with Bradford reagent (1:1 dilution) and

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2.5 ml of 0.5 M Na2CO3 at 40°C for 15 min. The keratinase activity was measured at 660 nm with a

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spectrophotometer (Beckman DU640, USA) and is expressed in keratinase units. One unit is defined as

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an increase of 0.01 OD value at 660 nm in 15 min.

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In order to estimate the amount of mature enzyme in the supernatant fractions of WT and its mutants,

the specific activity of supernatant was determined using keratin as substrate and calculated as U/mg protein (total protein) [17]. 2.6 Statistical analysis

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Data Analysis was used OriginPro 8.0 and the statistical method was T-test. The data were the average

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value of three independent experiments.

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3. Results and discussion

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3.1. Keratinase production of WT and its mutants

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All keratinase mutants, except the del-AHA mutation, secreted a mature keratinase (Fig. 1) with a size

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similar (30 kDa) to the WT enzyme. The keratinase specific activities of WT and its mutants were

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assessed (Fig. 2). Out of all mutants, only the specific activity of the L(P1)A mutant is higher (about

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50%) than that of the WT. In particular, the specific activities of L(P1)R, del-AHA, ins4GS and insPT

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mutants are only 10-30% compared with WT.

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3.2. Effects of Leu(P1) mutants on the production of keratinase

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“Pro-sequence engineering” is a useful tool for producing enzymes with novel beneficial functions

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by inducing conformational changes in the mature enzyme [14]. Mutation at appropriate sites in the

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propeptide can alter the rate of folding and thereby accelerate the maturation [18], improve the

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expression level [19] or alter the mature enzyme structure [20]. These results indicate that

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“pro-sequence engineering” would be useful for improving the functions of autoprocessing proteases

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[14].

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Based on these functional goals, propeptides with different amino acids substituted at the cleavage

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site at Leu(P1) were constructed to improve the expression level of mature keratinase. To optimize this

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residue, nine amino acid residues (differing in hydrophobicity, hydrophilicity, side chain length, and charge) were substituted. Specifically, Leu(P1), a hydrophobic amino acid, was replaced by a hydrophilic Arg residue (positively charged, long side chain), His residue (positively charged), Asp and Glu residues (negatively charged), Gly residue (uncharged, simplest structure), Ile residue

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(hydrophobic, long side chain), Ala residue (only one methyl in side chain), Phe as well as Tyr residues

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(aromatic, large side chain). All mutants performed normal autoprocessing and secreted mature enzyme

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(Fig. 1a). Thus, alteration of the residue at the propeptide cleave site (P1) did not disturbs the formation

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of mature keratinase. Leu(P1) residue is held in the S1 pocket of the mature region in a product-like

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manner [21]. The S1 binding pocket is a distinct, large, and elongated cleft that mainly consists of

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glycine residues [21], which may explain why large side chains (Arg, Ile, Phe, Tyr) or small side chains

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(Ala, Gly, Asp) could still cleave normally. However, different residues resulted in inconsistent

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production of mature keratinase. Interestingly, introduction of the Ala(P1) residue showed a 50%

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increase of keratinase specific activity compared with the WT (Fig. 2a).

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To investigated the changes of mature keratinase take place in the Leu(P1)Ala mutant, the amount of

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mature enzyme was compared by SDS-PAGE (Fig. 1a). The amount of mature keratinase (30 kDa) in

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the supernatant fraction of the Ala(P1) mutant was slightly higher than that of the WT. A previous

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study also found that the Ala(P1) mutant of subtilisin E caused a prominent increase in activity because

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a large amount of the mature enzyme were produced [22]. Conversely, introduction of Arg(P1) in this

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study remarkably decreased the specific activity (to 15%), and seven other substitutions also decreased

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specific activity to some extent (to 50-90%) compared with the WT (Fig. 2a). Although it has been

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reported that Arg and Phe residues at the P1 position are preferentially cleaved by keratinase such as

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those from Bacillus subtilis [23], Chryseobacterium sp [24] and Streptomyces lividans [17], for the

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keratinase from B. licheniformis, the corresponding mutations in this study were not effective. The possible reason is that the various sources of enzymes lead to the different preferences of substrate binding pocket on the residue of P1 position. Therefore, a suitable residue at P1 position could improve the cleavage efficiency of propeptide and produce more mature enzyme.

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3.3. Effects of linkers and residues deletion at the propeptide C-terminus on keratinase production

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The type of amino acid at the cleavage site (P1) of the propeptide seems to affect the efficiency of

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mature keratinase production. Thus, it is necessary to investigate whether changes in primary structure

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at the C-terminus of propeptide, would influence the production of mature enzyme. It is well known

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that linkers act as spacers between the domains of multi-domain proteins [25]. Alterations of the length

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and composition of linker peptides connecting different domains will affect the protein stability, folding

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rates [26]. A poly-glycine, serine-rich flexible sequence (GS linker peptide) or a rigid

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proline-threonine-threonine linker (PT linker peptide) was inserted into the propeptide C-terminus at P2

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position. Interestingly, all mutants could still secreted mature keratinase (Fig. 1b). Nevertheless,

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insertion of linkers decreased specific activity compared with the WT. The SDS-PAGE displayed a

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sufficient amount of pro-keratinase (37 kDa) in the supernatant (Fig. 1b), indicated that the maturation

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of pro-keratinase was not processing completely. More excitingly, we found that when the linker’s

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length increased, the specific activity decreased (Fig. 2b). It seems that the long linkers may weaken

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the interactions of the linked partners [25], thus reduce the maturation processing of precursor.

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The deletion of one residue at the P2 position had less effect on specific activity (90% remaining)

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(Fig. 2b). However, deletion of two residues, at P2 and P3 position, markedly decreased specific

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activity such that only half of the activity remained compared with the WT (Fig. 2b). The

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pro-keratinase was observed by SDS-PAGE (Fig. 1c), indicating incomplete cleavage of propeptide.

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However, deficiency of three residues (del-AHA mutant) resulted in none of the mature keratinase but the pro-keratinase (Fig. 1c).

The results showed that, without changing the cleavage site, variety in primary structure at the

C-terminus of propeptide could also affect the mature keratinase production. This may be attributed to

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the following reasons: (1) Changed in primary structure (without deletion) at the C-terminus of

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propeptide (such as insertion of linkers) affect the cleavage efficiency of propeptide. It is on the basis of

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our results that the mutants could secret mature enzyme normally, but are different in the amount of

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mature enzyme compared with the WT. (2) The secondary structure of the propeptide C-terminus

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consists of a coil (P1 to P5 position). As the residues of P3 and P4 are located in the core region (Fig.

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3), deletion of this region may destroy the secondary structure that likely causes two results. The first

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one is to inhibit the intramolecular chaperone function of propeptide, thus makes the precursor unable

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to fold correctly, and form an inactive molten-globule-like conformers [13]. The second one establishes

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that, although destroyed this region, the precursor can also folding correctly but unable to undergo

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autocatalytic processing (degradation of propeptide in cis) due to the poor conformation of propeptide,

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and the region of propeptide binding to the enzyme active site is impaired [27]. Early study shows that

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when added mature enzyme in vitro, the propeptides of inactive subtilisin carlsberg (deletion of

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propeptide at P1 to P5 position) can removed normally [28]. This suggests that the del-AHA mutant,

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which only produces pro-keratinase, probably dues to the latter effect.

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Early report revealed that the propeptide from the subtilisin family have two regions display

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significant sequence conservation (motifs N1 and N2). These two motifs and the non-conserved

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segments (variable region) between N1 and N2 may be important for the folding and maturation

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process of pro-subtilisin [29]. The C-terminus of propeptide (P1 and P2 position) is not located in the

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N1, N2 and variable region (Fig. 3), but our evidence shows that the primary structure of this region is also crucial for the production of mature keratinase (although only negative mutants were obtained in this study). What kind of primary structure in this region is beneficial for keratinase production still needs to be investigated further.

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3.4. Determination of N-terminal sequence of mutants

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To investigate whether the mutants were correctly cleaved at the P1 position, three mutants (Ala, insPT,

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del-AH) were examined at the N-terminal sequences. The results showed that these three mutants

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contained N-terminal sequences similar to the WT (Ala-Gln-Thr-Val), confirming that the

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pro-keratinase mutants were cleaved at the P1 site.

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4. Conclusion The effect of the keratinase propeptide C-terminus from B. licheniformis BBE11-1 on the production

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of keratinase in B. subtilis was functionally analyzed. The results presented here demonstrate that a

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suitable residue at P1 position could improve the cleavage efficiency of propeptide, and produce more

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mature enzyme. In addition, the primary structure of the C-terminus of propeptide is also crucial for the

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production of mature keratinase. In conclusion, our study provides a new insight of propeptide

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engineering at C-terminus to increase the yield of keratinase.

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Acknowledgements

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This project was financially supported by the National High Technology Research and Development

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Program of China (863 Program, 2011AA100905), the Program for Changjiang Scholars and

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Innovative Research Team in University (No.IRT1135), the China Postdoctoral Science

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Foundation(2013M540538)，the 111 project (111-2-06), and the Natural Science Foundation of Jiangsu

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Province (BK2012553).

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Figure caption

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Fig. 1 SDS-PAGE of wild-type and mutant keratinases. The supernatants of 24 h culture of wild-type

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and its mutant keratinases were analyzed by SDS-PAGE. All the samples were applied at the same

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concentration of protein. (a): Lane M: molecular weight marker; Lane 1-9: WT, L(-1)H, L(-1)R, L(-1)A,

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L(-1)G, L(-1)E, L(-1)D, L(-1)F, L(-1)Y; (b): Lane M: molecular weight marker; Lane 1-5: WT, ins3G,

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ins4G, ins4GS, insPT; (c): Lane 1-3: del-A, del-AH, del-AHA; (d): SDS-PAGE of purified wild-type

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and mutant keratinases. Lane M: molecular weight marker; Lanes 1-9: WT, L(-1)H, L(-1)R, L(-1)A,

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L(-1)G, ins4GS, insPT, del-A, del-AH.

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Fig. 2 Specific activities of wild-type and mutant keratinases. (a) The specific activities of WT and

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thermolysin-like

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Bacillus

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independent of the primary structure of the cleavage site. Mol Microbiol 1992;6:1115-1119.

[29] Shinde U, Fu X, Inouye M. A pathway for conformational diversity in proteins mediated by

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intramolecular chaperones. J Biol Chem 1999;274:15615-15621.

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protein folding. Protein Eng 2002;15:871-879. [27] Wetmore DR, Wong SL, Roche RS. The role of the pro-sequence in the processing and secretion

mutant enzymes; The sample of His means Leu(P1)His mutant enzyme, and so on, for other samples. (b) The specific activities of WT and mutants of inserted linkers (ins3G, ins4G, ins4GS and insPT) and residue deletion (del-A, del-AH and del-AHA) at the propeptide C-terminus. Fig. 3 Amino acid sequence alignment of propeptides from KB (keratinase from B. licheniformis BBE11-1) and subtilisin E.

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Table 1 Oligonucleotide primers used for site-directed mutagenesis Directed mutation

Nucleotide sequence 5’-GATCATGTGGCCCATGCCTTG(GCA)GCGCAAACCGTTCCTTAC-3’

Leu(P1)Asp

5’-GATCATGTGGCCCATGCCTTG(GAC)GCGCAAACCGTTCCTTAC-3’

Leu(P1)Glu

5’-GATCATGTGGCCCATGCCTTG(GAA)GCGCAAACCGTTCCTTAC-3’

Leu(P1)Phe

5’-CATGTGGCCCATGCCTTG(TTC)GCGCAAACCGTTCCTTAC-3’

Leu(P1)Gly

5’-GATCATGTGGCCCATGCCTTG(GGC)GCGCAAACCGTTCCTTAC-3’

Leu(P1)His

5’-GATCATGTGGCCCATGCCTTG(CAC)GCGCAAACCGTTCCTTAC-3’

Leu(P1)Ile

5’-GATCATGTGGCCCATGCCTTG(ATC)GCGCAAACCGTTCCTTAC-3’

Leu(P1)Arg

5’-GATCATGTGGCCCATGCCTTG(CGT)GCGCAAACCGTTCCTTAC-3’

Leu(P1)Tyr

5’-GATCATGTGGCCCATGCCTTG(TAC)GCGCAAACCGTTCCTTAC-3’

Ins4G

cr

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5’-GGCGGCGGCGGCTTGGCGCAAACCGTTCC-3’

5’-GGCGGCGGCGGCAGCTTGGCGCAAACCGTTCC-3’

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Ins4GS

5’-GGCGGCGGCTTGGCGCAAACCGTTCC-3’

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Ins3Gb

ip t

Leu(P1)Alaa

InsPT

5’-CCGACGCCGCCGACGACGCCGACGTTGGCGCAAACCGTTCC-3’

del-Ac

3’-ATGGGCCACATGATCCTCTTC-5’

del-AH

3’-GGCCACATGATCCTCTTCCAC-5’

del-AHA

3’-CACATGATCCTCTTCCACATAAGC-5’

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a: Nucleotides underlined correspond to the codons chosen for mutation. Nucleotides in parentheses

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replace the underlined nucleotides. The primers sequences (3’→5’) of Leu(P1)Aaa were the reverse

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compliment of forward ones. b: Nucleotides underlined correspond to the codons of linkers chosen for

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mutation. The primers sequence (3’→5’) of inserted linkers (ins3G, ins4G, ins4GS and insPT) was

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GGCATGGGCCACATGATC. c: The primers sequence (5’→3’) of residue deletion (del-A, del-AH

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and del-AHA) was TTGGCGCAAACCGTTCC.

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(Fig. 1)

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ip t cr us an

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(Fig. 2)

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(Fig. 3)

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