Improvement of the Activity and Stability of Starch-Debranching

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Biotechnology and Biological Transformations

Tailoring of active sites lining the catalytic pocket improves the activity and stability of starch-debranching pullulanase from Bacillus naganoensis Xinye Wang, Yao Nie, and Yan Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06002 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Journal of Agricultural and Food Chemistry

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Tailoring of active sites lining the catalytic pocket improves the activity and stability of

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starch-debranching pullulanase from Bacillus naganoensis

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Xinye Wang†, Yao Nie†,‡*, Yan Xu†,‡

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†School

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Education, Jiangnan University, Wuxi 214122, China

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‡Suqian

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China

of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of

Industrial Technology Research Institute of Jiangnan University, Suqian 223814,

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*Correspondence and requests for materials should be addressed to Y.N. (email:

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[email protected]).

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Address: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry

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of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China

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E-mail: [email protected]

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Tel.: +86-510-85197760; Fax: +86-510-85918201

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ACS Paragon Plus Environment


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ABSTRACT

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Pullulanases are well-known debranching enzymes that hydrolyze α-1,6-glycosidic

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linkages in starch and oligosaccharides. However, most of the pullulanases exhibit limited

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activity for practical applications. Here, two sites (787 and 621) lining the catalytic pocket of

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Bacillus naganoensis pullulanase were identified to be critical for enzymatic activity by

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triple-code saturation mutagenesis. Subsequently, both sites were subjected to NNK-based

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saturation mutagenesis to obtain positive variants. Among the variants showing enhanced

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activity, the enzymatic activity and specific activity of D787C were 1.5-fold higher than

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those of the wild-type (WT). D787C also showed a 1.8-fold increase in kcat and 1.7-fold

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increase in kcat/Km. In addition, D787C maintained higher activity compared to WT at

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temperatures over 60 °C. All the positive variants showed higher acid resistance, with D787C

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maintaining 90 % residual activity at pH 4.0. Thus, enzymes with improved properties were

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obtained by saturation mutagenesis at the active site.

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Keywords: pullulanase; directed evolution; saturation mutagenesis; catalytic pocket; activity;

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stability

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INTRODUCTION Pullulanase (EC 3.2.1.41), an enzyme with a large molecular weight and a multi-domain

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structure, is an important member of the glycoside hydrolase family 1. Pullulanase

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specifically catalyzes the hydrolysis of α-1,6-glycosidic linkages of unmodified substrates

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and is widely used in medicine, materials, organometallic chemistry, and the saccharification

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industry 2. In the saccharification process, pullulanase, combined with glucoamylase, can

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efficiently break down starch into monosaccharides. However, the specific activity of

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pullulanase needs to be further improved for the saccharification reaction, while maintaining

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its thermostability and acid resistance.

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Directed evolution is an important method employed to engineer protein to obtain desired

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characteristics. The most commonly used methods for directed evolution are epPCR, DNA

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shuffling, and saturation mutagenesis (SM). Instead of iterative cycles of random amino-acid

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changes in a protein, more attention is focused on improving the efficiency of directed

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evolution by creating ‘small-and-smart’ libraries 3-10. The combinatorial active-site saturation

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test and iterative SM have been developed to create mutant libraries with reduced amino acid

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alphabets 3, 5, 7-8, 11. In addition, Sun et al. developed a structure-guided triple-code saturation

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mutagenesis (TCSM) method for efficiently improving the stereoselectivity of limonene

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epoxide hydrolase and enantioselectivity of alcohol dehydrogenase 9-10. This method involved

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introduction of three rationally chosen amino acids as building blocks for SM at the

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randomization sites lining the catalytic/binding pocket of pullulanase. These sites were

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randomly grouped into smaller fragments, i.e., 3–4 sites in one group, to reduce screening

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efforts to a minimum for 95 % library coverage. After grouping, TCSM was applied on the

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first group to obtain a mutant, whose characteristics were superior to those of the original, as

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a template for the next mutation of the second group, and so on 10. Although only three amino

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acids were used as the degenerate codons for replacing native residues in groups, TCSM was

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useful to identify the sites in the catalytic pocket that have a marked influence on enzyme

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function.

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Pullulanase has a complex structure with multiple domains 12-15. Based on the TCSM

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method, it was feasible to construct a limited library of variants that focused on the catalytic

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pocket of the enzyme and identify key sites affecting enzymatic activity and/or other

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characteristics. Then, SM could be applied at the key sites for obtaining positive mutants.

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Here, we utilized the TCSM approach to identify the key sites in the catalytic pocket that

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significantly influence the catalytic activity of Bacillus naganoensis pullulanase (BnPUL).

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Then, we applied NNK (N: adenine/cytosine/guanine/thymine; K: guanine/thymine) codon

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degeneracy for encoding all 20 canonical amino acids for SM at the key sites to improve

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enzymatic activity and stability.

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MATERIALS AND METHODS

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Materials

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The recombinant plasmid pET-28a-PelB-pul bearing the pullulanase encoding gene pul

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(GenBank Accession No. JN872757) from B. naganoensis was constructed as described

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previously 16. Oligonucleotides for polymerase chain reaction (PCR) were synthesized by

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Genewiz (Suzhou, China). The plasmid preparation kit I (D6943-02) was purchased from

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Omega Bio-tek (Norcross, GA, USA). The ClonExpress II One Step Cloning Kit was

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purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). DNA sequencing was

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performed by Genewiz (Suzhou, China). HisTrap HP affinity column and disposable PD-10

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desalting columns were purchased from GE Healthcare Life Sciences (Beijing, China). All

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commercial chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), Takara

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(Shiga, Japan), or Sinopharm Chemical Reagent (Shanghai, China). Pullulan was purchased

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from Tokyo Chemical Industry (Chuo-ku, Tokyo).

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Primer design and library construction

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The recombinant plasmid pET-28a-PelB-pul was used as a template for TCSM at the key

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sites in the catalytic pocket. The involved nine residues were divided into three groups: (A)

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W504, Y506, and W650; (B) T585, S731, and N734; and (C) M621, D787, and Y790. The

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short fragments of plasmids containing the target mutation sites were amplified using mixed

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primers F1/R1, F2/R2, and F3/R3 for libraries I, II, and III, respectively (Figure S1). The

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long fragments of plasmids were amplified by PCR as well. Next, the short and long

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fragments were linked by homologous recombination using the ClonExpress II One Step

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Cloning Kit (Figure S1). Primers used for plasmid construction are listed in Table S1.

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Primers for SM at D787 and M621 sites were used with recombinant plasmid

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pET-28a-PelB-pul as the template. The short fragments of plasmids containing the target

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mutation site were amplified using mixed primers 787F/787R and 621F/621R for library

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D787 and M621, respectively. The long fragments of plasmids were amplified by PCR as

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well. Next, the short and long fragments were linked by homologous recombination using the

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ClonExpress II One Step Cloning Kit. All of the reaction mixture was then used to transform

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Escherichia coli BL21 (DE3) cells. After plating, individual colonies were selected and their

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plasmids were sequenced. Primers used for plasmid construction are listed in Table S2.

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Screening and expression of recombinant proteins

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Cell colonies containing mutant plasmids were picked and spotted at 45 positions on plates

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containing solid auto-induction Studier 17 medium with 2 % agar, 1 % red pullulan, and 50

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μg·mL–1 kanamycin, and then the plates were incubated overnight at 37 °C. Colonies forming

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obvious transparent zones on the screening plates were selected for activity assay and

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inoculated in 5 mL LB medium containing kanamycin (50 μg·mL–1). After incubation at

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37 °C and 200 rpm for 10 h, 1 mL of the culture was transferred to a 250 mL flask containing

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50 mL auto-induction medium (10 g·L–1 α-lactose, 1.0 g·L–1 glucose, 5.0 g·L–1 glycerol, 6.8

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g·L–1 KH2PO4, 0.25 g·L–1 MgSO4, 10 g·L–1 tryptone, 5.0 g·L–1 yeast extract, 7.1 g·L–1

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Na2HPO4, 0.71 g·L–1 Na2SO4, and 2.67 g·L–1 NH4Cl, pH 7.5) supplemented with kanamycin

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(50 μg·mL–1). After cultivation at 37 °C and 200 rpm for the first 2–3 h, the culture was

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incubated at 17 °C and 200 rpm for another 60 h for target protein expression.

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Purification of recombinant proteins

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The cells were lysed by sonication. The supernatant of the cell lysate, containing the crude

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enzyme, was collected by centrifugation at 18,514 × g at 4 °C for 40 min, and purified by an

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AKTAxpress system using HisTrap HP affinity column. Elution was carried out using 500

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mM imidazole in the same buffer at a flow rate of 2.0 mL·min−1. Then, the purified fractions

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were transferred into low salt buffer [10 mM Tris-HCl (pH 6.5), 0.1 M NaCl, 0.02 % NaN3,

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and 5 mM DL-dithiothreitol] using disposable PD-10 desalting columns.

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The molecular weight and concentration of the recombinant enzyme were estimated using 10 % (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels

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were stained with Coomassie Brilliant Blue R250, and a protein ladder ranging from 10 to

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200 kDa was used for electrophoresis.

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Pullulanase activity assay

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The supernatant obtained after cell lysis was assayed for pullulanase activity by measuring

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the amount of aldehyde released during the enzymatic reaction from a mixture containing

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pullulan solution and diluted enzyme sample. The reaction mixture, containing 0.2 mL 2 %

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(w/v) pullulan in 0.1 M sodium acetate buffer (pH 4.5) and 0.2 mL enzyme solution diluted

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in 0.1 M sodium acetate buffer (pH 4.5), was incubated at 60 °C for 20 min. Next, the amount

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of released aldehyde was determined using dinitrosalicylic acid and then by measuring the

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absorbance at 540 nm. One unit of pullulanase activity was defined as the amount of

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pullulanase that released 1 μmol reducing sugar equivalents per min under the reaction

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conditions. Enzymatic activity refers to the units of activity per volume of enzyme solution,

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and specific activity is the units of activity per milligram protein that is measured after

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protein purification. All assays were performed in triplicates.

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Determination of Michaelis-Menten parameters

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Km and kcat of the purified enzymes were determined based on a previously described

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method 18 at the following pullulan concentrations: 0.1, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75,

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2.0, 2.25, 2.5, 2.75, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 15.0, and 20.0 mg·mL–1. The kcat and Km

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values were obtained by fitting the initial rate data to the Michaelis-Menten equation using

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nonlinear regression with the GraphPad Prism 19. Values are shown as means from three

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replicates with standard deviation.

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Homology modeling

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The homologous structure of the wild-type (WT) BnPUL was modeled using

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SWISS-MODEL (https://www.swissmodel.expasy.org). The X-ray structure of pullulanase

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from B. acidopullulyticus (PDB ID: 2WAN), which has 64 % sequence identity with BnPUL,

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was used as the structural template.

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Amino acid sequence analysis by DisMeta server

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The DisMeta server (www-nmr.cabm.rutgers.edu/bioinformatics/disorder) employs a wide

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range of disorder prediction tools and several sequence-based structural prediction tools 20.

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The disorder regions in the amino acid sequence of the BnPUL were predicted using

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DisMeta.

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RESULTS AND DISCUSSION

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Selection of sites in the catalytic pocket for TCSM

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To date, there have been only a few reports focusing on engineering the catalytic pocket of

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BnPUL 21-22. We employed the TCSM method to try to improve the catalytic efficiency of

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BnPUL. Except for the catalytic triad, D619, E648, and D733, the functional residues were

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still unclear even in the active center of BnPUL. In the Protein Data Bank (PDB), there are

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four pullulanases with known protein structures showing high identity with BnPUL,

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including B. acidopullulyticus pullulanase (64 % identity, PDB ID: 2WAN), Anoxybacillus

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sp. LM18-11 pullulanase (43 % identity, PDB ID: 3WDJ), B. subtilis strain 168 pullulanase

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(40 % identity, PDB ID: 2E8Y), and Klebsiella pneumoniae pullulanase (28 % identity, PDB

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ID: 2FH8) 12-14, 18. Although B. acidopullulyticus pullulanase and B. subtilis strain 168

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pullulanase share high identities with BnPUL, their apo structures, without the ligand for the

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oligosaccharide molecule, make it difficult to identify the functional sites associated with

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substrate binding 12, 18. Thus, Anoxybacillus sp. LM18-11 pullulanase and K. pneumoniae

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pullulanase were selected to align with BnPUL to find the active sites possibly involved in

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ligand binding and molecular interactions (Figure 1a). There were several residues in the

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active sites that consistently appeared in 3WDJ and 2FH8, while some residues were unique

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to each of the two pullulanases. Based on the conservation of the residues lining the catalytic

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pocket, we chose nine residues (W504, Y506, T585, M621, W650, S731, N734, D787, and

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Y790) for TCSM. They were randomly divided into three groups: (A) W504, Y506, and

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W650; (B) T585, S731, and N734; and (C) M621, D787, and Y790 (Figure 1b and 1c).

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In order to minimize the library size of variants and improve screening efficiency, three

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conserved amino acids, as well as degeneracy of codons, needed to be determined for the

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employment of TCSM. The amino acid sequence of the WT BnPUL was analyzed using

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BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) by aligning the first 100 homologous

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pullulanases (Figure S2). Based on the frequency of amino acids at the nine sites in

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homologous sequences and the corresponding codon degeneracy, we chose tryptophan (W),

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tyrosine (Y), and asparagine (N) as the triplet codon for SM (Figure S1).

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Construction and screening of TCSM pullulanase libraries

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For the three groups, A, B, and C, comprising nine residues, the corresponding SM

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libraries were constructed as libraries I, II, and III, respectively. Catalytically active variants

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were rapidly identified using the red pullulan plate assay (Figure 2). Library III contained

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more variants that exhibited enzymatic activity as compared to the other two libraries.

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Moreover, all of the variants in the library I failed to form transparent zones. It was presumed

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that the three amino acids in group A were rigidly conserved, and their mutations had a

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negative impact on the enzymatic reaction.

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Next, 5 and 14 colonies showing obvious transparent zones in the plate assay were picked

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from libraries II and III, respectively, for further sequencing and activity assay. The

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enzymatic activity of the variant was assayed in the supernatant obtained after cell lysis. The

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variants from library II only maintained about half of the enzymatic activity of the WT, while

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the variants from library III possessed about 70–80 % of the enzymatic activity of the WT.

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Because the variants in library III were either active or inactive, the three sites of group C

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might not be highly conserved and related to enzymatic reactions (Figure S3). Among the

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three residues from group C, a mutation in Y790 was supposed to generate inactive variants,

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and D787 and M621 could be potential sites exerting positive effects on enzymatic activity

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and catalytic efficiency.

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SM at potential active sites

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We performed SM at the two potential sites, D787 and M621. For the mutation at site 621,

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three variants showing larger transparent zones in the plate compared to other variants were

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fermented by shake flask fermentation. The three variants exhibited about 400 U/mL activity,

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which was not very different from that of WT. In the case of mutation at site 787, ten variants

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showing larger transparent zones in the plate compared to other variants were fermented by

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shake flask fermentation. Then, their enzymatic activity was assayed in the supernatant

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obtained after cell lysis. Four variants, D787S, D787N, D787F, and D787C, exhibited higher

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enzymatic activity than that of the WT (Figure 3). Among them, D787C exhibited the highest

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enzymatic activity of 684 U·mL–1, which was 1.5-fold higher than that of the WT.

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SDS-PAGE analysis of the cell-free extracts after expression indicated that the mutation in

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D787 did not have a remarkable effect on protein expression (Figure 4).

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Additionally, the variants D787S, D787N, D787F, and D787C were purified by Ni-NTA

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affinity chromatography to measure specific activity and Michaelis-Menten parameters. The

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four variants all exhibited higher specific activity than that of the WT, and among them

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D787C showed the highest specific activity of 547 U·mg–1, which was 1.5-fold higher than

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that of the WT (Figure 3). Moreover, the values of kcat and kcat/Km of the four variants all

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showed more than 1.4-fold increase, compared to those of the WT (Figure 5). Among them,

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the variant D787C exhibited a 1.8-fold increase in kcat and 1.7-fold increase in kcat/Km relative

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to the WT (Figure 5). Therefore, the catalytic efficiency of the enzyme was improved by a

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mutation in the residue D787.

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According to the requirements of industrial saccharification process involving pullulanase,

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the optimal pH and temperature of pullulanase should fit the industrial working condition.

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Therefore, the effects of pH and temperature on enzymatic activity were investigated for

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these four variants and the WT. All the tested variants exhibited the highest activities at pH

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4.5 and 60 °C (Figure 6). Among them, the variant D787C maintained a higher activity than

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the WT when the temperature was above 60 °C. On the other hand, all four variants showed

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higher acid resistance than the WT, especially the variant D787C, retaining 90 % activity at

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pH 4.0. Thermostability and acid resistance are known to be critical for the application of

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pullulanase in starch saccharification. As compared to pullulanase from B. acidopullulyticus

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(BaPUL), which has been used for commercial application in the starch industry 12, the

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variant D787C has 75 % relative activity at 70 °C relative to 65 °C, while the variant from

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BaPUL has at least 60 % relative activity 23. The enzymatic activity of variants generated

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from BaPUL was measured at pH 5.0 24, while in an even more acidic environment, the

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variant D787C was assayed at pH 4.5 and was found to exhibit 85–90 % relative activity at

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pH 4.0–5.0. Hence, an improved pullulanase variant was obtained with a potential for future

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industrial application 22.

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With respect to the change in enzymatic properties of BnPUL through mutations in the

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functional sites identified by TCSM, we further analyzed the role of D787 in

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structure-function relationship. Corresponding to the active site 889 of 2FGZ, D787 of

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BnPUL could be one of the pullulanase-specific binding sites, which recognizes the

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non-reducing ends of the main-chain sugar residues 13. The enhanced specific activity and

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catalytic efficiency of the enzyme further confirmed the importance of D787 in the catalytic

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reaction. As shown in Figure 7, after replacing aspartic acid with cysteine at site 787, the

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hydrogen bond between Y735 and D787 as well as between Y735 and G785 was broken,

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while a new bond was formed between Y735 and Q743. Y735 and one of the catalytic triad,

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D733, are located in the same loop. The alteration in hydrogen bonds between Y735 and

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D787 as well as between Y735 and Q743 induced conformational changes in the adjacent

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loop and α-helix structures, leading to alterations in the steric conformation of D733 and

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consequent molecular interactions between catalytic sites and ligands.

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The amino acid sequence of the BnPUL was then analyzed using the Disorder Prediction

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Meta-Server (DisMeta). Except for the N- and C-terminus of the BnPUL predicted to be

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disordered by DisMeta, D787 was relatively more flexible than the other residues lining the

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catalytic pocket of the enzyme (Figure S4). However, after the substitution of aspartic acid

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with cysteine, the loop region close to the mutation site exhibited lower flexibility, compared

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with the WT (Figure S5). As already known, the flexibility of the enzyme affects its stability.

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Therefore, the improved stability of the variant D787C may be caused by the increased

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rigidity. Based on the sequence alignment of homologous pullulanases (1,422 sequences) that

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displayed more than 30% identity to the WT BnPUL (Figure S6), the conserved residues

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corresponding to the site 787 in BnPUL are serine (S) and asparagine (N), but not

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phenylalanine (F) and cysteine (C), suggesting that the obtained variant with improved

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properties has not yet been generated by natural evolution.

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In this work, we identified the active sites lining the catalytic pocket of BnPUL based on

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the TCSM approach. Using red pullulan plate assay, two non-conserved sites, D787 and

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M621, were discovered to have a positive effect on enzymatic activity. The improved

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variants were then successfully obtained by further SM at these two sites. Among them,

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D787S, D787N, D787F, and D787C showed enhanced specific activities and catalytic

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efficiencies compared with the WT. Furthermore, these variants exhibited a broadened

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temperature and pH profile. Hence, an improved enzyme variant exhibiting enhanced

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performance in terms of both activity and stability was obtained, which is critical for the

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industrial application of pullulanase.

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ASSOCIATED CONTENT

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Supporting Information

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Construction of recombinant plasmids; Sequence alignment of B. naganoensis pullulanase; enzymatic activity of the WT and TCSM variants from libraries II and III; disorder region

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and secondary structure prediction of the WT and the variant D787C; and the list of primers.

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ACKNOWLEDGMENT

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We would like to thank Editage (www.editage.cn) for English language editing. Funding Resources

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Financial support from the National Natural Science Foundation of China (NSFC)

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(21336009, 21676120, 31872891), the Natural Science Foundation of Jiangsu Province

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(BK20151124), the 111 Project (111-2-06), the High-end Foreign Experts Recruitment

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Program (GDT20183200136), the Program for Advanced Talents within Six Industries of

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Jiangsu Province (2015-NY-007), the National Program for Support of Top-notch Young

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Professionals, the Fundamental Research Funds for the Central Universities (JUSRP51504),

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the Project Funded by the Priority Academic Program Development of Jiangsu Higher

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Education Institutions, the Jiangsu province "Collaborative Innovation Center for Advanced

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Industrial Fermentation" industry development program, the Postgraduate Research &

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Practice Innovation Program of Jiangsu Province, Top-notch Academic Programs Project of

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Jiangsu Higher Education Institutions, and the National First-Class Discipline Program of

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Light Industry Technology and Engineering (LITE2018-09) is greatly appreciated.

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Conflict of Interest

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The authors declare no competing financial interest.

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REFERENCES

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1. Henrissat, B.; Bairoch, A. New families in the classification of glycosyl hydrolases based

300

on amino acid sequence similarities. Biochem. J. 1993, 293, 781-788.

301

2. Bertoldo, C.; Antranikian, G. Starch-hydrolyzing enzymes from thermophilic archaea and

302

bacteria. Curr. Opin. Chem. Biol. 2002, 6, 151-160.

303

3. Reetz, M. T.; Bocola, M.; Carballeira, J. D.; Zha, D. X.; Vogel, A. Expanding the range of

304

substrate acceptance of enzymes: Combinatorial active-site saturation test. Angew. Chem. Int.

305

Edit. 2005, 44, 4192-4196.

306

4. Reetz, M. T.; Wang, L. W.; Bocola, M. Directed evolution of enantioselective enzymes:

307

Iterative cycles of CASTing for probing protein-sequence space. Angew. Chem. Int. Edit.

308

2006, 45, 1236-1241.

309

5. Reetz, M. T.; Wu, S. Greatly reduced amino acid alphabets in directed evolution: making

310

the right choice for saturation mutagenesis at homologous enzyme positions. Chem. Commun.

311

2008, 5499-5501.

312

6. Kille, S.; Acevedo-Rocha, C. G.; Parra, L. P.; Zhang, Z. G.; Opperman, D. J.; Reetz, M. T.;

313

Acevedo, J. P. Reducing Codon Redundancy and Screening Effort of Combinatorial Protein

314

Libraries Created by Saturation Mutagenesis. ACS Synth. Biol. 2013, 2, 83-92.

315

7. Parra, L. P.; Agudo, R.; Reetz, M. T. Directed evolution by using iterative saturation

316

mutagenesis based on multiresidue sites. Chembiochem 2013, 14, 2301-2309.

317

8. Sun, Z. T.; Lonsdale, R.; Kong, X. D.; Xu, J. H.; Zhou, J. H.; Reetz, M. T. Reshaping an

318

enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino

319

acid alphabets in directed evolution. Angew. Chem. Int. Edit. 2015, 54, 12410-12415.

15



320

9. Sun, Z.; Lonsdale, R.; Ilie, A.; Li, G.; Zhou, J.; Reetz, M. T. Catalytic asymmetric

321

reduction of difficult-to-reduce ketones: Triple-code saturation mutagenesis of an alcohol

322

dehydrogenase. ACS Catal. 2016, 6, 1598-1605.

323

10. Sun, Z.; Lonsdale, R.; Wu, L.; Li, G.; Li, A.; Wang, J.; Zhou, J.; Reetz, M. T.

324

Structure-guided triple-code saturation mutagenesis: Efficient tuning of the stereoselectivity

325

of an epoxide hydrolase. ACS Catal. 2016, 6, 1590-1597.

326

11. Sun, Z.; Salas, P. T.; Siirola, E.; Lonsdale, R.; Reetz, M. T. Exploring productive

327

sequence space in directed evolution using binary patterning versus conventional mutagenesis

328

strategies. Bioresour. Bioprocess. 2016, 3, 44.

329

12. Turkenburg, J. P.; Brzozowski, A. M.; Svendsen, A.; Borchert, T. V.; Davies, G. J.;

330

Wilson, K. S. Structure of a pullulanase from Bacillus acidopullulyticus. Proteins 2009, 76,

331

516-9.

332

13. Mikami, B.; Iwamoto, H.; Malle, D.; Yoon, H. J.; Demirkan-Sarikaya, E.; Mezaki, Y.;

333

Katsuya, Y. Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides

334

in the active site. J. Mol. Biol. 2006, 359, 690-707.

335

14. Xu, J.; Ren, F.; Huang, C. H.; Zheng, Y.; Zhen, J.; Sun, H.; Ko, T. P.; He, M.; Chen, C.

336

C.; Chan, H. C.; Guo, R. T.; Song, H.; Ma, Y. Functional and structural studies of pullulanase

337

from Anoxybacillus sp. LM18-11. Proteins 2014, 82, 1685-93.

338

15. Janecek, S.; Majzlova, K.; Svensson, B.; MacGregor, E. A. The starch-binding domain

339

family CBM41-An in silico analysis of evolutionary relationships. Proteins 2017, 85,

340

1480-1492.

341

16. Wang, X.; Nie, Y.; Mu, X.; Xu, Y.; Xiao, R. Disorder prediction-based construct

16


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Page 17 of 27


342

optimization improves activity and catalytic efficiency of Bacillus naganoensis pullulanase.

343

Sci. Rep. 2016, 6, 24574.

344

17. Studier, F. W. Protein production by auto-induction in high-density shaking cultures.

345

Protein Expres. Purif. 2005, 41, 207-234.

346

18. Malle, D.; Itoh, T.; Hashimoto, W.; Murata, K.; Utsumi, S.; Mikami, B. Overexpression,

347

purification and preliminary X-ray analysis of pullulanase from Bacillus subtilis strain 168.

348

Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2006, 62, 381-4.

349

19. Swift, M. L. GraphPad prism, data analysis, and scientific graphing. J. Chem. Inf. Comp.

350

Sci. 1997, 37, 411-412.

351

20. Huang, Y. J.; Acton, T. B.; Montelione, G. T. DisMeta: a meta server for construct design

352

and optimization. Methods Mol. Biol. 2014, 1091, 3-16.

353

21. Wang, Q.-Y.; Xie, N.-Z.; Du, Q.-S.; Qin, Y.; Li, J.-X.; Meng, J.-Z.; Huang, R.-B. Active

354

hydrogen bond network (AHBN) and applications for improvement of thermal stability and

355

pH-sensitivity of pullulanase from Bacillus naganoensis. PLoS One 2017, 12.

356

22. Chang, M.; Chu, X.; Lv, J.; Li, Q.; Tian, J.; Wu, N. Improving the thermostability of

357

acidic pullulanase from Bacillus naganoensis by rational design. PLoS One 2016, 11,

358

e0165006.

359

23. Tomoko, M.; Akihiko, Y. Pullulanase variants and polynucleotides encoding same. U.S.

360

Patent 10,030,237, July 24, 2018.

361

24. Tomoko, M.; Suzanne, C.; Aki, T. Polypeptides having pullulanase activity suitable for

362

use in liquefaction. U.S. Patent 2,018,208,917, July 26, 2018.

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364

Figure Captions

365

Figure 1. (a) Sequence alignment of BnPUL with K. pneumoniae pullulanase (PDB ID:

366

2FGZ) and Anoxybacillus sp. LM18-11 pullulanase (PDB ID: 3WDH). (b, c) The spatial

367

location of the nine residues (W504, Y506, T585, M621, W650, S731, N734, D787, and

368

Y790) lining the catalytic pocket in the homology model of BnPUL that were identified

369

as mutational sites for TCSM. (a) The nine residues (W504, Y506, T585, M621, W650,

370

S731, N734, D787, and Y790) selected for TCSM are indicated by blue triangles. (b)

371

Backbone representations of the three groups: (A) W504, Y506, and W650; (B) T585, S731,

372

and N734; (C) M621, D787, and Y790 are shown in green, yellow, and blue, respectively.

373

The residues in the catalytic triad in the active site of the enzyme (D619, E648, and D733)

374

are indicated using red sticks. (c) Surface representation of BnPUL in the same orientation

375

and with the same color-coding as in panel (b). All images were obtained using the program

376

PyMOL.

377

Figure 2. Screening of variants with auto-induction of red pullulan plates. The substrate

378

is partially depolymerized pullulan, which is dyed with Procion Red MX-5B to an extent of

379

approximately one dye molecule per 30 sugar residues. When the enzyme variants are

380

incubated with red pullulan, the substrate is depolymerized to produce low-molecular-weight

381

dyed fragments, forming transparent zones and half-quantitatively determining the enzymatic

382

activity. (a) All of the variants in library I failed to form transparent zones. (b) Variants

383

forming transparent zones were selected for further activity measurement.

384

Figure 3. Enzymatic activity (purple bars, left scale) and specific activity (yellow bars,

385

right scale) of WT BnPUL and the variants. The values were measured in triplicates and

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386

the standard deviations are indicated as error bars.

387

Figure 4. SDS-PAGE analysis of WT BnPUL and the variants. Lane M: the protein

388

marker.

389

Figure 5. kcat (purple bars, left scale) and kcat/Km (yellow bars, right scale) of WT

390

BnPUL and the variants. The values were measured in triplicates and the standard

391

deviations are indicated as error bars.

392

Figure 6. Effects of (a) temperature and (b) pH on the enzymatic activity of WT BnPUL

393

and the variants. Enzymatic activity was measured in 0.1 M sodium acetate buffer with pH

394

ranging from 3 to 5.5 or at temperatures ranging from 45 to 70 °C. The highest activity is

395

shown as 100 %. Error bars represent the standard deviations of three replicates.

396

Figure 7. The homology model of (a) WT BnPUL and (b) the variant D787C. The

397

enzyme backbone is represented as a cartoon in gray. The catalytic triad (D619, E648, and

398

D733) is represented by red lines. The substrate isomaltose is indicated by purple sticks. The

399

hydrogen bonds are indicated by yellow dashes. (a) The residue D787 is indicated by violet

400

lines. (b) The residue D787C is indicated by orange lines. All images were obtained using the

401

program PyMOL.

19



Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Table of Contents Graphic

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Improvement of the Activity and Stability of Starch-Debranching

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