# Patent

## Title
Dual-functional hybrid glucanases

## Abstract

Described herein is a fusion polypeptide that contains (a) a first segment containing a Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase) or a fragment thereof; (b) a second segment containing a first Thermotoga maritima 1,3-β-D-glucanase (TmLam) or a fragment thereof; and (c) an optional third segment containing a second Thermotoga maritima 1,3-β-D-glucanase (TmLam) or a fragment thereof; wherein the fusion polypeptide has a glucanase activity. Also described are a nucleic acid molecule encoding the fusion polypeptide and a method of using the polypeptide.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Provisional Application No. 61/789,804, filed on Mar. 15, 2013, the content of which is hereby incorporated herein in its entirety.


BACKGROUND
Endo-β-D-glucanases are fibrolytic enzymes that play an important role in the hydrolysis of polysaccharide components. Several industrial applications of glucanase enzymes have been reported. For instance, 1,3-1,4-β-D-glucanases (EC 3.2.1.73, lichenase) can be used in the brewing industry and in the animal feeds industry. 1,3-β-D-Glucanases (laminarinases) have potential for use in commercial yeast extract production and for the conversion of algal biomass to fermentable sugars in the generation of bioenergy. The enzymes also have antimycotic activity for disease protection in plants.
SUMMARY
Described herein is a fusion polypeptide that includes (a) a first segment containing a Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase) or a fragment thereof; (b) a second segment containing a first Thermotoga maritima 1,3-β-D-glucanase (TmLam, e.g., SEQ ID NO:1) or a fragment thereof; and (c) an optional third segment containing a second Thermotoga maritima 1,3-β-D-glucanase (TmLam, e.g., SEQ ID NO:1) or a fragment thereof; wherein the fusion polypeptide has a glucanase activity. For example, the first segment can contain TFSW203F (e.g., residues 1-248 of SEQ ID NO:3), the second segment and the optional third segment can each contain a first carbohydrate binding module of TmLam (CBM1, e.g., residues 19-176 of SEQ ID NO:1), a second carbohydrate binding module of TmLam (CBM2, e.g., residues 495-642 of SEQ ID NO:1), or a catalytic domain (CD) of TmLam (e.g., residues 211-488 of SEQ ID NO:1). In one embodiment, the fusion polypeptide can have an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5, 7, 9, 11, or 13.
Also described herein is an isolated nucleic acid molecule that has a nucleic acid sequence encoding the above-described fusion polypeptide. An expression vector containing the nucleic acid molecule and a host cell harboring the expression vector are also described.
The fusion polypeptide, which has glucanase activity, can be used to degrade a substrate, e.g., lichenin and larmarine.
The details of one or more embodiments are set forth in the accompanying drawing and the description below. Other features, objects, and advantages will be apparent from the description and drawing, and from the claims.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1. is a schematic representation showing the structures of TmLam (e.g., SEQ ID NO:1), TFsW203F (having residues 1-248 of SEQ ID NO:3), and the hybrid glucanases described herein. The scale of the figure indicates amino acid residues.
FIG. 2 is a set of SDS-PAGE gels showing the electrophoretic mobility of TFsW203F (having residues 1-248 of SEQ ID NO:3) and the hybrid glucanases on 12% SDS-PAGE gel without the addition of any substrate (left panel) and with 0.1% lichenan (right panel).
FIG. 3 is a set of graphs showing log MW vs. relative migration distance Rf0 (without substrate; left panel) and Rf (with substrate; right panel) of protein standards and test enzymes. Protein standards (M); 1: TFsW203F-TmLamCD (e.g., SEQ ID NO:9); 2: TFsW203F-TmB2 (e.g., SEQ ID NO:7); 3: TmB1-TFsW203 (e.g., SEQ ID NO:13); 4: TmB1-TFsW203F-TmB2 (e.g., SEQ ID NO:11); 5: TmLamCD (e.g., residues 211-488 of SEQ ID NO:1); and 6: TFsW203F (having residues 1-248 of SEQ ID NO:3).
FIG. 4 is a graph showing the effect of temperature on the enzymatic activity of TFsW203F (having residues 1-248 of SEQ ID NO:3) and the hybrid glucanases. Purified enzymes were incubated for ten minutes at 30, 40, 45, 50, 55, 60, 70 and 80° C. in sodium phosphate buffer (50 mM, pH 7.0). Each assay was performed either in triplicate or in quadruplicate.
FIG. 5 is a set of graphs showing circular dichroism (CD) spectra of TFsW203F (having residues 1-248 of SEQ ID NO:3) (left panel) and TFsW203F-TmLamCD (e.g., SEQ ID NO:9) (right panel) under heat treatment at the temperatures indicated.
FIG. 6 is a set of graphs showing fluorescence emission spectra at 310-340 nm for TFsW203F (having residues 1-248 of SEQ ID NO:3) (left panel) and TFsW203F-TmLamCD (e.g., SEQ ID NO:9) (right panel). Each enzyme comprised 30 μg/mL in sodium phosphate buffer (50 mM, pH 7.0) or in an 8 M urea-phosphate buffer (denatured). The excitation wavelength was 295 nm.
FIG. 7 is a set of graphs showing recovery of enzymatic activity and fluorescence emission intensity of TFsW203F (having residues 1-248 of SEQ ID NO:3) (left panel) and TFsW203F-TmLamCD (e.g., SEQ ID NO:9) (right panel) at ambient temperature after heat treatment at 90° C. for ten minutes. The residual enzyme activity of heat-treated protein was measured every 18 seconds from when it was transferred from the incubator at 90° C. to ambient temperature (25° C.), in a ten-minute period. The fluorescence emission spectra of the treated enzymes were taken immediately after treatment at 90° C. and monitored for ten minutes.



DETAILED DESCRIPTION
Described herein are fusion polypeptides each containing at least a Fibrobacter succinogenes 1,3-1,4-β-D-glucanase (Fsβ-glucanase) or fragment thereof, and at least a Thermotoga maritima 1,3-β-D-glucanase (TmLam, e.g., SEQ ID NO:1) or a fragment thereof. The fusion polypeptides exhibit dual substrate specificities toward lichenin and larmarine, and also increased catalytic efficiencies and thermotolerance as compared to the parental single domain enzymes.
Shown below are a TmLam amino acid sequence and a TmLam nucleic acid sequence.












TmLam amino acid sequence (SEQ ID NO: 1)


catalytic domain TmLamCD - residues 211-488; bold black


carbohydrate binding module (CBM) TmB1 - residues 19-176, highlighted in grey


carbohydrate binding module (CBM) TmB2 - residues 495-642, highlighted in grey


linkers - residues 177-210 and 489-494; underlined


signal peptide SP - residues 1-18








TmLam nucleic acid sequence (SEQ ID NO: 2)


GATGGCCATGGCTCAAAACATCCTTGGCAACGCTTCTTTCGATGAACCAATTCTCATCGCAGGTGTGGATATAGACC


 


CACCCGCAGAGGATGGCTCTATAGACACAGGAGGAAACTGGGTATTCTTCACCAATTCAAACGGTGAGGGAACGGCT


 


CGAGTCGAAAACGGCGTTCTCGTGGTTGAGATAACAAACGGAGGAGATCACACCTGGTCGGTTCAGATCATACAGGC


 


TCCCATACGTGTTGAGAAACTCCACAAGTACAGAGTTTCTTTCCGAGCCAAGGCTTCCTCTCAAAAGAACATCGGGG


 


TGAAGATAGGAGGAACGGCCGGAAGAGGATGGACCGCGTACAACCCCGGTACCGACGAATCCGGCGGCATGGTCTTC


 


GAGCTCGGAACAGATTGGCAGAAGTACGAGTTCGAATTCGTCATGAGACAGGAGACCGATGAAAATGCTCGTTTCGA


 


GTTTCAGCTTGGAAGGTATACCGGCACGGTCTGGATAGACGACGTAGTGATGGAGGACATCGGTGTTCTCGAGGTAA


 


GCGGTGAGGAAAACGAAATCTACACCGAGGAGGATGAAGACAAAGTGGAAGACTGGCAGCTCGTTTGGAGTCAGGAG


 


TTCGATGACGGTGTTATCGATCCGAACATCTGGAACTTCGAGATAGGAAACGGTCATGCAAAAGGTATTCCAGGCTG


 


GGGTAACGGGGAACTCGAGTACTATACAGACGAAAACGCGTTCGTTGAGAACGGCTGTCTTGTGATTGAGGCAAGAA


 


AAGAACAGGTTTCCGATGAGTACGGAACCTACGACTACACCTCAGCCAGGATGACCACAGAAGGAAAATTCGAAATA


 


AAGTACGGAAAAATCGAAATAAGGGCAAAACTTCCAAAAGGAAAAGGTATCTGGCCCGCTCTCTGGATGCTCGGAAA


 


CAACATAGGAGAGGTCGGATGGCCCACCTGTGGTGAGATAGACATCATGGAAATGCTTGGCCACGACACCAGAACCG


 


TTTATGGAACAGCACACGGTCCGGGATATTCTGGTGGTGCGAGTATAGGTGTTGCCTATCATCTTCCAGAAGGAGTT


 


CCTGATTTCTCCGAAGACTTCCACATTTTCTCCATCGAGTGGGACGAAGACGAAGTGGAGTGGTACGTGGACGGACA


 


GCTCTACCACGTCCTCAGCAAGGATGAACTGGCCGAACTCGGTCTTGAGTGGGTTTTCGACCATCCGTTCTTCCTCA


 


TTCTGAACGTTGCCGTGGGAGGCTACTGGCCGGGTTATCCCGACGAAACCACCCAATTCCCGCAGAGAATGTACATC


 


GACTACATCAGAGTCTATAAAGATATGAATCCGGAAACAATCACCGGGGAAGTGGATGACTGCGAATATGAACAAGC


 


ACAGCAGCAGGCAGGTCCCGAGGTGACCTATGAACAGATAAATAACGGCACTTTCGACGAACCTATTGTGAACGATC


 


AGGCCAACAACCCGGACGAATGGTTCATTTGGCAGGCGGGAGATTACGGGATCAGCGGTGCCAGGGTCTCCGATTAC


 


GGTGTCAGGGATGGCTACGCTTATATCACGATAGCCGATCCTGGAACTGACACGTGGCATATTCAGTTCAACCAGTG


 


GATAGGTCTTTACAGAGGAAAAACCTACACCATTTCTTTCAAAGCAAAAGCGGATACACCAAGACCTATAAATGTGA


 


AAATTCTGCAGAATCACGATCCCTGGACCAACTATTTTGCTCAAACGGTGAATCTCACAGCGGACTGGCAGACGTTC


 


ACGTTCACCTACACGCATCCAGACGATGCGGATGAGGTCGTTCAGATCAGTTTCGAACTCGGAGAAGGAACGGCAAC


 


TACGATTTATTTCGATGATGTCACGGTGAGCCCTCAAGCGGCCGCACTCGAGCACCACCACCACCACCACTGA









Fsβ-glucanases and variants thereof (e.g., TFsSW203F having the sequence of SEQ ID NO:3) are known in the art. See, e.g., U.S. Pat. No. 7,527,958. The amino acid sequence and nucleic acid sequence of TFSW203F are shown below. The amino acid sequence shown includes an optional C-terminal tag sequence (underlined).








TFsw2o3F amino  



acid sequence
(SEQ ID NO: 3)







M V S A K D F S G A E L Y T L E E V Q Y G K F E A


 


R M K M A A A S G T V S S M F L Y Q N G S E I A D


 


G R P W V E V D I E V L G K N P G S F Q S N I I T


 


G K A G A Q K T S E K H H A V S P A A D Q A F H T


 


Y G L E W T P N Y V R W T V D G Q E V R K T E G G


 


Q V S N L T G T Q G L R F N L W S S E S A A W V G


 


Q F D E S K L P L F Q F I N W V K V Y K Y T P G Q


 


G E G G S D F T L D W T D N F D T F D G S R W G K


 


G D F T F D G N R V D L T D K N I Y S R D G M L I


 


L A L T R K G Q E S F N G Q V P R D D E P A P N S


 


S S V D K L A A A L E H H H H H H


 








TFsw2o3F nucleic 



acid sequence 
(SEQ ID NO: 4)







ATGOTTAGCGCAAAGGATTTTAGCGGTGCCGAACTCTACACGTTAGAAGA


 


AGTTCAGTACGGTAAGTTTGAAGCCCGTATGAAGATGGCAGCCGCATCOG


 


GAACAGTCAGTTCCATOTTCCTCTACCAGAATGOTTCCGAAATCGCCGAT


 


GGAAGGCCCTOGGTAGAAGTGGATATTGAAGTTCTCGGCAAGAATCCOGG


 


CAGTTTCCAGTCCAACATCATTACCGGTAAGGCCGGCGCACAAAAGACTA


 


GCGAAAAGCACCATGCTOTTAGCCCCGCCGCCGATCAGGCTTTCCACACC


 


TACGOTCTCGAATGGACTCCGAATTACGTCCGCTGGACTOTTGACGOTCA


 


GGAAGTCCGCAAGACGGAAGGTGGCCAGGTTTCCAACTTGACAGGTACAC


 


AGGGACTCCGTTTTAACCTTTGGTCGTCTGAGAGTGCGGCTTGGGTTGGC


 


CAGTTCGATGAATCAAAGCTTCCGCTTTTCCAGTTCATCAACTOGGTCAA


 


GGTTTATAAGTATACGCCOGGCCAGGGCGAAGGCGGCAGCGACTTTACGC


 


TTGACTGGACCGACAATTTTGACACGTTTGATGGCTCCCGCTOGGGCAAG


 


GGTGACTTCACATTTGACGGTAACCGTOTCGACCTCACCGACAAGAACAT


 


CTACTCCAGAGATGGCATOTTGATCCTCGCCCTCACCCGCAAAGGTCAGG


 


AAAGCTTCAACGGCCAGGTTCCGAGAGATGACGAACCTGCTCCGAATTCG


 


AGCTCCGTCGACAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCA


 


CTGA






For example, the N-terminal CBM domain TmB1 (e.g., residues 19-176 of SEQ ID NO:1), the catalytic domain TmLamCD (e.g., residues 211-488 of SEQ ID NO:1), and the C-terminal CBM domain TmB2 of TmLam (e.g., residues 495-642 of SEQ ID NO:1), and TFSW203F (residues 1-248 of SEQ ID NO:3) can be combined in different configurations to generate novel fusion proteins. The various segments of the fusion polypeptides can be linked directly to each other or indirectly via a linker (e.g., a peptide including 1 to 100 amino acids).
The amino acid sequences and nucleic acid sequences of exemplary fusion polypeptides are shown below. Each amino acid sequence shown includes an optional C-terminal tag sequence (underlined).








TmLamcD-TFsw2o3F amino



acid sequence
(SEQ ID NO: 5)







M E D E D K V E D W Q L V W S Q E F D D G V I D P


 


N I W N F E I G N G H A K G I P G W G N G E L E Y


 


Y T D E N A F V E N G C L V I E A R K E Q V S D E 


 


Y G T Y D Y T S A R M T T E G K F E I K Y G K I E


 


I R A K L P K G K G I W P A L W M L G N N I G E V 


 


G W P T C G E I D I M E M L G H D T R T V Y G T A 


 


H G P G Y S G G A S I G V A Y H L P E G V P D F S 


 


E D F H I F S I E W D E D E V E W Y V D G Q L Y H 


 


V L S K D E L A E L G L E W V F D H P F F L I L N 


 


V A V G G Y W P G Y P D E T T Q F P Q R M Y I D Y


 


I R V Y K D M N P E T I T G E V D D C E Y E Q A Q 


 


Q Q A G P E V T Y E Q I N N A M V S A K D F S G A 


 


E L Y T L E E V Q Y G K F E A R M K M A A A S G T 


 


V S S M F L Y Q N G S E I A D G R P W V E V D I E 


 


V L G K N P G S F Q S N I I T G K A G A Q K T S E 


 


K H H A V S P A A D Q A F H T Y G L E W T P N Y V 


 


R W T V D G Q E V R K T E G G Q V S N L T G T Q G 


 


L R F N L W S S E S A A W V G Q F D E S K L P L F 


 


Q F I N W V K V Y K Y T P G Q G E G G S D F T L D 


 


W T D N F D T F D G S R W G K G D F T F D G N R V


 


D L T D K N I Y S R D G M L I L A L T R K G Q E S


 


F N G Q V P R D D E P A P N S S S V D K L A A A L 


 


E H H H H H H


 








TmLamcD-TFsw2o3F nucleic



acid sequence
(SEQ ID NO: 6)







ATGGCCATGGAGGATGAAGACAAAGTGGAAGACTGGCAGCTCGTTTGGAG


 


TCAGGAGTTCGATGACGGTOTTATCGATCCGAACATCTGGAACTTCGAGA


 


TAGGAAACGOTCATGCAAAAGGTATTCCAGGCTGGGGTAACGOGGAACTC


 


GAGTACTATACAGACGAAAACGCGTTCGTTGAGAACGGCTGTCTTGTGAT


 


TGAGGCAAGAAAAGAACAGGTTTCCGATGAGTACGGAACCTACGACTACA


 


CCTCAGCCAGGATGACCACAGAAGGAAAATTCGAAATAAAGTACGGAAAA


 


ATCGAAATAAGGGCAAAACTTCCAAAAGGAAAAGGTATCTGGCCCGCTCT


 


CTGGATGCTCGGAAACAACATAGGAGAGGTCGGATGGCCCACCTGTGGTG


 


AGATAGACATCATGGAAATGCTTGGCCACGACACCAGAACCGTTTATGGA


 


ACAGCACACGOTCCGGGATATTCTGGTGGTGCGAGTATAGGTOTTGCCTA


 


TCATCTTCCAGAAGGAGTTCCTGATTTCTCCGAAGACTTCCACATTTTCT


 


CCATCGAGTOGGACGAAGACGAAGTGGAGTGGTACGTGGACGGACAGCTC


 


TACCACGTCCTCAGCAAGGATGAACTGGCCGAACTCGOTCTTGAGTOGGT


 


TTTCGACCATCCGTTCTTCCTCATTCTGAACGTTGCCGTGGGAGGCTACT


 


GGCCOGGTTATCCCGACGAAACCACCCAATTCCCGCAGAGAATGTACATC


 


GACTACATCAGAGTCTATAAAGATATGAATCCGGAAACAATCACCOGGGA


 


AGTGGATGACTGCGAATATGAACAAGCACAGCAGCAGGCAGGTCCCGAGG


 


TGACCTATGAACAGATAAATAACGCCATGOTTAGCGCAAAGGATTTTAGC


 


GGTGCCGAACTCTACACGTTAGAAGAAGTTCAGTACGGTAAGTTTGAAGC


 


CCGTATGAAGATGGCAGCCGCATCOGGAACAGTCAGTTCCATGTTCCTCT


 


ACCAGAATGOTTCCGAAATCGCCGATGGAAGGCCCTOGGTAGAAGTGGAT


 


ATTGAAGTTCTCGGCAAGAATCCOGGCAGTTTCCAGTCCAACATCATTAC


 


CGGTAAGGCCGGCGCACAAAAGACTAGCGAAAAGCACCATGCTGTTAGCC


 


CCGCCGCCGATCAGGCTTTCCACACCTACGOTCTCGAATGGACTCCGAAT


 


TACGTCCGCTGGACTOTTGACGOTCAGGAAGTCCGCAAGACGGAAGGTGG


 


CCAGGTTTCCAACTTGACAGGTACACAGGGACTCCGTTTTAACCTTTGGT


 


CGTCTGAGAGTGCGGCTTGGGTTGGCCAGTTCGATGAATCAAAGCTTCCG


 


CTTTTCCAGTTCATCAACTOGGTCAAGGTTTATAAGTATACGCCOGGCCA


 


GGGCGAAGGCGGCAGCGACTTTACGCTTGACTGGACCGACAATTTTGACA


 


CGTTTGATGGCTCCCGCTOGGGCAAGGGTGACTTCACATTTGACGGTAAC


 


CGTOTCGACCTCACCGACAAGAACATCTACTCCAGAGATGGCATGTTGAT


 


CCTCGCCCTCACCCGCAAAGGTCAGGAAAGCTTCAACGGCCAGGTTCCGA


 


GAGATGACGAACCTGCCCCGAATTCGAGCTCCGTCGACAAGCTTGCGGCC


 


GCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGC


 


CCGAAAGAAGCTGAAGTTCTCGGT


 








TFsw2o3F-TmB2 amino



acid sequence
(SEQ ID NO: 7)







M V S A K D F S G A E L Y T L E E V Q Y G K F E A


 


R M K M A A A S G T V S S M F L Y Q N G S E I A D


 


G R P W V E V D I E V L G K N P G S F Q S N I I T


 


G K A G A Q K T S E K H H A V S P A A D Q A F H T


 


Y G L E W T P N Y V R W T V D G Q E V R K T E G G


 


Q V S N L T G T Q G L R F N L W S S E S A A W V G


 


Q F D E S K L P L F Q F I N W V K V Y K Y T P G Q


 


G E G G S D F T L D W T D N F D T F D G S R W G K


 


G D F T F D G N R V D L T D K N I Y S R D G M L I


 


L A L T R K G Q E S F N G Q V P R D D E P A P N S


 


G T F D E P I V N D Q A N N P D E W F I W Q A G D


 


Y G I S G A R V S D Y G V R D G Y A Y I T I A D P


 


G T D T W H I Q F N Q W I G L Y R G K T Y T I S F


 


K A K A D T P R P I N V K I L Q N H D P W T N Y F


 


A Q T V N L T A D W Q T F T F T Y T H P D D A D E


 


V V Q I S F E L G E G T A T T I Y F D D V T V S P


 


Q A A A L E H H H H H H


 








TFsw2o3F-TmB2 nucleic



acid sequence
(SEQ ID NO: 8)







CCCCTTTTTAAAGGGCACATTCCCCTCTAGAATAATTTTOTTTAACTTTA


 


AGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTC


 


TGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGOTTAGCGCAAAGGAT


 


TTTAGCGGTGCCGAACTCTACACGTTAGAAGAAGTTCAGTACGGTAAGTT


 


TGAAGCCCGTATGAAGATGGCAGCCGCATCGGGAACAGTCAGTTCCATOT


 


TCCTCTACCAGAATGOTTCCGAAATCGCCGATGGAAGGCCCTOGGTAGAA


 


GTGGATATTGAAGTTCTCGGCAAGAATCCOGGCAGTTTCCAGTCCAACAT


 


CATTACCGGTAAGGCCGGCGCACAAAAGACTAGCGAAAAGCACCATGCTO


 


TTAGCCCCGCCGCCGATCAGGCTTTCCACACCTACGOTCTCGAATGGACT


 


CCGAATTACGTCCGCTGGACTOTTGACGOTCAGGAAGTCCGCAAGACGGA


 


AGGTGGCCAGGTTTCCAACTTGACAGGTACACAGGGACTCCGTTTTAACC


 


TTTGGTCGTCTGAGAGTGCGGCTTGGGTTGGCCAGTTCGATGAATCAAAG


 


CTTCCGCTTTTCCAGTTCATCAACTOGGTCAAGGTTTATAAGTATACGCC


 


OGGCCAGGGCGAAGGCGGCAGCGACTTTACGCTTGACTGGACCGACAATT


 


TTGACACGTTTGATGGCTCCCGCTOGGGCAAGGGTGACTTCACATTTGAC


 


GGTAACCGTGTCGACCTCACCGACAAGAACATCTACTCCAGAGATGGCAT


 


OTTGATCCTCGCCCTCACCCGCAAAGGTCAGGAAAGCTTCAACGGCCAGG


 


TTCCGAGAGATGACGAACCTGCCCCGAATTCCGGCACTTTCGACGAACCT


 


ATTGTGAACGATCAGGCCAACAACCCGGACGAATGGTTCATTTGGCAGGC


 


OGGAGATTACGGGATCAGCGGTGCCAGGGTCTCCGATTACGGTGTCAGGG


 


ATGGCTACGCTTATATCACGATAGCCGATCCTGGAACTGACACGTGGCAT


 


ATTCAGTTCAACCAGTGGATAGGTCTTTACAGAGGAAAAACCTACACCAT


 


TTCTTTCAAAGCAAAAGCGGATACACCAAGACCTATAAATGTGAAAATTC


 


TGCAGAATCACGATCCCTGGACCAACTATTTTGCTCAAACGGTGAATCTC


 


ACAGCGGACTGGCAGACGTTCACGTTCACCTACACGCATCCAGACGATGC


 


GGATGAGGTCGTTCAGATCAGTTTCGAACTCGGAGAAGGAACGGCAACTA


 


CGATTTATTTCGATGATGTCACGGTGAGCCCTCAAGCGGCCGCACTCGAG


 


CACCACCACCACCACCACTGA


 








TFsw2o3F-TmLamcD amino



acid sequence
(SEQ ID NO: 9)







M V S A K D F S G A E L Y T L E E V Q Y G K F E A


 


R M K M A A A S G T V S S M F L Y Q N G S E I A D


 


G R P W V E V D I E V L G K N P G S F Q S N I I T


 


G K A G A Q K T S E K H H A V S P A A D Q A F H T


 


Y G L E W T P N Y V R W T V D G Q E V R K T E G G


 


Q V S N L T G T Q G L R F N L W S S E S A A W V G


 


Q F D E S K L P L F Q F I N W V K V Y K Y T P G Q


 


G E G G S D F T L D W T D N F D T F D G S R W G K


 


G D F T F D G N R V D L T D K N I Y S R D G M L I


 


L A L T R K G Q E S F N G Q V P R D D E P A P N S


 


E D W Q L V W S Q E F D D G V I D P N I W N F E I


 


G N G H A K G I P G W G N G E L E Y Y T D E N A F


 


V E N G C L V I E A R K E Q V S D E Y G T Y D Y T


 


S A R M T T E G K F E I K Y G K I E I R A K L P K


 


G K G I W P A L W M L G N N I G E V G W P T C G E


 


I D I M E M L G H D T R T V Y G T A H G P G Y S G


 


G A S I G V A Y H L P E G V P D F S E D F H I F S


 


I E W D E D E V E W Y V D G Q L Y H V L S K D E L


 


A E L G L E W V F D H P F F L I L N V A V G G Y W


 


P G Y P D E T T Q F P Q R M Y I D Y I R V Y K D M


 


N P E T I T G E V D D C E Y E Q A Q Q Q A G P E V


 


T Y E Q I N N A A A L E H H H H H H


 








TFsw2o3F-TmLamcD nucleic



acid sequence
(SEQ ID NO: 10)







GAATOGGATAACGGAAATTCCCTCTAGAATAATTTTGTTTAACTTTAAGA


 


AGGAGATATACATATGAAATACCTGCTGGCGACCGCTGCTGCTGGTCTGC


 


TGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGTTAGCGCAAAGGATTTT


 


AGCGGTGCCGAACTCTACACGTTAGAAGAAGTTCAGTACGGTAAGTTTGA


 


AGCCCGTATGAAGATGGCAGCCGCATCOGGAACAGTCAGTTCCATOTTCC


 


TCTACCAGAATGOTTCCGAAATCGCCGATGGAAGGCCCTOGGTAGAAGTG


 


GATATTGAAGTTCTCGGCAAGAATCCOGGCAGTTTCCAGTCCAACATCAT


 


TACCGGTAAGGCCGGCGCACAAAAGACTAGCGAAAAGCACCATGCTOTTA


 


GCCCCGCCGCCGATCAGGCTTTCCACACCTACGOTCTCGAATGGACTCCG


 


AATTACGTCCGCTGGACTGTTGACGOTCAGGAAGTCCGCAAGACGGAAGG


 


TGGCCAGGTTTCCAACTTGACAGGTACACAGGGACTCCGTTTTAACCTTT


 


GGTCGTCTGAGAGTGCGGCTTGGGTTGGCCAGTTCGATGAATCAAAGCTT


 


CCGCTTTTCCAGTTCATCAACTGGGTCAAGGTTTATAAGTATACGCCOGG


 


CCAGGGCGAAGGCGGCAGCGACTTTACGCTTGACTGGACCGACAATTTTG


 


ACACGTTTGATGGCTCCCGCTOGGGCAAGGGTGACTTCACATTTGACGGT


 


AACCGTGTCGACCTCACCGACAAGAACATCTACTCCAGAGATGGCATOTT


 


GATCCTCGCCCTCACCCGCAAAGGTCAAGAAAGCTTCAACGGCCAGGTTC


 


CGAGAGATGACGAACCTGCTCCGAATTCGGAAGACTGGCAGCTCGTTTGG


 


AGTCAGGAGTTCGATGACGGTOTTATCGATCCGAACATCTGGAACTTCGA


 


GATAGGAAACGOTCATGCAAAAGGTATTCCAGGCTGGGGTAACGOGGAAC


 


TCGAGTACTATACAGACGAAAACGCGTTCGTTGAGAACGGCTGTCTTGTG


 


ATTGAGGCAAGAAAAGAACAGGTTTCCGATGAGTACGGAACCTACGACTA


 


CACCTCAGCCAGGATGACCACAGAAGGAAAATTCGAAATAAAGTACGGAA


 


AAATCGAAATAAGGGCAAAACTTCCAAAAGGAAAAGGTATCTGGCCCGCT


 


CTCTGGATGCTCGGAAACAACATAGGAGAGGTCGGATGGCCCACCTGTGG


 


TGAGATAGACATCATGGAAATGCTTGGCCACGACACCAGAACCGTTTATG


 


GAACAGCACACGOTCCOGGATATTCTGGTGGTGCGAGTATAGGTOTTGCC


 


TATCATCTTCCAGAAGGAGTTCCTGATTTCTCCGAAGACTTCCACATTTT


 


CTCCATCGAGTOGGACGAAGACGAAGTGGAGTGGTACGTGGACGGACAGC


 


TCTACCACGTCCTCAGCAAGGATGAACTGGCCGAACTCGOTCTTGAGTOG


 


GTTTTCGACCATCCGTTCTTCCTCATTCTGAACGTTGCCGTGGGAGGCTA


 


CTGGCCGGGTTATCCCGACGAAACCACCCAATTCCCGCAGAGAATGTACA


 


TCGACTACATCAGAGTCTATAAAGATATGAATCCGGAAACAATCACCOGG


 


GAAGTGGATGACTGCGAATATGAACAAGCACAGCAGCAGGCAGGTCCCGA


 


GGTGACCTATGAACAGATAAATAACGCGGCCGCACTCGAGCACCACCACC


 


ACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGAAGCTAAGTGCG


 


GGAA


 








Tins 1-TFSW203F- TMB2 amino



acid sequence
(SEQ ID NO: 11)







M A Q N I L G N A S F D E P I L I A G V D I D P P


 


A E D G S I D T G G N W V F F T N S N G E G T A R


 


V E N G V L V V E I T N G G D H T W S V Q I I Q A


 


P I R V E K L H K Y R V S F R A K A S S Q K N I G


 


V K I G G T A G R G W T A Y N P G T D E S G G M V


 


F E L G T D W Q K Y E F E F V M R Q E T D E N A R


 


F E F Q L G R Y T G T V W I D D V V M E D I G V L


 


E V S G E E N E I Y T M V S A K D F S G A E L Y T


 


L E E V Q Y G K F E A R M K M A A A S G T V S S M


 


F L Y Q N G S E I A D G R P W V E V D I E V L G K


 


N P G S F Q S N I I T G K A G A Q K T S E K H H A


 


V S P A A D Q A F H T Y G L E W T P N Y V R W T V


 


D G Q E V R K T E G G Q V S N L T G T Q G L R F N


 


L W S S E S A A W V G Q F D E S K L P L F Q F I N


 


W V K V Y K Y T P G Q G E G G S D F T L D W T D N


 


F D T F D G S R W G K G D F T F D G N R V D L T D


 


K N I Y S R D G M L I L A L T R K G Q E S F N G Q


 


V P R D D E P A P N S G T F D E P I V N D Q A N N


 


P D E W F I W Q A G D Y G I S G A R V S D Y G V R


 


D G Y A Y I T I A D P G T D T W H I Q F N Q W I G


 


L Y R G K T Y T I S F K A K A D T P R P I N V K I


 


L Q N H D P W T N Y F A Q T V N L T A D W Q T F T


 


F T Y T H P D D A D E V V Q I S F E L G E G T A T


 


T I Y F D D V T V S P Q A A A L E H H H H H H


 








Tms 1-TFSW203F- TMB2 amino



acid sequence
(SEQ ID NO: 12)







CGCTCTATACCCGAAAAAATTTCCCTTCTAGACTAATTTTOTTTAACTTT


 


AAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGT


 


CTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGCTCAAAACATCCT


 


TGGCAACGCTTCTTTCGATGAACCAATTCTCATCGCAGGTGTGGATATAG


 


ACCCACCCGCAGAGGATGGCTCTATAGACACAGGAGGAAACTOGGTATTC


 


TTCACCAATTCAAACGGTGAGGGAACGGCTCGAGTCGAAAACGGCGTTCT


 


CGTGGTTGAGATAACAAACGGAGGAGATCACACCTGGTCGOTTCAGATCA


 


TACAGGCTCCCATACGTOTTGAGAAACTCCACAAGTACAGAGTTTCTTTC


 


CGAGCCAAGGCTTCCTCTCAAAAGAACATCGOGGTGAAGATAGGAGGAAC


 


GGCCGGAAGAGGATGGACCGCGTACAACCCCGGTACCGACGAATCCGGCG


 


GCATGGTCTTCGAGCTCGGAACAGATTGGCAGAAGTACGAGTTCGAATTC


 


GTCATGAGACAGGAGACCGATGAAAATGCTCGTTTCGAGTTTCAGCTTGG


 


AAGGTATACCGGCACGOTCTGGATAGACGACGTAGTGATGGAGGACATCG


 


GTOTTCTCGAGGTAAGCGGTGAGGAAAACGAAATCTACACCATGOTTAGC


 


GCAAAGGATTTTAGCGGTGCCGAACTCTACACGTTAGAAGAAGTTCAGTA


 


CGGTAAGTTTGAAGCCCGTATGAAGATGGCAGCCGCATCOGGAACAGTCA


 


GTTCCATOTTCCTCTACCAGAATGOTTCCGAAATCGCCGATGGAAGGCCC


 


TOGGTAGAAGTGGATATTGAAGTTCTCGGCAAGAATCCOGGCAGTTTCCA


 


GTCCAACATCATTACCGGTAAGGCCGGCGCACAAAAGACTAGCGAAAAGC


 


ACCATGCTOTTAGCCCCGCCGCCGATCAGGCTTTCCACACCTACGOTCTC


 


GAATGGACTCCGAATTACGTCCGCTGGACTOTTGACGOTCAGGAAGTCCG


 


CAAGACGGAAGGTGGCCAGGTTTCCAACTTGACAGGTACACAGGGACTCC


 


GTTTTAACCTTTGGTCGTCTGAGAGTGCGGCTTGGGTTGGCCAGTTCGAT


 


GAATCAAAGCTTCCGCTTTTCCAGTTCATCAACTOGGTCAAGGTTTATAA


 


GTATACGCCOGGCCAGGGCGAAGGCGGCAGCGACTTTACGCTTGACTGGA


 


CCGACAATTTTGACACGTTTGATGGCTCCCGCTOGGGCAAGGGTGACTTC


 


ACATTTGACGGTAACCGTGTCGACCTCACCGACAAGAACATCTACTCCAG


 


AGATGGCATOTTGATCCTCGCCCTCACCCGCAAAGGTCAGGAAAGCTTCA


 


ACGGCCAGGTTCCGAGAGATGACGAACCTGCCCCGAATTCCGGCACTTTC


 


GACGAACCTATTGTGAACGATCAGGCCAACAACCCGGACGAATGOTTCAT


 


TTGGCAGGCOGGAGATTACGGGATCAGCGGTGCCAGGGTCTCCGATTACG


 


GTGTCAGGGATGGCTACGCTTATATCACGATAGCCGATCCTGGAACTGAC


 


ACGTGGCATATTCAGTTCAACCAGTGGATAGGTCTTTACAGAGGAAAAAC


 


CTACACCATTTCTTTCAAAGCAAAAGCGGATACACCAAGACCTATAAATG


 


TGAAAATTCTGCAGAATCACGATCCCTGGACCAACTATTTTGCTCAAACG


 


GTGAATCTCACAGCGGACTGGCAGACGTTCACGTTCACCTACACGCATCC


 


AGACGATGCGGATGAGGTCGTTCAGATCAGTTTCGAACTCGGAGAAGGAA


 


CGGCAACTACGATTTATTTCGATGATGTCACGGTGAGCCCTCAAGCGGCC


 


GCACTCGAGCACCACCACCACCACCACTGA


 








Tinsi-TFsw2o3F amino 



acid sequence
(SEQ ID NO: 13)







M A Q N I L G N A S F D E P I L I A G V D I D P P


 


A E D G S I D T G G N W V F F T N S N G E G T A R


 


V E N G V L V V E I T N G G D H T W S V Q I I Q A


 


P I R V E K L H K Y R V S F R A K A S S Q K N I G


 


V K I G G T A G R G W T A Y N P G T D E S G G M V


 


F E L G T D W Q K Y E F E F V M R Q E T D E N A R


 


F E F Q L G R Y T G T V W I D D V V M E D I G V L


 


E V S G E E N E I Y T M V S A K D F S G A E L Y T


 


L E E V Q Y G K F E A R M K M A A A S G T V S S M


 


F L Y Q N G S E I A D G R P W V E V D I E V L G K


 


N P G S F Q S N I I T G K A G A Q K T S E K H H A


 


V S P A A D Q A F H T Y G L E W T P N Y V R W T V


 


D G Q E V R K T E G G Q V S N L T G T Q G L R F N


 


L W S S E S A A W V G Q F D E S K L P L F Q F I N


 


W V K V Y K Y T P G Q G E G G S D F T L D W T D N


 


F D T F D G S R W G K G D F T F D G N R V D L T D


 


K N I Y S R D G M L I L A L T R K G Q E S F N G Q


 


V P R D D E P A P N S S S V D K L A A A L E H H H


 


H H H


 








Tins i-TFsw2o3F nucleic



acid sequence
(SEQ ID NO: 14)







TTTAAGAAGGAGATATACATATGAAATACCTOCTOCCGACCGCTOCTOCT


 


GOTCTOCTOCTCCTCGCTOCCCAGCCOGCGATGGCCATGGCTCAAAACAT


 


CCTTGGCAACGCTTCTTTCGATGAACCAATTCTCATCGCAGGTOTGGATA


 


TAGACCCACCCGCAGAGGATGGCTCTATAGACACAGGAGGAAACTOGGTA


 


TTCTTCACCAATTCAAACGOTGAGGGAACGGCTCGAGTCGAAAACGGCGT


 


TCTCGTGOTTGAGATAACAAACGGAGGAGATCACACCTGOTCGOTTCAGA


 


TCATACAGGCTCCCATACGTOTTGAGAAACTCCACAAGTACAGAGTTTCT


 


TTCCGAGCCAAGGCTTCCTCTCAAAAGAACATCGOGGTGAAGATAGGAGG


 


AACGOCCOGAAGAGGATGGACCGCGTACAACCCCGOTACCGACGAATCCG


 


GCGOCATGOTCTTCGAGCTCGGAACAGATTGGCAGAAGTACGAGTTCGAA


 


TTCGTCATGAGACAGGAGACCGATGAAAATOCTCGTTTCGAGTTTCAGCT


 


TGGAAGGTATACCGOCACGOTCTGGATAGACGACGTAGTGATGGAGGACA


 


TCGOTGTTCTCGAGGTAAGCGOTGAGGAAAACGAAATCTACACCATGOTT


 


AGCGCAAAGGATTTTAGCGGTOCCGAACTCTACACGTTAGAAGAAGTTCA


 


GTACGOTAAGTTTGAAGCCCGTATGAAGATGGCAGCCGCATCOGGAACAG


 


TCAGTTCCATOTTCCTCTACCAGAATGOTTCCGAAATCGCCGATGGAAGG


 


CCCTOGGTAGAAGTOGATATTGAAGTTCTCGOCAAGAATCCOGGCAGTTT


 


CCAGTCCAACATCATTACCGOTAAGGCCGGCGCACAAAAGACTAGCGAAA


 


AGCACCATOCTOTTAGCCCCGCCGCCGATCAGGCTTTCCACACCTACGOT


 


CTCGAATGGACTCCGAATTACGTCCGCTGGACTOTTGACGOTCAGGAAGT


 


CCGCAAGACGGAAGGTGGCCAGGTTTCCAACTTGACAGGTACACAGGGAC


 


TCCOTTTTAACCTTTGOTCGTCTGAGAGTOCGGCTTGGGTTGGCCAGTTC


 


GATGAATCAAAGCTTCCGCTTTTCCAGTTCATCAACTOGGTCAAGGTTTA


 


TAAGTATACGCCOGGCCAGGGCGAAGGCGOCAGCGACTTTACGCTTGACT


 


GGACCGACAATTTTGACACGTTTGATGGCTCCCGCTOGGGCAAGGGTGAC


 


TTCACATTTGACGOTAACCGTOTCGACCTCACCGACAAGAACATCTACTC


 


CAGAGATGGCATOTTGATCCTCGCCCTCACCCGCAAAGGTCAGGAAAGCT


 


TCAACGOCCAGGTTCCGAGAGATGACGAACCTOCCCCGAATTCGAGCTCC


 


GTCGACAAGCTTGCGOCCGCACTCGAGCACCACCACCACCACCACTGAGA


 


TCCGGCTOCTAACAAAGCCCGAAAGAAGCTAGGGTTTTTCGTC






The fusion polypeptides and nucleic acid molecules described herein can be generated using methods known in the art, e.g., recombinant technology.
The nucleic acid molecules can be used to express the polypeptides and fusion polypeptides described herein. Each nucleic acid molecule can be linked to suitable regulatory sequences to generate an expression vector.
Examples of the vector include a plasmid, cosmid, or viral vector. The vector includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. A regulatory sequence includes promoters, enhancers, and other expression control elements (e.g., T7 promoter, cauliflower mosaic virus 35S promoter sequences or polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vector can be introduced into host cells to produce the polypeptide or fusion protein of this invention.
Host cells include E. coli cells, insect cells (e.g., using baculovirus expression vectors), plant cells, yeast cells, and mammalian cells. See e.g., Goeddel, (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif.
Host cells containing an expression vector for expressing a fusion polypeptide described herein can be cultured under conditions allowing expression of the polypeptide. The expressed polypeptide can then be isolated from the host cells or culture medium.
The isolated fusion polypeptide can be used for various purposes. Thus, described herein are also methods of using the fusion polypeptides. For example, the fusion polypeptide can be used to degrade a substrate, e.g., a polysaccharide. The fusion polypeptide can also be used in industrial applications.
The specific example below is to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present disclosure to its fullest extent. All publications cited herein are herein incorporated by reference in their entirety.
A truncated and mutated 1,3-1,4-β-D-glucanase gene (encoding TFsW203F having residues 1-248 of SEQ ID NO:3) from Fibrobacter succinogenes, and a 1,3-β-D-glucanase gene (encoding TmLam having the sequence of SEQ ID NO:1) from hyperthermophilic Thermotoga maritima were used to generate hybrid enzymes. The substrate binding domains (TmB1 having residues 19-176 of SEQ ID NO:1 and TmB2 having residues 495-642 of SEQ ID NO:1) and the catalytic domain (TmLamCD having residues 211-488 of SEQ ID NO:1) of TmLam (SEQ ID NO:1) were linked to the N- or C-terminus of TFsW203F (having residues 1-248 of SEQ ID NO:3) to create four hybrid enzymes, TmB1-TFsW203F (having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq), TFsW203F-TmB2 (having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq), TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq), and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq). The results obtained from kinetic studies show that increased specific activities and turnover rate for lichenan and laminarin were observed in TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq), respectively. Furthermore, fluorescence and CD spectrometric analyses indicated that the hybrid TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) was structurally more stable and more thermal tolerant than the parental TFsW203F (having residues 1-248 of SEQ ID NO:3).
Materials and Methods
(1) Bacterial Strains and Cultivation
Thermotoga maritima MSB8 (Huber et al., 1986, Arch. Microbiol., 144, 324-333) was purchased from ATCC (DSM 3109) and used as the source for the cloning of the 1,3-β-D-glucanase (TmLam) gene. T. maritima cells were grown anaerobically at 70° C. in a growth medium (ATCC #2114 broth) composed of 25% artificial sea water (v/v) supplemented with the following components (g/L): soluble starch, 5.0; yeast extract, 0.5; NaCl, 20; KH2PO4, 0.5; NiCl2.6H2O, 0.002; Na2S.9H2O, 0.5; resazurin, 0.001; and Wolfe's mineral solution (1.5%, v/v). E. coli strain XL-1 Blue (Stratagene) was used for the purpose of cloning, and BL21(DE3) (Novagen) competent cells were employed for the overexpression of cloned genes. The conditions for cultivation of recombinant cells, media and overexpression were in accordance with previously-published methods (Wen et al., 2005, Biochemistry, 44, 9197-9205), and plasmid pET26b(+) was used for the cloning and expression of recombinant glucanase genes.
(2) Construction of Plasmids for Expression of Parental and Hybrid Glucanases
The chromosomal DNA of T. maritima was isolated from cultured cell using QIAGEN Genomic-tip 20/G (QIAGEN). The DNA was used as the template for PCR amplification of the open reading frame of TmLam gene (without the presence of N-terminal signal sequence) by a pair of specific primers TmNcoI and TmNotI. See Table 1. The amplified DNA fragments were digested with NcoI and NotI and then subcloned into pET26b(+) vector to form pTmLam.
(3) Construction of Recombinant Glucanase Genes
The TFsW203F plasmid (pTFsW203F) previously created (Tsai et al., 2011, Biochem. Bioph. Res. Comm., 407, 593-598) was used to create hybrid enzymes with the CBM and catalytic domains of TmLam. The primers for the construction of hybrid glucanase genes are shown in Table 1.







TABLE 1







Oligonucleotide primers for the subcloning of 


specific protein domains in T. maritima


1,3-β-D-glucanase.









Primer 
SEQ ID



Name
NO
Sequence





TmNcoI
15
5′-CCTGTTTTCACCATGGCTCAAAACATCCT




TGGC-3′


 


TmNotI
16
5′-GCACCGGGGATGCGGCCGCTTGAGGGCTC-3′


 


TmB1(+)/
17
5′-GGATGAAGAGAATCGGAAGACTGGC-3′


EcoRI




 


TmB1(+)/
18
5′-CGAAATCTACACCATGGAGGATGAAGAC-3′


NcoI




 


TmB1(−)/
19
5′-GTCTTCATCCTCCATGGTGTAGATTTCG-3′


NcoI




 


TmB2(+)/
20
5′-CCTATGAACAGATGAATTCCGGCACTTTCG-3′


EcoRI




 


TmB2(−)/
21
5′-CGAAAGTGCCAAGCTTTATCTGTTCATAGG-3′


HindIII




 


TmB2(−)/
22
5′-CGACCATGGCGTTATTTATCTGTTCATAGG-3′


NcoI




 


TmB2(−)/
23
5′-GCGGCCGCGTTATTATCTGTTCATAGG-3′


NotI




 


5Aa
24
5′-AAATACCTGCTGCCGACCG-3′


 


3Ba
25
5′-GCTAGTTATTGCTCAGCGGTG-3′





aVector-specific primers






The DNA fragments of N-terminal CBM4-1 (TmB1) and C-terminal CBM4-2 (TmB2) from the TmLam gene were obtained by PCR amplification with specific primer pairs, 5A and TmB1(−)/NcoI and TmB2(+)/EcoRI and 3B, respectively. The amplified DNA products were digested with appropriate restriction enzymes and ligated into plasmids pTFsW203F, before being pre-digested with the same restriction enzymes to obtain new chimera plasmids pTmB1-TFsW203F and pTFsW203F-TmB2. Furthermore, the amplified TmB1 DNA fragments and plasmid pTFsW203F-TmB2 were all pre-digested with NcoI and then ligated together to create another gene construct, pTmB1-TFsW203F-TmB2. The chimera plasmids containing the catalytic domain of TmLam, pTmLamCD or the hybrid enzyme genes, pTFsW203F-TmLamCD and pTmLamCD-TFsW203F, in other fibrolytic enzymes, were created and obtained using specific pairs of primers: TmB1(+)/NcoI and TmB2(−)/HindIII, TmB1(+)/NcoI and TmB2(−)/NcoI as well as TmB1(+)/EcoRI and TmB2(−)/NotI, respectively.
All of the created gene constructs were verified by the automated sequencing method through the use of the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). The confirmed plasmid genes were transformed into E. coli BL21(DE3) host cells for protein overexpression.
(4) Expression and Purification of Recombinant Proteins
E. coli BL21(DE3) cells harboring the appropriate plasmids were grown in two liters of LB medium containing kanamycin at 33° C. until the OD600 reached 0.8-1.0. Protein expression was then induced by the addition of IPTG (0.6 mM) and the culture was allowed to shake at 28-30° C. for 16 hours. The supernatant, containing expressed proteins, was harvested from the culture medium, and PMSF (1 mM) and leupeptin (1 μg/ml) were added to avoid protein degradation. Subsequently, the supernatant was concentrated using a PelliconTM-2 Mini Cassette Holder with a BiomaxTM 10K filter assembled on a LabscaleTM TFF system (Millipore). The concentrate was dialyzed against sodium phosphate buffer (50 mM, pH 8.0) containing imidazole (10 mM) and NaCl (0.3 M) at 4° C., and the resulting solution was applied to a 1.5×20 cm column containing pre-equilibrated HIS-selectTM nickel affinity gel (Sigma). The column was then washed with sodium phosphate buffer (50 mM, pH 8.0) containing imidazole (20 mM) and NaCl (0.3 M), followed with a five-times column volume of imidazole gradient (20-250 mM) in the same buffer, for protein elution. The fractions were analyzed by 12% SDS-PAGE, and the target proteins were pooled together for dialysis against sodium phosphate buffer (50 mM, pH 7.0) at 4° C. Purified proteins were stored in the presence of glycerol (10%) at −20° C., and protein concentration was determined by the Bradford method (Bio-Rad), using BSA (Sigma) as the standard.
(5) Kinetic Studies
The enzymatic activities of the purified parental or hybrid glucanases were measured by determining the rate of reducing sugar production from the hydrolysis of substrates, lichenan and laminarin (Sigma). The reduction of sugars was quantified by the use of 3,5-dinitrosalicylic acid (DNS) reagent (Wood and Bhat, 1988, Methods in Enzymology, Vol. 160, Academic Press, pp. 87-112) with glucose as the standard. A standard enzyme activity assay was performed in a 0.3 ml reaction mixture, as described previously (Cheng et al., 2002, Biochemistry, 41, 8759-8766). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of reducing sugar (glucose equivalent) per minute, and the specific activity was expressed in μmol of glucose per minute per nmol of protein. Various amounts of purified enzymes were used in each kinetic assay reaction, depending on the enzymatic activity. The kinetic data was analyzed using either ENZFITTER (BIOSOFT).
(6) On Gel Substrate-Enzyme Binding Analysis
The substrate-binding capability of the single domain and hybrid glucanases was evaluated using affinity SDS-PAGE. Unheated protein samples in a lysis buffer, in which the enzymes were not denatured and remained active, were separated on a 12% SDS gel with or without the presence of 0.1% substrate lichenan, along with a set of molecular weight (MW) standards (PageRulerTM Prestained Protein Ladder 10-170-kDa, Fermentas) on the same gel. After electrophoresis, proteins were visualized by staining with Coomassie brilliant blue R-250. Rf0 and Rf were defined as the ratio of the migration distance moved by each protein sample to the migration distance of the dye front on gel without and with substrate, respectively.
(7) Effects of Temperature on Parental and Hybrid Glucanases
Purified parental and hybrid glucanases were incubated individually for ten minutes at temperatures within the range of 30° C. and 80° C., at intervals of 5° C. Residual enzyme activity was determined immediately after heat treatment in sodium citrate buffer (50 mM, pH 6.0) at 45° C. using lichenan as the substrate (Cheng et al., 2002, Biochemistry, 41, 8759-8766).
(8) Fluorescence Spectrometry
The fluorescence emission spectra of TFsW203F (having residues 1-248 of SEQ ID NO:3) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) were taken on an AMICO-Bowman Series 2 spectrometer (Spectronic Instruments) with a 10×10 mm quartz cuvette incubated at 20° C., 50° C. and 75° C., respectively. Emission spectra were recorded from 310 nm to 430 nm by excitation at 295 nm, with a 4-nm monochromator bandpass. A final protein concentration of 30 μm/ml in sodium phosphate buffer (50 mM, pH 7.0) was used for all assays. Glucanase samples denatured with urea (8 M), or urea-denatured and then renatured by dialysis against sodium phosphate buffer (50 mM, pH 7.0) at 4° C. for 24 hours, were also analyzed for their fluorescence emission spectra at 25° C. Each measurement was carried out in triplicate.
(9) Circular Dichroism (CD) Spectrometry
CD spectrometric studies on the TFsW203F (having residues 1-248 of SEQ ID NO:3) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) proteins were carried out on a Jasco J715 CD spectrometer with a 10-mm cell at a range of temperatures from 30° C. to 90° C. Spectra were collected from 200 nm to 260 nm in 0.1 nm increments. Each spectrum was blank-collected and smoothed using a software package provided with the instruments.
(10) Protein Reactivation Assays
Purified TFsW203F (having residues 1-248 of SEQ ID NO:3) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) enzymes (30 μg/ml) were pretreated at 90° C. for ten minutes and then transferred to room temperature (25° C.). Recovery of enzymatic activity and protein re-folding of the heat-treated proteins within a ten-minute time frame at room temperature were measured using standard enzyme activity assay and fluorescence spectrometry respectively.
(11) Structural Modeling
In order to facilitate the interpretation of the kinetic data of TFsW203F (having residues 1-248 of SEQ ID NO:3) and hybrid enzymes from a structural point of view, we created a protein model complexed with oligosaccharides using the 3D structures of TmB2 (1GUI_A), truncated Fsβ-glucanase, TFs (1ZM1_A) (Tsai et al., 2005, J. Mol. Biol., 354, 642-651), TFsW203F (3H0O_A) (Tsai et al., 2011, Biochem. Bioph. Res. Comm., 407, 593-598) and TmLamCD (3AZZ_A) (Jeng et al., 2011, J. Biol. Chem., 286, 45030-45040), along with a model of TmB1 newly created for this study, the structure of which has not yet been resolved. The structural model of TmB1 was generated by using the HHpred (Soding et al., 2005, Nucleic Acids Res., 33, W244-W248), a website for homology detection and structure prediction by HMM-HMM comparison, based on the top five templates with the highest scores (PDB accession code: 3K4Z_A, 1CX1_A, 3P6B_A, 1GU3_A and 1GUI_A). The secondary structure matching (SSM) algorithm, with default settings for multiple 3D alignment in the PDBeFold server (Krissinel and Henrick, 2004, Acta Crystallogr. D Biol. Crystallogr., 60, 2256-2268), was used to superimpose all protein structures. After this the proteins were docked with β-1,3-cellohexose (from 1GUI) or β-1,3-1,4-celloheptaose (modeled), which were generated and energy-minimized by Coot (Emsley and Cowtan, 2004, Acta Crystallogr. D Biol. Crystallogr., 60, 2126-2132) and REFMAC5 (Murshudov et al., 2011, Acta Crystallogr. D Biol. Crystallogr., 67, 355-367.) from the CCP4 program suite (Collaborative Computational Project, Number 4, 1994; Winn et al., 2011, Acta Crystallogr. D Biol. Crystallogr., 67, 235-242), with the β-1,3-1,4-cellotriose in 1ZM1 as the template. The structural figures were then produced using PyMOL (DeLano Scientific; world wide web at pymol.org).
Results
(1) Construction and Purification of Hybrid Glucanases
The N-terminal CBM domain TmB1, catalytic domain TmLamCD, and C-terminal CBM domain TmB2 of TmLam (see SEQ ID NO:1) were amplified using PCR, and then ligated to the TFsW203F enzyme in different combinations. See FIG. 1. The confirmed gene sequences were subcloned into the pET26b(+) vector, and four recombinant chimera proteins, TmB1-TFsW203F (50.3 kDa; having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq), TFsW203F-TmB2 (46.3 kDa; having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq), TmB1-TFsW203F-TmB2 (66.8 kDa; having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq) and TFsW203F-TmLamCD (61.3 kDa; having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq), were then successfully expressed in E. coli BL21(DE3) host cells as extracellular proteins. The recombinant proteins were purified from the culture media using a nickel affinity column, and protein purity was judged by SDS-PAGE (See FIG. 2) and zymography analyses (data not shown).
(2) Affinity SDS-PAGE Analysis of Substrate-Enzyme Binding
The substrate binding ability and behavior of the non-catalytic modules TmB1 (having residues 19-176 of SEQ ID NO:1) and TmB2 (having residues 495-642 of SEQ ID NO:1) from heterogeneous enzyme (TmLam) in the constructs of hybrid lichenases were evaluated by visualizing the relative mobility retardation of individual proteins in 12% SDS gel with or without the presence of 0.1% lichenan, along with a set of MW standards on the same gel. The relative mobility distances (Rf values) of the MW standards were used as the negative control of substrate retardation effect and as the reference for demonstrating the electrophoretic quality of the same set of protein samples separated in two independent gels with or without substrate. See FIG. 3. After electrophoresis, the proteins with a single or double catalytic domains, TFsW203F (having residues 1-248 of SEQ ID NO:3), TmLamCD (having residues 211-488 of SEQ ID NO:1) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq), only had little differences on their Rf0 and Rf values obtained from plain gel and substrate-containing gel electrophoresis, respectively. See Table 2. The calculated retardation coefficient Kr (>10) revealed no observable retardation (Zverlov, et al., 2001, Microbiology, 147, 621-629).







TABLE 2







Parental and hybrid proteins binding 


affinity to lichenan in 12% SDS-PAGE.











Without substrate
0.1% Lichenan



Enzyme
Rf0a
Rfa
Krb













TFsW203F-TmLamCD
0.39
0.36
>10


TFsW203F-TmB2
0.40
0.38
>10


TmB1-TFsW203F
0.43
0.29
2


TmB1-TFsW203F-TmB2
0.33
0.19
1


TmLamCD
0.70
0.67
>10


TFsW203F
0.66
0.64
>10





aRf0 and Rf were defined as the ratio of the migration distance moved by each protein sample to the migration distance of the dye front in gel without and with substrate, respectively.


bAccording to the Zverlov et al. method (2001), the retardation factor Kr was calculated using the relative migration distance Rf0 (without substrate) and Rf (with substrate): Kr = Rf × (Rf0 − Rf)−1. No retardation (Kr > 10); observable retardation (Kr 6.0 − 0.1); strong retardation (Kr < 0.1).






Notably, both TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq) and TmB1-TFsW203F (having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq) enzymes showed significant discrepancies between Rf0 and Rf values, and as a result, Kr values (1 and 2) were within the range representing retardation (6.0-0.1). Although little changes of both Rf0 and Rf values and high Kr value (>10) were observed for TFsW203F-TmB2 protein (having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq), a group of smeared tailing bands distributed at higher MW range than the original protein band of TFsW203-TmB2 (having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq) in lichenan-containing gel, suggesting retardation occurred. These results indicate that CBMs from thermophilic T. maritima could function normally for substrate binding when fused with TFsW203F (having residues 1-248 of SEQ ID NO:3) from mesophilic F. succinogenes. 
(3) Kinetic Properties of Parental and Hybrid Glucanases
The kinetic properties of purified parental TFsW203F (having residues 1-248 of SEQ ID NO:3), TmLamCD (having residues 211-488 of SEQ ID NO:1) and their hybrid glucanases were determined under their respective optimal pH and temperature, with lichenan and/or laminarin as the substrate. The values for the Michaelis constant (KM), turnover number (kcat), and catalytic efficiency (kcat/KM) are presented in Table 3.
To compare the specific activities among the enzymes with differing molecular mass in this study, the specific activity is expressed as unit per nmol (U/nmol) of the protein. When lichenan was used as the substrate, the specific activity, kcat, KM and kcat/KM of TFsW203F (having residues 1-248 of SEQ ID NO:3) were determined as 626±38 U/nmol, 10100 s−1, and 5.3±0.6 mg/ml, and 1908 s−1 (mg/ml)−1, respectively. Comparing the kinetic data of the truncated mutant TFsW203F (having residues 1-248 of SEQ ID NO:3) with the truncated wild-type PCR-TF-glucanase from the same organism Fibrobacter succinogenes (Wen et al., 2005, Biochemistry, 44, 9197-9205), TFsW203F (having residues 1-248 of SEQ ID NO:3) showed a 1.4-fold increase in catalytic efficiency [1908 vs. 1358 s−1 (mg/ml)−1]. TFsW203F (having residues 1-248 of SEQ ID NO:3) was thus used as the parental enzyme to create various hybrid enzymes in this study.
The kinetic data in Table 3 show that the hybrid enzymes, TmB1-TFsW203F (having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq), TFsW203F-TmB2 (having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq) and TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq), each had either a slight increased or very similar specific activity and turnover number ( ̃1.1-fold) as compared to that of TFsW203F (having residues 1-248 of SEQ ID NO:3), while a slight decrease (1.2-fold) was found in TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq). The KM for lichenan increased 2.0-fold in TmB1-TFsW203F (having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq) and 2.6-fold in TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq), and decreased slightly (1.3-fold) in TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq). Given this, the catalytic efficiency of the hybrid enzymes increased by 1.5-fold in TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq), but decreased, by 1.8-fold and 3.1-fold respectively, in TmB1-TFsW203F (having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) relative to that of TFsW203F (having residues 1-248 of SEQ ID NO:3) (see Table 3). Notably, TmLamCD (having residues 211-488 of SEQ ID NO:1) also revealed activity and affinity toward lichenan, with the specific activity, kcat, KM and kcat/KM determined as 2.3±0.1 U/nmol, 38 s−1, 8.3±0.9 mg/ml and 5 s−1 (mg/ml)−1, respectively.







TABLE 3







Kinetic properties of parental TFsW203F and hybrid glucanases

















Opt.



Specific


kcat/KM
Reaction



Activityg
kcat
KM
s−1 
Conditionse


Enzyme
U/nmol
s−1
mg/ml
(mg/ml)−1
° C./pH















PCR-TF-
233 ± 10 
3911
2.9 ± 0.1
1358
50/6.0


glucanasea,b







TFsW203Fa
626 ± 38 
10100
5.3 ± 0.6
1908
45/6.0


TFsW203Fc
N.D.f
N.D.f
N.D.f
N.D.f
45/7.0


TmB1-TFsW203Fa,d
690 ± 8 
11500
10.7 ± 0.2 
1075
45/6.0


TFsW203F-TmB2a,d
658 ± 2 
10960
5.5 ± 0.0
1997
45/6.0


TmB1-TFsW203F-
685 ± 1 
11420
4.0 ± 0.0
2834
45/6.0


TmB2a,d







TFsW203F-
514 ± 1 
8570
14.0 ± 0.1 
611
45/6.0


TmLamCDa







TFsW203F-
23.3 ± 0.0 
389
5.0 ± 0.0
78
95/8.0


TmLamCDc 







TmLamCDa
2.3 ± 0.1
38
8.3 ± 0.9
5
95/6.0


TmLamCDc
6.5 ± 0.3
109
1.3 ± 0.2
82
95/6.0





aThe kinetic study was performed with lichenan used as a substrate.


bData from Wen et al. 2005.


cThe kinetic study was performed with laminarin used as a substrate.


dThe kinetic study performed with laminarin used as a substrate showed no detectable activity.


eThe enzymatic reaction was performed at the respective optimal temperatures and pHs as indicated.


f N.D.: Activity not detected.


gOne unit of enzyme activity was defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute.






When laminarin was used as the substrate in the activity assays, TFsW203F (having residues 1-248 of SEQ ID NO:3) showed no activity, even with a protein concentration four hundred times higher than that where lichenan was used as the substrate. The specific activity, kcat, KM and kcat/KM of TmLamCD (having residues 211-488 of SEQ ID NO:1) were 6.5±0.3 U/nmol, 109 s−1, 1.3±0.2 mg/ml, and 82 s−1 (mg/ml)−1 respectively. Although the TFsW203F (having residues 1-248 of SEQ ID NO:3) enzyme did not show any activity in the presence of laminarin, when it was fused to TmLamCD (having residues 211-488 of SEQ ID NO:1) to form the hybrid TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) enzyme, it displayed superior specific activity and kcat (3.6-fold increase) to TmLamCD (having residues 211-488 of SEQ ID NO:1), with a value of 23.3±0.0 U/nmol and 389 s−1, though the kcat/KM of the hybrid enzyme remained similar to that of TmLamCD (having residues 211-488 of SEQ ID NO:1) [78 vs. 82 s−1 (mg/ml)−1], owing to an increase in KM value (5.0±0.0 mg/ml). See Table 3.
(4) Temperature and pH Effects on Parental and Hybrid Glucanases
The effects of temperature and pH on the enzymatic activity of the purified parental and hybrid glucanases were also examined. The optimal temperature for TFsW203F (having residues 1-248 of SEQ ID NO:3), TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq), TmB1-TFsW203F (having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq), TFsW203F-TmB2 (having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq) and TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq) was between 45° C. and 50° C., and an optimal pH of 6.0 or 7.0 was observed. The optimal temperature of TmLamCD (having residues 211-488 of SEQ ID NO:1) was approximately 95° C. All of the enzymes exhibited similar pH response profiles in terms of their activities when the individual enzymes were pre-incubated at room temperature for one hour in buffers with pH values ranging from pH 3.0 to 9.0. All of the enzymes showed <20% residual activity after pH 3.0 pre-treatment, and little or no difference (85-100% activity) was found in the tested enzymes pre-incubated at pH 4.0-9.0 (data not shown).
To investigate the influence of the newly-introduced protein domain(s) on the thermal stability of TFsW203F (having residues 1-248 of SEQ ID NO:3), the proteins were incubated individually for ten minutes at temperatures between the range of 30° C. and 80° C. in 5° C. steps, and the residual enzyme activities measured using lichenan as the substrate. As shown in FIG. 4, little difference in temperature sensitivity was observed at 30-45° C. in all test enzymes. When the temperature was increased to 50° C., TFsW203F (having residues 1-248 of SEQ ID NO:3) only retained 38% of its activity, whereas the TFsW203F (having residues 1-248 of SEQ ID NO:3) fused with TmLamCD (having residues 211-488 of SEQ ID NO:1) enzyme remained 76% active. Other hybrid enzymes were shown to retain between 50% and 62% of their original activity under the same treatment. In pre-treatment at ≧55° C., on the other hand, the enzymatic activity of all the tested enzymes fell significantly, to below 20%.
(5) CD and Fluorescence Spectrometric Analyses
Because TFsW203F (having residues 1-248 of SEQ ID NO:3) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) exhibited the most differing temperature sensitivity at 50° C. out of the enzymes compared, we therefore examined their secondary structures at temperatures between 30° C. and 90° C. at 10° C. intervals, using CD spectroscopy. The CD spectral profile of TFsW203F protein (having residues 1-248 of SEQ ID NO:3) revealed no difference at 30° C. and 40° C., but protein unfolding and a substantial loss of structural integrity appeared when temperatures were elevated to 50° C. or 70° C., and protein denaturated at temperatures between 80° C. and 90° C., with random coiled spectra occurring. See FIG. 5. Surprisingly, only minor alterations were observed in the CD spectra of TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) when the temperatures were increased from 30° C. to 90° C., which suggests that TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) possesses superior thermal stability to TFsW203F (having residues 1-248 of SEQ ID NO:3)
Furthermore, fluorescence spectrometry was employed to investigate the structural integrity of TFsW203F (having residues 1-248 of SEQ ID NO:3) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) glucanases under native (25° C.), heat-treated (50° C. and 75° C.), 8 M urea-denatured, and denatured/renatured conditions. At 25° C. and 50° C., the emission spectra of both glucanases showed similar profiles, with a maximum emission peak of 336 nm. See FIG. 6. When emission spectra were monitored at 75° C., however, the profile of TFsW203F (having residues 1-248 of SEQ ID NO:3) exhibited a significant bathochromic (red) shift, peaking at 344 nm, whereas the TFsW203F-TmB2 protein (having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq) displayed little or no difference between 25° C., 50° C. and 75° C. When the enzymes were treated with denaturant 8 M urea, significant red shifts, with a maximum peak at 352 nm and a slight shoulder at 382 nm, were observed. The urea-denatured enzymes were then dialyzed against sodium phosphate buffer (50 mM, pH 7.0) for 24 hours in order to remove the denaturant and allow the protein to refold. The renatured enzymes were then analyzed for their fluorescence emission spectra under the same conditions.
As shown in FIG. 6, the maximum spectra of both denatured and renatured proteins were shifted back to 336 nm and superimposed on the native protein spectrum, with few or no differences visible. These results indicate that, although the structure of TFsW203F (having residues 1-248 of SEQ ID NO:3) displays greater sensitivity to high temperatures than the hybrid TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq), it was possible to effectively refold both enzyme structures to a native-like structure after urea denaturing.
(6) Protein Reactivation Profile of TFsW203F and TFsW203F-TmLamCD 
To evaluate the recovery efficiency of enzymatic activity after high-temperature treatment (90° C., ten minutes), the activities of TFsW203F (having residues 1-248 of SEQ ID NO:3) and hybrid TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) recovered at room temperature at different time intervals were examined, in parallel to monitoring the fluorescence emission spectra of the enzymes. As is shown in FIG. 7, within 110 seconds both TFsW203F (having residues 1-248 of SEQ ID NO:3) and hybrid TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) recovered approximately 27-34% activity, and after a ten-minute recovery time, parental TFsW203F (having residues 1-248 of SEQ ID NO:3) and hybrid TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) reached 48% and 63% of their original activities respectively. Meanwhile, the relative fluorescence intensity recovered 88% and 83% in TFsW203F (having residues 1-248 of SEQ ID NO:3) and TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) respectively. Thermal stability is an important criterion for enzymes, especially as regards their applications in the brewing and animal feeds industry, where malting or feed pelleting processes usually operate at high temperatures (>90° C.) (Hong et al., 2009, J. Microbiol. Biotechnol., 19, 818-822). Hybrid TFsW203F capable of enduring extreme conditions and gradually recovering its enzymatic activity at room temperature may therefore be desirable from an industrial point of view.
(7) Structural Modeling
By modeling, β-1,3-1,4-celloheptaose (representing the original substrate lichenan) fitted neatly into the catalytic cleft of TFsW203F (having residues 1-248 of SEQ ID NO:3), but laminarihexose (representative of laminarin) did not fit well, and indeed collided with the residues at the active site. The three sequential β-1,3-glucose moieties in the modeled laminarihexose did, however, fit neatly into the active site of TFsW203F (having residues 1-248 of SEQ ID NO:3). Because the TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) showed an improvement (a 3.6-fold increase) in specific activity against laminarin as compared to TmLamCD (having residues 211-488 of SEQ ID NO:1) alone, and because we have recently demonstrated that laminaritriose is the major product of TmLamCD (having residues 211-488 of SEQ ID NO:1) toward the substrate laminarin (Jeng et al., 2011, J. Biol. Chem., 286, 45030-45040), the TFsW203F domain in TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) may play a role in capturing the product laminaritriose, which results in the facilitation and enhancement of the catalytic activity of TFsW203F-TmLamCD (having the sequence of SEQ ID NO:9 with or without the optional C-terminal taq) toward laminarin. However, a slight decrease (2.6 ̃3.7-fold) in binding affinity with lichenan and laminarin was also observed in the hybrid enzyme as compared with the TFsW203F (having residues 1-248 of SEQ ID NO:3) and TmLamCD domain (having residues 211-488 of SEQ ID NO:1) alone. This suggest that there might be some subtle steric hindrance from other domains in the hybrid glucanases. On the other hand, in the modeled structures of TmLamCD (having residues 211-488 of SEQ ID NO:1) complexed with β-1,3-1,4-celloheptaose and laminarihexose, the oligosaccharides all fitted nicely into the catalytic cleft of the laminarinase, which in combination with the kinetic data indicated that TmLamCD (having residues 211-488 of SEQ ID NO:1) is capable of hydrolyzing both types of β-glucan substrate.
On the basis of the structural modeling, both TmB1 (having residues 19-176 of SEQ ID NO:1) and TmB2 (having residues 495-642 of SEQ ID NO:1) formed a concave catalytic-like open cleft like that of TFsW203F (having residues 1-248 of SEQ ID NO:3), which may contribute to holding the lichenan chain in place, and in turn to facilitating the efficient hydrolysis of lichenan in TFsW203F (having residues 1-248 of SEQ ID NO:3). Furthermore, in comparison to the TmB1 (having residues 19-176 of SEQ ID NO:1) and TmB2 (having residues 495-642 of SEQ ID NO:1) structures modeled, the narrow gate formed by two tryptophans in the binding cleft of TmB2 (having residues 495-642 of SEQ ID NO:1) (about 7.6-8.2 Å) is wider than the carbohydrate binding cleft of TmB1 (having residues 19-176 of SEQ ID NO:1) (about 6.3-7.3 Å) for polysaccharide binding, which may explain why TmB1-TFsW203F (having the sequence of SEQ ID NO:13 with or without the optional C-terminal taq) had a lower lichenan-binding affinity than TFsW203F-TmB2 (having the sequence of SEQ ID NO:7 with or without the optional C-terminal taq). The co-existence of both CBMs in TFsW203F (having residues 1-248 of SEQ ID NO:3) do, however, compensate somewhat for the weaker substrate binding, with a consequent slight decrease in KM value, and a corresponding improvement in the overall catalytic efficiency of TmB1-TFsW203F-TmB2 (having the sequence of SEQ ID NO:11 with or without the optional C-terminal taq) as compared to the single domain enzyme TFsW203F (having residues 1-248 of SEQ ID NO:3) [2834 vs. 1908 s−1 (mg/ml)−1].
OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

## Claims

1. A fusion polypeptide comprising:
(a) a first segment containing a TFsW203F polypeptide having residues 1-248 of SEQ ID NO:3; and
(b) a second segment containing a catalytic domain of TmLam having a sequence that is at least 90% identical to residues 211-488 of SEQ ID NO:1;
wherein the fusion polypeptide has a glucanase activity and an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:5 or 9.

2. The fusion polypeptide of claim 1, wherein the polypeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:5 or 9.

3. The fusion polypeptide of claim 1, wherein the polypeptide has the amino acid sequence of SEQ ID NO:5 or 9.

4. The fusion polypeptide of claim 1, wherein the polypeptide has residues 1-532 of SEQ ID NO:9.

5. The fusion polypeptide of claim 1, wherein the polypeptide has residues 1-538 of SEQ ID NO:5.