MMBT 1683

UNIVERSITI TEKNOLOGI MALAYSIA

FACULTY OF SCIENCE
......................................................................

FINAL EXAMINATION

SEMESTER I SESSION 2021/2022

COURSE CODE : MMBT 1683

COURSE NAME : PROTEIN ENGINEERING

PROGRAMME : MMBT

LECTURER : P.M. DR. GOH KIAN MAU

DATE : 13 FEBRUARY 2021

DURATION : 4 HOURS

INSTRUCTIONS : ANSWER ALL 3 QUESTIONS IN THIS

BOOK.

IC or STUDENT ID : ......................................................................

NAME : ......................................................................

___________________________________________________________________
(THIS EXAMINATION BOOKLET CONSISTS OF 15 PRINTED PAGES, APPENDIX
IS NOT COUNTED)
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INSTRUCTION
1. Answer all questions. Type your answer below each question. When needed, refer to the PDF version of the
question paper to ensure that you’ve not accidentally deleted any information.
2. Refer to ‘Guidelines for Protein Engineering Final Examination’ for more instruction.
3. The link for the online attendance form is provided below. The link will be disabled after 9.15 am 13/2/2021.
https://forms.gle/qH2mDXmD5JEz8ZEJ9
4. Quick guideline for submission. Type ‘yourstudentname_protein engineering’ in the “subject” of the email.
Convert the .docx file to .pdf format. Attached the answer-file (.pdf format only) and email to
gohkianmau@fbb.utm.my
5. Due date: 1.00 pm 13/2/2021 Malaysia time.

QUESTION 1 (5 MARKS)

Each protein has important amino acids or stretches related to its biochemical or structural
function. Knowing the position of these amino acids is a prerequisite for successful protein
engineering research. Assuming you are a protein engineer working on a new recombinant
enzyme.

a) How would you find the position (amino acid numbering) of catalytic residues and ligand
binding sites of the closest homologue enzyme that is well-studied? (1 mark)
b) Apply suitable approaches for a plan to find the positions of important amino acids or stretches
of amino acid in the new recombinant enzyme that you are working with. (4 marks)

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QUESTION 2 (5 MARKS)

Download and read the research article ‘The effects of reaction conditions on the production of g-
cyclodextrin from tapioca starch by using a novel recombinant engineered CGTase’. A copy of the
PDF file is available at the end of the question paper (PDF format) and available in UTM
eLearning. After reading the whole article, answer the following question.

a) Explain in your own words the difference between catalytic sites and subsite -3. (2 marks)
b) Examine Table 1 embedded in the research article and shown below here. Amino acid K, R,
H, and T may be directly related to the product specificity of CGTase. Demonstrate how you
would show your understanding of the relationship of this amino acid to product specificity.
(3 marks)

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QUESTION 3 (30 MARKS)

Starch and pullulan-degrading enzymes are essential industrial biocatalysts. Pullulan-

degrading enzymes are grouped into pullulanases (types I and type II) and pullulan hydrolase
(types I, II, and III). Type I pullulanase (EC 3.2.1.41) acts on α-1,6 glycosidic bonds in pullulan to
produce maltotriose (Figure 1). Types I pullulanase is also active against short branches (G1‒G7)
of starch, amylopectin, glycogen, and β-limit dextrin, producing reducing sugars (i.e., glucose,
maltose, and maltotriose). Moreover, this enzyme group does not hydrolyse α-1,4 glycosidic bonds
in the polysaccharides mentioned earlier.
Pullulan-degrading enzymes are multi-domain proteins. Generally, these enzymes contain
several domains such as carbohydrate-binding module (CBM), catalytic domain, C-terminal
domain (i.e., domain C or amyC domain), and fibronectin type III (FnIII) domain. CBM, a non-
catalytic ancillary domain, assists protein attachment to polysaccharide's surface (i.e., pullulan or
starch). CBM also facilitates the degradation process by distorting the conformation or the packing
of the polysaccharides. Based on the CAZy database, CBMs are currently grouped into 88 primary
sequence-based families [58]. CBMs with affinity for starch (CBM20, CBM21, CBM25, CBM26,
CBM34, CBM41, CBM45, CBM48, CBM53, CBM58, CBM68, CBM69, CBM82, and CBM83)
are commonly known as starch-binding domains (SBDs). Figure 2 shows the comparison of
domains and structure architecture for each group of pullulan-degrading enzymes. Type I
pullulanases are members of GH13. Members within this family have consensus key features such
as (i) they cleave the α-glycosidic bonds, (ii) the amino acid sequences contain four conserved
regions, (iii) the enzymes possess a TIM barrel structure, and (iv) Asp, Glu, and Asp are the
enzymes catalytic residues.

Figure 1. The degradation action pattern of (a) pullulanases (types I and II) on pullulan, starch,
or other polysaccharides, and (b) pullulan hydrolases (types I, II, and III) on pullulan.

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Figure 2 Schematic representation of conserved domains in pullulan-degrading enzymes.

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Table 2 Codon table

Codon Full Name Abbreviation (3 Letter) Abbreviation (1 Letter)
TTT Phenylalanine Phe F
TTC Phenylalanine Phe F
TTA Leucine Leu L
TTG Leucine Leu L
TCT Serine Ser S
TCC Serine Ser S
TCA Serine Ser S
TCG Serine Ser S
TAT Tyrosine Tyr Y
TAC Tyrosine Tyr Y
TAA Termination (ochre) Ter X
TAG Termination (amber) Ter X
TGT Cysteine Cys C
TGC Cysteine Cys C
TGA Termination (opal or umber) Ter X
TGG Tryptophan Trp W
CTT Leucine Leu L
CTC Leucine Leu L
CTA Leucine Leu L
CTG Leucine Leu L
CCT Proline Pro P
CCC Proline Pro P
CCA Proline Pro P
CCG Proline Pro P
CAT Histidine His H
CAC Histidine His H
CAA Glutamine Gln Q
CAG Glutamine Gln Q
CGT Arginine Arg R
CGC Arginine Arg R
CGA Arginine Arg R
CGG Arginine Arg R
ATT Isoleucine Ile I
ATC Isoleucine Ile I
ATA Isoleucine Ile I
ATG Methionine Met M
ACT Threonine Thr T
ACC Threonine Thr T
ACA Threonine Thr T
ACG Threonine Thr T
AAT Asparagine Asn N
AAC Asparagine Asn N
AAA Lysine Lys K
AAG Lysine Lys K
AGT Serine Ser S
AGC Serine Ser S
AGA Arginine Arg R
AGG Arginine Arg R

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GTT Valine Val V

GTC Valine Val V
GTA Valine Val V
GTG Valine Val V
GCT Alanine Ala A
GCC Alanine Ala A
GCA Alanine Ala A
GCG Alanine Ala A
GAT Aspartate Asp D
GAC Aspartate Asp D
GAA Glutamate Glu E
GAG Glutamate Glu E
GGT Glycine Gly G
GGC Glycine Gly G
GGA Glycine Gly G
GGG Glycine Gly G

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atg ctt cac atc agc cga acg ttt gcc gcc tat ttg gac gag atg gat caa atc gtt gtg
M L H I S R T F A A Y L D E M D Q I V V
ctt gcg ccg aaa tcg ctc ggc ttt gat ggg atg gcg ccg ttg acg ctc gta gcg ccg agc
L A P K S L G F D G M A P L T L V A P S
ggc gag gag att ccg ctg tcc gtg cag cac gtc gag gat tgc ggg gag acg gtg aaa tat
G E E I P L S V Q H V E D C G E T V K Y
gtg tgc cgg ttt gca tcc gcg ttc gag ttt gga gcg aca tac tgg gtg cgt tct tgc cgc
V C R F A S A F E F G A T Y W V R S C R
ggg gag gag acc gat gtt caa atc ggc gcc gtt gtg cgc act cct gca ttt gat gat cgg
G E E T D V Q I G A V V R T P A F D D R
ttt ttc tat gat ggg ccg tta ggc gtg gag tat tcc aaa gaa cag gcg gta ttt cgc gta
F F Y D G P L G V E Y S K E Q A V F R V
tgg gcg ccg act gcc acc gcg gtc aac gtc aag ctt gtt cat ccg cat ctc ggc gag atc
W A P T A T A V N V K L V H P H L G E I
cgc tgc gtg ccg ctt gag cgc ggc gag tgc ggc gta tgg tca gcc gcc gtc ccc ggc gat
R C V P L E R G E C G V W S A A V P G D
tgg gaa cga gcg tgt tac acg tat atc gcc tgc atc aac cgc gta tgg cgc gag gcg gtg
W E R A C Y T Y I A C I N R V W R E A V
gac ccg tat gca act gct gtg tcg atc aat ggc gag ttc ggc gtc gtg atc gac tgg gag
D P Y A T A V S I N G E F G V V I D W E
aaa acg aag ctg acg ccg ccc tct tcg ccg ctt ccg ccg ctc tgt tcg ccg acg gat gcc
K T K L T P P S S P L P P L C S P T D A
atc ctt tat gaa ctg agc atc cgc gac ttt aca agc cat ccg gac agc ggc gcc gtc cat
I L Y E L S I R D F T S H P D S G A V H
aaa ggg aag tat ctc ggg ttg gct gaa acg aac aca agc ggg cca aac ggg acg gcc act
K G K Y L G L A E T N T S G P N G T A T
ggg ctt tcg tat gtc aaa gag ctg ggc gtc acc cat gtg cag ctc atg ccg ttt atg gac
G L S Y V K E L G V T H V Q L M P F M D
ttt gca ggc gtt gat gag cgc gac cca caa gca gca tac aac tgg gga tac aat ccc ctt
F A G V D E R D P Q A A Y N W G Y N P L
cat cta tat gcg ccg gaa ggg agt tat gcg acc gat cca gcg gat cca tac gca cgc att
H L Y A P E G S Y A T D P A D P Y A R I
gta gaa ttg aag cag gcg atc cac acg ctg cac gaa aat gga ttg cgc gtc gtg atg gat
V E L K Q A I H T L H E N G L R V V M D
gcg gtc tac aac cat gtc tac gat cgg gag caa tcg ccg ctt gag aag ctc gtt ccc ggc
A V Y N H V Y D R E Q S P L E K L V P G
tat tac ttc cgc tac gac gcc tat ggc caa ccg gcc aac ggc aca ggc gtc ggc aac gac
Y Y F R Y D A Y G Q P A N G T G V G N D
atc gct tcg gag cgg cgg atg gcg cgc cgt tgg atc gtc gat tcg gtt gtg ttt tgg gcg
I A S E R R M A R R W I V D S V V F W A
aaa gaa tac ggc ctt gat ggg ttc cgc ttt gat ttg atg ggc gtg cac gat atc gag acg
K E Y G L D G F R F D L M G V H D I E T
atg aag gcg gtg cgc gat gcc ctc gac acc atc gat cca tcg atc ctt gtg tat ggg gaa
M K A V R D A L D T I D P S I L V Y G E
ggg tgg gac ttg ccg aca ccc ctt ccg ccg gaa caa aag gcg acg atg gcc aac gcc aat
G W D L P T P L P P E Q K A T M A N A N
cag ttg ccg cgc ttc gcg tat ttt aat gac cgg ttt cgc gat gcg gtg aaa ggg agc acc
Q L P R F A Y F N D R F R D A V K G S T
ttt cat ttg ccg gat cgg gga ttc gcc ctc ggc aac cca ggc ggg cga gaa cag gtg aag
F H L P D R G F A L G N P G G R E Q V K
ctc gcc att gcc ggg agc ttg cga gcg ctc ggc ggg ctg ttt tgc cac ccg cgt cag tcg
L A I A G S L R A L G G L F C H P R Q S
atc aat tac gtc gaa tgc cat gac aac cat acg ttt tgg gat aag atg gag gcg gcc aac
I N Y V E C H D N H T F W D K M E A A N
cat gat gag ccg gaa tgg ctc cgg cgg aag cgg caa aag ctg gcg acg gcg atc gtt ctg
H D E P E W L R R K R Q K L A T A I V L
ttg gcg caa ggc att ccg ttt ttg cac agc ggc caa gag ttt tat cgg acg aaa ggc ggc
L A Q G I P F L H S G Q E F Y R T K G G
gat ggg aac agc tac cga tcg ccg gat gcg gtc aat cag ctg gat tgg ggg cgg aaa agc
D G N S Y R S P D A V N Q L D W G R K S
cgc tat gaa gac gac gtc cgc tac gtt caa gga ttg atc gct ctt cgc cgc gcg cat ggc
R Y E D D V R Y V Q G L I A L R R A H G
gcg ttc cgc ctc gcc acg gaa gcg gaa gtg ctg cgc cat ttg acg ttt ctt gag ccg ctg
A F R L A T E A E V L R H L T F L E P L
ccg ccc tcg gtc atc gcc tac cga ttg cat gat gtc gcc gtc tat ggg cca tgg gat gag
P P S V I A Y R L H D V A V Y G P W D E
atc atc gtt ctt cat cat aac gaa gaa aaa aaa gag gcc atc cac ctt cca gac gaa cgg
I I V L H H N E E K K E A I H L P D E R
gaa tgg gac atc gta tgc gac gga cag cgg agc gga gcg gcg ccg ttt cgc cga gtg cgt
E W D I V C D G Q R S G A A P F R R V R
ggc agg ctt gag ctt gac ggc att ggc aca tgg gtg ctc gtc aaa acg gac gct
G R L E L D G I G T W V L V K T D A

Figure 3 Gene and protein sequence of pullulanase type I (PulGT) from Geobacillus
thermocatenulatus

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atg ctt cac atc agc cga acg ttt gcc gcc tat ttg gac gag atg gat caa atc gtt gtg
M L H I S R T F A A Y L D E M D Q I V V
ctt gcg ccg aaa tcg ctc ggc ttt gat gga atg gcg ccg ttt acg ctc gtg gcg ccg agc
L A P K S L G F D G M A P F T L V A P S
ggc gag gag att ccg ctg tcc gtg cag cac gtc gag gat gtt ggg gag acg gtg aaa tat
G E E I P L S V Q H V E D V G E T V K Y
gtg tgc cgg ttt gca tcc gcg ttc gag ttt gga gcg aca tac tgg gtg cgt tct tgc cgc
V C R F A S A F E F G A T Y W V R S C R
ggg gag gag acc gat gtt caa atc ggc gcc gtt gtg cgc act cct gca ttt gat gat cgg
G E E T D V Q I G A V V R T P A F D D R
ttt ttc tat gat gga ccg tta gga gcg gag tat ctc aaa gaa cag acg gta ttt cgc gta
F F Y D G P L G A E Y L K E Q T V F R V
tgg gcg ccg acc gcc acc gcg gtt agc gtc aag ctg gtt cat ccg cat ctc gac gag atc
W A P T A T A V S V K L V H P H L D E I
cgc tgc gtg ccg ctt gtg cgc ggc gaa cgc ggc gta tgg tca gcc gtc gtc ccc ggc gat
R C V P L V R G E R G V W S A V V P G D
tgg gag cga gcg cgt tac aca tat atc gcc tgc atc aac cgc gta tgg cgc gag gca gtg
W E R A R Y T Y I A C I N R V W R E A V
gac ccg tat gcg acc gcg gtt tcg gtc aat ggc gag ttc ggc gtc gtg atc gac tgg gag
D P Y A T A V S V N G E F G V V I D W E
aaa acg aag ctg gcg ccg ccc tct ttg ccg ctt ccg ccg ctc tgt tcg ccg acg gat gcc
K T K L A P P S L P L P P L C S P T D A
atc att tat gag ctg agc atc cgc gac ttt acc agc cac ccg gac agc ggc gcc gtc cat
I I Y E L S I R D F T S H P D S G A V H
aaa ggg aag tat ctc ggg ctg gcc gaa acg aac acg agc ggg ccg aac ggg acg gcc acc
K G K Y L G L A E T N T S G P N G T A T
ggg ctt tcg tat gtc aaa gag ctg ggc gtc acc cat gtg cag ctc atg ccg ttt atg gac
G L S Y V K E L G V T H V Q L M P F M D
ttt gcg ggc gtc gat gag cgc gac cca caa gcg gct tac aac tgg gga tac aat ccc ctt
F A G V D E R D P Q A A Y N W G Y N P L
cat cta tat gcg ccg gaa ggg agt tat gcg acc gat cca gcg gat cca tac gcg cgc att
H L Y A P E G S Y A T D P A D P Y A R I
gta gaa ttg aag cag gcg atc cac acg ctg cac gaa aat ggc ttg cgc gtc gtg atg gat
V E L K Q A I H T L H E N G L R V V M D
gcg gtc tac aac cat gtc tat gac cgg gag caa tcg ccg ctt gag aag ctc gtt ccc ggt
A V Y N H V Y D R E Q S P L E K L V P G
tat tac ttc cgc tac gac gcc tat ggc caa ccg gcc aac ggc acc ggc gtc ggc aac gac
Y Y F R Y D A Y G Q P A N G T G V G N D
atc gct tcg gag cgg cgg atg gcg cgc cgc tgg atc gtc gat tcg gtg gtg ttt tgg gcg
I A S E R R M A R R W I V D S V V F W A
aaa gaa tat ggc att gac ggg ttc cgc ttt gat ttg atg ggc gtg cac gat atc gag acg
K E Y G I D G F R F D L M G V H D I E T
atg aaa gcg gtg cgc gat gcc ctc gac gcc atc gat ccg tcg atc ctt gtg tat ggg gaa
M K A V R D A L D A I D P S I L V Y G E
ggg tgg gat ttg ccg acg cct ctt cca ccg gaa caa aag gcg acg atg gcc aac gcc aag
G W D L P T P L P P E Q K A T M A N A K
cag ctg ccg cgc ttc gct tat ttc aat gac cgg ttt cgc gat gcg gtg aaa ggg agc acc
Q L P R F A Y F N D R F R D A V K G S T
ttt cat ttg ccg gac cgt ggg ttc gcc ctc ggc aac cca ggc ggg cga gaa cag gtg aag
F H L P D R G F A L G N P G G R E Q V K
ctc gcc att gcc ggg agc ttg cga gcg ctc ggc ggg ctg ttt tgc cac ccg cgt cag tca
L A I A G S L R A L G G L F C H P R Q S
atc aat tac gtc gaa tgt cat gac aac cat acg ttt tgg gat aag atg gag gcg gcc aac
I N Y V E C H D N H T F W D K M E A A N
cat gat gag ccg gaa tgg ctc cgg cga aag cgg caa aag ctg gcg acg gcg atc gtt ctg
H D E P E W L R R K R Q K L A T A I V L
ttg gcg caa ggc att ccg ttt ttg cac agc ggc caa gag ttt tat cgg acg aaa ggc ggc
L A Q G I P F L H S G Q E F Y R T K G G
gat ggg aac agc tac cga tcg ccg gat gcg gtc aat cag ctg gat tgg gag cgg aaa agc
D G N S Y R S P D A V N Q L D W E R K S
cgc tat gaa gac gac gtc cgc tac gtt caa gga ttg atc gcc ctt cgc cgt gcg cat ggc
R Y E D D V R Y V Q G L I A L R R A H G
gca ttt cgc ctc gcc acg gag gcg gaa gtg ctg cgt cat ttc acg ttt ctt gag ccg ctg
A F R L A T E A E V L R H F T F L E P L
ccg ccg tcg gtc atc gcc tac cga ttg cat gat gcc gcc gtc tat ggg cct tgg gag gac
P P S V I A Y R L H D A A V Y G P W E D
atc atc gtc gtg cat cat aac gag gag aaa gag acc gcc att gcg ctc cct gac gag cgc
I I V V H H N E E K E T A I A L P D E R
gag tgg gcg gtt gta tgc gac gga cag cga tgc ggg aca acg ccc ttt ggc caa gcg cgc
E W A V V C D G Q R C G T T P F G Q A R
ggc atg ctt cgg ctt gac ggc atc ggc aca tgg gtg ctc gtc cat cct gca ggg
G M L R L D G I G T W V L V H P A G

Figure 4 Gene and protein sequence of pullulanase type I (PulBT) from Bacillus
thermoleovorans

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PulGT MLHISRTFAAYLDEMDQIVVLAPKSLGFDGMAPLTLVAPSGEEIPLSVQHVEDCGETVKY
PulBT MLHISRTFAAYLDEMDQIVVLAPKSLGFDGMAPFTLVAPSGEEIPLSVQHVEDVGETVKY
*********************************:******************* ******

PulGT VCRFASAFEFGATYWVRSCRGEETDVQIGAVVRTPAFDDRFFYDGPLGVEYSKEQAVFRV
PulBT VCRFASAFEFGATYWVRSCRGEETDVQIGAVVRTPAFDDRFFYDGPLGAEYLKEQTVFRV
************************************************.** ***:****

PulGT WAPTATAVNVKLVHPHLGEIRCVPLERGECGVWSAAVPGDWERACYTYIACINRVWREAV
PulBT WAPTATAVSVKLVHPHLDEIRCVPLVRGERGVWSAVVPGDWERARYTYIACINRVWREAV
********.********.******* *** *****.******** ***************

PulGT DPYATAVSINGEFGVVIDWEKTKLTPPSSPLPPLCSPTDAILYELSIRDFTSHPDSGAVH
PulBT DPYATAVSVNGEFGVVIDWEKTKLAPPSLPLPPLCSPTDAIIYELSIRDFTSHPDSGAVH
********:***************:*** ************:******************

PulGT KGKYLGLAETNTSGPNGTATGLSYVKELGVTHVQLMPFMDFAGVDERDPQAAYNWGYNPL
PulBT KGKYLGLAETNTSGPNGTATGLSYVKELGVTHVQLMPFMDFAGVDERDPQAAYNWGYNPL
************************************************************

PulGT HLYAPEGSYATDPADPYARIVELKQAIHTLHENGLRVVMDAVYNHVYDREQSPLEKLVPG
PulBT HLYAPEGSYATDPADPYARIVELKQAIHTLHENGLRVVMDAVYNHVYDREQSPLEKLVPG
************************************************************

PulGT YYFRYDAYGQPANGTGVGNDIASERRMARRWIVDSVVFWAKEYGLDGFRFDLMGVHDIET
PulBT YYFRYDAYGQPANGTGVGNDIASERRMARRWIVDSVVFWAKEYGIDGFRFDLMGVHDIET
********************************************:***************

PulGT MKAVRDALDTIDPSILVYGEGWDLPTPLPPEQKATMANANQLPRFAYFNDRFRDAVKGST
PulBT MKAVRDALDAIDPSILVYGEGWDLPTPLPPEQKATMANAKQLPRFAYFNDRFRDAVKGST
*********:*****************************:********************

PulGT FHLPDRGFALGNPGGREQVKLAIAGSLRALGGLFCHPRQSINYVECHDNHTFWDKMEAAN
PulBT FHLPDRGFALGNPGGREQVKLAIAGSLRALGGLFCHPRQSINYVECHDNHTFWDKMEAAN
************************************************************

PulGT HDEPEWLRRKRQKLATAIVLLAQGIPFLHSGQEFYRTKGGDGNSYRSPDAVNQLDWGRKS
PulBT HDEPEWLRRKRQKLATAIVLLAQGIPFLHSGQEFYRTKGGDGNSYRSPDAVNQLDWERKS
******************************************************** ***

PulGT RYEDDVRYVQGLIALRRAHGAFRLATEAEVLRHLTFLEPLPPSVIAYRLHDVAVYGPWDE
PulBT RYEDDVRYVQGLIALRRAHGAFRLATEAEVLRHFTFLEPLPPSVIAYRLHDAAVYGPWED
*********************************:*****************.******::

PulGT IIVLHHNEEKKEAIHLPDEREWDIVCDGQRSGAAPFRRVRGRLELDGIGTWVLVKTDA
PulBT IIVVHHNEEKETAIALPDEREWAVVCDGQRCGTTPFGQARGMLRLDGIGTWVLVHPAG
***:******: ** ******* :******.*::** :.** *.**********:. .

Figure 5 Protein sequence alignment of pullulanase type I PulGT and PulBT. The colour coding
at the base of the alignment refers to four typical domains. Red=Domain N1 (CBM41), green=
Domain N2 (CBM48), yellow= Domain A (catalytic domain, where active sites are located) and
blue= Domain C. Refer to Figure 1 for detail.

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Assume you have the pullulanase type I PulGT gene originated from G. thermocatenulatus. You
have all the needed laboratory equipment, chemicals, and reagents. The gene that encodes for
PulGT has been cloned earlier in a plasmid. However, you do not have the plasmid construct with
the PulBT gene.

a) Analyze the information provided above. To generate the mutant PulGT genes, which
approach should you choose to generate the following mutants? (4 marks)

Table 3
Single Chosen method Reason
mutation
G138D

V652A

b) Evaluate what are the best primers design that should be used to conduct the PCR
amplifications to obtain the complete mutated gene for G138D and V652A (Note: please
underline the locations of primers in Figure 3). Provide the primer sequence below. (5 marks)

Mutant G138D:
External forward primer (restricted to 21 bp for this primer):

5'-type within this space 3'

External reverse primer (restricted to 21 bp for this primer):

5'-type within this space 3'

Other primer(s), if applicable:

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Mutant V652A:
External forward primer (restricted to 21 bp for this primer):

5'-type within this space 3'

External reverse primer (restricted to 21 bp for this primer):

5'-type within this space 3'

Other primer(s), if applicable:

c) After performing several rounds of PCRs to generate the mutant in Question (b), you are asked
to load your PCR products into separate wells on a 0.7% agarose gel. The agarose gel was
casted with a standard 8-comb (well) according to the standard protocol. Well number 1 was
loaded with a DNA ladder (DNA marker). Using a DNA ladder as reference, write the size of
the expected bands for each PCR rounds in Table 4.
(Remarks: Each well is meant for one PCR round) (2 marks)

Figure 6

Provide the answers in the Table 4 below. Do not draw line in the gel image.

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Table 4
Well numbering Sample loaded Size (bp)
1 DNA ladder (marker) 250-10000 (as shown in Figure 6)
2
3
4
5
6
7
8

d) The overlap extension PCR technique can generate double point mutation T205A/S209. One
can choose either (i) generate one mutation site at a time in a total of six PCR rounds or (ii)
simultaneously generate two mutation sites in three PCR rounds. Draw each process stated
above, and do you agree that approach (ii) is better? (6 marks)

Table 5
(i) Six PCR rounds to generate (ii) Three PCR rounds to generate
T205A/S209L T205A/S209L

Suggestion: Suggestion:
1. The drawing shall be prepared in scale. 1. The drawing shall be prepared in scale.
2. You can draw using a pencil or pen, take a 2. You can draw using a pencil or pen, take a
photo and place the image here. photo and place the image here.
3. Label the illustration. 3. Label the illustration.
4. No primer design is required. 4. No primer design is required.
5. Evaluate and explain which approach is 5. Evaluate and explain which approach is
better. better.
6. You may delete the text in grey after 6. You may delete the text in grey after viewing
viewing it. it.

13
 MMBT 1683

e) After performing several rounds of PCRs to generate the mutant in Question 3(d) using the
fastest approach, you are asked to load your PCR products into separate wells on a 0.7%
agarose gel. The agarose gel was casted with a standard 8-comb (well) according to the
standard protocol. Well number 1 was loaded with a DNA ladder (DNA marker). Using a
DNA ladder as reference, examine and write the sizes of expected bands for each PCR rounds
in the Table 6. (3 marks)
(Remarks: Each well is meant for one PCR round)

Figure 7

Provide the answers in the Table 6 below. Do not draw line in the gel image.

Table 6
Well numbering Sample loaded Size (bp)
1 DNA ladder (marker) 250-10000 (as shown in Figure 5)
2
3
4
5
6
7
8

14
 MMBT 1683

f) Please refer to Figures 2 and 5. A research article wrote ‘The scientific community has
understood sufficiently the biochemical role of CBMs and catalytic domain. However, the role
of C-terminal domain (Domain C) is uncertain’.
(i) Evaluate the information stated above. What does it mean? (3 marks)
(ii) How would you apply what you learned to evaluate the role of this Domain C of
pullulanase PulGT? Use proper illustration. Primer design is not required. (7 marks)

END OF QUESTIONS

15
 Available online at www.sciencedirect.com

Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126

The effects of reaction conditions on the production of

␥-cyclodextrin from tapioca starch by using a novel
recombinant engineered CGTase
Kian Mau Goh a,b , Nor Muhammad Mahadi c , Osman Hassan d ,
Raja Noor Zaliha Raja Abdul Rahman e , Rosli Md Illias a,∗
aDepartment of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering,
Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia
b Faculty of Bioscience and Bioengineering, Universiti Teknologi Malaysia,

81310 Skudai, Johor, Malaysia

c Malaysia Genome Institute, UKM-MTDC Smart Technology Center,

43600 Bangi, Selangor, Malaysia

d School of Chemical Science and Food Technology, Faculty of Science and Technology,

Universiti Kebangsaan Malaysia, 43600 Selangor, Malaysia

e Enzyme and Microbial Technology Research Group, Faculty of Biotechnology and Biomolecular Sciences,

Universiti Putra Malaysia, 43400 Selangor, Malaysia

Received 18 April 2007; received in revised form 17 August 2007; accepted 6 September 2007
Available online 12 September 2007

Abstract
A novel mutant enzyme namely H43T CGTase can produce up to 39% ␥-cyclodextrin (␥-CD) compared to the native enzyme which produces
only 10% ␥-CD. The effect of the reaction conditions on ␥-CD production was studied using this mutant CGTase. The effects of substrate–buffer
combination, starch pretreatment and concentration, pH, additives and finally the use of a debranching enzyme improved the ␥-CD ratio further. The
tapioca–acetate pair gave the highest conversion (16% conversion) among four types of starch and four buffer system combinations. Gelatinized
starch was preferred compared to raw tapioca starch in producing a high percentage of ␥-CD and conversion rate. Higher pH especially pH 8–9
led to a higher proportion of ␥-CD, and was relatively more apparent when the concentration of starch was increased. Forty-six percent ␥-CD was
produced using 2.5% gelatinized tapioca starch at pH 8. Pullulanase enzyme was found to be useful in reducing the viscosity of tapioca starch
paste thus increasing the efficiency of utilization of starch by CGTase by at least 20- to 30-fold. Up to 48% ␥-CD can be produced when 4%
pullulanase-pretreated tapioca starch was reacted with the CGTase mutant. It was also found that the supplementation of the reaction mixture with
glucose, toluene, or cyclododecanone improved the ␥-CD yield by 42.2, 46.4, 43.4, and 43.4%, respectively. All the parameters involved have been
shown to affect the product specificity of the mutant H43T CGTase transglycosylation mechanism.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Bacillus sp. G1; Cyclodextrin glucanotransferase; Gamma-cyclodextrin; Pullulanase; Site-directed mutagenesis; Tapioca starch

1. Introduction respectively, joined by ␣-1,4-glucosidic bonds. The doughnut-

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is
an industrially important enzyme that produces cyclodextrins
(CDs) from starch or starch-like substrates. ␣-, ␤- and ␥-CD con-
tain closed ring structures with six, seven and eight glucose units,
∗ Corresponding author. Tel.: +60 7 5535564; fax: +60 7 5581463.
E-mail address: r-rosli@utm.my (R.M. Illias).
shaped CDs have an interior portion that easily form inclusion
complexes with many organic substances or drugs which can
change the physicochemical properties of the latter molecule,
such as its solubility and stability. In practical terms, due to its
relatively larger cavity volume ␥-CD can form inclusion com-
plexes with larger compounds in cases where the volume of the
interior cavity of ␣-CD and ␤-CD is not sufficiently large to do
so. Another advantage of ␥-CD is its high solubility (23.2%, w/v,
at 25 ◦ C) in water, which is almost double and 10 times that of

1381-1177/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2007.09.011
 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126
␣-CD and ␤-CD, respectively [1]. The major disadvantage of ␥-
CD production by CGTase is that ␣-, ␤- and ␥-CDs are produced
as a mixture where the amount of ␥-CD is generally small and
the ratio of CDs greatly depends on the bacterial source of the
enzyme [2], and the reaction time and conditions [3]. CGTase
catalyzes four reactions (cyclization, coupling, disproportional
and hydrolysis) which occur simultaneously through different
kinetic [4] and thermodynamic mechanisms [5]. The net contri-
bution of these reactions determines the overall yield and product
specificity in a prolonged reaction [4].
The source of the CGTase greatly influences the production of
cyclodextrin since the amino acid sequence and the folding of the
protein structure determine the outcome of the kinetic equilib-
rium. Enzyme engineering of CGTase at central active site cleft
[6], subsite-3 and subsite-7 [4] were reported to be important
for enriching the production of certain types of CD. Alterna-
tively, modifying the enzymatic reaction conditions could also
help in enriching the yield of the target type of CD; such param-
eters include starch source [7,8], starch concentration [9], buffer
type [3,10], reaction pH [10], presence of additives [11–13] and
presence of precipitants [14]. Some of the studied parameters
are reported to have a beneficial effect on CD production while
contradictory results have been reported in other publications.
Thus, it can be concluded that reaction condition studies are still
essential for each case of interest.
This is the first report on the effects of reaction parameters
on the ratio of ␥-CD to the total amount of CDs produced by the
utilization of a mutant CGTase (mutant H43T). A recombinant
form of the enzyme derived from the predominant ␤-CGTase G1
[8] was protein tailored at subsite-3 of the protein structure. The
mutant exhibited the ability to produce a higher percentage of
␥-CD. This study shows that combining protein engineering and
reaction manipulation, enriches the production of ␥-CD signif-
icantly, and is relatively more effective than altering one single
mode alone.
2. Materials and method
2.1. Bacterial strain, DNA manipulation, and purification
of mutant CGTase H43T
The alkalophilic bacteria identified as Bacillus sp. G1
was isolated from soil and the nucleotide sequence of the
CGTase gene was submitted to the National Center for
Biotechnology Information (NCBI) database (accession num-
ber AY770576). The Megaprimer-PCR method [15] was used to
construct mutant CGTase H43T. Internal mutagenized primer 5 -
CCACCACAATACTTGGTAAGATCTATACAG-3 was used to
generate the “megaprimer” which was subsequently used as
the forwarding primer to complete the whole gene amplifica-
tion. The PCR product was digested with restriction enzyme
Xba1 and EcoR1 (Promega) and ligated to plasmid pUC19 that
was digested with the same enzymes. Mutagenesis was con-
firmed by DNA sequencing. The mutant protein was expressed
by E. coli JM109 in 1 l LB/amp media, and the crude enzyme
was harvested after overnight incubation at 37 ◦ C, 200 rpm. The
method used to purify the mutant CGTase H43T is similar to
119
the procedure used to purify the wild type CGTase [8]. The
␥-CD forming activity of the purified mutant CGTase was deter-
mined by the bromocresol green (BCG) method [16]. One unit
of ␥-CD forming activity is defined as the amount of enzyme
that produces 1 ␮mol of ␥-CD per minute. In all the following
reactions (Sections 2.2–2.6), approximately 0.012 U of diluted
purified enzyme was used per ml of reaction mixture. Under
normal conditions, this corresponded to 1.2 U of enzyme per
gram of starch. The enzymatic reaction was carried out for 18 h
based on the initial screening process which showed that the
enzymatic reaction had reached steady state after 18 h (data not
shown). Two or more runs were conducted for all the enzymatic
reactions.
2.2. Screening of substrates and buffers on γ-CD
production
Corn and potato starch were purchased from Merck, solu-
ble starch from Goodrich Chemical Enterprise (GCE), amylose
and glycogen were from Sigma. The tapioca starch used was of
food grade and sourced from a local manufacturer. Substrates
were boiled for 10 min in each 20 mM buffer system (phosphate,
acetate, MES, citric), cooled to room temperature before adding
diluted CGTase. Purified mutant CGTase H43T was reacted with
1 ml of 1% (w/v) substrate in selected buffer systems at pH 6 and
60 ◦ C for 18 h. Enzymatic reactions were later stopped by boil-
ing for 10 min. A concentration of 1% substrate was chosen for
the basis of comparison because certain starch solutions (typ-
ically tapioca starch) at high concentrations are very viscous,
difficult to handle and result in lower yield as reported in a study
using the wild type CGTase G1 [8].
2.3. Effects of raw and gelatinized starch on γ-CD
production
Raw starch slurry was freshly prepared before use by sus-
pending 1–4% (w/v) tapioca starch in 20 mM acetate buffer of
pH 6. Gelatinized starch was prepared by heat treatment in a
boiling water bath for 10 min. After cooling to room tempera-
ture, mutant CGTase H43T was added. Reactions were carried
out as described earlier.
2.4. Effects of pH on γ-CD production
Mutant CGTase H43T was reacted with 1 and 2.5% gela-
tinized tapioca starch in 20 mM buffers ranging from pH 6 to 10.
The buffers used were acetate buffer (pH 6), phosphate buffer
(pH 7–8) and glycine–NaOH buffer (pH 9–10).
2.5. Effects of starch pretreatment using a debranching
enzyme on γ-CD production
Debranching of amylopeptin in 1, 2.5 and 4% (w/v) gela-
tinized tapioca starch–acetate buffer were carried out using
pullulanase (Promozyme 400 l, Sigma). Concentrations of 0.1
and 0.3% (v/v) pullulanase were added to the starch and
incubated for 30 min at 50 ◦ C. Mutant CGTase H43T was
120 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126

added after the debranching activity had been stopped by boil- Table 1
ing. Comparison of amino acid sequence at subsite-3 of various CGTase
CGTase source Sequence at Main Accession
2.6. Effects of additives, precipitants and solvent on γ-CD subsite-3 region product number
production Bacillus macerans HS-NLKLYF ␣ P04830
T. thermosulfurigenes EM1 HT-SLKKYF ␤/␣ P26827
Mutant CGTase was reacted with 1 ml of 1% gelatinized Bacillus licheniformis CS-NLKLYC ␣/␤ P14014
tapioca starch–acetate buffer supplemented with additives Bacillus circulans No. 8 CS-NLKLYC ␤ CAA48404
Alkalophilic B. sp. 17.1 CT-NLRLYC ␤ P30921
(5–20% CaCl2 , 10–50 mM glucose), solvent (10–30% ethyl Bacillus circulans 251 CT-NLRLYC ␤ P43379
alcohol) and precipitants (5–40% toluene, 2.5–8% limonene, Alkalophilic B. sp. 1011 CT-NLRLYC ␤ P05618
0.0057–0.115 mM cyclododecanone). These chemicals were
Alkalophilic B. sp. 1.1 CI-DLHKYC ␤ (No ␣) P31746
only added once at the beginning of the reaction. Alkalophilic B. sp. G1a CI-DLHKYC ␤ (No ␣) AAV38118
Mutant CGTase H43T+ CI-DLTKYC ␤/␥ This study
2.7. Analysis of cyclodextrins by HPLC
Bacillus firmus/lentus 290-3 CL-DLTKYC ␥/␤ CAA01436
Bacillus clarkii 7364 CS-DLTKYC ␥ BAB91217
The ratio of cyclodextrins produced was analyzed using
Alkalophilic B. sp. G-825-6 CL-DLTKYC ␥ BAE87038
a Waters HPLC system with separation carried out using
an Econosphere NH2 (5 ␮m, 250 mm × 4.6 mm) column. The
a The native CGTase used to construct mutant H43T CGTase+ .
peaks were eluted with 70:30 acetonitrile–water at a rate of
1 ml/min. An RI detector was used to detect the reaction prod- which affects the cyclization activity to allow the formation of
ucts. The standards used for calibration were glucose, maltose, a larger cyclodextrin molecule in the extra space at subsite-3.
maltotriose, maltotetraose and CDs and were of high purity This finding is in agreement with the results reported by van
grade purchased from Sigma and Supelco. der Veen et al. [17] which suggested that a relatively short side
chain at the same position is accompanied by a clear preference
2.8. Determination of enzyme kinetic parameters for the production of the larger size cyclodextrin. In addition to
its importance for product specificity, the residue at subsite-3 is
The enzyme kinetic values for the purified enzyme were also involved in the binding of starch and cyclodextrin during
determined by incubating 0.2 ml of samples with soluble the cyclization process. Shortening the side chain after mutation
starch in 1 ml of 0.1 M phosphate buffer (pH 6.0). The data to threonine may cause the changes in position of the sugars
was produced according to the ␥-CD cyclization assay. Km which bind during the cyclization reaction [18].
and Vmax values were then determined from a Hanes–Woof The novel mutant enzyme namely H43T CGTase can produce
plot. The turnover number (kcat ) for CGTase G1 was calcu- up to 39% ␥-CD and 61% ␤-CD when reacted with 1% tapioca
lated by dividing Vmax by the molar concentration of CGTase starch. The increment in ␥-CD production was approximately
(kcat = Vmax /(E)0 ). four times that obtained using the native CGTase and was higher
than that of other subsite-3 mutants described in other publica-
3. Results and discussion tions. Table 2 shows the spectrum of CD production by various
CGTase mutants reported in previous studies. Moreover, another
The study conducted by Sian et al. [8] showed that a puri- advantage of mutant CGTase H43T is that it does not produce
fied CGTase from Bacillus sp. G1 produced about 10% ␥-CD ␣-CD which facilitates isolation of the other types of CD. The
and 90% ␤-CD when reacted with 1% tapioca starch in phos- wild type CGTase from Bacillus sp. G1 produces approximately
phate buffer (pH 6). With tapioca starch as the substrate, no 0.85 mg/ml total CDs when 1% tapioca starch in phosphate
␣-CD was formed using CGTase produced by Bacillus sp. G1. buffer is used as the substrate. Under the same reaction con-
In this study, an engineered enzyme from the predominant ␤- ditions and enzyme concentration, mutant CGTase H43T was
CD producer CGTase from Bacillus sp. G1 was constructed by only able to generate 0.55 mg CDs/ml. The shorter side chain of
mutation at subsite-3 of the recombinant enzyme. A comparison threonine compared to histidine at residue 43 caused the inter-
of the amino acid sequence of CGTase G1, mutant H43T and action between the enzyme and starch–CD complex to become
other CGTases is shown in Fig. 1. The varying patterns of residue weaker as the distance between them is greater and this lead
at subsite-3 can be clearly distinguished between the different to a reduction in the total CD produced. Kinetic study revealed
groups of CGTase. For ␣-CD and ␣-/␤-CD producers, lysine (K) that the Km and kcat values for the wild type CGTase G1 was
or arginine (R) is mainly found at subsite-3 whereas for the ␤-CD 2.9 mg/ml and 2349 s−1 , respectively. However, the values for
producer either in the absence or presence of minor quantities mutant CGTase H43T were lower, 1.9 mg/ml and 2103 s−1 for
of ␣-CD, histidine (H) is found at this site while ␥-CD pro- Km and kcat , respectively. Uitdehaag et al. [18] reported that
ducers have threonine (T) at corresponding location (Table 1). mutation at Arg47 (corresponding to residue 43 in CGTase G1
Therefore, a site-directed mutagenesis based on rational design numbering) can cause a change in binding affinity (Km ) and
was conducted in this work. Threonine has a shorter and less cyclization turn-over number.
complex side chain than histidine. The mutation at this position Although mutant CGTase H43T was able to produce 39% ␥-
in recombinant CGTase of Bacillus sp. G1 created extra space CD and 61% ␤-CD, the percentage production of ␥-CD is still
 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126 121

Fig. 1. Comparison of amino acid sequence of CGTase Bacillus circulan 251 (BC251), CGTase Bacillus sp. G1 (G1), CGTase mutant H43T (H43T) and CGTase
Bacillus firmus/lentus 290-3 (Firmus 290-3). The catalytic residues for CGTase are indicated by *. The location of subsite-7 is shown in the square box. All the
amino acids at subsite-3 are highlighted and the residue mutated in this study is indicated by the arrow.

lower compared to the wild type ␥-CGTases, typically CGTase
from Bacillus firmus/lentus 290-3 [9] which can produce almost
the same quantity of ␤-CD and ␥-CD. Therefore, the effect of
reaction conditions on the performance of mutant CGTase H43T
was studied with the aim of improving the percentage of ␥-CD
formed.
3.1. Effects of substrates and buffers on γ-CD production
The effects of various substrates (tapioca, potato, solu-
ble, corn starch, amylose, glycogen) in different buffers were
studied to identify the best substrate–buffer combination for
␥-cyclodextrin production, using the engineered CGTase. As

Table 2
Ratio of cyclodextrins produced by parent and mutants CGTase from various sources
CGTase strain Mutation site CD spectrum (␣-CD:␤-CD:␥-CD) Reference

Before mutation After mutation

Bacillus sp. G1 H43T 0:90:10 0:61:39 This study

Bacillus firmus var. alkalophilus H59Q and (154–160) 0:83:17a No changes [31]
Thermococcus sp. B1001 Y267W 85:8:7a 70:17:13a [32]
Bacillus circulans No. 8 Y195W 11:68:21a 6:39:55a [33]
(145–151)D 6:54:40a
Bacillus circulans 251 Y195W 13:64:23 18:63:19 [34]
Bacillus ohbensis Y188W 0:85:15a 0:57:43a [6]
a Numbers were estimated from graph.
122 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126

Fig. 2. Effect of substrate selectivity and buffer systems at pH 6; 1% gelatinized substrates were used throughout.

shown in Fig. 2, the use of tapioca starch in a different buffer sys- portional activity would compete with cyclization reactions, and
tem leads to a significant improvement in the ␥-CD ratio: acetate may lead to a reduction in the synthesis of CDs. Overactivation
buffer (41% ␥-CD), MES (43% ␥-CD) and the highest percent- of the coupling reaction will cause the CD ring to be opened up
age of ␥-CD was found using citric buffer (46% ␥-CD). Despite to linear oligosaccharides and hence will decrease cyclodextrin
enhancing the formation of ␥-CD from 39% in phosphate buffer yield [20]. Furthermore, small oligosaccharides such as glucose
to 46% ␥-CD when citric buffer was used, conversion to total and maltose that are formed from by-reactions are inhibitors of
CDs, however, was relatively low as only half the amount of total CGTase leading to a reduction in CDs formed [21].
CDs could be produced in comparison to the tapioca–phosphate
system as shown in Fig. 2. 3.2. Effects of raw and gelatinized tapioca starch on γ-CD
When the ␥-CD ratio and conversion capability factor were production
compromised, the tapioca–acetate system gave the numerically
highest conversion rate (16%) while maintaining a high ratio of Since the tapioca–acetate buffer combination gave good
␥-CD (41%) which is almost three times the amount of cyclodex- conversion (16%) and a high percentage of ␥-CD (41%), it
trins (16% conversion) produced under normal conditions. In was chosen as the basis for comparison in all the following
fact, by using the tapioca–acetate system, conversion to CDs was experiments. A higher percentage of ␥-CD was produced from
the highest compared to all the substrate–buffer combinations
studied in this work (Fig. 2).
It was observed that there were fewer short oligosaccharides
formed when tapioca starch was used (Fig. 3). Conversion to
CDs was almost four times less for potato starch compared
to tapioca starch, whereas the formation of linear oligosac-
charides was relatively higher when potato starch was utilized
(Fig. 3). HPLC analysis showed that potato starch used in
this experiment was slightly richer in free short linear malto-
oligosaccharides. The presence of short oligosaccharides can
interfere with the efficiency of the cyclization reaction as these
molecules can promote more disproportional and coupling activ-
ities than cyclization activity, the latter being responsible for
the formation of CD. Disproportional reactions will only result
in various lengths of linear oligosaccharides and those that are
less than eight glucose units in length are unsuitable as sub-
strates for the formation of cyclodextrin since the cyclization
reaction follows the equation Gn ↔ Gn−x + cGx , (where n ≥ 8,
Fig. 3. Typical HPLC chromatograms for the different sources of starch. (A)
9 ≥ x ≥ 6), Gn are ␣,1-4-glucopyranosyl chains of length n, and tapioca starch; (B) potato starch; (C) corn starch; (D) soluble starch; G1 , glucose;
cGx are cyclodextrins of ring size x [19]. Triggering of dispro- G2 , maltose; G3 , maltotriose; G4 , maltotetraose.
 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126
Fig. 4. Effects of raw and gelatinized tapioca starch in sodium acetate buffer
(pH 6).
gelatinized starch compared to the native form (Fig. 4). The
total amount of CDs produced was also higher when gelatinized
tapioca starch was used compared to raw starch.
Raw starch has a compact crystalline structure which is not
easily degraded by common starch degrading enzymes [22] due
to the weak interaction of CGTase with raw tapioca starch gran-
ules. The native crystalline structure of starch is disrupted by
heating in the presence of water, a phenomenon known as gela-
tinization. Gelatinized starch swells irreversibly to many times
its original size and creates a large surface to volume area for
enzymatic reaction [22,23]. Another reason for the superior ␥-
CD production in the presence of gelatinized tapioca starch is
that during gelatinization, heat leaches out amylose [24] provid-
ing more contact sites between the substrate and enzyme.
Increasing the concentration of gelatinized starch will signif-
icantly reduce the conversion rate of starch to CDs as shown in
Fig. 4. Tapioca starch appears to be ‘cloudy’ when gelatinized
and becomes more viscous as the concentration increases. This
is a common problem in the starch industry especially when
high starch concentrations are used. Normally high temperature
is required to liquefy the starch. An alternative approach fre-
quently used in industry to liquefy starch slurry is to use amylase,
however, it was found that low molecular weight oligosaccha-
Fig. 5. Effects of different concentrations of gelatinized tapioca starch at pH 6–10.
123
rides produced by the hydrolytic reaction inhibits CD production
[20].
3.3. Effects of pH on γ-CD production
A relatively higher percentage of ␥-CD was achieved under
alkaline conditions with a maximum percentage of ␥-CD (46%)
observed at pH 8 and pH 9 and the ratio was noticeably
higher when the starch concentration (2.5% starch) was elevated
(Fig. 5). Although the percentage of ␥-CD was enhanced at a
higher pH, the amount of total CDs produced however, was less
than that produced at pH 6 which appears to be the optimum
pH for the mutant CGTase enzyme activity (data not shown). It
has been reported that the ability of CGTase to produce ␥-CD
can be enhanced by using alkaline conditions and improved to a
more obvious level using higher starch concentrations [10,21].
This could be due to the different binding modes of the enzyme
and substrate at different pHs [10].
3.4. Effects of debranching enzyme on γ-CD production
The content of amylose in tapioca starch was reported to be
16.27 ± 0.32% [25]. Compared to other starches such as corn
and potato, the content of amylopectin (the branched portion)
in tapioca starch is higher than that of amylose [24,25]. CGTase
efficiently degrades starch to CD by its action on the amylose
portion of the molecule and not on the amylopectin moiety. The
effect of the debranching enzyme pullulanase to improve the
ratio of ␥-CD produced using this mutant H43T CGTase was
studied.
Fig. 6 shows that the amount of ␥-CD produced was greater
when tapioca starch was treated with pullulanase. When 2.5%
tapioca starch was treated with 0.1 and 0.3% pullulanase, there
was a reduction in the total CDs produced while the percentage of
␥-CD increased. This was also observed for 1% tapioca starch.
In both cases (1 and 2.5% tapioca starch), although the ratio
of ␥-CD produced was improved when pullulanase was used,
124 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126
Fig. 6. Effects of starch pretreatment with pullulanase. Gelatinized tapioca starch was digested with 0.1 or 0.3% (v/v) pullulanase; pull., pullulanase.
the total conversion of starch to CDs decreased. Production of
cyclodextrin using CGTase is influenced by substrate quantity
(concentration), substrate quality (amylose–amylopectin ratio
and viscosity), and the presence of inhibitors (glucose produced
from pullulanase action on starch) where these parameters are
related to each other in this context. Equilibrium between these
factors determines the final ratio of ␥-CD and total CD pro-
duction. Our initial analysis on starch treated with pullulanase
using HPLC showed that a high level of glucose was produced
(data not shown). Glucose has been reported to be an inhibitor
of CGTase since both glucose and starch bind at the same posi-
tion in CGTase molecule, i.e. at the maltose binding site (MBS)
which affects the activity of the enzyme [19,26].
An increment in the total CDs produced was observed when
pullulanase was used in 4% tapioca starch as shown in Fig. 6.
The overall conversion to CDs for the control was 5% while 6.2
and 5.4% conversions were observed with 0.1 and 0.3% pullu-
lanase pretreatment of the starch, respectively. Interestingly the
percentage of ␥-CD produced was increased from 42 to 48%
when 0.3% pullulanase was used. Pullulanase reduced the vis-
cosity of the starch slurry and produced abundant quantities of
free linear ␣-1-4-glucopyranosyl as substrate for CD produc-
tion. In the presence of a rich substrate concentration, more
CDs are produced and this effect is greater than the negative
effect caused by glucose inhibition. In low starch concentrations
(e.g. in the case of 1 and 2.5%) as shown in Fig. 6, the use of
the debranching enzyme only leads to excess of glucose which
inhibits CGTase activity and this negative effect of pullulanase
overrides its positive effects in reducing viscosity and increas-
ing the amylose content. From the results obtained with different
starch concentrations and the varying degree of debranching, it
can be concluded that the amount of pullulanase used is critical
since it controls the variable of substrate concentration, viscosity
and the inhibition effect. Only in an appropriate concentration
where the pros outperform the cons would pullulanase be useful
in ␥-CD production.
In studies conducted by Pishtiyski and Zhekova [7], Sze-
jtli [27] and Saha and Zeikus [28], pullulanase was also used
together with CGTase in cyclodextrin production, however, no
enhancement of the ␥-CD ratio was observed by these authors.
Pishtiyski and Zhekova [7] reported that preliminary treatment
of starch with ␣-amylase and pullulanase was inefficient and
unnecessary because it did not lead to an increase in the yield
of CDs. In addition, no significant diversity in CD profile was
observed. The results from this study, however, contradict the
results of previous researchers [7].
3.5. Effects of additives, precipitants and solvent on γ-CD
production
The effects of supplements on ␥-CD production using 1%
gelatinized tapioca starch in acetate buffer (pH 6) are shown in
Table 3. The addition of CaCl2 is known to increase the ther-
mostability of CGTase. Calcium ion binds at two locations in the
protein structure as shown by X-ray crystallography [29]. Addi-
tion of CaCl2 to the reaction mixture did not lead to a significant
difference in ␥-CD ratio or total conversion (Table 3).
Table 3 also summarizes the effect of ethyl alcohol on the pro-
duction of ␥-CD using the H43T mutant CGTase. Reactions at
60 ◦ C (which is the optimum temperature) using mutant CGTase
H43T and the addition of 10, 20 or 30% ethyl alcohol showed
no improvement in ␥-CD specificity, but reduced the conversion
rate progressively as the concentration of alcohol increased. In
this study, the activity of mutant H43T is inversely proportional
to the percentage of ethyl alcohol added. This could be due to
inactivation of enzyme since reactions were carried out using the
optimum temperature of 60 ◦ C. Mori et al. [12] reported that the
presence of 20% ethyl alcohol in the reaction mixture at 40 ◦ C
greatly enhanced the proportion of ␥-CD. In another example,
the yield of ␥-CD increased with ethyl alcohol concentration up
to 30% [11]. In both successful examples, reactions were carried
out at 40 ◦ C since the enzyme is inactivated at temperature higher
than 50 ◦ C in the presence of ethyl alcohol [12]. All the reported
studies were conducted at reaction temperatures lower than the
actual optimum temperature of the CGTases used. Therefore, a
further study of the effect of ethyl alcohol was carried out at
 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126 125

Table 3 middle of the reaction, a portion of the ␥-CD will be precipitated

Relative comparison of the effects of selected additives, precipitants and solvent causing an imbalance in the kinetics equilibrium, and driving the
on cyclodextrin production and ␥-CD ratio
reaction towards producing more ␥-CD. In this study, some of
Additives and concentration ␥-CD ␤-CD Total CDs Conversion the commonly used precipitants such as toluene, limonene, and
(%) (%) (mg) (%) cyclododecanone were added to a 1% tapioca–acetate buffer
No additives 41.0 59.0 1.60 16.0 system to improve ␥-CD production using the mutant H43T
CaCl2 (mM) CGTase. All additives were added only once at the beginning of
5 40.6 59.4 1.61 17.0 each reaction.
10 40.6 59.4 1.61 16.1 Toluene at a concentration of 40% (v/v) was added to the
20 40.4 59.6 1.61 16.1 reaction mixture and the results show that it increases the pro-
Ethyl alcohol (60 ◦ C) (%) duction of ␥-CD up to 46.4%. The ␥-CD ratio was relatively
10 39.4 60.6 0.99 9.9 higher than the control as shown in Table 3. The decline in the
20 40.2 59.8 0.42 4.2 total amount of CDs produced was less than 10%, probably due
30 23.3 76.7 0.23 2.3
to some enzymatic inactivation caused by the solvent. The high
Ethyl alcohol (40 ◦ C) (%) volatility of toluene makes it a useful precipitant because the
0 35.0 65.0 0.93 9.3
toluene–␥-CD complex is not tightly bound and can easily be
10 26.7 73.3 0.93 9.3
20 30.3 69.7 0.67 6.7 disrupted by simple heat treatment. However, the use of toluene
30 20.5 79.5 0.61 6.1 is not recommended when the CDs produced are to be used in
Glucose (mM)
food and drugs.
10 40.3 59.7 1.47 14.7 In a patent file, Ammeraal [30] described the use of limonene
30 42.0 58.0 1.40 14.0 for purifying ␤-CD from a reaction mixture. It was reported that
50 42.2 57.8 1.33 13.3 limonene preferentially precipitated ␤-CD and in a second step
Toluene (%) precipitated ␥-CD from a mixture of CDs. It was also reported
5 41.9 58.1 1.52 15.2 that the overall yield of ␤-CD was increased in the presence of
25 41.2 58.8 1.48 14.8 limonene. Unfortunately, utilizing low concentration (2.5–8%)
40 46.4 53.6 1.45 14.5
of limonene together with mutant H43T did not result in an
Limonene (%) increase in the production of ␥-CD (Table 3).
2.5 37.5 62.5 1.56 15.6 2-Butanone used to dissolve cyclododecanone is itself inert
5 37.7 62.3 1.73 17.3
8 37.4 62.6 1.63 16.3
to product specificity, nevertheless it inactivates mutant CGTase
H43T. Only half the amount of total CDs were formed in the
2-Butanone (%)
presence of 2-butanone compared to the control as shown in
4 40.0 60.0 0.77 7.7
Table 3. Approximately 43.4% ␥-CD was produced when the
2-Butanone + 0.0057 mM 41.9 58.1 0.78 7.8 reaction was supplemented with 0.115 mM cyclododecanone.
cyclododecanone
2-Butanone + 0.028 mM 41.9 58.1 0.78 7.8
Nevertheless, Rendleman [14] managed to change the product
cyclododecanone specificity from an ␣-CD:␤-CD:␥-CD ratio of 11.2:15.6:2.8 to
2-Butanone + 0.115 mM 43.4 56.6 0.77 7.7 3.7:37.6:41.8 in the presence of cyclododecanone by adding 10
cyclododecanone incremental aliquots of fresh CGTase from Bacillus macerans
to the reaction mixture. The percentage of ␥-CD was increased
to 72.2% using 35 increments of CGTase.
40 ◦ C which is lower than the optimum temperature for H43T
CGTase. The results show that at 40 ◦ C, the percentage of ␥-CD 4. Conclusion
dropped to 35% and that only a quarter of total CDs generated
at 60 ◦ C were produced. The activity and product specificity of Mutant H43T CGTase produced 39% ␥-CD while the par-
mutant H43T is greatly influenced by reaction temperature. This ent enzyme produced only 10% in the tapioca–phosphate (pH
study shows that ethyl alcohol increases ␤-CD yield rather than 6) system. Manipulating reaction parameters increased the per-
␥-CD as shown in Table 3. centage of ␥-CD produced. These parameters include acetate
Table 3 indicates that at 10 and 50 mM glucose, inhibition of buffer (41% ␥-CD), pH 8–9 (46% ␥-CD), 0.3% pullulanase pre-
mutant CGTase H43T was found to be 8 and 17%, respectively. treatment of starch (48% ␥-CD), 50 mM glucose (42.2% ␥-CD),
Looking at the individual amounts of ␤-CD and ␥-CD produced 40% toluene (46.4% ␥-CD), and 0.115 mM cyclododecanone
by mutant H43T in the presence of glucose (data not shown), it (43.4% ␥-CD). The improvement was achieved as a result of the
appears that as the concentration of glucose increases the quan- effects of all the parameters used on the structure of the mutant
tity of ␤-CD produced clearly falls significantly compared with H43T. This has led to product specificity and changes in total
that of ␥-CD whose percentage yield was enhanced. In this study CD production of the mutant CGTase H43T via an intermolec-
it has been shown that glucose might have a greater inhibitory ular transglycosylation. Our findings with regard to this mutant
effect on the formation of ␤-CD than of ␥-CD. enzyme are compatible with those of other researchers [7–14]
␥-CD forms inclusion complexes with certain solvents and who also showed that the use of different starches, pH, solvents,
chemicals. If the precipitants are applied at the beginning or etc., together with native CGTase can affect the transglycosyla-
126 K.M. Goh et al. / Journal of Molecular Catalysis B: Enzymatic 49 (2007) 118–126
tion reaction, which leads to the different product specificities.
We have demonstrated that the enhancement of ␥-CD produc-
tion can be achieved via the combination of protein engineered
CGTase and manipulation of the reaction conditions. Using both
these methods in combination provides better results than the use
of either one alone. It is therefore concluded that mutant CGTase
H43T serves as a potential enzyme for use in the manufacture
of ␥-CD.
Acknowledgements
The authors would like to thank Madihah Salleh for mak-
ing the HPLC system available for product analysis. This work
was financially supported by National Biotechnology Divi-
sion, Malaysia Ministry of Science, Technology and Innovation
(MOSTI) under project number 09-02-05-006 BTK/ER/34.
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