(Microbial and Enzyme Technology)

Cloning, characterization and molecular docking of a highly thermostable β-1,4-glucosidase from Thermotoga petrophila

Ikram ul Haq1*, Mahmood Ali Khan1*, Bushra Muneer1, Zahid Hussain1, Sumra Afzal1, Sana Majeed1, Naeem Rashid2, Muhammad Mohsin Javed1 and Ishtiaq Ahmad1

1Institute of Industrial Biotechnology, GC University, Lahore-54000, Pakistan

2School of Biological Sciences, Quaid-i-Azam campus, PunjabUniversity, Lahore-54590, Pakistan

Mahmood Ali Khan e-mail:

Bushra Muneer e-mail:

Zahid Hussain e-mail:

Sumra Afzal e-mail:

Sana Majeed e-mail:

Naeem Rashid e-mail:

Muhammad Mohsin Javed e-mail:

Ishtiaq Ahmad e-mail:

*Correspondingauthors. Mailing address: Institute of Industrial Biotechnology, GC University, Lahore54000, Pakistan.

Phone: +92 42 99211634

E-mail: (I.U.Haq)

1* Both author contributed equally

Supplementary Fig. 1 SDS-PAGE of purified BglAfractions. Lane M: Molecular size marker; lane 1, 2, 3: purified BglAfractions after Ion exchange and hydrophobic interaction chromatography. Gel was stained with Coomassie Brilliant blue R-250 by standard protocol


Supplementary Fig. 2 pH stability was analyzed by pre-incubating 10µl of BglA in 90µl of citrate/ phosphate buffer (closed diamond, pH 3-7,20 mM), Tris/HCl buffer (closed square, pH 7-9, 50 mM), HEPES (dash, pH 8, 25 mM) and CAPS (closed triangle, pH 9-11, 25 mM) for 30 min and the residual activity was determined at 90oC for 10 min in citrate/phosphate buffer pH 7. Blanks were run under the same conditions without the added enzyme. The highest activity (30.4 kUmg-1), observed at pH 7, is defined as the 100% activity to calculate the residual activity. All values are the average of experiments done in triplicate

Supplementary Fig. 3

Sequence comparisonCLUSTAL W (1.83) multiple sequence alignment

S.I
T.petrophila
Thermotoga sp.RQ2 99%
Thermotoga maritima MSB8 99%
Thermotoga neapolitana 91%
Petrotoga mobilis 68%
Halothermothrix orenii 53%
Clostridium thermocellum 52%
Bacillus sp. 50%
Streptomyces sp. 50% / ------MN----VKKFPEGFLWGVATASYQIEG 23
------
------MN----VKKFPEGFLWGVATASYQIEG
------MM---IVKKFPEGFLWGVATASYQIEG
------MS----EKVFPKDFMWGAATASYQIEG
------MA----KIIFPEDFIWGAATSSYQIEG
MFPLGYNYIITLFANNILKGVVNMS----KITFPKDFIWGSATAAYQIEG
------MA----SIQFPKDFVWGTATASYQIEG
------MTDGLDYAALPEGFTWGVATAAYQVEG
T.petrophila
Thermotoga sp.RQ2
Thermotoga maritima MSB8
Thermotoga neapolitana
Petrotoga mobilis
Halothermothrix orenii
Clostridium thermocellum
Bacillus sp.
Streptomyces sp. / KAYRFSISWPRILPEGTGRVNQKGLDFYNRIIDTLLEKGITPFVTIYHWD 123
KAYRFSISWPRILPEGTGRVNQKGLDFYNRIIDTLLEKGITPFVTIYHWD
KAYRFSISWPRILPEGTGRVNQKGLDFYNRIIDTLLEKGITPFVTIYHWD
KAYRFSISWPRILPEGTGKVNQKGLDFYNRIIDTLLEKNITPFITIYHWD
KAYRFSISWSRIFPNGKGKINEKGVDFYNRLVDELLKANITPFVTLYHWD
RSYRFSTSWPRILPEGKGRVNQKGLDFYKRLVDNLLKANIRPMITLYHWD
KSYRFSISWPRIFPEGTGKLNQKGLDFYKRLTNLLLENGIMPAITLYHWD
KAYRFSIAWPRIYPDGDGELNQKGLDYYAKVIDGLLAAGIEPCVTLYHWD
GAYRFSVAWPRVVPGGDGPVNAKGLDFYDKLVDGLLAEGIEPFVTLYHWD
T.petrophila
Thermotoga sp.RQ2
Thermotoga maritima MSB8
Thermotoga neapolitana
Petrotoga mobilis
Halothermothrix orenii
Clostridium thermocellum
Bacillus sp.
Streptomyces sp. / 166
LPFALQLKGGWANREIADWFAEYSRVLFENFGDRVKNWITLNEPWVVAIV 173
LPFALQLKGGWANREIADWFAEYSRVLFENFGDRVKNWITLNEPWVVAIV
LPFALQLKGGWANREIADWFAEYSRVLFENFGDRVKNWITLNEPWVVAIV
LPFSLQLKGGWANRDIADWFAEYSRVLFENFGDRVKHWITLNEPWVVAIV
LPAALQDLGGWTNRDIAYWYTDYADYMFQRLGDRVKNWITLNEPWVMAFV
LPQALQDKGGWTNRDTAKYFAEYARLMFEEFNGLVDLWVTHNEPWVVAFE
LPQKLQDKGGWKNRDTTDYFTEYSEVIFKNLGDIVPIWFTHNEPGVVSLL
LPQALQDKGGWDNRDTIRAFVRYAETAFKAFGGKVKQWITFNETWCVSFL
LPQALQDRGGWPNRETAEHFARYAEVVAGRLGDRVKRWATLNEPLCSAWI
*
T.petrophila
Thermotoga sp.RQ2
Thermotoga maritima MSB8
Thermotoga neapolitana
Petrotoga mobilis
Halothermothrix orenii
Clostridium thermocellum
Bacillus sp.
Streptomyces sp. / 351
LPKTAMGWEIVPEGIYWILKKVKEEYNPPEVYITENGAAFDDVVSEDGRV 366
LPKTAMGWEIVPEGIYWILKKVKEEYNPPEVYITENGAAFDDVVSEDGRV
LPKTAMGWEIVPEGIYWILKKVKEEYNPPEVYITENGAAFDDVVSEDGRV
LPKTAMGWEIVPEGIYWILKGVKEEYNPQEVYITENGAAFDDVVSEGGKV
LPKTEMGWEIYPEGFYKILKGVQEEYNPKEVYVTENGAAFDDSVVN-QEV
RPSTEMGWEIYPQGLYDILVRVNKEYTDKPLYITENGAAFDDKLTEEGKI
FEKTDMGWIIYPEGLYDLLMLLDRDYGKPNIVISENGAAFKDEIGSNGKI
FDKTFMDWNVYAEGLYKVLSWVHEEYGDVPIYITENGACYEDELTQEGRV
VPRTGMDWEIDANGIERMLLRLTEEYGARKIYVTENGSAWPDVVGSDGSV
*
T.petrophila
Thermotoga sp.RQ2
Thermotoga maritima MSB8
Thermotoga neapolitana
Petrotoga mobilis
Halothermothrix orenii
Clostridium thermocellum
Bacillus sp.
Streptomyces sp. / 405
HDQNRIDYLKAHIGQAWKAIQEGVPLKGYFVWSLLDNFEWAEGYSKRFGI 416
HDQNRIDYLKAHIGQAWKAIQEGVPLKGYFVWSLLDNFEWAEGYSKRFGI
HDQNRIDYLKAHIGQAWKAIQEGVPLKGYFVWSLLDNFEWAEGYSKRFGI
HDQNRIDYLRAHIEQVWRAIQDGVPLKGYFVWSLLDNFEWAEGYSKRFGI
HDENRIDYLKQHLEQALRAIQNGVTLKGYFVWSLLDNFEWALGYSKRFGI
HDEKRINYLGDHFKQAYKALKDGVPLRGYYVWSLMDNFEWAYGYSKRFGL
EDTKRIQYLKDYLTQAHRAIQDGVNLKAYYLWSLLDNFEWAYGYNKRFGI
HDAKRADYFKKHFIQCHRLIESGVPLKGYFAWSLLDNFEWAEGYVKRFGI
DDPERIRYLEDHLAACARAARKGAPLAGYFAWSLMDNFEWAYGYDKRFGL
*

Sequence alignment of family 1 β-1,4-glucosidases. Partial sequences of β-1,4-glucosidase with highlighted yellow blocks indicate conserved and highly conserved residues surrounding the active site. Black stars indicate the probable Glu residue involved in catalysis (166, 351, 405). The numbering is based on T.petrophila β-1,4-glucosidaseamino acid sequence

S.I = Sequence Identity

1