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Molecular and biochemical characterizations of a new low-temperature active mannanase

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Abstract

A mannanase-coding gene was cloned from Sphingobacterium sp. GN25 isolated from the feces of Grus nigricollis. The gene encodes a 371-residue polypeptide (ManAGN25) showing less than 74 % identity with a number of hypothetical proteins and putative glucanases and mannanases. Before experiment’s performance, ManAGN25 was predicted to be a low-temperature active mannanase based on the molecular characterization, including (1) ManAGN25 shared the highest identity of 41.1 % with the experimentally verified low-temperature active mannanase (ManAJB13) from Sphingomonas sp. JB13; (2) compared with their mesophilic and thermophilic counterparts, ManAGN25 and ManAJB13 had increased number of amino acid residues around their catalytic sites; (3) these increased number of amino acid residues built longer loops, more α-helices, and larger total accessible surface area and packing volume. Then the experiments of biochemical characterization verified that the purified recombinant ManAGN25 is a low-temperature active mannanase: the enzyme showed apparently optimal activity at 35–40 °C and retained 78.2, 44.8, and 15.0 % of its maximum activity when assayed at 30, 20, and 10 °C, respectively; the half-life of the enzyme was approximately 60 min at 37 °C; the enzyme presented a K m of 4.2 mg/ml and a k cat of 0.4/s in McIlvaine buffer (pH 7.0) at 35 °C using locust bean gum as the substrate; and the activation energy for hydrolysis of locust bean gum by the enzyme was 36.0 kJ/mol. This study is the first to report the molecular and biochemical characterizations of a mannanase from a strain.

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Abbreviations

manAGN25 :

Mannanase-coding gene from Sphingobacterium sp. GN25

ManAGN25:

Mannanase from Sphingobacterium sp. GN25

rManAGN25:

Recombinant ManAGN25

ManAJB13:

The low-temperature active mannanase from Sphingomonas sp. JB13

ManA:

Thermophilic mannanase from Dictyoglomus thermophilum Rt46B.1

Man26BCD:

Mesophilic mannanase from Paenibacillus sp. BME-14

PMAN:

Low-temperature active mannanase from Glaciozyma antarctica PI12

G. nigricollis :

Grus nigricollis

E. coli :

Escherichia coli

S. thalpophilum :

Sphingobacterium thalpophilum

GH:

Glycosyl hydrolase family

IUCN:

International Union for Conservation of Nature

PCR:

Degenerate polymerase chain reaction

TAIL-PCR:

Thermal asymmetric interlaced PCR

IPTG:

Isopropyl-β-d-1-thiogalactopyranoside

SP primers:

Nested insertion-specific primers

ASA:

Accessible surface area

TPV:

Total packing volume

pET-manAGN25 :

Recombinant plasmid of pET-28a(+) vector and coding sequence of the mature protein of manAGN25

LB:

Luria–Bertani

Buffer A:

20 mmol/l Tris–HCl 0.5 mol/l NaCl, pH 7.2

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

MALDI-TOF MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

DNS:

3,5-Dinitrosalicylic acid

U:

Unit

McIlvaine buffer:

A mixture of 0.1 mol/l citric acid and 0.2 mol/l Na2HPO4

P1–P4:

Positions 1–4 shown in Figs. 1 and 2

T m :

Annealing temperature

NR:

Not reported

Ea:

Activation energy

ΔG * :

Free energy of activation

ΔH * :

Enthalpy of activation

ΔS * :

Entropy of activation

Q10 :

Temperature coefficient

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Acknowledgments

This work was supported by the Key Technologies Research and Development Program of China (2013BAD10B01), the National Natural Science Foundation of China (31460694), and the Applied and Basic Research Foundation of Yunnan Province (2011FB048 and 2013FZ045).

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    Authors and Affiliations

    1. Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People’s Republic of China

      Rui Zhang, Junpei Zhou, Junjun Li, Xianghua Tang, Bo Xu, Junmei Ding & Zunxi Huang

    2. College of Life Sciences, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People’s Republic of China

      Rui Zhang, Junpei Zhou, Yajie Gao, Yaping Guan, Junjun Li, Xianghua Tang, Bo Xu, Junmei Ding & Zunxi Huang

    3. Key Laboratory of Yunnan for Biomass Energy and Biotechnology of Environment, Kunming, 650500, People’s Republic of China

      Rui Zhang, Junpei Zhou, Junjun Li, Xianghua Tang, Bo Xu, Junmei Ding & Zunxi Huang

    4. Key Laboratory of Enzyme Engineering, Yunnan Normal University, No. 1 Yuhua District, Chenggong, Kunming, Yunnan, 650500, People’s Republic of China

      Rui Zhang, Junpei Zhou, Junjun Li, Xianghua Tang, Bo Xu, Junmei Ding & Zunxi Huang

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    1. Rui Zhang
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    Correspondence to Zunxi Huang.

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    Rui Zhang and Junpei Zhou contributed equally to this work.

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    Zhang, R., Zhou, J., Gao, Y. et al. Molecular and biochemical characterizations of a new low-temperature active mannanase. Folia Microbiol 60, 483–492 (2015). https://doi.org/10.1007/s12223-015-0391-1

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