Improvement of the Optimum pH of Aspergillus niger Xylanase towards an Alkaline pH by Site-Directed Mutagenesis = Improvement of the Optimum pH of Aspergillus niger Xylanase towards an Alkaline pH by Site-Directed Mutagenesis

다국어 초록 (Multilingual Abstract)

In an attempt to shift the optimal pH of the xylanase B (XynB) from Aspergillus niger towards alkalinity, target mutation sites were selected by alignment between Aspergillus niger xylanase B and other xylanases that have alkalophilic pH optima that highlight charged residues in the eight-residues-longer loop in the alkalophilic xylanase. Multiple engineered XynB mutants were created by site-directed mutagenesis with substitutions Q164K and Q164K+D117N. The variant XynB-117 had the highest optimum pH (at 5.5), which corresponded to a basic 0.5 pH unit shift when compared with the wild-type enzyme. However, the optimal pH of the XynB- 164 mutation was not changed, similar to the wild type. These results suggest that the residues at positions 164 and 117 in the eight-residues-longer loop and the cleft``s edge are important in determining the pH optima of XynB from Aspergillus niger.

참고문헌 (Reference)

1 Collins T, "Xylanases, xylanase families and extremophilic xylanases" 29 : 3-23, 2005

2 Polizeli ML, "Xylanases from fungi: properties and industrial applications" 67 : 577-591, 2005

3 Valls C, "Using both xylanase and laccase enzymes for pulp bleaching" 100 : 2032-2039, 2009

4 Miller GL, "Use of dinitrosalicylic acid reagent for determination of reducing sugar" 31 : 426-428, 1959

5 Li F, "Synonymous condon usage bias and overexpression of a synthetic xynB gene from Aspergillus niger NL-1 in Pichia pastoris" 7 : 2330-2343, 2012

6 Yang JH, "Shifting pH optimum of Bacillus circulans xylanase based on molecular modeling" 133 : 294-300, 2008

7 Bardford, M., "Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding" 72 : 248-, 1976

8 Wohlfahrt G, "Probing pH-dependent functional elements in proteins:modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A" 42 : 10095-10103, 2003

9 Kim SH, "Mutation of nonconserved amino acids surrounding catalytic site to shift pH optimum of Bacillus circulans xylanase" 55 : 130-136, 2008

10 Ahmed S, "Molecular cloning of fungal xylanases: an overview" 84 : 19-35, 2009

11 Kulkarni N, "Molecular and biotechnological aspects of xylanases" 23 : 411-456, 1999

12 Oh DK, "Modification of optimal pH in L-arabinose isomerase from Geobacillus stearothermophilus for D-galactose isomerization" 43 : 108-112, 2006

13 Fenel F, "Increased alkali stability in Trichoderma reesei endo-1,4-β-xylanase by site directed mutagenesis" 121 : 102-107, 2006

14 De LemosEsteves F, "Improving the alkalophilic performances of the Xyl1 xylanase from Streptomyces sp. S38: structural comparison and mutational analysis" 14 : 292-302, 2005

15 Sriprang R, "Improvement of thermostability of fungal xylanase by using site-directed mutagenesis" 126 : 454-462, 2006

16 Umemoto H, "Improvement of alkaliphily of Bacillus alkaline xylanase by introducing amino acid substitutions both on catalytic cleft and protein surface" 73 : 965-967, 2009

17 Kanokratana P, "Identification and expression of cellobiohydrolase (CBH) gene from an endophytic fungus, Fusicoccum sp. (BCC4124) in Pichia pastoris" 58 : 148-153, 2008

18 Baba T, "Identification and characterization of clustered genes for thermostable xylandegrading enzymes, β-xylosidase and xylanase of Bacillus stearothermophilus 21" 60 : 2252-2258, 1994

19 Bim MA, "Extraction in aqueous two-phase system of alkaline xylanase produced by Bacillus pumilus and its application in kraft pulp bleaching" 743 : 349-356, 2001

20 Bergquist P, "Expression of xylanase enzymes from thermophilic microorganisms in fungal host" 6 : 177-184, 2002

21 Mchunu NP, "Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli" 141 : 26-30, 2009

22 Wang Q, "Enhancement of the activity and alkaline pH stability of Thermobifida fusca xylanase A by directed evolution" 30 : 937-944, 2008

23 Chipeta ZA, "Effect of cultivation pH and agitation rate on growth and xylanase production by Aspergillus oryzae in spent sulphite liquor" 35 : 587-594, 2008

24 Chen YL, "Directed evolution to produce an alkalophilic variant from a Neocallimastix patriciarum xylanase" 47 : 1088-1094, 2001

25 Xia T, "Directed evolution of Streptomyces lividans xylanase B toward enhanced thermal and alkaline pH stability" 25 : 93-100, 2009

26 Huang JL, "Cloning, sequencing and expression of the xylanase gene from a Bacillus subtilis strain B10 in Escherichia coli" 97 : 802-808, 2006

27 Li F, "Cloning, expression and characterization of xylanase gene XYNB from Aspergillus niger in Pichia pastoris" ISETPP 637-640, 2010

28 Luttig M, "Cloning of two b-xylanase-encoding genes from Aspergillus niger and their expression in Saccharomyces cerevisiae" 19 : 411-415, 1997

29 Laemmli UK, "Cleavage of structural proteins during the assembly of the head of bacteriophage T4" 227 : 680-685, 1970

30 Asghari SM, "Acid-induced conformational changes in Bacillus amyloliquefaciens alpha-amylase: appearance of a molten globule like state" 35 : 51-57, 2004