title: Cloning and biochemical characterization of an endo-1,4-β-mannanase from the coffee berry borer Hypothenemus hampei.
abstract: BACKGROUND
  The study of coffee polysaccharides-degrading enzymes from the coffee berry borer Hypothenemus hampei, has become an important alternative in the identification for enzymatic inhibitors that can be used as an alternative control of this dangerous insect. We report the cloning, expression and biochemical characterization of a mannanase gene that was identified in the midgut of the coffee berry borer and is responsible for the degradation of the most abundant polysaccharide in the coffee bean.
  
  
  METHODS
  The amino acid sequence of HhMan was analyzed by multiple sequence alignment comparisons with BLAST (Basic Local Alignment Search Tool) and CLUSTALW. A Pichia pastoris expression system was used to express the recombinant form of the enzyme. The mannanase activity was quantified by the 3,5-dinitrosalicylic (DNS) and the hydrolitic properties were detected by TLC.
  
  
  RESULTS
  An endo-1,4-β-mannanase from the digestive tract of the insect Hypothenemus hampei was cloned and expressed as a recombinant protein in the Pichia pastoris system. This enzyme is 56% identical to the sequence of an endo-β-mannanase from Bacillus circulans that belongs to the glycosyl hydrolase 5 (GH5) family. The purified recombinant protein (rHhMan) exhibited a single band (35.5 kDa) by SDS-PAGE, and its activity was confirmed by zymography. rHhMan displays optimal activity levels at pH 5.5 and 30°C and can hydrolyze galactomannans of varying mannose:galactose ratios, suggesting that the enzymatic activity is independent of the presence of side chains such as galactose residues. The enzyme cannot hydrolyze manno-oligosaccharides such as mannobiose and mannotriose; however, it can degrade mannotetraose, likely through a transglycosylation reaction. The K(m) and k(cat) values of this enzyme on guar gum were 2.074 mg ml(-1) and 50.87 s(-1), respectively, which is similar to other mannanases.
  
  
  CONCLUSIONS
  This work is the first study of an endo-1,4-β-mannanase from an insect using this expression system. Due to this enzyme's importance in the digestive processes of the coffee berry borer, this study may enable the design of inhibitors against endo-1,4-β-mannanase to decrease the economic losses stemming from this insect.
authors: Disciplina de Mejoramiento Genético, Centro Nacional de Investigaciones de Café (CENICAFE), Planalto, Km 4 vía antigua, Chinchiná-Manizales, Colombia.
authors: Carolina
authors: C
authors: Aguilera-Gálvez
authors: Juan J
authors: JJ
authors: Vásquez-Ospina
authors: Pablo
authors: P
authors: Gutiérrez-Sanchez
authors: Ricardo
authors: R
authors: Acuña-Zornosa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular / Q000379:methods; D003069:Coffee / Q000469:parasitology*; D004591:Electrophoresis, Polyacrylamide Gel; D005638:Fruit; D005685:Galactans / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives; D006790:Host-Parasite Interactions; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008351:Mannans / Q000378:metabolism; D008361:Mannosidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D053149:Plant Gums / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D048889:Weevils / Q000201:enzymology* / Q000235:genetics
chemical_list: D003069:Coffee; D005685:Galactans; D019476:Insect Proteins; D008351:Mannans; D009844:Oligosaccharides; D053149:Plant Gums; D011994:Recombinant Proteins; C012990:galactomannan; C039922:mannotetraose; C007894:guar gum; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase; D005690:Galactose
references: J Biotechnol. 1998 Aug 27;63(3):199-210
references: 9803534
references: J Microbiol Biotechnol. 2009 Oct;19(10):1184-90
references: 19884778
references: Microb Cell Fact. 2010 Apr 11;9:20
references: 20380743
references: Biochem Biophys Res Commun. 1990 Jun 29;169(3):1035-9
references: 2363713
references: Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
references: 18838391
references: Curr Opin Plant Biol. 2008 Jun;11(3):338-48
references: 18430603
references: Planta. 2006 Oct;224(5):1091-102
references: 16649044
references: J Biotechnol. 2002 Jan 18;92(3):267-77
references: 11689251
references: Biochim Biophys Acta. 2004 Nov 1;1674(3):239-50
references: 15541293
references: Planta. 2001 Jun;213(2):296-308
references: 11469596
references: Microb Cell Fact. 2009 Nov 13;8:59
references: 19912637
references: BMC Res Notes. 2012 Jan 10;5:23
references: 22233686
references: Nature. 1970 Aug 15;227(5259):680-5
references: 5432063
references: Comp Biochem Physiol B Biochem Mol Biol. 2010 Sep;157(1):137-43
references: 20639136
references: Comp Biochem Physiol B Biochem Mol Biol. 2008 Sep;151(1):32-40
references: 18579426
references: FEBS Lett. 1993 Jan 25;316(2):137-40
references: 8093602
references: Obesity (Silver Spring). 2010 May;18(5):995-9
references: 19798073
references: Appl Microbiol Biotechnol. 2008 May;79(2):165-78
references: 18385995
references: J Biotechnol. 2006 Sep 1;125(2):269-80
references: 16621092
references: Insect Biochem Mol Biol. 2000 Mar;30(3):207-13
references: 10732988
references: Nucleic Acids Res. 2006 Jan 1;34(Database issue):D247-51
references: 16381856
references: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W563-8
references: 20507913
references: Appl Biochem Biotechnol. 2009 Oct;159(1):85-94
references: 18820840
references: Mol Microbiol. 1988 Jan;2(1):159-64
references: 2835589
references: Bull Entomol Res. 2006 Jun;96(3):223-33
references: 16768810
references: J Mol Biol. 2008 Jun 6;379(3):535-44
references: 18455734
references: Nucleic Acids Res. 1997 Dec 15;25(24):4876-82
references: 9396791
references: J Mol Biol. 1990 Oct 5;215(3):403-10
references: 2231712