PMID- 23965285
OWN - NLM
STAT- MEDLINE
DCOM- 20131022
LR  - 20211203
IS  - 1756-0500 (Electronic)
IS  - 1756-0500 (Linking)
VI  - 6
DP  - 2013 Aug 22
TI  - Cloning and biochemical characterization of an endo-1,4-beta-mannanase from the 
      coffee berry borer Hypothenemus hampei.
PG  - 333
LID - 10.1186/1756-0500-6-333 [doi]
AB  - 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-beta-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-beta-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 degrees 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. CONCLUSION: This work is the first study of an endo-1,4-beta-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-beta-mannanase to decrease the economic losses 
      stemming from this insect.
FAU - Aguilera-Galvez, Carolina
AU  - Aguilera-Galvez C
AD  - Disciplina de Mejoramiento Genetico, Centro Nacional de Investigaciones de Cafe 
      (CENICAFE), Planalto, Km 4 via antigua, Chinchina-Manizales, Colombia.
FAU - Vasquez-Ospina, Juan J
AU  - Vasquez-Ospina JJ
FAU - Gutierrez-Sanchez, Pablo
AU  - Gutierrez-Sanchez P
FAU - Acuna-Zornosa, Ricardo
AU  - Acuna-Zornosa R
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130822
PL  - England
TA  - BMC Res Notes
JT  - BMC research notes
JID - 101462768
RN  - 0 (Coffee)
RN  - 0 (Galactans)
RN  - 0 (Insect Proteins)
RN  - 0 (Mannans)
RN  - 0 (Oligosaccharides)
RN  - 0 (Plant Gums)
RN  - 0 (Recombinant Proteins)
RN  - 11078-30-1 (galactomannan)
RN  - 3634-02-4 (mannotetraose)
RN  - E89I1637KE (guar gum)
RN  - EC 3.2.1.- (Mannosidases)
RN  - EC 3.2.1.78 (endo-1,4-beta-D-mannanase)
RN  - X2RN3Q8DNE (Galactose)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Chromatography, Thin Layer
MH  - *Cloning, Molecular/methods
MH  - Coffee/*parasitology
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Fruit
MH  - Galactans/metabolism
MH  - Galactose/analogs & derivatives
MH  - Host-Parasite Interactions
MH  - Hydrogen-Ion Concentration
MH  - Hydrolysis
MH  - Insect Proteins/genetics/isolation & purification/*metabolism
MH  - Kinetics
MH  - Mannans/metabolism
MH  - Mannosidases/genetics/isolation & purification/*metabolism
MH  - Molecular Weight
MH  - Oligosaccharides/metabolism
MH  - Pichia/genetics
MH  - Plant Gums/metabolism
MH  - Recombinant Proteins/metabolism
MH  - Sequence Analysis, DNA
MH  - Substrate Specificity
MH  - Weevils/*enzymology/genetics
PMC - PMC3765340
EDAT- 2013/08/24 06:00
MHDA- 2013/10/23 06:00
PMCR- 2013/08/22
CRDT- 2013/08/23 06:00
PHST- 2013/05/23 00:00 [received]
PHST- 2013/08/19 00:00 [accepted]
PHST- 2013/08/23 06:00 [entrez]
PHST- 2013/08/24 06:00 [pubmed]
PHST- 2013/10/23 06:00 [medline]
PHST- 2013/08/22 00:00 [pmc-release]
AID - 1756-0500-6-333 [pii]
AID - 10.1186/1756-0500-6-333 [doi]
PST - epublish
SO  - BMC Res Notes. 2013 Aug 22;6:333. doi: 10.1186/1756-0500-6-333.