Agriculture Reference
In-Depth Information
of cellulase activity. The 1176-kb ORF encoding a putative endo-glucanase of 392
amino acids was associated with high cellulase activity and was designated as CelA.
The recombinant CelA was over-expressed (85 mg/l) in
E. coli
. Activity staining
was used to confirm the enzymatic activity of purified CelA with a molecular weight
of 43 kDa. The cellulase was found to be active over a broad temperature range
(45-75 °C), retained over 80 % activity for 4 h at 75 °C and was optimally active at
65 °C and pH 5.0. The enzyme retained 80 % activity after 16 h incubation over a
broad pH range from 5.0 to 9.0. The endo-glucanase activity was found to be inhib-
ited by Cu
2+
and Zn
2+
ions; however Mn
2+
, Co
2+
, Ca
2+
stimulated the enzyme activ-
ity. The authors also reported a ten-fold greater specific activity for CelA endoglu-
canases when compared to the commercially available
T. reesei
endo-glucanases.
The CelA endo-glucanase showed resemblance to the GH5 family of glycosyl hy-
drolases; it has been considered a novel enzyme, as the highest sequence identity of
CelA was 45.1 % to that of the CelN from
Pectobacterium atrosepticum
. The unique
properties of this cellulase, including a broad range of thermostability and pH stabil-
ity, make it an ideal candidate for application in biofuel and food industry. Yang
et al. (
2010
) reported the isolation and identification of a novel cellulase producing
Bacillus subtilis
strain 115 from long-term thermal compost containing rich cellu-
lose materials in a factory of Zhengzhou in China. The cellulases exhibited optimum
activity at pH 6.0 and 60 °C. The enzyme was thermostable as it retained more than
90 % activity at 65 °C after 2 h incubation. The cellulase gene was cloned and ex-
pressed in
E. coli
BL21 (DE3) and purified using a Sephadex G-100 column. Mo-
lecular weight of the purified protein was found to be 52 kDa using SDS-PAGE
electrophoresis. Cloning and expression resulted in three-fold higher cellulase pro-
duction with no change in properties compared to the wild-type protein. Zambare
et al. (
2011
) reported the isolation of a thermophilic microbial consortium (TMC)
producing cellulolytic and xylanolytic enzymes from yard waste following enrich-
ment with CMC and birchwood xylan. High titres of cellulases and xylanases were
observed following production of the TMC cellulases on lignocellulosic substrates
(corn stover and prairie grass). Characterization studies with extracellular TMC cel-
lulases showed the presence of pH optima peaks at 4.0, 7.0, and 10.0. The tempera-
ture optimum for cellulase was found to be at 60 °C, although the enzyme was active
over a broad range of temperature (40-80 °C). The TMC cellulases retained 98 %
activity after 1 h incubation at 50 °C, and 77 % after incubation at 60 °C for 3 h.
SDS-PAGE of the crude TMC cellulases resulted in several bands with different
molecular weights, however zymogram analysis revealed the presence of three
bands corresponding to 60, 35 and 27 kDa proteins that exhibited cellulase activity.
The substrate specificities of the TMC cellulases were the highest for Avicel fol-
lowed by microcrystalline cellulose, filter paper, pinewood saw dust and CMC. The
authors also reported the higher hydrolytic activities of the TMC cellulases com-
pared to the cellulase produced by isolated strains from the microbial consortium.
They also suggested the similarity of cellulases in TMC to that of
Bacillus
sp. re-
ported elsewhere (based on the enzyme characteristics). The versatility and high
activities of TMC cellulases on different lignocellulosic substrates suggests a robust
and compatible system for the cost-effective breakdown of lignocellulosic biomass.
