ANNUAL REPORT - Department of Biotechnology

The conditions for the preparation of crude bromealin
extract from pineapple wastes (peel, core, stem and
crown) have been standardized. The storage studies
under different conditions (25±2ºC and 4±2ºC) are
carried out to examine the stability of enzyme. The
crude extract preparation of β-galactosidase from
plant sources such as tomato, radish seeds, cow pea
and spinach are attempted. The work on
standardizing the extraction conditions for βgalactosidase
from other plant sources is under
progress. The processing conditions for the forward
and back extraction of bromelain from the crude
extract (from pineapple wastes) are being varied to
optimize the enzyme activity recovery and
purification. A purification factor of 5.2 with nearly
130% activity recovery has been achieved with the
crude extract obtained from pineapple core. The
bromelain activity in the pineapple peel is found to be
very less (~17%) as compared to that of core. The
RME of peel extract resulted in an activity recovery of
50% with 2 fold purification. The processing
conditions have been suitably modified to selectively
separate the bromelain from polyphenol oxidase
(PPO). The selective separation of enzyme has been
achieved based on the iso-electric point of the two
enzymes. The work on integration of RME with
membrane processing has been taken up and
preliminary results are found to be encouraging
Large Cardamom - Product Plan
Evaluation of the performance of tissue culture
raised large cardamom (Amomum subulatum) vis-àvis
open pollinated (OP) seedlings in farmers' field
over a total area of 50 ha has been continued in four
districts Chamoli, Uttarkashi, Champawat and
Pithoragarh of Uttarakhand state jointly by the Herbal
Research and Development Institute, Gopeshwar
and Regional Research Station of the Indian
Cardamom Research Institute (Spices Board),
Gangtok, Sikkim. About 1.00 lakh OP seedlings were
raised in two secondary nurseries located at
Dewalthal Pithoragarh and Kothiyalsain Chamoli. A
total of 47, 898 tissue culture plantlets of large
cardamom were supplied by M/S Sunglow Biotech,
Coimbatore. These were hardened at Kothiyalsain
nursery and field planted during the planting season
2006. A total of about 34.45 area has been planted
during 2006 season involving 222 beneficiaries
using 90,000 OP seedlings (22.50 ha) and 47,818
tissue culture plantlets (11.95 ha). Accordingly, a total
area of 50.45 ha has been covered in the project so
far with OP seedlings (38.50 ha) and tissue culture
plantlets (11.95 ha) involving 276 beneficiaries.
Four training programmes for project personnel and
farmers on scientific cultivation and management
practices of large cardamom were organized during
the year at Pithoragarh, Gopeshwar and Gangtok.
Microbial and Industrial Biotechnology
Microbial Enzymes for Industrial Use
In an ongoing project at Himachal Pradesh
University, Shimla, 11 fold purification of nitrile
hydratase enzyme (NHase, EC 4.2.1.84) with a yield
of 52 % has been achieved from cell free extract of
Rhodococcus rhodochrous PA-34. The molecular
weight of holoenzyme of NHase was found to be 86
kDa by native-PAGE. Studies also revealed that the
enzyme consisted of two subunits of 25.04 kDa and
30.6 kDa. The Km and Vmax values were 167 mM
and 250 µmole/min/mg respectively. A DNA
sequence of 1.7 kb containing NHase gene was
cloned and expressed in E. coli JM109 using specific
primers based PCR techniques from genomic DNA
of R. rhodochrous and the gene sequence had 99%
homology with low molecular weight NHase gene of
R. rhodochrous J1 (Fig.5).
At NIO, Goa, among the 14 marine fungi identified for
laccase activity, a basidiomycetous fungus, NIOCC #
2a, isolated from mangrove wood was found to be the
best producer of laccase enzyme when grown in
seawater of 25-30 ppt salinity. The enzyme with
optimum activity at 60ºC with pH 3 and 6
decolorized several synthetic dyes, effluents from
textile mills, paper and pulp mills and alcohol
distilleries. In an ongoing project at IIT, Delhi, on
lipases from haloalkalophilic and organic solvent
tolerant microbes for industrial applications, a
solvent tolerant strain of Pseudomonas aeruginosa
has been isolated which secrets very good protease
and lipase. These enzymes were purified and it also
exhibited stability in alkaline range and in presence of
surfactant and detergents. The gene responsible for
expression of protease was identified and
characterized as lasB gene and the phylogenetic tree
based on the gene sequence was deduced.
147 Bioprocess and Product Development