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Homework Title / No.

: 3 Course Code : BTY 461

Course Instructor : Mr Himanshu Singh Course Tutor (if applicable) : ____________

Date of Allotment : __ Date of submission : 1 Nov 2010

Student’s Roll No. RA7801B24 Section No. : A7801

I declare that this assignment is my individual work. I have not copied from any other
student’s work or from any other source except where due acknowledgment is made
explicitly in the text, nor has any part been written for me by another person.

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1Ans Response surface methodology (RSM) was employed to optimize culture medium for
production of lipase with Candida sp. 99-125. In the first step, a Plackett–Burmen design was
used to evaluate the effects of different components in the culture medium. Soybean oil,
soybean powder and K2HPO4 have significant influences on the lipase production. The
concentrations of three factors were optimized subsequently using central composite designs
and response surface analysis. The optimized condition allowed the production of lipase to be
increased from 5000 to 6230 IU/ml in shake flask system. The lipase fermentation in 5 l
fermenter reached 9600 IU/ml.

Current studies about lipase production by solid-state fermentation involve the use of agro-
industrial residues towards developing cost-effective systems directed to large-scale
commercialization of enzyme-catalyzed processes. In this work, lipase production and partial
characterization of the crude enzymatic extracts obtained by Penicillium verrucosum using
soybean bran as substrate was investigated. Different inductors were evaluated and the results
showed that there is no influence of this variable on the lipase production, while temperature
and initial moisture were the main factors that affected enzyme production. The optimized
cultivation temperature (27.5 °C) and initial moisture of substrate (55%) were determined
using the response surface methodology. Kinetics of lipase production was followed at the
optimized growth conditions. Optimum lipase yield was 40 U/g of dry bran. The crude
enzymatic extract showed optimal activity in the range from 30 to 45 °C and in pH 7.0

2. Ans. Whenever enzymes are immobilised, may be on a support or a matrix, several
changes are noticed in the enzyme conformities. For instance when enzymes are immobilised
in a charged matrix as a result of change in the microenvironment of the enzyme the apparent
bulk pH optimum of the immobilised enzyme will shift from that of the soluble one.

The charged matrix will either repel or attract the substrates , products, co factors and
protons and ofcourse depends on the type of surface charge and also on the quantum of
charge. If the substrate is of the high molecular weight , the activity of an enzyme towards it
is usually reduced upon immobilisation.

The reason is mainly the steric hindrance by the support e.g. starch, a carbohydrate
witha high molecular weight will not be able to penetrate to the active sites of immobilised
enzymes. Also immobilisation affects the thermal stability of enzymes.

Thermal stability usually increases upon immobilisation and is primarily due to the
presence of thermal diffusion barriers and the constraints on protein unfolding. But in a few
cases decrease in thermal stability is also noticed. As far the pH is concerned it increases
upon immobilisation.

3 Ans Enzymes may be immobilized by using very different protocols (eg., by physical or
chemical fixation on pre-existing supports). The main goal of immobilization is the re-use of
enzymes for very long times. a.- The just fixation of enzymes on porous supports prevents
them from aggregation, proteolysis, interaction with hydrophobic interfaces, etc. b.- The
multipoint covalent attachment of the enzyme (e.g., through its region containing the highest
density in Lys residues) on highly activated supports (e.g., with epoxy or glyoxyl groups)
may promote an intense rigidification of the 3D structure of enzymes. The relative distances
among all residues involved in the multipoint covalent immobilization have to be preserved
unaltered during any conformational change induced by any distorting agent. Thus, these
conformational changes may become strongly reduced and these immobilized enzymes are
much more stable that the one-point immobilized ones. c.- Multi-subunit immobilization of
multimeric enzymes should promote dramatic stabilization under conditions where
dissociation of subunits is the main mechanism of inactivation. After a correct orientation of
the enzyme on the support surface, involving the maximum amount of enzyme subunits, , an
additional rigidification of each subunit (eg., by multipoint covalent attachment) may exert
additional beneficial effects for the enzyme stability. d.- Selectivity of enzymes can be
greatly improved by promoting multipoint covalent immobilization on different enzyme
regions (eg., in the proximity of the active center).

In spite of their excellent catalytic properties, enzyme properties have to be usually improved
before their implementation at industrial scale (where many cycles of high yield processes are
desired). Generally, soluble enzymes have to be immobilized to be reused for long times in
industrial reactors and, in addition to that, some other critical enzyme properties have to be
improved like stability, activity, inhibition by reaction products, selectivity towards non-
natural substrates. Some strategies to improve these enzyme properties during the
performance of tailor-made enzyme immobilization protocols are here reviewed. In this way,
immobilized enzymes may also exhibit much better functional properties than the
corresponding soluble enzymes by very simple immobilization protocols. For example,
multipoint and multisubunit covalent immobilization improve the stability of monomeric or
multimeric enzymes. Moreover, enantioselectivity of different enzymes (e.g., lipases) may be
also dramatically improved (from E = 1 to >100) by performing different immobilization
protocols of the same enzyme. In all cases, enzyme engineering via immobilization
techniques is perfectly compatible with other chemical or biological approaches to improve
enzyme functions and the final success depend on the availability of a wide battery of
immobilization protocols.

4. Ans. Residence time in case of continuous flow reactor:-

D = F/V
F is flow rate of medium and V is volume of reactor.
It is the time spend by any substrate molecule within the reactor during the continuous
operation. It is reciprocal of the dilution rate.
Tou= 1/D = V/F = hr ms-1
Continuous flow rate reactors are based on immobilized enzymes. They offer the following
1. Greater productivity per unit amount of enzyme;
2. Can be used for substrates having low solubility;
3. Uniform product quality, i.e, lack of batch to batch variation. These reactors may be of the
following three types; (a) Continuous flow stirred tank reactor, (b) packed bed reactor, (c)
fluidized bed reactor.
A vertical cylindrical packed-bed plug-flow reactor employed for a substance degradation is
modeled. The question of its best performance is mathematically formulated, and the
existence of the corresponding optimal flow axial velocity is proven. A way to compute this
optimal velocity is exhibited as well. Results are obtained under a rather general hypothesis
on the reaction velocity: it may be applied to first- and second-order reactions,
Michelis−Menten reactions, and others. An application to lactose hydrolysis on a β-
galactosidase-immobilized chitosan bed is presented.

ANS 5: km for immobilised enzyme =1.82 kg/m3

Vmax= 45 kg/m3/h1
The lactose concentration in the feed stream i.e. Si =9.5 kg/m3

Sf= 9.5(1-0.98)
= 9.5*0.02
= 0.19 kg/m3
(a) D(Si – Sf) = nT VmS/(Km+S)
D(9.5 – 0.19) = 1*45*0.19/ (1.82+ 0.19)
D(9.31) = 8.55/ 2.01
D = 8.55/(2.01*9.31)
D = 0.456/s
As acc. to formula
D = F/V
0.456 = F/0.5
F= 0.228 m3/hr
(b) In a year there are 365 days;
Glucose produced= Column operated for a plug flow rated/ Lactose conc * 365 days
= 310/ 9.5 * 365
= 11910.525 kgs.

ANS 6: The electrochemical detection of physiologically important molecules using enzyme-

based biosensors has been an area of intense activity for a number of years, the most
successful application being determination of glucose.
Enzyme Electrodes for Determination of Glucose
The concept involved in amperometric enzyme biosensors is conversion of a chemical signal
(in this case, the enzyme reaction) to an analytical signal (a current) using the working
electrode as the transducer. The enzyme is immobilized on the surface of an electrode, and
this immobilized layer is covered by a membrane. The function of the membrane is to
provide stability, and it can also be used to prevent potential interferants from reacting with
the enzyme. The electrode assembly is placed in the solution containing the analyte, which
can readily diffuse through the membrane, and into the immobilized enzyme layer. The
enzyme most widely studied for biosensors is glucose oxidase (used for the determination of
glucose). The active site of this enzyme is a flavin adenine nucleotide (FAD), which exists in
one of two forms: oxidized (FAD) or reduced (FADH ). FAD oxidizes glucose to gluconic
acid, and the FADH generated by this reaction can be oxidized to FAD by oxygen.
(Hydrogen peroxide is a by-product of this reaction.)


EzNET system for eliminating nitrate pollution .THIS system include 3 enzymes .All are
immobilized on beads with an electron carrying diode. The enzymes are nitrate reductase
,nitrite reductase and nitrous oxide reductase.This device is used to measure the concentration
of nitrate pollution in soil or water.This device converts nitrate into nitrogen.The reduction of
nitrate into non-toxic nitrogen is given by the low voltage current.This may be recorded and
analysed by the transducer.Current produced due to physical changes is directly proportional
to nitrate in the sample.