Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral cyanohydrins

Article abstract of DOI:10.1016/j.bioorg.2018.11.017

Hydroxynitrile lyase (HNL) catalyzed enantioselective C–C bond formation is an efficient approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is known to provide robustness, reusability and in some cases also enhances activity and selectivity. We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL) by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters were: 20 min of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, that synthesized (S)-mandelonitrile in ～99% ee and ～60% conversion. Addition of organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive cycles without loss of conversion or product formation and five cycles with a little loss in enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.

Full text of DOI:10.1016/j.bioorg.2018.11.017

Accepted Manuscript
Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis
of chiral cyanohydrins
Nisha Jangir, Santosh Kumar Padhi
PII:
S0045-2068(18)30542-X
https://doi.org/10.1016/j.bioorg.2018.11.017
YBIOO 2625
DOI:
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
15 August 2018
14 November 2018
15 November 2018
Please cite this article as: N. Jangir, S. Kumar Padhi, Immobilized Baliospermum montanum hydroxynitrile lyase
catalyzed synthesis of chiral cyanohydrins, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2
018.11.017
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Immobilized Baliospermum montanum hydroxynitrile lyase catalyzed synthesis of chiral
cyanohydrins
Nisha Jangir, and Santosh Kumar Padhi*
Biocatalysis and Enzyme Engineering Laboratory, Department of Biochemistry, School of Life
Sciences, University of Hyderabad, Hyderabad – 500 046, India.
*
Email id: skpsl@uohyd.ernet.in
Abstract
Hydroxynitrile lyase (HNL) catalyzed enantioselective C-C bond formation is an efficient
approach to synthesize chiral cyanohydrins which are important building blocks in the synthesis
of a number of fine chemicals, agrochemicals and pharmaceuticals. Immobilization of HNL is
known to provide robustness, reusability and in some cases also enhances activity and
selectivity.
We optimized the preparation of immobilization of Baliospermium montanum HNL (BmHNL)
by cross linking enzyme aggregate (CLEA) method and characterized it by SEM. Optimization
of biocatalytic parameters was performed to obtain highest % conversion and ee of (S)-
mandelonitrile from benzaldehyde using CLEA-BmHNL. The optimized reaction parameters
were: 20 minutes of reaction time, 7 U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate
buffer pH 4.2, that synthesized (S)-mandelonitrile in ~99% ee and ~60% conversion. Addition of
organic solvent in CLEA-BmHNL biocatalysis did not improve in % ee or conversion of product
unlike other CLEA-HNLs. CLEA-BmHNL could be successfully reused for eight consecutive
cycles without loss of conversion or product formation and five cycles with a little loss in
enantioselectivity. Eleven different chiral cyanohydrins were synthesized under optimal
biocatalytic conditions in up to 99% ee and 59% conversion, however the % conversion and ee
varied for different products. CLEA-BmHNL has improved the enantioselectivity of (S)-

mandelonitrile synthesis compared to the use of purified BmHNL. Nine aldehydes not tested
earlier with BmHNL were converted into their corresponding (S)-cyanohydrins for the first time
using CLEA-BmHNL. Among the eleven (S)-cyanohydrins syntheses reported here, eight of
them have not been synthesized by any CLEA-HNL. Overall, this study showed preparation,
characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL and its
biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins.
Keywords: Hydroxynitrile lyase, chiral cyanohydrins, cross-linked enzyme aggregates,
asymmetric synthesis, biocatalysis, immobilization, recyclability.
1
. Introduction
Hydroxynitrile lyase (HNL) are known to catalyze the synthesis of chiral cyanohydrins[1–3].
Optically pure cyanohydrins are useful molecules in the preparation of pharmaceutical
intermediates, agrochemicals and fine chemicals [1,2,4]. The importance of these enantiopure
cyanohydrins and the environment friendly method that the HNL catalysis provides, both
enhances the significance of HNL research. Thus, there is a constant demand to discover new
HNLs, [3,5–9] enzymatic methods to synthesize chiral cyanohydrins, improving enzyme’s
biocatalytic properties etc. Among the several /β hydrolase fold HNLs reported, BmHNL has
shown unique biocatalytic features in terms of its substrate preference [10,11]. It prefers
aromatic aldehydes as substrates because of the presence of hydrophobic residues in its binding
pocket. However, BmHNL has not been explored in biocatalysis unlike its other HNL
counterparts of /β hydrolase fold family.
One of the common problem in HNL biocatalysis is spontaneous formation of racemic
cyanohydrin at pH 5 or above. To avoid the formation of racemic cyanohydrin which contributes
in decreasing the enantiomeric excess (ee) of the biocatalytically produced chiral cyanohydrin,

usually HNL biocatalysis is carried out at (i) low pH, (ii) low temperature and (iii) in presence of
organic solvent. Use of organic solvent helps in (i) lowering the substrate concentration in
aqueous phase[12] which could be a reason for minimizing the racemic cyanohydrin formation,
(
ii) extraction of product, and (iii) may minimize degradation of product. However, use of
organic solvent and low pH may reduce the stability of the enzyme. Stability of BmHNL in
organic solvent has not been investigated while Asano and coworkers have reported its pH
stability for 1 h even at lower pH i.e. 3.5 [11]. Possible solutions to both these issues are (i)
engineering BmHNL to improve its pH stability and organic solvent tolerance, or (ii)
immobilization of BmHNL which may improve these two properties. Another important
character of a biocatalyst is its reusability. Reusability makes the process more economic.
Considering all these important properties, we have studied immobilization of BmHNL using
cross-linking method.
Cross-linked enzyme aggregates (CLEA) is an important method of immobilizing enzymes
which has several advantages e.g. high enzyme loading, less leaching, no requirement of purified
protein and cost effective etc [13]. CLEA involves cross-linking protein molecules with each
other without the interference of carrier and hence the enzymes get better access to the substrate.
CLEA of many HNLs e.g. Prunus amygdalus (PaHNL), Manihot esculenta (MeHNL), Hevea
brasiliensis (HbHNL), Linum usitatissimum (LuHNL), Prunus dulcis (PdHNL), and Davallia
tyermannii (DtHNL) has been reported [12,14–19]. CLEA-HNLs are known to improve several
biocatalytic properties e.g. reusability, organic solvent tolerance, activity, and enantioselectivity
in the chiral cyanohydrin synthesis. We describe here for the first time preparation and
characterization of CLEA of BmHNL and also its biocatalytic application in the synthesis of
several (S)-cyanohydrins.

2
2
. Experimental
.1. Chemicals and materials
BmHNL (LOCUS: AB505969) synthetic gene cloned into pUC57 was procured from Gene
Script, USA and sub-cloned into pCold1 plasmid at BamHI and SalI. Culture media and
ampicillin were purchased from HiMedia laboratory Pvt. Ltd, India. Isopropyl-β-D-1-
thiogalactopyranoside (IPTG) was purchased from BR-BIOCHEM Pvt. Ltd, India.
Glutaraldehyde was obtained from Molychem, India. Aldehydes and mandelonitrile were
purchased from Sigma Aldrich, AVRA, SRL and Alfa-Aesar. HPLC grade solvents were
obtained from RANKEM, Molychem, FINAR, and SRL.
2
.2. Preparation of crude enzyme extract
A single colony of E.coli BL21 (DE3) containing the recombinant plasmid pCold1-BmHNL was
inoculated into 15 mL of LB broth containing 100 μg/mL ampicillin and incubated at 37°C, 200
rpm for 16 h. This primary culture was transferred into a 1 L of fresh culture medium in 1:100
ratio, incubated at 37 ºC until OD600 reached 0.5-0.6. Then cold-shock at 4°C for 2 h was
performed followed by induction with IPTG to a final concentration of 1 mM and incubated at
1
8°C, 200 rpm for 24 h. Cells were harvested, the cell pellet was suspended in 80 mL of 20 mM
potassium phosphate buffer (pH 7.0) and then disrupted by a sonicator (Sonics & materials INC
USA/VCX500) for 5x6 min at 25 KHz. The lysed cells were centrifuged at 12857g, 4°C for 45
(
minutes. The resulting supernatant was used as crude enzyme. The protein content was measured
by a Nanodrop.
2
.3. Preparation of BmHNL aggregates

BmHNL cell lysate (1 volume) was precipitated by adding 9 volumes of different precipitating
agents e.g. ammonium sulfate (AS), t-butyl methyl ether (TBME), acetone, acetonitrile (AcN),
methanol, isopropyl alcohol (IPA), dimethylformamide (DMF) and 1,2-dimethoxyethane
(
DME). Each of the nine mixtures were incubated at 4°C for 30 minutes on a rocker and then
centrifuged at 12857g, 4°C for 30 minutes. The supernatant and pellet were separated. Each
pellet was re-suspended in 20 mM potassium phosphate buffer (pH 7.0). The protein content in
supernatant and pellet was measured by Nanodrop followed by their HNL activity.
2
.4. Optimization of ratio of cross-linking agent
Five best precipitating agents i.e. AS, TBME, AcN, IPA and DME of section 2.3 were selected
and tested in the process of optimization of volume of cross linking agent. To the mixture of
crude BmHNL and precipitating agent (1:9 v/v), the cross-linker i.e. 25% solution of
glutaraldehyde was added in 0 to 6 volumes. This resulted in BmHNL: precipitating agent: cross-
linker in 1:9:0 to 1:9:6 v/v. Each of these mixtures were shook on a rocker at low speed at 4°C
for 6 h and then centrifuged at 12857g, 4°C for 30 minutes. The supernatant was removed and
pellet was washed with 20 mM potassium phosphate buffer (pH 7.0). HNL activity of the pellet
i.e. CLEA-BmHNL was measured.
2
.4.1. Preparation of CLEA-BmHNL under optimized conditions
CLEA-BmHNL was prepared in a preparative scale by adding 1 volume of cell lysate of
BmHNL, 9 volumes of IPA and 4 volumes of 25% (v/v) solution of glutaraldehyde. The mixture
after shaking at 4°C for 6 h over a rocker, centrifuged at 12857g, 4°C for 30 minutes. The pellet
was washed twice with 20 mM potassium phosphate buffer (pH 7.0). The CLEA-BmHNL thus

obtained was re-suspended in 20 mM potassium phosphate buffer (pH 7.0) and used for
experiments.
2
.5. Characterization of CLEA-BmHNL by scanning electron microscope
The CLEA-BmHNL sample was air-dried over slide and coated with Au metal using sputter for
scanning electron microscopy (SEM) using ZEISS, Merlin compact 30 kVA microscope.
Scanning electron microspore images were taken for CLEA-BmHNL at various magnifications.
2
.6. HNL assay
o
HNL activity was monitored using a Multiskan GO UV-Visible spectrophotometer at 25 C. The
assay was carried out in a cuvette with 1 mL of total reaction volume. The reaction mixture
contained 850 µL of 50 mM citrate-phosphate buffer pH 5.0, 50 µL of diluted CLEA-BmHNL
(
200 μg) and 100 µL of 70 mM of racemic mandelonitrile in 5 mM citrate buffer pH 3.15. The
assay measured the formation of benzaldehyde resulted by enzymatic cleavage of mandelonitrile
at 280 nm. A control experiment was carried out in an identical manner except the enzyme was
replaced with 20 mM potassium phosphate buffer pH 7.
2
.7. Synthesis of racemic cyanohydrins
Racemic cyanohydrins were synthesized in order to use them as analytical standards. More than
one method was used to synthesize different racemic cyanohydrins that involved KCN, TMSCN
and acetone cyanohydrin as a cyanide donor. Two racemic cyanohydrins i.e. cyanohydrins of
2
,4-dimethoxybenzaldehyde and trans-cinnamaldehyde were prepared using KCN as a cyanide
donor [20]. Five mmoles of an aldehyde was dissolved in 10 mL of glacial acetic acid in a 100
mL round bottom flask, 3 equivalents of KCN in 10 mL of water was added to the mixture. The
reaction mixture was stirred at 4°C and reaction progress was monitored by TLC. Finally, 25 mL

of water was added to the reaction mixture and the acid was neutralized by the addition of
saturated NaHCO solution. The reaction mixture was extracted by diethyl ether with
consecutive washes of water and saturated NaCl solution. The organic extract was dried over
anhydrous Na SO followed by evaporation of the solvent under reduced pressure. Purification
3
2
4
of products was done by flash chromatography (hexane/ethyl acetate: 85/15). Racemic
cyanohydrins of 2-phenyl acetaldehyde, 3-phenoxybenzaldehyde and 3-benzyloxybenzaldehyde
were prepared by using trimethylsilyl cyanide (TMSCN) as a cyanide source and lithium
chloride (LiCl) as a catalyst in solvent-free medium [21,22]. To a pre-cooled 50 mL round
bottom flask containing 5 mmoles of an aldehyde, 3 equivalents of TMSCN (caution: low
temperature is must to avoid vigorous reaction) was added followed by 2-3 drops of 100 mM
LiCl pre-dissolved in THF. Reaction was continued by stirring at 25°C and monitored by TLC.
After completion, 5 mL of ethyl acetate was added, subsequently the TMS group was cleaved by
adding 1 N HCl with vigorous stirring at 25°C. The reaction mixture was extracted using ethyl
acetate with consecutive washes of saturated NaHCO
3
and brine. The organic extract was dried
over anhydrous Na SO , evaporated the solvent under reduced pressure and products purified by
2
4
flash chromatography (hexane/ethyl acetate: 85/15). Four aromatic aldehydes e.g. 3,5-
dimethoxybenzaldehyde, 2,5-dimethoxybenzaldehyde, 4-benzyloxybenzaldehyde, and 4-
hydroxybenzaldehyde were converted into corresponding racemic cyanohydrins using acetone
cyanohydrin as a cyanide donor by a modified protocol [23]. The modification includes the
addition of NaHCO as a base catalyst, aldehydes pre-dissolved in dichloromethane (DCM) and
3
using water instead of the aliphatic amine as a solvent. Ten mmol of an aldehyde and 100 mmol
of acetone cyanohydrin (10 equivalents) were added to a 100 mL round bottom flask containing
1
0 mL of 5% w/v aqueous NaHCO solution. A 20 mL of DCM was also added to minimize the
3

dissociation of the cyanohydrin product formed. The reaction mixture was stirred at 25°C and
monitored by TLC. The reaction was stopped by acidifying the mixture with 1N HCl, extracted
using DCM with consecutive washes of water. The organic extract was dried over anhydrous
Na
2
SO , solvents evaporated under reduced pressure and the mixture was purified by flash
4
chromatography (eluent hexane/ethyl acetate: 85/15; acidified by dil. HCl). All racemic
1
13
cyanohydrins synthesized were characterized by H and C NMR spectroscopy (BRUKER 400
MHz NMR). The racemic cyanohydrins were used as analytical HPLC standards.
2
.8. Effect of reaction time in the enantioselective synthesis of (S)-mandelonitrile
Effect of time of biotransformation in the enantioselective synthesis of mandelonitrile was
studied. The reaction mixture contained 7 U of CLEA-BmHNL (48 mg CLEA-BmHNL), 100 µL
of 1 M KCN in double distilled water (ddH O), 40 µL of 20 mM of benzaldehyde (0.8 mM final
2
concentration) in dimethyl sulfoxide (DMSO) and 768 µL of 300 mM citrate buffer pH 4.2. The
reaction was carried out in a thermomixer by incubating at 22 ºC, 1000 rpm for 45 minutes.
The % of ee of a product was calculated using formula
ꢀ
ꢁꢂꢃꢄꢅꢆꢇꢈꢂꢀꢁꢂꢃꢄꢅꢉꢊꢈꢋꢆꢊꢌꢍꢂ ꢀꢎꢂꢃꢄꢅꢆꢇꢈꢂꢀꢎꢂꢃꢄꢅꢉꢊꢈꢋꢆꢊꢌꢍ
%
%
of ee of a product ={ ꢁꢂꢃꢄꢅꢆꢇꢈꢂꢀꢁꢂꢃꢄꢅꢉꢊꢈꢋꢆꢊꢌꢍꢏ ꢀꢎꢂꢃꢄꢅꢆꢇꢈꢂꢀꢎꢂꢃꢄꢅꢉꢊꢈꢋꢆꢊꢌꢍ}*100
ꢀ
ꢀ
ꢁꢂꢃꢄꢅꢏꢀꢎꢂꢃꢄꢅ
ꢀꢁꢂꢃꢄꢅꢏꢀꢎꢂꢃꢄꢅ
conversion ={[ꢀ
]
rxn  [
]control }*100.
ꢀꢐꢑꢅꢏꢀꢁꢂꢃꢄꢅꢏꢀꢎꢂꢃꢄꢅ
ꢐꢑꢅꢏꢀꢁꢂꢃꢄꢅꢏꢀꢎꢂꢃꢄꢅ
(
S-MN)rxn and (S-MN)control represent the area of (S)-mandelonitrile peak in the
biotransformation and control respectively. (R-MN)rxn and (R-MN)control represent similarly
for the (R)-mandelonitrile peak. BA: peak area of benzaldehyde.
2
.9. Optimization of substrate concentration

Optimum substrate concentration for the enantioselective synthesis of mandelonitrile was
determined by varying the concentration of benzaldehyde in the biotransformation. The reaction
mixture of 1 mL total contained 7 U of CLEA-BmHNL, 40 µL of benzaldehyde of 10 to 55 mM
stock solution in DMSO, equivalent to a final concentration 0.4 mM to 2.2 mM, 100 µL of 1 M
KCN in ddH O and 768 µL of 300 mM citrate buffer pH 4.2. Biocatalysis was carried out in a
2
thermomixer by incubating the reaction mixture at 22 ºC, 1000 rpm. After 20 min, 1 mL of
hexane: IPA (90:10) was added to it. The organic extract was analyzed by chiral HPLC in a
Chiralpak IE column using hexane: IPA (table S1) to find the % conversion and ee of
mandelonitrile synthesized.
2
.10. Optimization of amount of CLEA-BmHNL
Optimum amount of CLEA-BmHNL in the enantioselective synthesis of (S)-mandelonitrile was
found out by varying number of enzyme units against two substrate concentrations i.e. 0.4 mM
and 1.2 mM. Two different sets of reactions were carried out for different substrate
concentrations. The reaction mixture contained CLEA-BmHNL (5-15 U), 40 µL of 10 or 30 mM
stock solution of benzaldehyde in DMSO, 100 µL of 1 M KCN in ddH O and 768 µL of 300
2
mM citrate buffer pH 4.2. Biocatalysis conditions and HPLC analysis of products was as
described earlier.
2
.11. Effect of organic solvents in the biotransformation
Effect of different organic solvents in the enantioselective synthesis of mandelonitrile was
studied. To a 368 µL of 300 mM citrate buffer pH 4.2, 7 U of CLEA-BmHNL, 40 µL of 30 mM
benzaldehyde in DMSO, 100 µL of 1 M KCN in ddH
2
O, and 400 µL of an organic solvent (40%
v/v) were added. Six different organic solvents e.g. hexane, toluene, n-butyl acetate, di-isopropyl

ether (DIPE), TBME and AcN were used in this optimization study. Biocatalysis conditions and
HPLC analysis of products were same as described earlier.
2
.12. Effect of ratio of organic solvents
The effect of ratio of organic solvent to buffer in the biotransformation mixture was studied in
the CLEA-BmHNL catalyzed enantioselective synthesis of (S)-mandelonitrile. Among the
organic solvents used in 2.11, the one showed best result i.e. toluene was chosen for this study.
The biocatalysis conditions were kept identical to the previous experiment, except the % volume
of toluene in the biotransformation was varied from 30 to 78 of the total volume.
2
.13. Effect of buffer pH
Optimum pH for the CLEA-BmHNL catalyzed enantioselective synthesis of (S)-mandelonitrile
was investigated by varying the pH of the reaction buffer. The reaction mixture composed of 7 U
of CLEA-BmHNL (7 U), 1.2 mM benzaldehyde in DMSO, 100 µL of 1 M KCN in ddH
68 µL of 300 mM citrate buffer of different pH i.e. 3, 3.5, 4.2, 5, 5.5, and 6. Biocatalysis
conditions and HPLC analysis of products was as described earlier.
.14. Reusability of CLEA-BmHNL
2
O and
7
2
Reusability of CLEA-BmHNL was determined by repeated use of the biocatalyst in optimized
condition to synthesize (S)-mandelonitrile. The optimal biocatalytic condition includes: 7 U of
CLEA-BmHNL, 1.2 mM benzaldehyde, 100 µL of 1 M KCN in ddH O (100 mM) and 768 µL
2
of 300 mM citrate buffer pH 4.2 incubated in a thermomixer at 22 ºC, 1000 rpm for 20 minutes.
After 20 minutes, the reaction mixture was centrifuged at 14,500 g, 4 ºC for 1 min. The
supernatant was extracted using 1 mL of hexane: isopropanol (90:10). The pellet thus remained
was used for the subsequent cycle of biocatalysis that was performed keeping all other

parameters identical to the optimal condition. Ten cycles of biocatalysis was performed. In each
cycle the reaction composition was kept same while the CLEA-BmHNL of previous cycle was
used for each successive round without wash.
2
.15. Synthesis of (S)-cyanohydrins using CLEA-BmHNL
The CLEA-BmHNL was used in the enantioselective synthesis of different cyanohydrins under
optimized conditions i.e. 7 U of CLEA-BmHNL, 1.2 mM of an aldehyde, 100 µL of 1 M KCN in
ddH O and 768 µL of 300 mM citrate buffer pH 4.2. Separate biocatalysis was performed using
2
different aldehydes as substrates to prepare their corresponding chiral cyanohydrins. As the
reaction time of different substrates would differ from benzaldehyde, the corresponding
biocatalysis were carried out for 20-120 minutes. Percentage ee and conversion of each
biocatalysis at different time was determined by chiral HPLC. Detail of HPLC analysis with
varied hexane: IPA and flow rate for different cyanohydrins is given in table S1.
3
3
3
. Results and discussion
.1. CLEA-BmHNL preparation
.1.1. Preparation of BmHNL aggregates
The first step in the preparation of CLEA of an enzyme is to prepare enzyme aggregates.
Selection of precipitant for enzyme aggregation is crucial because achieving active enzyme after
precipitation is more important than getting just enzyme aggregates. The enzyme aggregates
resulted from different precipitants do not show same amount of catalytic activity. We studied
eight different precipitants and tested the corresponding aggregates for their HNL activity.
Among the different precipitants i.e. AS, TBME, acetone, AcN, methanol, IPA, DMF and DME
used, AS based enzyme aggregate has showed highest BmHNL activity i.e. 1.51 U/mg (Fig 1).

Ammonium sulfate is considered as a common precipitant and often used during protein
purification. BmHNL aggregates resulted from other precipitating agents such as AcN, TBME,
IPA, and DME showed specific activity 1.43, 1.41, 1.2 and 1.19 U/mg respectively (Fig 1).
1
1
1
1
1
0
0
0
0
0
.80
.60
.40
.20
.00
.80
.60
.40
.20
.00
Different precipitants
Figure 1: Effect of different precipitating agents
3
.1.2. Preparation of cross-linked enzyme aggregate of BmHNL
The second step is to cross-link the aggregated enzyme molecules. BmHNL cross-linked enzyme
aggregates have not been reported earlier. We optimized the preparation of BmHNL-CLEA using
the common cross-linker glutaraldehyde. The carbonyl groups of glutaraldehyde makes covalent
linkage with amino group of a lysine residue present in the surface of an enzyme linking it to its
neighbor, via Schiff base formation. Optimization of ratio of glutaraldehyde to protein is
important in CLEA preparation to determine the best ratio that provides HNL activity to the
CLEA. Use of high volume of glutaraldehyde can result in enzyme inactivation. Five best
precipitants of section 3.1 were selected i.e. AS, TBME, AcN, IPA and DME for this study. In
case of each of the five precipitants, to a total volume of 1 mL consisting of BmHNL cell lysate

and precipitant in 1:9 v/v, different volume of glutaraldehyde i.e. 0 to 600 µL was added (Fig 2).
Among them, IPA has showed highest specific activity. Although IPA as a precipitant has
showed less activity than AS in Fig 1, however after cross-linking, the corresponding CLEA has
showed highest activity. The probable reason for this could be due to formation of some inactive
CLEA or loss of some enzymatic activity in case of CLEA resulted from AS.
1
1
1
1
1
0
0
0
0
0
.8
.6
.4
.2
.0
.8
.6
.4
.2
.0
AS IPA DME AcN TBME
Precipitants:
0
100
200
300
400
500
600
Volume of glutaraldehyde (µL) with different precipitants
Figure 2: Effect of volume of glutaraldehyde along with different precipitants
Under optimal cross-linking conditions i.e. 1:9:4 v/v of BmHNL cell lysate: IPA: glutaraldehyde,
cross-linking time: 6 h and at 4°C CLEA-BmHNL was prepared. CLEA-BmHNL preparation
was repeated several time. CLEA-BmHNL with protein content 78.56 mg/mL and specific
activity of 1.38 U/mg was used in successive experiments.
3
3
.2. Characterization of CLEA-BmHNL
.2.1. Scanning electron microscope analysis of CLEA-BmHNL:

A
B
Figure 3 (A-B): SEM image of CLEA-BmHNL prepared under optimized conditions
The FESEM images of CLEAs at different crosslinking times are shown in Figure 3A-B, which
shows the aggregation of spherical CLEA particles. The porous structure with cavities was
formed wherein few spherical particles were embedded. The amorphous structure with high
surface area gradually increased as a consequence of extended crosslinking time.
3
.2.2. Yield, efficiency and activity recovery of CLEA-BmHNL
The success of preparation of immobilized enzyme can be described in terms of yield, efficiency
and activity recovery as elaborated by Sheldon and Pelt [13]. Yield, efficiency and activity
recovery were calculated using the following three equations.
%
%
%
yield = (immobilized activity/starting activity)*100
efficiency = (observed activity/immobilized activity)*100
activity recovery = (observed activity/starting activity) *100
Note: Immobilized activity = crude BmHNL activity – activity of the supernatant resulted during
CLEA preparation, starting activity = crude BmHNL activity, observed activity = CLEA activity

Enzyme or
supernatant
Total volume
(mL)
BmHNL protein
concentration (mg/mL)
Specific activity Total activity
(U/mg)
(U)
342.72
142.78
ve
Crude BmHNL
CLEA-BmHNL
12
6
21
1.36
23.33
0.0719
1.02
Supernatant after
CLEA prepared
98
ve
Yield = 100%, efficiency = 41.66% and activity recovery = 41.66%.
.3. Optimization of biocatalytic parameters for CLEA-BmHNL catalyzed enantioselective
synthesis of (S)-mandelonitrile
.3.1. Reaction time
3
3
Chiral HPLC analysis of CLEA-BmHNL catalyzed enantioselective synthesis of mandelonitrile
at different time intervals showed 46.68% conversion and 90% ee of product in 20 min, which
found to be the highest among different time points (Fig 4). In 25 min, 41.57% conversion and
8
7.4% ee of (S)-mandelonitrile was observed from benzaldehyde. Beyond 25 min, % ee and
conversion both were decreased. Decrease in % ee could be due to the cleavage of (S)-
mandelonitrile to benzaldehyde i.e. the reverse reaction, with longer reaction time. Such cleavage
could also be a possible reason for decrease in conversion observed in Fig 4. MeHNL-CLEA in a
biphasic system is reported to show such decrease in % ee with increase in time [15].
Stereoselective synthesis of cyanohydrins by CLEA of other HNLs has been reported earlier.
Reaction time of CLEA of different HNLs differ in the synthesis of chiral cyanohydrins.
DtHNL-CLEA has showed 99% conversion and 98% ee of (R)-mandelonitrile in 24 h [19].
PdHNL-CLEA synthesized (R)-mandelonitrile in 99% yield and 99% ee in 72 h [24]. MeHNL-
CLEA synthesized (S)-mandelonitrile in 97% ee and 96% conversion in 2 h while HbHNL-
CLEA in 72 h synthesized the same in 67% ee and 55% conversion [15].

100
60
%ee
%conv
90
80
70
60
50
40
30
20
10
0
5
4
0
0
30
2
1
0
0
0
5
10
15
20
25
30
45
Time (min)
Figure 4: Time of biotransformation
.3.2. Substrate concentration
3
Effect of different benzaldehyde concentrations in the CLEA-BmHNL catalyzed synthesis of (S)-
mandelonitrile was investigated (Fig 5) by performing the biotransformation for 20 minutes as
per 3.3.1. Benzaldehyde concentration was varied from 0.4 to 2.2 mM in different biocatalysis.
In case of 0.4 mM benzaldehyde, CLEA-BmHNL showed highest i.e. ~93% ee and 42.8%
conversion. With 1 and 1.2 mM concentrations, also high % ee was observed i.e. 91.2 and 90.2%
respectively while % conversion was 42.7 and 45.5 respectively. Beyond 1.2 mM substrate
concentration, % ee and conversion both decreased. However the reason for low % ee of product
with 0.6 mM benzaldehyde is not clear.
Yildirim et al reported almost 0.056 M benzaldehyde (100 µL of 1 M benzaldehyde) for the
synthesis of (R)-mandelonitrile using CLEA-PdHNL [18]. Alagöz et al also reported the same
concentration of benzaldehyde in CLEA-PdHNL biocatalysis [24]. In CLEA-PaHNL based
biotransformations, 0.2 M aldehyde was used by Langen et al [25]. Torrelo et al reported the

recyclability of MeHNL-CLEA by using 0.5 M benzaldehyde [26]. Cabirol et al reported the
synthesis of cyanohydrins catalyzed by CLEA-HbHNL and CLEA-MeHNL by using 0.5 M
substrate concentration [15]. However for these different CLEA-HNL biocatalysis, enzyme
concentration also differs. Therefore, a comparison of optimal substrate concentration between
different CLEA-HNLs would be difficult.
%
ee
% conv
100
60
90
80
70
60
50
40
30
20
10
0
5
4
0
0
30
2
1
0
0
0
0
.4
0.6
0.8
1
1.2
1.4
1.8
2.2
Benzaldehyde concentration (mM)
Figure 5: Effect of benzaldehyde concentration in the synthesis of (S)-mandelonitrile
3
.3.3. Amount of enzyme
Two best substrate concentrations from the previous experiment i.e. 0.4 and 1.2 mM
benzaldehyde were selected for the current study. Different amount of enzyme i.e. 5 to 15 U
were used in the enantioselective synthesis of mandelonitrile (Fig 6). With 1.2 mM
benzaldehyde, 92.7% ee and 60.5% conversion was observed in case of 7 U of CLEA-BmHNL.
The % ee decreased with increasing enzyme units such as with 15 U the % ee was 66 while the
%
conversion increased up to 71%. In case of 0.4 mM substrate concentration, the % ee and
conversion was also found to be highest with 7 U of enzyme i.e. 88.2 and 35.4 respectively. Both

the substrate concentrations showed similar trend of decrease in % ee with increase in amount of
enzyme. A possible explanation for this could be the dehydrocyanation of product. At higher
enzyme concentration, cleavage of (S)-mandelonitrile could be active apart from its synthesis.
Since cleavage of cyanohydrin is a favorable reaction than its synthesis, this could be a reason
for the decreased ee.
1
00
100
80
60
40
20
0
80
60
40
20
0
5
7
9
11
13
15
Enzyme units
%
%
ee with 0.4 mM benzaldehyde
conv with 0.4 mM benzaldehyde
% ee with 1.2 mM benzaldehyde
%conv with 1.2 mM benzaldehyde
Figure 6: Effect of different CLEA-BmHNL units in the synthesis of (S)-mandelonitrile
3
.3.4. Different organic solvents
Application of biphasic system in HNL biocatalysis is well known. Use of organic solvent helps
in minimization of spontaneous formation of racemic cyanohydrin and also in product
extraction[27] [12,14,24]. In order to find out the effect of an organic solvent in the CLEA-
BmHNL biocatalysis, we have selected six different organic solvents which had been reported
with other HNLs. They are hexane, toluene, n-butyl acetate, DIPE, TBME and AcN. The
biocatalysis was performed using 1.2 mM benzaldehyde and 7 U of the enzyme (Fig 7). A 40%
v/v of each of the mentioned organic solvents were used separately in biocatalysis. Another

biocatalysis without adding any organic solvent was also performed. None of the organic
solvents improved % ee and % conversion of product compared to without organic solvent.
Only in case of toluene, 96.9% ee of (S)-mandelonitrile was observed while in aqueous medium
it was 96.8%. However, the % conversion was highest in the aqueous system i.e. ~38%
compared to 9.3% in toluene. In case of DIPE, 32% conversion was seen but the % ee was 74
only. With n-butyl acetate, the % ee and % conversion were 82 and 12 respectively. Acetonitrile
showed negative results in the CLEA-BmHNL catalyzed the synthesis of (S)-mandelonitrile, as
both the % ee and % conversion were less than 1. Compared to CLEA of other HNLs, this result
was found to be different as it did not show any improve in % ee and conversion in synthesis of
(
S)-mandelonitrile.
100
40
%
ee
% conv
3
3
2
2
1
1
5
0
5
0
5
0
5
0
8
6
4
2
0
0
0
0
0
-
-
20
40
-5
Organic solvents
Figure 7: Effect of different organic solvents in the synthesis of (S)-mandelonitrile
3
.3.5. Ratio of organic solvent to buffer
The effect of ratio of organic solvent in the biocatalysis was investigated. We have selected
toluene as the best organic solvent among all (Fig 7) and different percentage by volume i.e. 30

to 78 of it was used in biocatalysis. With increasing percentage of toluene, decrease in % ee was
observed (Fig 8). Although highest % ee i.e. 72.5 was observed with 40% toluene but the %
conversion was only 14.6%. In contrary, highest % conversion i.e. 26.8, was observed with 60%
of toluene, while the % ee was 0.9. With further increase in % of toluene resulted in decrease of
both % ee and conversion.
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
30
%
ee
% conv 25
20
15
10
5
30
40
50
60
70
78
-
10
0
%
of toluene
Figure 8: Effect of different ratio of toluene in the synthesis of (S)-mandelonitrile
.3.6. Buffer pH
3
pH is a key factor in the synthesis of enantioselective cyanohydrins. At higher pH spontaneous
formation of racemic cyanohydrin occurs by chemical reaction that limits the enantiomeric yield
of cyanohydrins [28]. To elucidate the effect of pH in the CLEA-BmHNL catalyzed synthesis of
(
S)-mandelonitrile, pH of the aqueous system was varied from 3 to 6 (Fig 9). The biocatalysis
was performed in aqueous system only, because no improvement in % conversion or ee was
observed by using any of the organic solvents in a biphasic system. In pH 4.2, CLEA-BmHNL
showed highest % ee of (S)-mandelonitrile i.e. 98.76 while the % conversion was almost 50 (Fig

1
0). Further increase in pH resulted in decreased % ee and conversion because at high pH
racemization takes place. At pH less than 4.0, decreased % ee was also observed. It could be due
to enzyme instability at low pH. Optimum pH of BmHNL was reported to be 5.0 [11]. While a
lower pH is preferred for HNL catalysis, at pH 4.2 the HNL activity of BmHNL was almost half
of its maximum activity [11]. CLEA-BmHNL in contrary has showed optimum pH at 4.2.
Another important property observed by CLEA-BmHNL is in the improvement of % ee of (S)-
mandelonitrile i.e. 98.76% compared to 54% by purified BmHNL [11]. Similar observation of
change in pH optima by CLEA has been reported [29,30]. CLEA of Pichia pastoris alcohol
oxidase [31] and Roystonea regia peroxidase [29] has showed higher activity at low pH
compared to their corresponding free enzymes.
1
00
70
60
50
40
30
20
10
0
90
80
70
60
50
40
30
20
10
0
%
ee
% conv
-10
-20
-30
3.0
3.5
4.2
5.0
5.5
6.0
pH
Figure 9: Effect of pH in the synthesis of (S)-mandelonitrile using CLEA-BmHNL

Figure 10: HPLC chromatogram of CLEA-BmHNL catalyzed synthesis of (S)-mandelonitrile in
3
00 mM citrate-phosphate buffer pH 4.2. (Black: control; Pink: Reaction).
3
.4. Reusability of CLEA-BmHNL
One of the important reason of doing enzyme immobilization is to reuse the biocatalyst. To
investigate the reusability of CLEA-BmHNL, it was used for 10 successive cycles in the
synthesis of (S)-mandelonitrile (Fig 11). The biocatalysis was performed according to protocol
mentioned in section 2.15. After each cycle, the reaction mixture and enzyme were separated by
centrifugation followed by addition of freshly prepared reaction mixture into CLEA-BmHNL to
carry out the successive cycle. In the first three cycles, the % ee and conversion was almost
th
th
similar. The ee was above 95% while the % conversion was from 59 to 66. From 4 to 8 cycle,
there was almost no decrease in product formation. The % conversion was maintained between
th
th
5
8
8 and 72. The % ee of (S)-mandelonitrile has decreased a little in the 4 and 5 cycle; they are
th
th
8 and 81 respectively. However in case of 5 to 8 cycle, the % ee was from 66 to 68. In the
th
th
last two cycles i.e. 9 and 10 , both % ee and conversion were decreased. They are ~55% ee and
44-45% conversion. The possible reason for the gradual decrease in % ee in successive cycles
could be due to the loss of catalyst during each cycle in the operational process or enzyme
~

inhibition by aldehyde. Nevertheless, this process has showed reusability of CLEA-BmHNL for
eight successive cycles without loss in conversion or product formation and five cycles with a
little loss in enantioselectivity.
Torrelo et al reported that CLEA-MeHNL can be reused for seven cycles without wash. The %
ee remained constant >98% but the % conversion was decreased from 98 to 95% in first three
th
cycles. At the end of the 7 cycle, the % conversion decreased up to 55% [26]. While this is
th
~
45% loss in % conversion to product at 7 cycle, in the present study CLEA-BmHNL showed
th
nearly no loss in % conversion until 8 cycle. CLEA-PaHNL was used for 10 successive cycles
without any loss in % conversion with substrate 2-methyl benzaldehyde. The enzyme was
washed with water after each cycle [25]. The recyclability of CLEA-PdHNL was tested for eight
cycles towards the synthesis of (R)-mandelonitrile in a biphasic system at 5°C [24]. At the end of
th
the 8 cycle, although the % of ee of product was 99 but activity has decreased to 29% of its
original activity. CLEA-LuHNL in the synthesis of (R)-2-butanone cyanohydrin at 30°C showed
recyclability for four times [17]. Compared to the first cycle (81% ee and 84% conversion) at the
th
end of 4 cycle it showed 78% ee and 56% conversion.
100
80
%
ee
% conv
90
80
70
60
50
40
30
20
10
0
7
6
5
0
0
0
40
3
2
1
0
0
0
0
I
II
III
IV
V
VI VII VIII IX
X
Cycle number

Figure 11: Recyclability of CLEA-BmHNL towards the synthesis of (S)-mandelonitrile
.5. Synthesis of (S)-cyanohydrins using CLEA-BmHNL
3
CLEA-BmHNL was used to synthesize different (S)-cyanohydrins using substrates other than
benzaldehyde (Scheme 1). Eleven different aromatic aldehydes were used to synthesize the
corresponding (S)-cyanohydrins using CLEA-BmHNL under optimized reaction conditions.
Selection of aromatic aldehydes is because of the preference of BmHNL toward aromatic
substrates. The amount of CLEA-BmHNL used for all the biotransformations was only 7 U. The
reaction time was different with respect to different substrates. The % ee and % conversion of
the products obtained are summarized in Table 1.
O
OH
CN
CLEA-BmHNL
+
KCN
R
H
R
(
S)
Scheme1: CLEA BmHNL catalyzed synthesis of (S)-cyanohydrins.
Table 1: CLEA-BmHNL catalyzed synthesis of different chiral cyanohydrins
Substrate no
Aldehydes (R)
Reaction time
% ee
% conv
(
min)
20
1
2
3
4
5
6
7
C
6
H
5
99.8
91.4
96.3
76.3
97.6
75.6
87.9
59.8
12.5
1.3
3,5-di MeOC
2,4-di MeOC
2,5-di MeOC
6
6
6
H
H
H
3
3
3
100
60
40
3.2
4-CH
2
=CH-CH
-CH
4-PhCH OC
2
C
6
H
4
40
32.3
15
C
6
H
5
2
60
2
6
H
4
60
3.5

8
9
3-PhOC
trans-PhCH=CH
3-PhCH OC
4-OHC
6
H
4
60
100
60
97.64
98.6
92.8
18.3
47.9
3.5
10
2
6
H
4
48.7
14.3
11
6
H
4
20
Using CLEA-BmHNL, (S)-mandelonitrile was synthesized in 99.8% ee and 59.8% conversion.
While purified BmHNL has been reported to synthesize (S)-mandelonitrile in 54% ee [11],
CLEA of BmHNL has improved the enantioselectivity of this transformation. Kawahara and
Asano have engineered BmHNL to improve its enantioselectivity [32]. They reported BmHNL-
H103C-N156G catalyzed synthesis of (S)-mandelonitrile in 93% ee. 3,5-Dimethoxy
benzaldehyde was converted to its corresponding (S)-cyanohydrin in 91.4% ee and 12.5%
conversion by CLEA-BmHNL in 100 minutes while with purified BmHNL it’s synthesis is
reported with 85% ee [11]. The other nine substrates (No 3 to 11, Table 1) used in this study
were not tested with BmHNL earlier. Here we have converted these aldehydes into their
corresponding (S)-cyanohydrins for the first time using CLEA-BmHNL. Among the nine, five
aldehydes i.e. 2,4-dimethoxybenzaldehyde, 4-allyloxybenzaldehyde, 3-phenoxybenzaldehyde,
trans-cinnamaldehyde, and 3-benzyloxybenzaldehyde were converted to their corresponding (S)-
cyanohydrins with very high % of ee i.e. 93 to 99 (No 3, 5, 8, 9 and 10, Table 1). Three among
them have showed reasonable conversion i.e. 32 to 49% to their products. For example, 3-
phenoxy benzaldehyde, 4-allyloxybenzaldehyde and 3-benzyloxybenzaldehyde (No 8, 5 and 10,
Table 1) have produced their corresponding cyanohydrins in 48, 32.3, and 49% respectively. In
case of 2,4-dimethoxybenzaldehyde and trans-cinnamaldehyde (No 3 and 9, Table 1) the %
conversion was very less i.e. 1.3 and 3.5% respectively. CLEA-BmHNL converted three other

aromatic aldehydes i.e. 2,5-dimethoxybenzaldehyde, 4-benzyloxybenzaldehyde and 2-phenyl
acetaldehyde (No 4, 6 and 7, Table 1) to their respective (S)-cyanohydrins in 76.3, 88 and 75.6%
of ee respectively. Although the % ee was moderate to high, the % conversion for these three
substrates was very low i.e. 3 to 15% only. CLEA-BmHNL catalyzed the synthesis of (S)-2-
hydroxy-2-(4-hydroxyphenyl) acetonitrile from its corresponding aldehyde however with only
1
8.3% ee and 14.3% conversion. Among the eleven (S)-cyanohydrins syntheses reported here,
eight of them (No, 2, 3, 4, 5, 6, 7, 10, and 11) have not been reported to be synthesized by any
CLEA-HNL. The low % conversion observed in case of the synthesis of several mandelonitrile
derivatives may be caused by product inhibition, however this is a speculation only and we do
not have any evidence to explain the reasons behind it.
Synthesis of (S)-cyanohydrins has been reported by CLEA-HbHNL [15] and CLEA-MeHNL
[
14,33]. The (S)-selective CLEA-HbHNL is reported to synthesize (S)-mandelonitrile in 55%
conversion and 67% ee in 72 h [15]. Cabirol et al synthesized (S)-m-phenoxybenzaldehyde
cyanohydrin using CLEA-MeHNL in 81% conversion and 83% ee in 72 h. They also described
the preparation of (S)-enantiomers of hexanal cyanohydrin in 92% conversion and 81% ee in 3 h,
2
-furaldehyde cyanohydrin in 94% conversion and 94% ee in 30 min; and mandelonitrile in 96%
conversion and 97% ee in 2 h [15]. Chmura et al showed CLEA-MeHNL catalyzed synthesis of
three (S)-cyanohydrins in 55 to 99% ee and 86 to 96% conversion [14]. They also reported the
preparation of cyanohydrins of two ketones i.e. acetophenone and 1-phenylpropanone with 99%
ee and 7% conversion and 96% ee and 90% conversion respectively. Other HNLs as CLEA has
also been reported to synthesize chiral cyanohydrins but in (R)-form [16,17,24].
4
. Conclusion

Preparation of CLEA-BmHNL was optimized with different precipitants, and amount of cross
linking agent i.e. glutaraldehyde. CLEA-BmHNL was prepared using optimal conditions and
characterized by SEM. Under optimized biocatalytic parameters i.e. 20 minutes of reaction time,
7
U of CLEA-BmHNL, 1.2 mM substrate, and 300 mM citrate buffer pH 4.2, benzaldehyde was
converted to (S)-mandelonitrile in high (~99%) ee and ~60% conversion. While CLEA of other
HNLs show tolerance to organic solvent during their biocatalysis in a biphasic system, in case of
CLEA-BmHNL, addition of organic solvent did not improve in % ee or conversion of product.
We have successfully demonstrated the re-usability of CLEA-BmHNL for eight consecutive
cycles without loss in conversion or product formation and five cycles with a little loss in
enantioselectivity. CLEA of BmHNL showed improved enantioselectivity in synthesis of (S)-
mandelonitrile compared to the use of purified BmHNL that showed only 54% ee. This
observation also holds good with the second substrate i.e. 3,5-dimethoxy benzaldehyde, found
common between our study and that reported with purified BmHNL. However varied %
conversion and ee was observed in the synthesis of different (S)-cyanohydrins, similar to the
catalytic behavior of purified BmHNL. We synthesized here eleven different chiral cyanohydrins
using CLEA-BmHNL, among them eight have not been reported to be synthesized by any
CLEA-HNL and nine substrates were not tested earlier with BmHNL. We showed here the
preparation, characterization of a stable, robust and recyclable biocatalyst i.e. CLEA-BmHNL
and its biocatalytic application in the synthesis of different (S)-aromatic cyanohydrins. However
preparative scale synthesis and practical application of CLEA-BmHNL to synthesize enantiopure
cyanohydrins still remains a challenge because of the low substrate concentration used here.
Acknowledgements

We are thankful to Dheeraj Sanjogi for helping in the synthesis of few substrates, University of
Hyderabad for fellowship to Nisha Jangir and infrastructure facility support provided by DST-
FIST. We thank T. Revathi for her initial experiments on CLEA-BmHNL. The authors gratefully
acknowledge the financial support by SERB grant number EMR/2016/003801, and UGC-SAP-
DRS-1 to the Biochemistry Department. We are indebted to Professor Akhila Kumar Sahoo,
School of Chemistry, UoH for helping with NMR, Professor K. Muralidharan School of
Chemistry, UoH and Mr Muvva Durga Prasad for their kind help in SEM analysis.
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