(S)-hydroxynitrilelyase from Hevea brasiliensis

Details (S)-hydroxynitrilelyase from Hevea brasiliensis (S)-hydroxynitrilelyase from Hevea brasiliensis

1997-12-22

2000-04-04

Achutamurthy, Ponnathapu

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing nitrogen-containing organic compound

435232, 4352523, 4353201, 435280, 435419, 536 232, C12P 1300, C12N 988, C12N 120, C07H 2104

Patent

active

060460423

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The carrying out of chemical reactions with the assistance of biological catalysts is becoming increasingly important, especially in those areas of application in which it is possible to exploit the property, which is frequently marked among enzymes, of preferentially converting one of the two enantiomers in reactions with chiral or prochiral components.
One of the enzymes used is the (S)-hydroxy-nitrile-lyase (Hnl) from Hevea brasiliensis which catalyzes not only the formation of aromatic but also the formation of aliphatic (S)-cyanohydrins from the corresponding aldehydes or ketones with HCN or HCN donors (EP-A-0 632 130). This is important inasmuch as it is not possible to prepare aliphatic (S)-cyanohydrins with other (S)-hydroxy-nitrile-lyases such as, for example, that from Sorghum bicolor (Tetrahedron Letters, 31: 1249-1252, 1990).
2. Description of the Related Art
The previously known Hnl is prepared from the leaves of Hevea brasiliensis by the method of Selmar (Physiologia plantarum 75: 97-101, 1989) and has a molecular weight of 46 kDa (J. E. Poulton in: Cyanide Compounds in Biology [Ciba Foundation Symposium 140], pp 67-91, 1988). However, the enzyme isolated in this way is insufficiently pure for obtaining specific anti-Hnl antibodies or determining amino-acid sequences of the Hnl protein. All attempts to isolate pure HNL [sic] enzyme using other conventional chromatographic purification steps have failed. In all attempts to obtain the enzyme in pure form by ion exchange chromatography with sodium chloride gradient elution, no Hnl activity was detectable in the column eluate. This was successful only after ammonium sulfate was used, in place of the sodium chloride gradient which is otherwise customary, for the elution. The invention accordingly relates to a (S)-hydroxy-nitrile-lyase in purified and isolated form. The Hnl isolated and purified in this way has a molecular weight of 30.+-.1 kDa, a specific activity of 19 IU/mg of protein and comprises the following amino-acid part-sequences:


Part-sequence 1: ...-leu-met-glu-val-phe-pro-... (SEQ ID NO:1) Part-sequence 2: ...-gly-ser-leu-phe-gln-asn-... (SEQ ID NO:2) Part-sequence 3: ...-glu-ile-ala-glu-ile-leu-gln-glu-val-ala [sic] (SEQ ID NO:3)
This made it possible subsequently, after reverse transcription of mRNA from Hevea brasiliensis, to clone a cDNA copy of the hnl gene, which has the following nucleotide sequence, the amino-acid sequence derived therefrom for the Hnl protein being indicated underneath, and the part-sequences determined from the Hnl protein being indicated by underlining.


(-43)G AAG AGC ACA TAT CGA TAG TAA AGA GTA AGA TAT CAT CAG AAA (SEQ ID NO:4) - 1/1 31/11 ATG GCA TTC GCT CAT TTT GTT CTT ATT CAT ACC ATA TGC CAC GGT GCA TGG ATT TGG CAC Met ala phe ala his phe val leu ile his thr ile cys his gly ala trp ile trp his (SEQ ID NO:12) - 61/21 91/31 AAG CTC AAA CCC CTC CTT GAG GCA CTT GGC CAC AAG GTT ACT GCA CTG GAC CTT GCA GCA lys leu lys pro leu leu glu ala leu gly his lys val thr ala leu asp leu ala ala - 121/41 151/51 AGC GGC GTT GAC CCA AGG CAA ATT GAG GAG ATT GGC TCA TTT GAT GAG TAT TCT GAA CCC ser gly val asp pro arg gln ile glu glu ile gly ser phe asp glu tyr ser glu pro - 181/61 211/71 TTG TTG ACG TTC TTG GAG GCA CTC CCT CCA GGG GGA AAG GTG ATT CTG GTT GGC GAG AGC leu leu thr phe leu glu ala leu pro pro gly glu lys val ile leu val gly glu ser - 241/81 271/91 TGT GGA GGA CTC AAT ATA GCA ATT GCT GCT GAT AAA TAC TGT GAA AAG ATT GCA GCT GCT cys gly gly leu asn ile ala ile ala ala asp lys tyr cys glu gly ile ala ala ala - 301/101 331/111 GTT TTC CAC AAT TCA GTA TTG CCA GAC ACC GAG CAC TGC CCA TCT TAC GTC GTG GAT AAG val phe his asn ser val leu pro asp thr glu his cys pro ser tyr val val asp lys - 361/121 391/131 CTC ATG GAG GTG TTT CCC GAC TGG AAA GAC ACC ACG TAT TTT ACG TAC ACT AAA GAT GGC leu met glu val phe pro asp trp lys asp thr thr tyr phe thr t

REFERENCES:
patent: 5346816 (1994-09-01), Griengl et al.
Selmar et al. Physiologia Plantarum, 75: 97-101, 1989.

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