Mskfdtsaleafvrhipqnykgpggvvavvkdgevvlqh Mfkttlcallitascstfaapqqindivhrtitplieqqkipgmavaviyqgkpyyf

dd 71 SEGVADRATGRAITTTDRFRVGSVTKSFSAVVLLQLVDEGKLDLDASVNTYLPGLLPDDRITVRQVMSHR dap 40 AWGFADLRTRTPMTLDTRMPICSVSKQFTCAVLLDAVGEPEL-LDDALEAYL-DKFEDERPAVRDLCNNQ bla 58 TWGYADIAKKQPVTQQTLFELGSVSKTFTGVLGGDAIARGEIKLSDPTTKYW-PELTAKQWNGITL----

dd 141 SGLYDYTNDMFAQTVPGFESVRNKVFSYQDLITLSLKHGVTNAPGAAYSYSNTNFVVAGMLIEKLTGHSV

dap 107 SGLRDY----WALSVLCGADPEGVFLPAQAQSLLRRLKTTHFEPGSHYS YCNGNFRILADLIEAHTGRTL

bla 123 LHLATY-----TAGGLPLQVPDEVKSSSDLLRFYQNWQPA-WAPGTQRLYANSSIGLFGALAVKPSGLSF

dd 211 ATEYQNRIFTPLNLTDTFYVHPDTVIPGTHANGYLTPDEAGGALVDSTEQTVSW-AQSAGA-VISSTQDL

dap 174 VDILSERIFAPAGMKRAELI-SDTALFD-ECTGY—EGDTVRGFLPATNR-IQWMGDAGICASLNDMIAW

bla 187 EQAMQTRVFQPLKLNHTWINVPPAEEKN-YAWGY—REGKAVHVSPGALDAEAYGVKSTIEDMARWVQSN

dd 279 DTFFSALMSGQLMSAAQLAQMQQWTTVNSTQGYGLGLRRRDLSCGISVYGHTGTVQGYYTYAFASKDGKR

dap 239 EQFIDATRDDESGLYRRLSGPQTFKD-GVAAPYGFGLNLHETG-GKRLTGHGGALRGWRCQRWHCADERL

bla 254 LKPLDINEKTLQQGIQLAQSRYWQTGDMYQGLGWEMLDWPVNPDSIINGSDNKIALAARPVKAITPPTRA

dd 349 SVTALANTSNNVNVLNTMARTLESAFCGKPTTAKLRSATSSATTVERHEDIAPGIARD

dap 307 STIAMFNFEGGASEVAFKLMNIALGVSSSEVSRVEADSAWFGSWLDDETGLVLSLEDAGHGRMKARFGTS

bla 324 VRASWVHKTGATGGFGSYVAFIPEKELGIVMLANKNYPNPARVDAAWQILNALQ

dap 377 PEMMDVVSANEARSAVTTIRRDGETIELVRASENLRLSMKRVKGEAKHDIIGRYHSDELDADLLLVSEGG dap 447 AIYGAFEGFLGKSDMYPLYSVGSDVWLLPVQRSMDAPSPGEWKLVFRRDDKGEITGLSVGCWLARGVEYR dap 517 RVQP

Figure 3. Comparison of the amino acid sequences of the dap gene from O. anthropi SCRC C1-38, carboxypeptidase DD from Streptomyces R61, and class C j?-lactamase from E. coli: asterisk (*), identical residue; single dot (•) similarity of functional group.The first amino acid sequence (dd) is the sequence of carboxypeptidase DD from Streptomyces R61 (sequence size: 406) (30). The second amino acid sequence (dap) is that of d-aminopeptidase from O. anthropi (sequence size: 521) (28). The third amino acid sequence (bla) is that of class C j?-lactamase from E. coli K12 (sequence size: 377) (32).

in water-saturated toluene with 250 mM of D-alanine methyl ester HCl and 750 mM of triethylamine (Table 2). The kcat of the reaction was calculated to be 19,500 (min-1), which is several tens of thousands times greater than that of the known enzymatic syntheses of amino acid oligomers (26,27).

The gene for the D-aminopeptidase was cloned in Escherichia coli JM109 to overproduce the enzyme (28). An expression plasmid pC138DP was constructed. The amount of the enzyme in a cell-free extract of E. coli JM109/ pC138DP was elevated to 288,000 units per liter of culture, which is about 3,600-fold over that of the wild strain (29). The intact cells of E. coli transformant were used as a catalyst for the D-stereospecific hydrolysis of several racemic amino acid amides HCl. Complete hydrolysis of D-alanine amide was achieved in a short time (4.5 h) from 5.0 M of racemic alanine amide HCl using the cells of E. coli transformant (Fig. 2). The concentration of D-alanine reached up to 220 g/L. The cells or the cell-free extract catalyzed the synthesis of D-a-aminobutyric acid, D-methionine, D-norvaline, and D-norleucine from their amides in a similar manner.

Reference

Brew Your Own Beer

Brew Your Own Beer

Discover How To Become Your Own Brew Master, With Brew Your Own Beer. It takes more than a recipe to make a great beer. Just using the right ingredients doesn't mean your beer will taste like it was meant to. Most of the time it’s the way a beer is made and served that makes it either an exceptional beer or one that gets dumped into the nearest flower pot.

Get My Free Ebook


Post a comment