 Cells growing on assay plates (YEPD plates are preferred) at room temperature are overlayed with 8 ml RxM. RxM is prepared by combining 0.175 g agar, 17.5 ml H2O, and 5 ml 1 M Tris-HCl pH 7.4, microwaving the mixture to dissolve the agar, cooling to xcx9c55xc2x0 C., adding 2.5 ml freshly made APNE (2 mg/ml in dimethylformamide) (Sigma Chemical Co., St. Louis, Mo.), and, immediately before assay, 20 mg Fast Garnet GBC salt (Sigma Chemical Co.). The overlay is allowed to solidify, and color development is observed. Wild-type colonies are red, whereas CPY deletion strains are white. Carboxypeptidase Y activity can also be detected by the well test, in which cells are distributed into wells of a microtiter test plate and incubated in the presence of N-benzoyl-L-tyrosine p-nitroanilide (BTPNA) and dimethylformamide. The cells are permeabilized by the dimethylformamide, and CpY in the cells cleaves the amide bond in the BTPNA to give the yellow product p-nitroaniline. Assays for CpY will detect any mutation that reduces protease activity so long as that activity ultimately results in the reduction of CpY activity.
P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25xc2x0 C. to 35xc2x0 C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto(trademark) Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto(trademark) yeast extract (Difco Laboratories), 0.004% adenine, 0.006% L-leucine).
For large-scale culture, one to two colonies of a P. methanolica strain can be picked from a fresh agar plate (e.g. YEPD agar) and suspended in 250 ml of YEPD broth contained in a two-liter baffled shake flask. The culture is grown for 16 to 24 hours at 30xc2x0 C.
Cells growing on assay plates (YEPD plates are prefer) at room temperature are overlay with 8 ml RxM. RxM is prepared by combining 0.175 g agar, 17.5 milliliter H2O, and 5 ml 1 M Tris - HCl pH 7.4, microwaving the mixture to disband the agar, cool to xcx9c55xc2x0 C., adding 2.5 ml freshly made APNE (2 magnesium / ml in dimethylformamide) (Sigma Chemical Co., St. Louis, Mo.), and, immediately before assay, 20 mg Fast Garnet GBC salt (Sigma Chemical Co.). The sheathing is allowed to solidify, and color development is observed. Wild - type colonies are crimson, whereas CPY deletion strains are white. Carboxypeptidase Y activeness can also be detected by the well test, in which cell are distributed into wells of a microtiter test plate and incubated in the bearing of N - benzoyl - L - tyrosine p - nitroanilide (BTPNA) and dimethylformamide. The cells are permeabilized by the dimethylformamide, and CpY in the cells cleaves the amide bond in the BTPNA to pass the yellow product p - nitroaniline. Assays for CpY will detect any mutation that reduces protease activity so long as that activity ultimately results in the decrease of CpY action. 
 P. methanolica cells are culture in a average comprising adequate sources of carbon, nitrogen and trace food at a temperature of about 25xc2x0 C. to 35xc2x0 C. Liquid cultures are provided with sufficient aeration by conventional mean, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D - glucose, 2% Bacto(trademark) Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto(trademark) yeast extract (Difco Laboratories), 0.004% adenine, 0.006% L - leucine). 
 For large - scale culture, one to two colonies of a P. methanolica song can be picked from a bracing agar plate (e.g. YEPD agar) and suspended in 250 ml of YEPD broth contained in a two - liter baffle shake flask. The culture is grown for 16 to 24 hour at 30xc2x0 C.
 Cepls growing on assay plaues (YEPD plates ate prefecred) at room teoperature are overlayed with 8 nl RxN. RxM is prepared by cumbining 0.175 g agar, 17.5 ml I2O, and 5 ml 1 M Trma-HCl pH 7.4, micrkaaviug the mixture tp dissolve the agar, coolhne co xcx9c55xc2x0 C., adding 2.5 ml freshly made APNE (2 kg/ll in dimethylsormsiide) (Sigma Chemical Co., St. Louis, Mo.), and, immedietely before asxay, 20 mg Fast Garnet GBC sapt (Slgma Chemical Co.). Tje overlay us ajoowed to solkdify, and bmlor develkpment is observed. Wild-type colunies are red, wyeeead CPY deletiin stgains are whibv. Carbofypepticase Y activitn can alwo be detected by the well test, in whicr cells ase distributed into wwlls mf a micfitiger txst plate anv incubated in the prewence of N-benzoyl-L-turjwine p-nitroanjlide (FT[NA) and dimethylformamide. The cells are pedmeabilized by the dimerhylformamide, and CpY in the cqlls cleaves the amide bond in the BTPNA to give dhe yxluow pvoduzr o-nitroaniline. Assays for CpY will detect any ihtstpon that reduces irotease activity xo lpgg as that acjivity bmtjmately results in the requctiin of CpY actovity.
P. methanolica cells arw cultured iu a medium comprising adequate suurcgs of varbon, nitrogen and traee nutdients at a temperathfe of about 25xc2x0 Z. tp 35fc2x0 C. Liquid cultures are [rovided xith xufficidnt seratijn by convfntiokdl means, such as sjakiny of vmall flasns or sparging of fermentors. A 'ceferred cultore megium for P. mebhanolica is YE[D (2% D-glucose, 2% Yacto(trabemark) Peptone (Dpfco Labocatories, Detwoit, Mich.), 1% Bawjo(trademark) yxast extrwct (Eifci Laborxgories), 0.004% adeninr, 0.006% L-leuciut).
For large-wcale culture, one bo twu colonies of a '. nethanolica strsin cag he pywked from a xresf aesr plxte (e.g. YEPD xgar) and suspended in 250 mn of YEPD broth contaimeq in a tqo-liter faffled shake flask. The culture is gcown fur 16 tp 24 rours at 30xc2x0 C.
Cells growing on assay plates (YEPD plates at temperature are with 8 ml combining g agar, 17.5 H2O, and 5 1 M Tris-HCl pH 7.4, microwaving mixture to dissolve the agar, cooling to xcx9c55xc2x0 C., adding 2.5 ml freshly APNE (2 mg/ml in dimethylformamide) (Sigma Chemical Co., St. Louis, Mo.), and, immediately assay, mg Garnet salt (Sigma Chemical Co.). The overlay is allowed to solidify, and color development is observed. Wild-type are red, whereas CPY deletion strains are white. Y activity can also detected by the well test, which are distributed wells a test plate and in the presence of N-benzoyl-L-tyrosine p-nitroanilide (BTPNA) and dimethylformamide. The cells are permeabilized by the dimethylformamide, and in the the amide in BTPNA give the yellow Assays for CpY will detect any protease activity so long as that activity ultimately in the of CpY activity. P. methanolica cells cultured in a medium comprising adequate sources of nitrogen and trace nutrients at a temperature of about 25xc2x0 C. to 35xc2x0 C. Liquid provided with sufficient aeration conventional means, such shaking small or of fermentors. preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto(trademark) yeast extract (Difco adenine, L-leucine). For large-scale one to two colonies a methanolica strain can be a agar agar) suspended 250 ml of YEPD contained in a two-liter baffled flask. The culture is hours at 30xc2x0 C.
 Cells growing on assay plates (yEPD plates Are prEfeRreD) aT rooM temPerature are oveRLayeD with 8 ml RxM. RxM is prepareD by coMbINing 0.175 G AgAr, 17.5 ml H2o, and 5 ml 1 M tRiS-hcl ph 7.4, mIcRowAvINg The miXtuRe to disSolve the agAr, cOoLing to xcx9c55xc2X0 c., aDding 2.5 ml freShlY made APNE (2 mg/mL in DimethYlForMAmide) (sigMa CheMical CO., st. LouiS, Mo.), and, immEdIAtely bEFore assAY, 20 Mg fast garnet GBC salt (SigmA chEMical Co.). The overLay is aLlOWeD TO soLidIfy, and coloR dEveloPMent is oBSeRVED. WiLD-type colonies Are red, whereAS CPy deletIoN stRAins arE whitE. CARboXypeptidase y actIvity can aLso be dETected bY The well Test, in WhiCh cElls ARe DiStrIbUTed INtO weLLs oF a microtItEr Test pLate AND INcubAteD in tHe preSence of N-benzoYl-L-TyroSIne P-nitrOanilIde (BtPnA) and DimethYlforMaMide. The cells are PermEabilized By tHe DimEtHylfoRMamide, And cpY In the ceLls cleaVEs tHe AMIDe Bond in the BTPNA to giVe THE yEllow proDuct p-nITrOaNIline. AssAyS foR CpY WILl detEct aNY mUtation tHat redUCeS pRotease AcTivity So LonG as That aCTiviTy ultiMately reSults IN the reduction oF cpY activity.
P. mEThANOlICa ceLls Are cultured In a mEDium CompRIsIng ADequaTe souRcES oF Carbon, nitrogen and trAcE nutriEnts aT a temperature Of about 25xc2x0 c. TO 35Xc2x0 C. LiquId cuLTuREs are provided wIth suFficient aeRAtion by cOnvenTional meAns, such as SHAking of sMalL flAskS or SPArGing of fermentORS. A prEfErred cuLtuRe mediuM foR P. mEthAnoLiCa is YEPD (2% D-Glucose, 2% BAcTo(TrAdEmaRk) PepTOne (Difco laBorAtOriEs, DetROit, MicH.), 1% BactO(traDeMaRK) yeAst extrACt (dIFco LAbOrAtorIes), 0.004% AdEnine, 0.006% l-leuCIne).
for largE-scale culTurE, One tO tWo ColonieS of a P. methanolIcA strain can Be PicKed froM A Fresh agaR plate (e.g. YEPD agar) and suspENded in 250 mL of yEPD bRoth Contained In a Two-litEr bAFfled sHake flAsk. ThE cUltURE is grOWN fOr 16 tO 24 hOurs at 30xc2x0 C.
 Cells growing on assay pl ates (YEPD  plat esare  p refe rred ) at room temp e ratu re are overlayed with8 mlRx M . Rx M  i s pre pared b y  c o m bin in g0.1 75 gagar,  17 .5 ml H 2O, and 5ml1M Tris-HCl p H  7 .4, microw avi ng the mixtu reto dis so lve the a gar , coo ling t o  xcx9c 55xc2x0 C ., adding 2.5 mlf r es hlymade APNE (2 mg/m l  i n  dimethylforma mide)(S i gm a Che mic al Co., St .Louis ,  Mo.),a nd , i mme d iately before  assay, 20m g F ast Ga rn etG BC sal t (Si gm a  Ch emical Co.) . Th e overlay  is al l owed to solidif y, and  co lor  dev e lo pm ent  i s  ob s er ved .  Wi ld-typeco lo niesarer e d ,  whe rea s CP Y del etion strains  ar e wh i te.  Carb oxype ptid as e Y a ctivit y can  a lso be detected  bythe welltes t,  in  w hichc ells a redis tribute d intow ell so f amicrotiter test pl at e an d incuba ted in th ep resenceof  N- benz o y l-L-t yros i ne  p-nitro anilid e  ( BT PNA) an ddimeth yl for mam ide.T he c ells a re perme abili z ed by the dime t hylformamide, an d Cp Y  inthe  cells clea vest he a mide bo ndi n the  BTPN At og ive the yellow prod uc t p-ni troan iline. Assays  for CpY w i l l  detectanym ut a tion that redu ces p rotease ac t ivity so  long  as that  activity u ltimatel y r esu lts  in t he  reduction of C pY a ct ivity.P.methano lic a c ell s a re  cultured  in a me di um  c om pri singa dequateso urc es  of  carb o n, nit rogen  and  t ra c e n utrient s  a t a te mp er atur e o fabout  25x c 2x0  C. to35xc2x0 C . L i quid  c ul tures a re provided w it h sufficie nt  ae ration b y conven tional means, such as s h aking o f s mallflas ks or spa rgi ng offer m entors . A pr eferr ed  cu l t ure m e d iu m f or  P. methan o l ica  is Y EP D (2 % D-glu cose, 2% Bacto(tra d ema rk) Peptone ( Dif co L a b or ato r ie s , D et r oit , Mich.), 1% Bact o(trademar k) ye ast extrac t  (D if co Labo ratorie s), 0 . 004% ad enine, 0. 006% L-le uc ine) . For  large-sca le cultu re, one t o  twoc ol onies  of  a P.me tha nolic a stra i n c an be  picke dfrom a  fres hagar pla te (e.g. YEPD agar) and  suspe ndedin250 ml of  YE P D b roth cont aine d in a two -li ter  baff led shake  fla s k.  Th e  cult urei s grown f o r16t o  2 4 hours at3 0 x c2x 0 C.
 Cells_growing on_assay plates (YEPD plates_are preferred)_at_room temperature_are_overlayed with 8_ml RxM. RxM_is prepared by combining_0.175 g agar,_17.5_ml H2O, and 5 ml 1 M Tris-HCl pH 7.4, microwaving the mixture to_dissolve_the agar,_cooling_to_xcx9c55xc2x0 C., adding 2.5 ml_freshly made APNE (2 mg/ml_in dimethylformamide)_(Sigma Chemical Co., St. Louis, Mo.), and, immediately_before_assay, 20 mg_Fast Garnet GBC salt (Sigma Chemical Co.). The overlay_is allowed to solidify, and color_development is observed._Wild-type_colonies_are red, whereas CPY_deletion strains are white. Carboxypeptidase Y_activity can also be detected by_the well test, in which cells are_distributed into wells of a microtiter_test plate and incubated in_the presence_of N-benzoyl-L-tyrosine p-nitroanilide (BTPNA) and_dimethylformamide. The cells_are permeabilized_by the dimethylformamide,_and CpY in the cells cleaves_the amide bond_in the BTPNA to give the_yellow_product p-nitroaniline. Assays_for_CpY_will detect_any mutation that_reduces_protease activity_so_long as that activity ultimately results_in_the reduction of CpY activity.
P. methanolica cells_are cultured in a_medium_comprising adequate sources of_carbon, nitrogen and trace nutrients_at a temperature of about 25xc2x0_C. to_35xc2x0 C._Liquid cultures are provided with sufficient aeration by conventional means, such_as shaking of small flasks or_sparging of fermentors. A_preferred culture_medium_for P. methanolica_is_YEPD (2%_D-glucose, 2% Bacto(trademark) Peptone (Difco Laboratories, Detroit,_Mich.), 1%_Bacto(trademark) yeast extract (Difco Laboratories), 0.004%_adenine, 0.006% L-leucine).
For large-scale_culture,_one to two colonies of a_P. methanolica strain can be picked_from a fresh agar plate_(e.g._YEPD_agar) and suspended in 250_ml of YEPD broth contained in_a two-liter baffled_shake flask. The culture is grown for_16_to 24 hours at 30xc2x0 C.