Papain preparation

Stereoselective acylahon of racemic hydroxyalkylsilanes 102 was achieved using a crude papain preparation and, surprisingly, 4-phenylpentanoic acid as the acyl donor. The ees of the products 103 and the recovered substrates varied from 30 to 99% (Equation 49). ... 

Figure C2.2.3 Relationship between amount of enzyme in reaction mixture (mg papain preparation from Sigma) and measured activity. Results were obtained using Basic Protocol 1 with Hammerstein casein substrate (ICN Biomedicals).

Enantioselective enzymatic transesterification has also been used for a kinetic racemate resolution75. Starting from the racemic (hydroxymethyl)silane rac-92 (analytical scale), transesterification with vinyl acetate in water-saturated 2,2,4-trimethylpentane, catalyzed by a commercial crude papain preparation (E.C. 3.4.22.2), yielded the corresponding optically active (acetoxymethyl)silane 93 (sign of optical rotation and absolute configuration not reported) (Scheme 21). The enantiomeric purity of the remaining dextrorotatory (hydroxymethyl)silane (+)-92 was moderate (49% ee). 

Some of the basic information on stabilizing sulfhydryl enzymes, has been responsible for their commercialization. Without the judicious use of reducing compounds throughout the processing of the papaya latex, it would not have been possible to maximize the proteolytic activity of commercial papain preparations. Examples of other studies of enzymes which have contributed to commercialization are the determination of calcium ion as a requirement for amylase stability at high temperature, the difference in properties of catalases derived from bacterial, fungal, or... 

Papain. Papain was first isolated in crystalline form from fresh latex by Balls et al. 22), but it is more conveniently isolated from commercially available dried latex by the procedure of Kimmel and Smith 23). Papain prepared by this procedure, however, is not fully active see Activation and Inhibition below) and usually contains only 0.5 mole SH per mole of protein. Fully active papain containing I mole SH per mole of protein may be prepared by affinity chromatography on a column of an inhibitor, Gly-Gly-Tyr (Bzl)-Arg, covalently linked to agarose 24). Two other techniques which have been used to purify papain involve... 

PanoL [Enzyme Development] Enzyme, papain preparation. 

Bradley, R., O Toole, D.T., Wells, D.E., Anderson, P.H., Hartley, P., Berrett, S., Morris, J.E., Insch, C.G. and Hayward, E.A. (1987) Clinical biochemistry and pathology of mature beef cattle following ante-mortem intravenous injection of a commercial papain preparation. Meat Sci. 19(1), 39-51. 

The activity of papain preparations may be estimated with either synthetic or protein substrates. 

DNP Amino Acids in Complete Htdroltzates of DPN-Papain Preparations... 

Pish protein concentrate and soy protein concentrate have been used to prepare a low phenylalanine, high tyrosine peptide for use with phenylketonuria patients (150). The process includes pepsin hydrolysis at pH 1.5 ptonase hydrolysis at pH 6.5 to Hberate aromatic amino acids gel filtration on Sephadex G-15 to remove aromatic amino acids incubation with papain and ethyl esters of L-tyrosine and L-tryptophan, ie, plastein synthesis and ultrafiltration (qv). The plastein has a bland taste and odor and does not contain free amino acids. Yields of 69.3 and 60.9% from PPG and soy protein concentrate, respectively, have been attained. 

Pish silage prepared by autolysis of rainbow trout viscera waste was investigated as a substrate for the plastein reaction using pepsin (pH 5.0), papain (pH 6—7), and chymotrypsin (pH 8.0) at 37°C for 24 h (152). Precipitation with ethanol was the preferred recovery method. Concentration of the protein hydrolysate by open-pan evaporation at 60°C gave equivalent yields and color of the final plastein to those of the freeze-dried hydrolysate. 

Recovery. The principal purpose of recovery is to remove nonproteinaceous material from the enzyme preparation. Enzyme yields vary, sometimes exceeding 75%. Most industrial enzymes are secreted by a microorganism, and the first recovery step is often the removal of whole cells and other particulate matter (19) by centrifugation (20) or filtration (21). In the case of ceU-bound enzymes, the harvested cells can be used as is or dismpted by physical (eg, bead mills, high pressure homogenizer) and/or chemical (eg, solvent, detergent, lysozyme [9001 -63-2] or other lytic enzyme) techniques (22). Enzymes can be extracted from dismpted microbial cells, and ground animal (trypsin) or plant (papain) material by dilute salt solutions or aqueous two-phase systems (23). 

Specimens for assay were prepared by suspending 500 mg of lyophilized brain in 50 ml of the buffer-papain mixture and hydrolyzing overnight at 37° C. The solution was then autoclaved for 10 minutes. This procedure yielded higher results than autolysis or prolonged enzymatic hydrolysis. After centrifuging off the debris, 0.5, 1.5, and 2.5 ml of the supernatant were used for assay. This represents 1, 3, and 5 mg of the original brain tissue per milliliter of final medium. 

Cysteine endopeptidases, like serine endopeptidases, can also catalyze peptide synthesis under preparative conditions [66-68]. Thus, papain has been used to synthesize enkephalins and angiotensin. 

The core protein was prepared by limited papain proteolysis from the intact enzyme preparation shown in Fig. 2... 

For the SAXS studies a CBH II sample was prepared by affinity chromatography from r. reesei QM 9414 to give the enzyme in a homogeneous form 27. In SDS-PAGE the protein had a size of 58 kDa and the isoelectric point was 4.9. Glycosy-lation was estimated as 8 to 18 % 36. The molar absorptivity at 280 nm was 75 000 M xm To obtain the core protein partial proteolytic hydrolysis with papain was per-... 

Sensitivity to papain or any other components of these preparations. 

Brocklehurst, K., Carlsson, J., Kierstan, M.P.J, and Crook, E.M., Covalent chromatography. Preparation of fully active papain from dried papaya latex, Biochem. J., 133, 573-584, 1973. 

An alternative to the synthesis of proteins by classical fragment synthesis in solution or by solid-phase synthesis on a support is the use of enzyme-catalyzed condensation of amino acids or peptides. This possibility was first demonstrated in 1938 91 with the synthesis of poorly soluble benzoyl-leucyl-leucine anilide by papain catalysis. After many years, this approach was extended to the preparation of peptide hormones such as Leu-enkephalin 92 and dynorphin(l -8).[93 This was made possible by the use of highly purified enzymes and by careful control of reaction conditions. The basic principles of protease-catalyzed peptide bond formation have been discussed.194 ... 

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