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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals

Definitions

• the present invention concerns a procedure for extracting endoproteolytic hatching enzymes (zonases) in waste water from hatcheries producing Atlantic salmon larvae, and in addition, it establishes a simple procedure for obtaining up to sequence-grade purity of these special endoproteases, which turn out to possess rather unique proteoiytic characteristics.
• zonases endoproteolytic hatching enzymes
• Proteases in purified states are increasingly used in research, in laboratory and clinical analysis, and in food production procedures.
• Demand is increasing, especially for enzymes with properties commensurate with specific applications. This has stimulated quests for new sources of proteases which allow safe, sustainable and economical modes of production.
• Putative hatching enzymes have also been reported in invertebrates, where again most such enzymes have been interpreted as being metalloproteases (e.g. Barrett & Edward 1976; Lepage & Gache 1989; Roe &. Lennarz 1990).
• metalloproteases e.g. Barrett & Edward 1976; Lepage & Gache 1989; Roe &. Lennarz 1990.
• a few strong cases for serine protease-Wat zonases have been reported (e.g. Post et al 1988).
• putative hatching enzymes have also been reported. For instance, both Urch & Hedrick (1981; concerning amphibians) and Yamazaki et al (1994; concerning mouse) reported zonases which appeared to be serine proteases. The biological and biochemical rationale behind two different types of zonases among hatching animals is at present not fully understood.
• An additional advantage is that the developmentally-staged salmon eggs may be transferred to minimal volumes of water prior to hatching.
• highly synchronous hatching is induced by elevated (room) temperatures, or by deoxygenaton (Oppen-Berntsen et al. 1990), this yields a small volume of highly concentrated preparation of crude zonases.
• a further essential aspect of this procedure is that, despite the increasing concentration of the proteoiytic zonases, the stability of its resident zonases was observed to remain intact. Furthermore, it is important to note that this procedure yields z ⁇ nase enzymes in a medium of almost pure water, containing at most 1 mM NaCl, but where zonases nevertheless possess and retain full enzymatic integrity over time. This preparation is therefore a valuable starting material for subsequent preparations of proteoiytic zonases in various degree of purification, up to sequence-grade purity.
• Example 1 Concentrated preparation of crude zonase from Atlanta «fl'm ⁇ m-
• the initial purification of zonases involves only filtration of batched salmon eggs through cheese cloth. Such a filtrate may be frozen for years without significant zonase degradation, before being thawed and employed for further zonase purification. This fact greatly simplifies production of a starting material for purifying salmon zonases.
• the next step involves adjusting the "zonase crude” to usually 4 M urea, which dissociates fragments of the salmon eggshell and allow their removal along with extraneous debris by low speed centrifugation (15,0005; 2 x 15 min).
• This material shows no sign of clogging columns, which is characteristics of crude materials prepared differently from what is described above.
• a li z ⁇ nase crude” preparation suitable for purification by conventional chromatographic techniques is thus available. It is noteworthy that the salmon zonases are stable and catalytically active in 4, or even 8 M urea. Furthermore, this preparation of salmon zonase is effectively inhibited only by inhibitors of serine protease-type of proteases.
• Example 2 Purified zonases from Atlantic salmon.
• the product extracted from the "zonase crude” preparation may be chosen in different stages of purification. However, already after one round of gel filtration, zonases are separated from the larger molecular components in the filtrate with a 12 fold purification with better than a 50 % yield. Larger components present in the "zonase crude” seem for the most part to be soluble fragments of the eggshell, to which some zonases are bound tightly. It is essential that the high molecular weight contaminants are discarded at this early stage of purification, as their presence will otherwise interfere with, and block, the success of subsequent purification steps. In other words, the sequence in which the conventional purification methods are undertaken, is an essential aspect of the process.
• the matrix utilized may vary, but Sephacryl SR-200 is our usual choice.
• the buffer was Tris-HCl pH 8.0 or pH 8.5(0.05 M) or Tris-Acetate (0.025 M, same pHs).
• the zonases obtained after gel filtration procedures account for the predominant zonase moieties in the "zonase crude", and the enzymatic activity was catalytically inhibited by benzamidine.
• the zonases account for about 10 % of the material already at this stage, This partly purified salmon zonase is again only inhibited by serine protease-type inhibitors.
• Example 3 Zonase as a homogeneous protein product
• the enzyme fractions retarded on gel filtration columns may be readily purified further by affinity chromatography on commercially available Benzamidine Sepharose 6B-columns. This steps allows a 7.5 fold purification for an overall 94 fold total purification over the "zonase crude", with a yield of 37 % of activity.
• the zonase preparation After affinty-purification, the zonase preparation exhibits one protein band on SDS- PAGE analysis, with a molecular weight of around 28 kDa. This moiety of zonase was strongly antigenic, allowing production of polyclonal antibodies which specifically recognize salmon zonases, but not other salmon serine proteases such as salmon trypsins. Conversely, polyclonal antibodies to salmon trypsins do not recognize salmon zonases, establishing salmon zonase as a distinct product of embryonic salmon. However, this zonase product is not of sequence-grade purity, as revealed by Edman procedures for its N-terminal sequence. However, beyond the initial dozen N-terminal steps of sequencing, the overall amino acid sequence of this protein was shown to be similar to pure zonases. This highly purified salmon zonase preparation is also specifically inhibited only by serine protease-type inhibitors.
• Gelfiltration-purified plus affinity-purified salmon zonases may be further purified to sequence-grade purity by one final chromatographic procedure.
• This procedure employs a PBE94 column, with a buffer of Tris-Acetate (10 mM, pH 9.0), where subsequent elution was with a salt gradient (up to 1 M NaCl salt) in this buffer.
• This step itself increases the catalytic activity of the zonases by a further 7.6 fold, for an overall purification of 714 fold , and with a yield, of 28 % from the starting material.
• This purification step leaves the protein identity of the zonases intact as a 28 kDa moiety.
• this step does not remove unrelated, major protein contaminants from the zonase preparation, as is custumary for protein purification, as also illustrated in Examples 2 & 3.
• the molecular weight of purified zonases is the same as observed by Western blotting technique for zonase moieties present in the hatching fluid and in the "zonase crude".
• Partial amino acid sequences from CNBr-generated peptides established the zonases as a distinct proteins. Strucutural analysis yielded indications that zonases may have distinct catalytic and substrate-binding domains, which may account for their sensitivity to calcium- chelating agents when acting on macromolecular (physiological) substrates (binding is inhibited, hence catalysis is inhibited indirectly), and also sensitivity to serine protease- inhibitors when acting on small substrates (catalysis is directly inhibited).
• zonases The catalytic action of zonases is unaffected by the presence of salt in molar concentration, being nearly as effective in destilled water as in 6M salt.
• the enzyme is essentially equally active between pH 7 and 9. However, zonase is inactivated below pH 4, and only weakly active at pH 6.
• Zonase is unaffected by the presence of 8 M urea. Zonase can be stored at room temperature (with and without urea) for fifty days with only minimal loss of enzymatic activity. Enzymatic activity is also not impeded by even 40 % (v/v) of organic solvents such as dioxane or propanol.
• the enzyme is easily inactivated by 10 % (v v) of 2- mercaptoethanol, which may subsequently be removed by evaporation at 50°C.
• Catalysis is maximal at 42°C (using the commercial substrate chromozym X (from Boehringer), with little, but significant catalytic action observed above 65°C, or after heating to up to 90 ⁇ C for 5 min, and subsequently cooled and assayed at room temperature.
• Example 6 Catalytic characteristics of salmon zonases
• the Vmax is equal to 1.3 ⁇ /min, and the Kcat / KM of 57 (/mM sec), compared to a value of about 1 for bovine and porcine (cationic) trypsins.
• zonases seem to possess excellent prospects in terms of accomplishing specific splits in various candidate proteins, as nowadays are achieved using commercial enzyme-preparations of enzymes possessing (other) site-specific properties, e.g. the Boehringer products Asp-N and Glu-C.
• enzymes possessing (other) site-specific properties e.g. the Boehringer products Asp-N and Glu-C.
• zonases rank alongside commercial enzymes which have found use in analytical work preparatory to protein sequenation, by yielding defined peptides from the large proteins to be sequenced.
• this trait of the enzymology of pure salmon zonases is commercially valuable.

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• 1997
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