Biochem.J.(2005)391,567–574(Printed in Great Britain)doi:10.1042/BJ20050718567 Diversi?cation in substrate usage by glutathione synthetases from
soya bean(Glycine max),wheat(Triticum aestivum)and maize(Zea mays)
Mark SKIPSEY*1,Benjamin G.DAVIS?and Robert EDWARDS*
*Crop Protection Group,School of Biological and Biomedical Sciences,University of Durham,South Road,Durham DH13LE,U.K.,and?Department of Chemistry,
University of Oxford,Mans?eld Road,Oxford OX13TA,U.K.
Unlike animals which accumulate glutathione(γ-glutamyl-L-cysteinyl-glycine)alone as their major thiol antioxidant,several crops synthesize alternative forms of glutathione by varying the carboxy residue.The molecular basis of this variation is not well understood,but the substrate speci?city of the respective GSs(glutathione synthetases)has been implicated.To investigate their substrate tolerance,?ve GS-like cDNAs have been cloned from plants that can accumulate alternative forms of glutathione, notably soya bean[hGSH(homoglutathione orγ-glutamyl-L-cysteinyl-β-alanine)],wheat(hydroxymethylglutathione orγ-glutamyl-L-cysteinyl-serine)and maize(γ-Glu-Cys-Glu).The respective recombinant GSs were then assayed for the incorpor-ation of differing C-termini intoγ-Glu-Cys.The soya bean enzyme primarily incorporatedβ-alanine to form hGSH,whereas the GS enzymes from cereals preferentially catalysed the form-ation of glutathione.However,when assayed with other sub-strates,several GSs and one wheat enzyme in particular were able to synthesize a diverse range of glutathione variants by incorporat-ing unusual C-terminal moieties including D-serine,non-natural amino acids andα-amino alcohols.Our results suggest that plant GSs are capable of producing a diverse range of glutathione homologues depending on the availability of the acyl acceptor. Key words:glutathione synthetase,Glycine max(soya bean), homoglutathione synthetase,hydroxymethylglutathione,Triticum aestivum(wheat),Zea mays(maize).
INTRODUCTION
The majority of higher plants contain the tripeptide glutathione
(γ-glutamyl-L-cysteinyl-glycine),which serves important func-
tions as an antioxidant,a scavenger of reactive chemical species
and a signalling agent[1,2].In several economically important
crops,glutathione is replaced either completely or in part by more
unusual thiols.For example,in legume species including soya
bean(Glycine max L.),hGSH(homoglutathione orγ-glutamyl-L-cysteinyl-β-alanine)is the dominant thiol[3].In other legumes such as Medicago truncatula,both glutathione and hGSH are
synthesized,accumulating in an organ-speci?c manner[4].In ce-
reals of the Poaceae family,such as wheat(Triticum aestivum L.),
hmGSH(hydroxymethylglutathione orγ-glutamyl-L-cysteinyl-
serine)co-accumulates with glutathione[5].Maize(Zea mays L.)
also has the capacity to accumulateγ-ECE(γ-glutamyl-L-cys-
teine-glutamic acid)on exposure to heavy metals[6].
Glutathione,hGSH and related variants are synthesized from γ-EC(γ-glutamyl-L-cysteine),which is formed from L-glutamate and L-cysteine in an ATP-dependent reaction catalysed byγ-ECS (γ-glutamyl-L-cysteine synthetase;EC6.3.2.2).Glutathione is then synthesized fromγ-EC and glycine by an ATP-dependent GS(glutathione synthetase;EC 6.3.2.3).Most GS enzymes selectively produce glutathione due to their tight acyl acceptor speci?city for glycine as a substrate,but in legumes the enzyme has evolved to preferentially incorporateβ-alanine,effectively becoming an hGS(homoglutathione synthetase).Thus the hGSs from M.truncatula andpea(Pisumsativum L.)selectively catalyse the synthesis of hGSH fromγ-EC andβ-alanine[7,8].The relative abundance of glutathione and hGSH in legumes is then
determined by the expression of either GS or hGS enzymes[9].
In wheat,the source of hmGSH is less clear-cut.Whileγ-EC
remains the direct precursor,the serine could either be incorpor-
ated directly by an ATP-dependent GS-like reaction or could be
derived through a post-synthetic modi?cation of the glutathione
molecule[5].Similarly,the source of the glutamate analogue,γ-ECE,in maize is not known,with the possibilities being that it is derived from an alternative GS activity or from the degrad-
ation of phytochelatins,which are polymers of(γ-Glu-Cys)n-
Glu-Cys-Gly[6].To address whether or not the GSs of maize
and wheat could determine the synthesis of alternative forms of
glutathione,we have cloned enzymes from the respective cereals
and determined their substrate preference,comparing the results
obtained with an hGS from soya bean.
MATERIALS AND METHODS
Cloning of thiol synthetase cDNAs
DNA probes were prepared by PCR using a speci?c primer(MS-3,
gCgAAgCCHCARMgAgARggHggAgg)based on aligned GS
sequences[10].PCR ampli?cation was performed on cDNA pre-
pared from total RNA prepared from either10-day-old soya bean
cell-suspension cultures(cv.Mandarin)[11],10-day-old wheat
shoots[12]or on plasmid DNA prepared from a mass excised
cDNA library prepared from etiolated10-day-old maize roots
[13].MS-3was used in combination with the non-speci?c primer
Abbreviations used:AABA,BABA and GABA,α-,β-andγ-aminobutyric acid respectively;ACV synthetase,L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine synthetase;BAIBA,β-aminoisobutyric acid;γ-EC,γ-glutamyl-L-cysteine;γ-ECE,γ-glutamyl-L-cysteine-glutamic acid;γ-ECS,γ-glutamyl-L-cysteine synthetase;ESI,electrospray ionization;GS,glutathione synthetase;hGS,homoglutathione synthetase;hGSH,homoglutathione;hmGSH,hydroxy-methylglutathione;TOF,time-of-?ight.
1To whom correspondence should be addressed(email mark.skipsey@https://www.doczj.com/doc/5b17669286.html,).
The nucleotide sequence data reported will appear in DDBJ,EMBL,GenBank?and GSDB Nucleotide Sequence Databases under the accession numbers AJ272035(Gm GS),AJ579380(Ta GS1),AJ579381(Ta GS2),AJ579382(Ta GS3)and AJ579383(Zm GS).
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OG9for soya bean[11],whereas,for maize and wheat,two speci?c primers were designed from GS sequences in the re-spective EST(expressed sequence tag)databases,namely Zm GS3 (gCgCTgAgCTTgCCAgTCAAgTATAggC)and Ta GS3 (gCggT-CgACCTCATCTgTgAggAgAATgC)respectively.Ampli?cation products were cloned into pGEM-T Easy(Promega,Chilworth, Southampton,U.K.)and sequenced using an Applied Biosystems 373DNA sequencer.For soya bean,the full-length Gm GS cDNA was cloned as described previously[10].The partial wheat (Ta MS3-1)and maize(Zm MS3-1)clones were PCR-labelled with digoxigenin(Roche,Lewes,East Sussex,U.K.)and used to screen cDNA libraries prepared from shoots[14]and roots of etiolated seedlings[13]respectively.In each case,200000plaque-forming units were screened and positive clones were puri?ed and sequenced on both strands.
Expression and puri?cation of recombinant enzymes
PCR was used to engineer restriction sites for direct cloning of the coding sequences into pET-24a or pET-24d(Novagen,Madison, WI,U.S.A.)essentially as described for Gm GS[10].For the maize and wheat clones,the following primer pairs were used in the presence of1M betaine:Zm GS,Zm GS5 Nde(CCAATAC-CTCATATgAgTgCCgCCATgCCg)and Zm GS3 (gCgCTCgA-gCTTgCCAgTCAAgTATAggC);Ta GS1either Ta GSBNdensp (CCCTgAggCATATgggAgCCgAggCgC)and Ta GS3 (gCggTC-gACCTCATCTgTgAggAgAATgC)or Ta GSSigNde(gCgCATA-TgTCCTCTTgCgTCTCCTCCTCCC)and Ta GS3 ;Ta GS2, Ta GSCNde(CCACTgCCgCATATgAgCACCACC)and Ta GSC3 (gCgCCTCgAgCTTgTCggTCAggTATAAgC).The pET-based plasmids were resequenced and used to transform Rosetta TM DE3(Novagen)bacteria.The production and puri?cation of the recombinant GSs was then performed as described for Gm GS [10].In each case,the purity of the His-tagged proteins was con-?rmed by SDS/PAGE(10%polyacrylamide)before use. Enzyme assays
Protein concentration was determined using the Bio-Rad dye-binding reagent,withγ-globulin standards,as recommended by the manufacturer.Enzyme assays were prepared in250mM Tris/ HCl(pH8.0)containing20mM MgCl2,5mM dithiothreitol, 10mM ATP,1mMγ-EC and100mM(unless otherwise stated) of the co-substrate to be assayed,in a total volume of100μl.The reaction mixtures were incubated at30?C for up to120min,with enzyme or substrate omitted for controls.At time intervals,20μl samples were derivatized for15min at room temperature(21?C) using200μl of0.2M Tris/HCl(pH8.0)containing0.24μmol of monobromobimane.After adding780μl of5%(v/v)acetic acid,50μl samples were analysed for?uorescent S-bimane deriv-atives by HPLC,with authentic glutathione and hGSH used to quantify tripeptide products[15].After ensuring time and pro-tein dependence for the assay,enzyme activity was expressed in pkat(=1pmol of product formed s?1).
Reaction products were also analysed directly before bimane derivatization using a TOF(time-of-?ight)mass spectrometer (Micromass LCT;Micromass,Manchester,U.K.)using ESI (electrospray ionization),in positive ion mode.The reaction mix-ture(20μl)was injected on to a Jupiter C18150mm×2mm 5μm column(Phenomenex,Maccles?eld,U.K.)in water/aceto-nitrile/formic acid(180:20:1,by vol.)at a?ow rate of0.2ml·min?1.The eluate was analysed for the expected mass of ions in the range100–800Da using the supplied MassLynx software, after calibration with sodium iodide.Determination of amino acid con?guration in hmGSH
Samples of S-bimane-derivatized hmGSH were puri?ed by HPLC from crude wheat leaf extracts or enzyme preparations after de-rivatization with monobromobimane,freeze-dried,resuspended in10mM Hepes buffer(pH7.9)containing0.2mM EDTA,and digested with1unit of bovineγ-glutamyltranspeptidase for2h at25?C.The reaction products were analysed by reverse-phase HPLC[15]or on a Chirobiotic V250mm×4.6mm column (Astec,Whippany,NJ,U.S.A.)using the same gradient con-ditions.The retention times of reaction products were compared with those for derivatized authentic L-Cys-L-Ser and L-Cys-D-Ser standards,prepared on an automated peptide synthesizer,and the identity of the peptides was con?rmed by ESI-MS.
RESULTS
Cloning of GS-like sequences from soya bean,wheat and maize As described previously for the cloning of Gm GS[10],PCR amp-li?cation generated a354bp sequence from wheat(Ta MS3-1)and a353bp sequence from maize(Zm MS3-1),which were found to be closely related to other plant GS sequences when subjected to BLAST searches[16].Following digoxigenin labelling of the PCR products,four positive clones(Ta GS1–Ta GS4)were ob-tained from a wheat library probed with Ta MS3-1.When probed with Zm MS3-1,17positive clones,Zm GS1–Zm GS17,were ob-tained from the maize library.Following restriction enzyme analysis,the wheat clones were fully sequenced on both strands.
A combination of restriction enzyme analysis and sequencing demonstrated all17maize clones to be essentially identical, except that some of the sequences were attenuated at the5 -end. Three different wheat clone sequences were obtained,since Ta GS1and Ta GS4(=Ta GS1)were identical.Ta GS2and Ta GS3 differed in13out of475amino acids,but had homologous nucleo-tide sequences in both5 -and3 -untranslated regions.Ta GS1was only77and79%identical at the amino acid level with the Ta GS2 and Ta GS3clones respectively.The amino acid sequences of the four GS clones isolated from wheat and maize are shown aligned with Gm GS for reference in Figure1.
The predicted amino acid sequences of the four cDNAs from the cereal species were more similar to GS rather than hGS sequences. Zm GS was most identical with a recently deposited maize cDNA putatively assigned to be a GS(EMBL accession no.AJ302784),which was predicted to encode a45.9kDa protein. However,Zm GS was84amino acids longer at its N-terminus than AJ302784and contained four in frame methionine residues upstream,all of which scored highly as predicted translation start sites using NetStart1.0[17].Ta GS1,Ta GS2and Ta GS3were most identical with an unassigned rice cDNA clone(EMBL accession no.AK068792).
In common with Gm GS,the Ta GS1protein contained an extended N-terminus in comparison with the other GS sequences cloned in the present study(Figure1),and was predicted to be tar-geted to the chloroplast by both PSORT[18]and ChloroP[19]. Similarly,the rice cDNA AK068792,which most closely re-sembled Ta GS1in the database,also contained a homologous N-terminal extension predicted to target the putative peptide to the chloroplast.The other GS sequences from the cereals were not predicted to be targeted to subcellular compartments and therefore would be deposited in the cytosol.
Expression of recombinant GS polypeptides
The plant GS-coding sequences were subcloned into pET24 for the expression of the C-terminal His-tagged fusion proteins,
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Figure1CLUSTAL W alignment of the predicted peptide sequences obtained for the GS cDNAs cloned from soya bean,wheat and maize Residues shaded black are identical or conserved in all?ve sequences and those shaded in grey, identical in only four of the?ve sequences.The methionine residues used as translation start codons in pET constructs have been underlined.The conserved alanine residues found within GS,but not within hGS protein sequences[7],can be seen at position530–531in the cereal crop sequences but not in the soya bean sequence.Putative N-terminal polypeptide extensions encoding cellular targeting signals are shown in lower-case letters.
using the?rst methionine residue in each case to initiate trans-lation.Ta GS3could not be subcloned due to the dif?culty in amplifying this cDNA with the required restriction enzyme sites. Similarly,dif?culty was initially experienced in PCR ampli?-cation of the other wheat and maize clones,although the correct products were eventually obtained after adding betaine to the PCR. Since Ta GS1was predicted to contain an N-terminal signal pep-tide,two pET constructs were employed that either encoded the protein with the signal peptide or utilized an endogenous methio-nine residue at position45,two amino acids downstream of the predicted cleavage site to initiate translation.When analysed by SDS/PAGE,the lysate from those induced bacteria harbouring the pET constructs were found to contain recombinant polypeptides with molecular masses between50and60kDa that were absent from control bacteria.As reported previously[8],considerable dif?culty was experienced in expressing the recombinant plant GS proteins.This expression problem was overcome using Rosetta TM (DE3)cells grown in a medium containing1%glucose with an overnight induction at15?C that improved both the solubility and yield of the recombinant GS proteins to between5and20%of the total soluble protein
extract.Figure2Analysis of puri?ed recombinant GS polypeptides
His-tagged proteins were puri?ed on Ni-chelate af?nity columns and analysed by Coomassie Blue staining after SDS/PAGE(10%polyacrylamide).From left to right:M,reference molecular-mass standards;Gm GS(minus leader sequence);Ta GS1+(plus leader sequence);Ta GS1(minus leader sequence);Ta GS2(clone has no leader sequence);and Zm GS(clone has no leader sequence).Contaminating polypeptides around60and30kDa were present in all preparations and were presumably from the E.coli extract.
Substrate selectivity of plant GSs
The His-tagged enzymes were puri?ed using Ni-chelate af?nity chromatography and purity was assessed by SDS/PAGE(Fig-ure2).When these puri?cation runs were repeated with Esch-erichia coli extracts that did not contain recombinant GS proteins, no thiol synthetase activity was determined,con?rming that the af?nity-puri?ed preparations were free of any contaminating bacterial GS enzyme.In the case of Gm GS,depending on extrac-tion conditions,the respective af?nity-puri?ed preparation could be resolved into a doublet of polypeptides.We presume that this was due to proteolytic processing of the recombinant Gm GS,but on the basis of speci?c activity determinations between batches, this truncation did not affect activity.Puri?ed protein(1μg)was incubated withγ-EC in the presence of a representative range of acyl donor substrates including L-α-amino acids,D-amino acids andα-amino alcohols in the presence of ATP.Enzyme activity was determined by quantifying the amount of product formed after derivatization to the?uorescent S-bimane adducts and analysis by HPLC,with the respective molecular masses of the underivatized tripeptides con?rmed by ESI-TOF MS(Table1).Under all assay conditions,GS-catalysed product formation was directly propor-tional to enzyme concentration and incubation time over 120min for all puri?ed enzymes.Under standard assay con-ditions,incubations were carried out for60min.In the case of the Ta GS1clone,no signi?cant differences were observed in the speci?c activities displayed when the recombinant protein was expressed with,or without,the signal peptide sequence.All activ-ity data presented with respect to Ta GS1was obtained with recombinant enzyme expressed without the signal peptide se-quence.
Despite the presence of hGSH and hmGSH in place of gluta-thione in several plant species,the substrate selectivity of the re-spective GSs has received only limited attention.Initial exper-iments focused on the use of glycine andβ-alanine as C-terminal substrates using a?xed concentration of1mMγ-EC.With the soya bean GS,the recombinant enzymes displayed a6-fold higher speci?c activity withβ-alanine as substrate compared with glycine(Table1).With Ta GS1,Ta GS2and Zm GS,glycine was the preferred substrate,although signi?cant activity was also ob-served withβ-alanine in each case.The puri?ed recombinant
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Table1Speci?c activities of puri?ed recombinant thiol synthetases towards a range of substrates
All assays were performed in triplicate with1mMγ-EC under standard assay conditions with100mM co-substrate,except for L-cysteine,which was used at10mM so as not to saturate the monobromobimane derivatization.Only those substrates with which an activity could be measured after correcting for enzyme-free controls are shown in the Table and the mass of the predicted reaction products,(M+H)+,was con?rmed by MS in each case using NaI as calibrant.Other substrates assayed for which no enzyme activity could be detected include L-serine,D-and L-glutamic acid,L-valine,L-threonine,L-lysine,L-cysteine,L-arginine,L-asparagine,DL-BABA,6-aminohexanoic acid and n-butylamine.Each value represents the mean+?S.D.for three replicate experiments. NA,no detectable activity.
Recombinant enzyme*Tripeptide product
Substrate Gm GS Ta GS1Ta GS2Zm GS HPLC retention time(min)(M+H)+
Glycine501+?751620+?132895+?3451781+?4168.5308.09
β-Alanine3306+?46956+?5403+?77591+?2910.9322.11
L-Alanine NA23+?5564+?169177+?1210.3322.11
D-Alanine NA NA118+?5168+?1716.7322.11
AABA NA NA29+?6NA16.9336.12
GABA2287+?107107+?34584+?130138+?2013.9336.12
BAIBA1641+?10610+?216+?3NA20.1336.12
L-2,3-Diaminopropionic acid1100+?18233+?358+?1065+?228.5337.12
Ethanolamine150+?11NA17+?1NA18.2294.11
3-Amino-1-propanol88+?8NA29+?7NA23.0308.13
D-Serine35+?2NA64+?13NA8.6338.10
L-Ornithine21+?1NA7+?3NA10.1365.15
*Activities are given in pkat(pmol of product formed·s?1).
Table2Kinetic characterization of puri?ed recombinant GS enzymes
K m data for bothβ-alanine and glycine were assayed with a constantγ-EC concentration (1mM).K m data forγ-glutamylcysteine were assayed with aβ-alanine concentration of10mM for Gm GS and a glycine concentration of10mM for all other enzymes.For V max,activities are expressed as pkat·(mg of pure protein)?1.K cat/K m values presented in the Table are with respect to the acyl acceptor.ND,kinetic constants could not be determined due to the very low activities determined.V max values are given in pkat·(mg of puri?ed GS)?1.
Co-substrate
Kinetic constant Glycineβ-Alanine Gm GS V max(pkat·mg?1)364+?362132+?152 K m(mM)19+?60.32+?0.08
K cat/K m(M?1·s?1)10737083
K m forγ-EC(mM)ND0.06+?0.04 Ta GS1V max(pkat·mg?1)663+?35ND
K m(mM)0.16+?0.04ND
K cat/K m(M?1·s?1)24003ND
K m forγ-EC(mM)0.18+?0.07ND
Ta GS2V max(pkat·mg?1)2817+?1725660+?3153 K m(mM)0.07+?0.02170+?136
K cat/K m(M?1·s?1)212404175
K m forγ-EC(mM)0.10+?0.02ND
Zm GS V max(pkat·mg?1)1304+?179372.4+?597 K m(mM)0.18+?0.08592+?1077
K cat/K m(M?1·s?1)38060 3.3
K m forγ-EC(mM)0.03+?0.02ND
enzymes were used for more detailed kinetic analysis to de?ne the observed substrate preference between glycine andβ-alanine (Table2).The puri?ed Gm GS showed a signi?cant speci?city forβ-alanine compared with glycine,as shown by an approx. 350-fold higher k cat/K m that is largely a consequence of an approx.60-fold lower K m,indicating a strong ground state binding af?nity discrimination.The kinetic constants determined were very similar to those reported for the respective enzyme partially puri?ed from soya bean and runner bean[20].In contrast,glycine was the preferred substrate with the Ta GS and Zm GS recombinant enzymes.The>1000-fold speci?city discrimination,as indicated by k cat/K m,for the maize and wheat GS enzymes was again largely a consequence of striking differences in K m.However,on the basis
of k cat,a different pattern in their utilization of these two amino
acids emerges(Table2).While Ta GS1showed negligible turnover
withβ-alanine,Ta GS2had,in fact,a much greater V max with this
substrate than with glycine.
In soya bean,the presence of aβ-alanine-selective GS cor-
related with the preferential accumulation of hGSH in this legume.
It was therefore of interest to determine whether or not the
accumulation of hmGSH in wheat andγ-ECE in maize could
be similarly explained by an unusual amino acid selectivity of
the respective GS enzymes.Therefore all enzymes were probed
with the diverse array of C-terminal substrates shown in Figure3.
None among Ta GS1,Ta GS2or Zm GS would accept L-serine or L-glutamate as co-substrate withγ-EC.When assayed with the other amino acid co-substrates,enzyme activity was only seen
with L-alanine,notably with Ta GS2.The utilization of L-alanine
by the cereal GS enzymes then prompted the question as to what
other amino derivatives(Figure3)could serve as GS substrates
for the wheat,maize and soya bean enzymes(Table1).Putative
reaction products were analysed by a combination of HPLC and
MS(Figure4).
Ta GS1showed a broadly similar selectivity for non-physio-logical amine substrates to Gm GS,albeit with lower activities determined.Ta GS2proved to be the most versatile enzyme,acting on most of the substrates tested including D-alanine and D-serine, and being the only GS to utilize AABA(α-aminobutyric acid). Zm GS also catalysed the incorporation of L-and D-alanine but otherwise resembled Ta GS1in its selectivity and did not utilize D-glutamic acid.For reference,none of the recombinant GSs could utilize DL-BABA(DL-β-aminobutyric acid),6-aminohexanoic acid or n-butylamine as substrates(Figure3).A clear difference in the selectivity of Gm GS and the wheat and cereal enzymes was the relative usage of GABA(γ-aminobutyric acid)and BAIBA (β-aminoisobutyric acid).All enzymes used GABA ef?ciently, but the introduction of a methyl group on the equivalentα-carbon in BAIBA provoked at least a10-fold decrease in activity with the cereal GS,but only a30%reduction in Gm GS.These results are in contradiction to a previous report utilizing partially puri?ed hGS from Phaseolus coccineus,which was5-fold more active in utilizing BAIBA than GABA[20].Although GABA has been
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Figure3Chemical structures of some potential substrates
(A)Structures of substrates utilized in vitro by recombinant GS proteins from either G.max L.,T.aestivum L.or Z.mays L.(B)Examples of substrates not utilized in vitro by any recombinant GS protein.
shown to be a substrate for several GS enzymes,it may not be a
universally acceptable substrate as previous reports showed that
this acyl acceptor was tolerated by a partially puri?ed GS from
mung bean(Vigna radiata)but was not utilized by the respective
enzyme from peas[21]or tobacco(Nicotiana tabacum)[22].
With the diamino acceptor2,3-diaminopropionic acid,the site
of peptide bond formation was not unequivocally demonstrated.
However,based on the activity seen with BAIBA,it would seem
most likely that where incorporation occurred theβ-amino group
was used.The utilization ofβ-andγ-amino groups was most
markedly observed with Gm GS,but in all cases further extension
of the amino-group-bearing side chain beyond C-3(ornithine)
resulted in a total loss of activity[i.e.extension to C-4(lysine)
or C-5(6-aminohexanoic acid)].Replacement of theα-carboxy
residue with an alcohol function(e.g.substitution ofβ-alanine
with3-amino-1-propanol and glycine with ethanolamine)also
reduced incorporation,with only Gm GS and Ta GS2capable of
utilizing these substrates.
Active-site model based on chemical mapping
The use of a broad range of acyl acceptors allowed an assessment
of the substrate speci?city of the S1 binding subsite in these
peptide synthetases.For Gm GS,three broad levels of selectivity
were seen.The?ve substrates showing the highest activ-
ity(Table1),namely glycine,β-alanine,GABA,BAIBA and L-2,3-diaminopropanoic acid,all have an amine substitution on the primary carbon.Unlike all the other GS enzymes examined,
while Gm GS was intolerant toα-substitution,β-substitution was
acceptable as seen with BAIBA and L-2,3-diaminopropanoic acid.
A secondary level of activity(21–150pkat·mg?1),roughly an
order of magnitude lower,was also observed for the simple substituted ethylamines[X-(CH2)2-NH2,where X=OH,CH2OH
or CH2CH(NH2)CO2H],indicating that a certain amount of
structural diversity and/or branching can be tolerated at theγ-
position relative to the NH2group.However,acceptors beyond
a C-4chain length(e.g.6-aminohexanoic acid,Lys)are too
large to occupy the Gm GS subsite.For Ta GS1,only one high-
level acyl acceptor was identi?ed in the screen,namely glycine
(1620pkat·mg?1).However,several moderate level acceptor
substrates were turned over at a rate that was one order of magni-
tude lower(10–107pkat·mg?1).From this pattern of substrate
usage,it was concluded that Ta GS1must have a relatively
short and narrow binding site since it could tolerate only minor α-branching tolerance(L-Ala),was stereoselective for the L-con?guration(L-Ala not D-Ala)and had weakβ-branching
tolerance(BAIBA and L-2,3-diaminopropanoic acid),but had
acceptance of unbranchedω-aminocarboxylic acids up to C-3
in length(Gly,β-Ala and GABA).For Ta GS2,a broad substrate
tolerance was seen.The optimal substrates(Table1)Gly,β-Ala, L-Ala and GABA demonstrated tolerance ofα-branching,even for the bulkier ethyl substituent,as seen in the activity of this enzyme towards AABA.Although an accompanyingα-stereoselectivity was observed,this was relatively weak,with the ratio of L-Ala turnover to D-Ala turnover being5:1.Limited activity (7–118pkat·mg?1)was seen towards all other amines tested except those beyond a C-4chain length,which were not tolerated, as was the case with all GS enzymes tested.Moderateβ-branching tolerance was observed although this was substantially less than that shown by Gm GS.Zm GS displayed high-level activity for just Gly andβ-Ala(Table1)with lesser activity(65–177pkat·mg?1) observed forα-branched L-and D-Ala.Moderate activity towards GABA but not towards longer carbon chain nucleophiles indicated a shorter C-3-chain-binding subsite.Some weak tolerance of
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Edwards
Figure 4HPLC and MS analyses of a GS assay with a non-natural co-substrate
(A )HPLC ?uorescence trace of S -bimane-derivatized reaction products of puri?ed recombinant Ta GS2assay using γ-GC and D -alanine as substrates.Peak 1,excess γ-EC in the assay.Peak 2,novel reaction product (γ-Glu-Cys-D -Ala),which was not observed when Ta GS2was incubated with γ-EC minus D -alanine.Peak 3,bromobimane degradation product.(B )Control incubation of (A ),with Ta GS2incubated with γ-GC only.(C )Upper and lower traces:mass spectral analysis of total reaction mixture shown in (A )and (B )respectively before derivatization with monobromobimane.The (M +H)+at 251.1corresponds to unchanged γ-GC,whereas the novel peak at 322.1present in the upper trace but not in the lower trace corresponds to γ-Glu-L -Cys-D -Ala in trace (A ).
β-branching was also observed with L -2,3-diaminopropanoic acid,but not with BAIBA.
D -Serine
as a substrate for Ta GS
The lack of activity of Ta GS1and Ta GS2with L -serine suggested that neither enzyme was responsible for catalysing the direct formation of the all L -con?guration hmGSH (i.e.γ-L -Glu-L -Cys-L -Ser).However,intriguingly,the activity seen with D -serine in Ta GS2demonstrated that it would be possible to synthesize the D -serine variant of hmGSH.Such a possibility could not be discounted,since the con?guration of serine in hmGSH was not reported during its original identi?cation in wheat plants [5].Alternatively,γ-L -Glu-L -Cys-L -Ser may be synthesized in wheat by the Ta GS after inversion of the D -serine to L -serine during or after ligation.An analogous co-ligation/epimerization is seen during tripeptide synthesis catalysed by ACV synthetase [L -δ-(α-aminoadipoyl)-L -cysteinyl-D -valine synthetase],a multi-functional enzyme,involved in penicillin biosynthesis [23].By analogy to the synthesis of glutathione,ACV synthetase combines both γ-ECS and GS activities by sequentially synthesizing L -δ-(α-aminoadipoyl)-L -cysteine and then incorporating valine via a peptide bond.During this latter incorporation,L -valine is inverted to the D -con?guration.The possibility that the incorpor-ation of D -serine was associated with its epimerization was
examined by preparing 1μmol of the putative tripeptide reaction product.After S -derivatization with bromobimane,the conjugate was digested with γ-glutamyltranspeptidase and the product co-chromatographed with S -bimane derivatives of authentic L -Cys-L -Ser and L -Cys-D -Ser.The digested Cys-Ser-bimane pro-duct formed by Ta GS2co-chromatographed with the correspond-ing L -Cys-D -Ser-bimane standard rather than the L -Cys-L -Ser derivative,con?rming that the serine had not undergone epimeriz-ation during ligation (Figure 5).hmGSH present in wheat plants was similarly S -bimane-derivatized and treated with γ-glutamyl-transpeptidase after its puri?cation by HPLC and was found to contain L -serine rather than D -serine.
DISCUSSION
GSs are members of the ATP-grasp superfamily of ligases forming amide bonds in post-translationally synthesized peptides [24,25].Family members include D -alanine:D -alanine ligase,ribosomal protein S6:glutamate ligase and an α-L -glutamate ligase involved in the biosynthesis of the coenzyme tetrahydrosarcinapterin [25].The usage of differing amino acids in peptide formation has arisen through gene evolution,but our studies suggest that there
c 2005Biochemical Society
Substrate diversi?cation of thiol synthetases
573
Figure5HPLC analysis of wheat hmGSH and Ta GS2D-serine reaction product
(A)HPLC trace ofγ-glutamyltranspeptidase digest,run on a Chirobiotic V column,of the puri?ed product of Ta GS2D-serine assay.MS identi?ed peak1as undigested parent compound (S-bimane-derivatizedγ-Glu-Cys-Ser:m/z+528.18),peak2as the S-bimane-derivatized Cys-Ser dipeptide product of the digest(m/z+399.13)and peak3as S-bimane-derivatized cys-teine(m/z+312.10).(B)HPLC trace ofγ-glutamyltranspeptidase digest,run on a Chirobiotic V column,of hmGSH puri?ed from wheat.MS con?rmed the identity of each peak to be of identical mass to that shown in(A).(C)HPLC trace of puri?ed peak2in(A),co-injected with S-bimane-derivatized L-Cys-D-Ser standard,on a Kingsorb C18column.(D)HPLC trace of puri?ed peak2in(B),co-injected with S-bimane-derivatized L-Cys-L-Ser standard,on a Kingsorb C18column.(E)HPLC trace of S-bimane-derivatized L-Cys-L-Ser standard and S-bimane-derivatized L-Cys-D-Ser standard co-injected on to a Kingsorb C18column. Peak1is S-bimane-derivatized L-Cys-L-Ser and peak2is S-bimane-derivatized L-Cys-D-Ser.is already latent diversity in the ability of the GS enzymes to synthesize diverse tripeptides.Thus,by expressing and assaying four recombinant GSs from three crop species,we have demon-strated differing substrate speci?cities and determined that these enzymes can catalyse the synthesis of a broad range of novel tripeptide products.This suggests that the nature of the thiol tri-peptides formed in planta must be determined primarily by acyl acceptor availability in speci?c compartments,namely a readily available supply of glycine in maize and wheat andβ-alanine in soya bean respectively.This is particularly relevant,since several of the amine acceptors tested in the present study are known to exist in plants and could therefore be incorporated into novel tripeptides.Thus GABA is a signi?cant component of the free amino acid pool in plant species[26]and has been reported at concentrations as high as1–2μmol·(g of FW)?1,in soya bean leaves after biotic stress[27].Similarly,AABA occurs in legumes, notably Lens sp.[28],and ethanolamine is known to accumulate in plants as an intermediate in choline synthesis after serine decarboxylation[29].It is therefore possible that,if present in suf?ciently high concentrations in a compartment along with a GS,these alternative amine acceptors could be incorporated into glutathione homologues in selected plant species.In the case of hGS,this diversi?cation in the use of the C-terminus has resulted in the preferential incorporation ofβ-alanine and the re-sultant accumulation of hGSH in many legume species.The reasons for the selective use ofβ-alanine over glycine in hGS have received some attention.All cloned hGS enzymes to date,includ-ing the GS cloned from soya bean in the present study(Figure1), have a substitution of leucine and proline for two alanine residues found in the putative glycyl binding domain of conventional GSs[30].It has recently been demonstrated that mutation of the leucine alone,or both the leucine and proline residues,to the re-spective alanine residues(Figure1)enhances the ability of an M.truncatula hGS to use glycine and synthesize glutathione[7]. However,as determined by comparison of the kinetic constants, Gm GS exhibits a much greater preference for utilizingβ-alanine over glycine as compared with the hGS of M.truncatula[7], suggesting that these two residues in the putative active site cannot be the only factors in determining hGS rather than GS speci?city. Interestingly,when assayed with four structural isomers of aminobutyric acid,Gm GS showed a marked preference for those substrates in which the amino group is on a terminal carbon rather than theα-carbon next to the carboxy group,which helps explain the preference forβ-alanine over L-alanine.The kinetic mechanism of the Arabidopsis thaliana GS has recently been determined and shown to have a random Ter-reactant mechanism [31].Studies with the Arabidopsis GS demonstrated that the order of binding of the?rst pair of substrates determined the af?nity of binding to the third substrate.It would now be of interest to extend these studies to other acyl acceptors using different plant GSs. Although it is possible that additional Ta GS and Zm GS clones, encoding enzymes that could catalyse the incorporation of serine and glutamic acid respectively could have been overlooked in our screen,the available evidence increasingly suggests that hmGSH andγ-ECE arise from post-synthetic modi?cations of gluta-thione rather than from unique GSs that catalyse the ligation of serine rather than glycine at the C-termini.In the case of wheat,it has been suggested that hmGSH could arise by transpeptidation on the basis of the demonstration of hmGSH formation when a yeast carboxypeptidase was incubated with glutathione and L-serine [32].Similarly,γ-ECE may well arise from the proteolytic trans-peptidation of phytochelatins,which are polymers of(γ-Glu-Cys)n formed by plants on exposure to heavy metals[6,33].
Our studies demonstrate that,among the ATP-grasp ligase superfamily[24,25],the plant GSs are?exible peptide synthases.
c 2005Biochemical Society
574M.Skipsey,B.G.Davis and R.Edwards
While these enzymes are limited to synthesizingγ-glutamyl linked glutathione analogues,this is of interest in regulating the activity of glutathione-dependent enzymes.For example,the overexpression of glutathione S-transferases in tumours is associ-ated with the onset of multidrug resistance,and as such there has been considerable research effort to prepare inhibitory glutathione analogues for therapy[34].It will now be of interest to screen the library of novel tripeptides produced by plant GSs for their ability to inhibit glutathione S-transferases and other glutathione-dependent proteins.
M.S.acknowledges?nancial support from the University of Durham through an Addison Wheeler Fellowship.We thank Dr J.Sanderson(Department of Chemistry,University of Durham)for the synthesis of L-Cys-L-Ser and L-Cys-D-Ser standards. REFERENCES
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Received3May2005/29June2005;accepted11July2005
Published as BJ Immediate Publication11July2005,doi:10.1042/BJ20050718 c 2005Biochemical Society
机械制造学 课程设计说明书 题目名称连接座加工工艺规程编制专业班级11级机械制造及自动化2班学生姓名 学号 指导教师王月英 机械与电子工程系 二○一四年六月二十日
目录 任务书----------------------------------------------------------------------------------------------3 指导教师评阅表----------------------------------------------------------------------------------4 一、序言----------------------------------------------------------------------------------------8 二、零件的分析--------------------------------------------------------------------------------9 三、工艺规程的设计----------------------------------------------------------------------------10 （1）. 确定毛坯的制造形式----------------------------------------------------------------12 （2）. 基面的选择-----------------------------------------------------------------------------15 （3）. 制订工艺路线--------------------------------------------------------------------------17 （4）. 机械加工余量、工序尺寸及毛坯尺寸的确--------------------------------------19 （5）. 确定切削用量及基本工时-----------------------------------------------------------20 四、设计心得与小结-----------------------------------------------------------------------------23 五、参考文献-------------------------------------------------------------------------------------23
机械加工工艺编制（阶梯轴加工工艺路线拟定） 系部：机械工程系教师：张春明 授课班级：大专机制专业科目：机械加工工艺编制 时间：2013年4月12日地点：306（1） 一、课题名称：阶梯轴加工工艺路线拟定 二、教学目标： 1、知识技能目标：复习轴类零件的材料、热处理及机械加工方法，学习轴的机 械加工、热处理和辅助工序的安排，理解何时安排热处理工序和辅助工序，并能正确安排阶梯轴加工工艺路线。 2、过程与方法：教师通过多种不同加工路线的讲解，学习正确合理安排机械加 工工艺路线，并掌握科学安排机械加工工艺路线的基本方法。 3、情感态度与价值观：通过学习让学生理解一个机器零件的加工要许多工序在 不同的车间才能完成，从而培养学生干工作做事情不能投机取巧，要脚踏实地团结合作，才能把事情做好。 三、教学重点：理解加工分段进行，以主要加工表面为主线，次要表面穿插其中。 教学难点：热处理工序的安排。 四、教学准备：挂图。 五、教学过程设计： （一）导入 1、简约板书上节课主要内容。 2、上节课我们讲了轴类零件的材料和热处理工艺，不同的材料热处理工艺也是有所不同。本节课我们来看一看这样一个轴类零件怎样来进行加工，如何安排它的加工工艺路线。出示挂图。 （二）教学新课 1、出示问题：（1）零件材料是什么？主要加工表面是那个？ （2）进行什么热处理？机械加工工序的安排？ 小组讨论。 2、指名回答问题： 图中零件材料是什么？根据学生回答指出。并根据零件材料说明应进行何种热处理。 图中零件主要加工表面是那个？根据学生回答指出并讲解，让学生直观感知和
加深理解。 根据零件主要加工表面的技术要求，详细讲解零件所要进行的机械加工工序，再进行分析、比较。 根据机械加工工序，合理安排热处理工序，再安排辅助工序。小组讨论，最后确定该零件加工工艺路线。 3、小结： 1）机器零件加工工艺路线，应合理科学的安排。只有这样才能保证机器零件的加工质量。 2）机器零件加工工艺路线，包含机械加工，热处理工序和辅助加工工序。各工序都是穿插进行的，应根据零件的材料、技术要求妥善安排，机器零件加工工艺路线也不是唯一的。
工艺规程的编制 在生产过程中，按本单位的实际情况，根据设计技术要求，需制订必要的工艺规程，以利于安排生产。零件加工的工艺规程就是一系列不同工序的综合。由于生产规模与具体情况的不同，对于同一零件的加工工序可能有很多方案，应当根据具体条件，采用其中最完善（从工艺上来说）和最经济的一个方案。 1.影响编制工艺规程的因素 a)生产规模是决定生产类型（单件、成批、大量）的主要因素，也是设备、 工夹量具、机械化与自动化程度等的选择。 b)制造零件所用到坯料或型材的形状、尺寸和精度是选择加工总余量和加 工过程中头几道工序的决定因素。 c)零件材料的性质（硬度、可加工性、热处理在工艺路线中排列的先后等） 是决定热处理工序和选用设备及切削用量的依据。 d)零件制造的精度，包括尺寸公差、形位公差以及零件图上所指定或技术 条件中所补充指定的要求。 e)零件的表面粗糙度是决定表面上光精加工工序的类别和次数的主要因素。 f)特殊的限制条件，例如工厂的设备和用具的条件等。 g)编制的加工规程要在既定生产规程与生产条件下达到多、快、好、省的 生产效果。 2.工艺规程的编制步骤 工艺规程的编制，可按下列步骤进行：
a)研究零件图及技术条件。如零件复杂、要求高，要先详细熟悉在机器中 所起的作用、加工材料及热处理方法、毛坯的类别与尺寸，并分析对零件制造精度的要求，然后选择毛基面，再选择零件重要表面加工所需的光基面。 b)加工的毛基面和光基面确定后，最初工序（由毛基面所决定的）和主要 表面的粗、精加工工序（在某种程度上由光基面决定）已很荆楚，也就能编制零件加工的顺序。 c)分析已加工表面的粗糙度，在已拟的加工顺序中增添光精加工的工序。 d)根据加工时的便利情况，确定并排列零件上下不重要表面加工所需的所 有其余工序（带自由尺寸的表面的加工、减小零件质量的工序、改善外观的工序、不重要的螺纹切削等）。这一类次要工序往往分配在已设计了的主要工序之间（或与之合并），也有时放在加工过程的末尾。这时必须考虑到，由于次要工序排列不当，在执行中会有损坏精密加工后的重要表面的可能性。 e)如果有限制加工工艺规程选择的特殊条件存在，通常要作补充说明，以 修正加工的顺序。 f)确定每一工序所需的机床和工具，填写工艺卡和工序卡。 g)详细拟定工艺规程时，必须进行全部加工时间的标定和单件加工时间的 结算，并计算每一工序所需的机床台数。但有时把已拟订好的工艺规程作某些修正（例如个别机床任务太少，则有必要把几个单独工序合并成一个工序）。
零件加工工艺的编制 课程作业 班级: 数控1班 姓名: 学号: 前言 机械制造工艺学课程设计，是以切削理论为基础、制造工艺为主线、兼顾工
艺装备知识的机械制造技术基本能力的培养；是综合运用机械制造技术的基本知识、基本理论和基本技能，分析和解决实际工程问题的一个重要教学环节；是对学生运用所掌握的“机械制造技术基础”知识及相关知识的一次全面训练。 机械制造技术基础课程设计，是以机械制造工艺及工艺装备为内容进行的设计。即以所选择的一个中等复杂程度的中小型机械零件为对象，编制其机械加工工艺规程，并对其中某一工序进行机床专用夹具设计。 机械制造工艺学课程设计是作为未来从事机械制造技术工作的一次基本训练。通过课程设计培养学生制定零件机械加工工艺规程和分析工艺问题的能力，以及设计机床夹具的能力。在设计过程中，我熟悉了有关标准和设计资料，学会使用有关手册和数据库。 1、能熟练运用机械制造工艺学课程中的基本理论以及在生产实践中学到的实践知识，正确地解决一个零件在加工中的定位、夹紧以及工艺路线安排、工艺尺寸确定等问题，保证零件的加工质量。 2、提高结构设计能力。学生通过夹具设计的训练，应获得根据被加工零件的加工要求，设计出高效、省力、经济合理而能保证加工质量的夹具的能力。 3、学会使用手册、图表及数据库资料。掌握与本设计有关的各种资料的名称、出处，能够做到熟练运用。 就我个人而言，我希望能通过这次课程设计锻炼自己分析问题、解决问题的能力，为今后所从事的工作打下基础。 由于本人能力有限，设计尚有许多不足之处，可请各位老师给予批评指正。 目录 前言 (1) 零件的工艺分析 (4)
轴类零件机械加工工艺编制 目录 ●任务１分析轴类零件的技术资料 ●任务2确定轴类零件的生产类型 ●任务3 轴类零件的毛坯类型及其制造方法 ●任务4 选择轴类零件的定位基准和加工装备 ●任务5 拟定轴类零件的工艺路线 ●任务6 设计轴类零件的加工工序 ●任务7 填写轴类零件的机械加工工艺文件
任务一分析轴类零件的技术资料 教学目标 ?能看懂轴类零件的零件图和装配图。 ?明确轴类零件在产品中的作用，找出其主要技术要求. ?确定轴类零件的加工关键表面. 一、看懂传动轴的结构形状 如图1,零件图采用了主视图和移出断面图表达其形状结构。从主视图可以看出,主体由四段不同直径的回转体组成,有轴颈、轴肩、键槽、挡圈槽、倒角等结构,由此可以想象出传动轴的结构形状,如图2所示。 二、明确传动轴的装配位置和作用 传动轴起支承齿轮、传递扭矩的作用. ? 30js６外圆(轴颈)用于安装轴承,? 35轴肩起轴承向定位作用。?２5ｆ7、? 2５g６及轴肩用于安装齿轮及齿轮的轴向定位,采用普通平键连接,左轴端有挡圈槽,用于安装挡圈，以轴向固定齿轮。 三、确定传动轴的加工关键表面 (1)? 25f7、?25g6轴头? 3０js6轴颈都具有较高的尺寸精度（IT７，ＩT６)和位 置精度(同轴度为0。02)要求,表面粗糙度(Ra值分别为0。８um)?35轴肩两端面虽然尺寸精度要求不高，但表面精糙度要求较高（Ra值为1.6ｕm)；所以?2５f7、?25g６轴头、? ３０ｊs6轴颈及? 3５轴肩两端均为加工关键表面。 (２）键糟侧面(宽度)尺寸精度(IT9）要求中等,位置精度（对称度0。0１2)要求比较高,表面粗糙度(Rａ值为３.2ｕm）要求中等，键槽底面（深度)尺寸精度（２1)和表面精糙度（Ra值为6。3um）要求都较低，所以键槽是次要加工表面。 (3）挡圈槽、左、右、倒角等其余表面，尺寸及表面精度要求都比较低,均为次要加工表面,如图3所示. 任务2 确定轴类零件的生产类型 教学目标 ?掌握轴类零件生产纲领的计算方法。
第一讲 机械加工工艺的编制 学习指南： 通过本次课程设计能熟练运用机械制造工艺课程中的基本理论，正确地解决一个零件在加工中的定位、夹紧以及工艺路线安排、工艺尺寸确定等问题，保证零件的加工质量。 本讲首先介绍了机械加工工艺编制的基本步骤，然后将每个步骤详细地进行讲解。 一、 加工工艺规程的设计步骤 分析零件工作图和产品装配图 阅读零件工作图和产品装配图，以了解产品的用途、性能及工作条件，明确零件在产品中的位置、功用及其主要的技术要求。 工艺审查 主要审查零件图上的视图、尺寸和技术要求是否完整、正确；分析各项技术要求制订的依据，找出其中的主要技术要求和关键技术问题，以便在设计工艺规程时采取措施予以保证；审查零件的结构工艺性。 确定毛坯的种类及其制造方法 常用的机械零件的毛坯有铸件、锻件、焊接件、型材、冲压件以及粉末冶金、成型轧制件等。零件的毛坯种类有的已在图纸上明确，如焊接件。有的随着零件材料的选定而确定，如选用铸铁、铸钢、青铜、铸铝等，此时毛坯必为铸件，且除了形状简单的小尺寸零件选用铸造型材外，均选用单件造型铸件。对于材料为结构钢的零件，除了重要零件如曲轴、连杆明确是锻件外，大多数只规定了材料及其热处理要求，这就需要工艺规程设计人员根据零件的作用、尺寸和结构形状来确定毛坯种类。如作用一般的阶梯轴，若各阶梯的直径差较小，则可直接以圆棒料作毛坯；重要的轴或直径差大的阶梯轴，为了减少材料消耗和切削加工量，则宜采用锻件毛坯。常用毛坯的特点及适用范围见 表1-1 。 拟定机械加工工艺路线 这是机械加工工艺规程设计的核心部分，其主要内容有：选择定位基准；确定加工方法；安排加工顺序以及安排热处理、检验和其它工序等。 确定各工序所需的机床和工艺装备 工艺装备包括夹具、刀具、量具、辅具等。机床和工艺装备的选择应在满足零件加工工艺的需要和可靠地保证零件加工质量的前提下，与生产批量和生产节拍相适应，并应优先考虑采用标准化的工艺装备和充分利用现有条件，以降低生产准备费用。对必须改装或重新设计的专用机床、专用或成组工艺装备，应在进行经济性分析和论证的基础上提出设计任务书。确定各工序的加工余量，计算工序尺寸和公差。 确定切削用量。 确定各工序工时定额。 评价工艺路线对所制定的工艺方案应进行技术经济分析，并应对多种工艺方案进行比较，或采用优化方法，以确定出最优工艺方案。 填写或打印工艺文件。 一、分析零件技术要求及其合理性 一般将零件图上提出的有关技术要求分为以下几类： 1. 加工表面本身的要求（尺寸精度、形状和粗糙度）：据其选择加工方法、加工步序； 2. 表面之间的相对位置精度（包括位置尺寸、位置精度）：与基准的选择有关； 3. 表面质量及镀层要求：涉及选材及热处理工艺的确定； 4. 其它要求：如等重、平衡、探伤等。
机械加工工艺流程是工件或者零件制造加工的步骤，采用机械加工的方法，直接改变毛坯的形状、尺寸和表面质量等，使其成为零件的过程称为机械加工工艺流程。 比如一个普通零件的加工工艺流程是粗加工-精加工-装配-检验-包装，就是个加工的笼统的流程。 总的来说，工艺流程是纲领，加工工艺是每个步骤的详细参数，工艺规程是某个厂根据实际情况编写的特定的加工工艺。 工艺规程是组成技术文件的主要部分，是工艺装备、材料定额、工时定额设计与计算的主要依据，是直接指导工人操作的生产法规，它对产品成本、劳动生产率、原材料消耗有直接关系。工艺规程编制的质量高低。对保证产品质量第一起着重要作用。 一个同样要求的零件，可以采用几种不同的工艺过程来加工，但其中总有一种工艺过程在给定的条件下是最合理的，人们把工艺过程的有关内容用文件的形式固定下来，用以指导生产，这个文件称为“工艺规程”。 **主要内容** 1．产品特征，质量标准。 2．原材料、辅助原料特征及用于生产应符合的质量标准。 3．生产工艺流程。 4．主要工艺技术条件、半成品质量标准。 5．生产工艺主要工作要点。 6．主要技术经济指标和成品质量指标的检查项目及次数。 7．工艺技术指标的检查项目及次数。 8．专用器材特征及质量标准。 **形式** 企业所用工艺规程的具体格式虽不统一，但内容大同小异。一般来说，工艺规程的形式按其内容详细程度，可分为以下几种； 工艺过程卡 这是一种最简单和最基本的工艺规程形式，它对零件制造全过程作出粗略的描述。卡片按零件编写，标明零件加工路线、各工序采用的设备和主要工装以及工时定额。 工艺卡 它一般是按零件的工艺阶段分车间、分零件编写，包括工艺过程卡的全部内容，只是更详细地说明了零件的加工步骤。卡片上对毛坯性质、加工顺序、各工序所需设备、工艺装备的要求、切削用量、检验工具及方法、工时定额都作出具体规定，有时还需附有零件草图。
Q/FH.G0907-2003 工艺路线编制规则 1范围： 本标准规定了工艺路线编写的原则，主加工单位确定的原则及其任务、工艺路线格式及批准。 本标准适用于我厂计划内的军品及名品。 2规范性引用文件 Q/FH6.2-2003工艺文件管理制度第2部分管理用工艺文件格式及其填写规则 Q/FH6.3-2003工艺文件管理制度第3部分工艺规程格式及编制规则 3管理职能 3.1工艺路线是指产品或零、部件再生产过程中，有毛坯准备到成品包装入库的全部工艺过程的先后顺序。 3.2工艺路线是设计工艺规程的依据，是提高产品质量，提高生产率，均衡组织生产，合理利用设备的保证。 3.3工艺路线规定了产品生产分工，协调单位之间周转关系，它是领取图纸、技术文件及下达生产任务的依据。 4一般规定 4.1工艺路线以产品为单位拟制，综合反映零、部件从加工到装配的全部工艺过程，着重反映每个件号在各加工中的流程顺序。
4.2工艺路线内容栏，按工艺方法填写，写下料，机加，热处理等字样，一般不写分厂代号。其表面处理涵义为电镀、涂覆其含义为喷涂4.3设计工艺路线时，要严格依据工艺总方案的规定，保证工艺路线合理、正确。 4.4下列情况不编入工艺路线： a)毛坯生产过程和主制单位内部加工的各工序； b)零、组件排故。 4.5重要件、关键件的标记 4.5.1关键件在零件序号前用“关键件”表示，可用“G”表示，并用粗方格括起来。 4.5.2重要件在零件序号前用“重要件”表示，可用“Z”表示，并用粗方格括起来。 5主加工单位确定原则及任务 5.1主加工单位是指产品零、部、组件在制造、装配、封存的全部工艺过程中，担负主要任务的单位。 5.2主加工单位确定的基本原则：“总体为主”、“质量控制”、“工序集中”三原则。 5.2.1依据全厂的工艺布局和分厂职责一般以负责产品总装任务的分厂担任该产品零部件的主加工单位。 5.2.2从控制质量的角度考虑，选择能有效的控制零部件生产过程中的质量关键的单位担任主加工单位。 5.2.3从工作量的角度考虑，以承担该零部件加工工序最集中，工作
（）1、工件在夹具中定位的任务是使同一工序中的一批工件都能在夹具中占据正确的位置。 （）2、正确、合理地选择工件的定位与夹紧方式，是保证零件加工精度的必要条件。（）3、为保证不加工表面与加工表面之间的相对位置要求，一般应选择加工表面的毛坯面为粗基准。 （）4、平面磨削的加工质量比刨削和铣削都高，还可以加工淬硬零件。 （）5、中间工序尺寸公差常按各自采用的加工方法所对应的加工经济精度来确定。（）6、选择外圆表面加工方法，一般先根据表面的精度和粗糙度选定精加工前加工方法。 （）7、粗基准可以在第一道工序中使用多次，一般能重复使用。 （）8、车削螺纹时能使用恒线切削速度功能。 （）9、辅助支承可以增加工件的刚性和稳定性，又可以起定位作用。 （）10、编制工艺规程不需考虑现有生产条件。 （）11、选择切槽切削用量时，进给量一般取0.05～0.3mm/r。 （）12、装夹薄壁零件时，必须采取相应的预防纠正措施，以免加工时引起变形。（）13、表面质量应包括表面层几何形状偏差和表面层物理力学性能两方面。 （）14、车削薄壁类零件时，车削刀具应选择较小的主偏角。 （）15、端铣平面的平面度决定于铣床主轴轴线与进给方向的垂直度。 （）16.刀具补偿工能包括刀补的建立、刀补的执行和刀补的取消三个阶段。 （）17、M代码可以分为模态G代码和非模态G代码。 （）18、G00指令中的快进速度不能用程序规定。（）19、螺纹指令G32 X41.0 W-43.0 F1.5是以每分钟1.5mm的速度加工螺纹。 （）20、标准立铣刀的螺旋角为40°～45°（粗齿）和30°～35°（细齿）。 （）21、FANUC OM系统中，攻左旋螺纹循环指令为G84。 （）22、切削小于或等于180度的圆弧，其圆弧半径“R”值要使用正值。 （）23、在补偿状态下，铣刀的直线移动量及铣削内侧圆弧的半径值不得大于刀具半径。（）24、指令刀尖半径补偿G41或G42后，刀具路径必须是单向递增或单向递减。 （）25、建立刀具补偿后，不能两个程序段无补偿面的移动指令，否则过切。 （）1、机械加工中使用最多的刀具材料为。 A、工具钢 B、硬质合金 C、陶瓷 D、高速钢 E、高速钢与硬质合金（）2、在车床上加工某零件，先加工其一端，再调头加工另一端，这应是。 A、两个工序 B、两个工步 C、两次装夹 D、两个工位 （）3、在车床上加工轴，用三爪卡盘安装工件，相对夹持较长，它的定位是 A、六点定位 B、五点定位 C、四点定位 D、三点定位 （）4、影响刀具磨损的最为显著的因素是。 A、切削速度 B、背吃刀量 C、进给量 D、刀具主偏角 （）5、编制加工中心的程序时应正确选择( )的位置，避免刀具交换时与工件或夹具产生干涉。 A、对刀点 B、工件原点 C、参考点 D、换刀点 （）6、粗车加工表面，经济精度等级（IT）可达。 A、11～13 B、9～10 C、7～8 D、6～7 （）7、为改善材料切削性能而进行的热处理工序（如退火、正火），通常安排在哪进行。 A、切削加工之前 B、磨削加工之前 C、粗加工后，精加工前 （）8、加工箱体类零件时常选用一面两孔作定位基准，这种方法一般符合 A、基准重合原则 B、基准统一原则 C、互为基准原则 D、自为基准原则（）9、( )是指机床上一个固定不变的极限点。 A、机床原点 B、工件原点 C、换刀点 D、对刀点 （）10、零件上孔径大于40mm的孔，精度要求为IT10，通常采用的加工方案为： A、钻-镗 B、钻-铰 C、钻-拉 D、钻-扩-磨 （）11、机床坐标系判定方法采用右手直角的笛卡尔坐标系。减小工件和刀具距离的方向是( )。 A、负方向 B、正方向 C、任意方向 D、条件不足不确定（）12、安装在机床主轴上，能带动工件一起旋转的夹具是。 A、钻床夹具 B、车床夹具 C、铣床夹具 D、镗床夹具 （）13、为消除一般机床主轴箱体铸件的内应力，应采用
机械加工工艺规程包括以下内容：工件加工的工艺路线、各工序的具体内容及所用的设备和工装设备、工件的检验项目及检验方法、切削用量、时间定额等。 制订工艺规程的原始资料： 1.产品全套装配图和零件图； 2.产品验收的质量标准； 3.产品的生产纲领（年产量）； 4.毛坯资料； 5.本厂的生产条件； 6.国内外先进工艺及生产技术发展情况； 7.有关的工艺手册及图册。 制订工艺规程的步骤： 1、计算年生产纲领，确定生产类型； 生产纲领计算——N=Qn(1+a%+b%)——N为零件的年生产纲领，件/年；Q为产品的年生产纲领，台/年；n为每台产品中该零件的数量；a为备料的百分率；b为废品百分率。 生产类型——单件生产、批量生产、大量生产。 2、分析零件图及产品装配图，对零件进行工艺分析； 承类零件 零件表面的组成及基本类型套类零件 零件结构分析主要表面和次要表面区分箱体类零件 零件的结构工艺性叉架类零件 零件的工艺 分析包括： 加工表面的尺寸精度、形状精度和表面粗糙度 零件技术要求分析各加工表面之间的相互位置精度 工件的热处理和其它要求，如动平衡、去磁电镀等 3、选择毛坯； 木模手工造型适用于单件小批生产或大型零件 砂型铸造 1. 铸件金属模机器造型适用于大批量生产的中小型铸件 压力铸造（特殊铸造适用于 特殊铸造离心铸造少量质量要求较 熔模铸造高的小型铸件） 手工锻打小型毛坯单件、小批 自由锻造锻件机械锤锻中型毛坯生产，以及 2. 锻件压力机压锻大型毛坯大型锻件 模锻件适用于大批量中小型锻件 毛坯的种类 热轧精度低、价格低 3. 型材 冷拉精度高、价格高、大批量生产、自动机床加工 4. 焊接件制造简单、周期短、抗振性差、变形大、加工前要时效处理 5. 冲压件 6. 冷挤压件 7. 粉末冶金
机械加工工艺编制基准知识 1、机械加工工艺总过程 机械加工工艺的整个过程是指用机械加工方法改变毛坯形状、尺寸、相对位置和性质，使其成为零件的全过程。在实施对零件的机械加工前，须对零件机械加工工艺的总过程、方法和加工目标进行规划，即制定机械加工工艺规程，制定它的主要依据是产品图纸、生产纲领生产类型、现场加工设备及生产条件等。制定机械加工工艺规程一般需要如下过程： 1.1 分析加工零件 要设计零件生产加工工艺，首先要分析加工零件，充分领会产品的使用要求和设计要求，在此基础上，进一步审查零件制造工艺的可行性和加工的经济性。 1.2毛坯的选择 在选择毛坯的种类和制造方法时，全面考虑机械加工成本和毛坯制造成本，以达到降低零件生产总成本的目的。 1.3 拟订零件机械加工工艺总过程： 选择零件的加工方法，划分工艺过程的各工序组成，安排各加工工序的先后顺序和工序的相互组合等。 1.4工序设计 对工艺过程中包含的各工序进行详细的工艺设计。 2、加工零件的分析 制定零件加工工艺规程前准确地对加工零件分析，是制定零件加工工艺规程的前提； 分析零件包括： 1）分析产品的装配图和零件的工作图，熟悉该产品的用途、性能及工作条件，明确被加工零件在产品中的装配位置和作用，进而了解零件上各项技术要 求制订的依据，找出主意技术要求和加工关键，以便在拟订工艺规程时采
用适当的工艺措施加以保证，对图纸的完整性、技术要求的合理性以及材 料选择是否适当等提出意见。 2）审查零件结构的工艺性，在充分领会产品的使用要求的前提下，审查零件制造工艺的可行性和加工的经济性，遇到工艺问题与设计问题有矛盾时与 设计人员共同磋商解决方法 2.1 分析零件要求 在通过分析装配图、产品说明书等熟悉了零件在产品中的作用、位置、装配关系和工作条件的前提下，还应进一步分析零件图样，搞清楚零件主要和关键的技术要求，理解各项技术要求对零件装配质量和使用性能的影响。 1）检查零件图的完整性和正确性 在了解零件形状和结构之后，应检查零件视图是否正确，尺寸、公差以及技术要求的标注是否齐全，合理等。 2）零件的技术要求 零件的技术要求包括下列几个方面：加工表面的尺寸精度，主要加工表面的形状精度，位置精度，粗糙度、热处理等，其它要求：动平衡、未标注圆角、或倒角、去毛刺、毛坯等要求。 要注意分析这些要求保证使用性能的前提下是否经济合理，在现有生产条件下能否实现；特别要分析主要表面技术要求，因为主要表面的加工确定了零件工艺过程的大致轮廓。 3）零件的材料分析 分析所提供的毛坯材质本身的机械性能和热处理状态，毛坯的品质和被加工部位的材料硬度，判断工件材料加工的难易程度，为选择刀具材料和切削用量提供依据。 4）分析尺寸标准 分析零件图上的重要尺寸的设计基础，是否在加工时用作工艺基准，分析设计
（ )1、工件在夹具中定位的任务是使同一工序中的一批工件都能在夹具中占据正确的位置。 （)2、正确、合理地选择工件的定位与夹紧方式，是保证零件加工精度的必要条件。(）3、为保证不加工表面与加工表面之间的相对位置要求,一般应选择加工表面的毛坯面为粗基准。 （)４、平面磨削的加工质量比刨削和铣削都高,还可以加工淬硬零件。 ()５、中间工序尺寸公差常按各自采用的加工方法所对应的加工经济精度来确定。（）６、选择外圆表面加工方法，一般先根据表面的精度和粗糙度选定精加工前加工方法。 ( )7、粗基准可以在第一道工序中使用多次,一般能重复使用。 （ )8、车削螺纹时能使用恒线切削速度功能。 （）9、辅助支承可以增加工件的刚性和稳定性,又可以起定位作用。 ( ）10、编制工艺规程不需考虑现有生产条件。 ()11、选择切槽切削用量时，进给量一般取0.０5～０.3mm/r。 ( ）1２、装夹薄壁零件时，必须采取相应的预防纠正措施，以免加工时引起变形。( )13、表面质量应包括表面层几何形状偏差和表面层物理力学性能两方面。 （）14、车削薄壁类零件时，车削刀具应选择较小的主偏角。 （）15、端铣平面的平面度决定于铣床主轴轴线与进给方向的垂直度。 (）16.刀具补偿工能包括刀补的建立、刀补的执行和刀补的取消三个阶段。 ( ）17、M代码可以分为模态Ｇ代码和非模态G代码。 ()18、Ｇ00指令中的快进速度不能用程序规定。( )1９、螺纹指令G32 Ｘ41.0 W－４3.0 Ｆ1.5是以每分钟1．5mm的速度加工螺纹。（)20、标准立铣刀的螺旋角为４０°~４５°（粗齿)和30°～3５°(细齿）。 （）21、FAＮUＣOＭ系统中，攻左旋螺纹循环指令为G84。 （）2２、切削小于或等于180度的圆弧,其圆弧半径“R”值要使用正值。 ( )２３、在补偿状态下，铣刀的直线移动量及铣削内侧圆弧的半径值不得大于刀具半径。 ( )2４、指令刀尖半径补偿G4１或G42后，刀具路径必须是单向递增或单向递减。( ）25、建立刀具补偿后，不能两个程序段无补偿面的移动指令,否则过切。 ( ）1、机械加工中使用最多的刀具材料为。 A、工具钢Ｂ、硬质合金Ｃ、陶瓷 D、高速钢E、高速钢与硬质合金( )2、在车床上加工某零件,先加工其一端，再调头加工另一端，这应是。 A、两个工序 B、两个工步 C、两次装夹 D、两个工位 ( ）3、在车床上加工轴，用三爪卡盘安装工件，相对夹持较长，它的定位是 A、六点定位Ｂ、五点定位Ｃ、四点定位 D、三点定位 ( )4、影响刀具磨损的最为显著的因素是。 A、切削速度 B、背吃刀量Ｃ、进给量 D、刀具主偏角 （）5、编制加工中心的程序时应正确选择( )的位置，避免刀具交换时与工件或夹具产生干涉。 A、对刀点 B、工件原点 C、参考点 D、换刀点 （)６、粗车加工表面，经济精度等级(IT）可达。 Ａ、11~13 B、9～1０ C、７~8 D、6~7 ( )7、为改善材料切削性能而进行的热处理工序(如退火、正火)，通常安排在哪进行。 A、切削加工之前 B、磨削加工之前 C、粗加工后，精加工前 （）8、加工箱体类零件时常选用一面两孔作定位基准，这种方法一般符合 A、基准重合原则 B、基准统一原则 C、互为基准原则 D、自为基准原则 ( ）9、( ）是指机床上一个固定不变的极限点。 A、机床原点 B、工件原点 C、换刀点 D、对刀点 ( )10、零件上孔径大于40mm的孔，精度要求为IT10，通常采用的加工方案为： A、钻-镗 B、钻-铰Ｃ、钻-拉 D、钻-扩－磨 （ )１1、机床坐标系判定方法采用右手直角的笛卡尔坐标系。减小工件和刀具距离的方向是( )。?Ａ、负方向Ｂ、正方向 C、任意方向Ｄ、条件不足不确定
例题 在成批生产条件下，加工如例题图所示零件，其机械加工工艺过程如下所述： ⑴在车床上加工整批工件的小端端面、小端外圆（粗车、半精车）、台阶面、退刀槽、小端孔（粗车、精车）、内外倒角； ⑵调头，在同一台车床上加工整批工件的大端端面、大端外圆及倒角； ⑶在立式钻床上利用分度夹具加工四个螺纹孔； ⑷在外圆磨床上粗、精磨1206h 外圆。 试列出其工艺过程的组成，并确定各工序的定位基准，画出各工序的工序简图，用符号标明加工面，标明定位基准面，用数字注明所消除的不定度（自由度）数，其它用文字说明、工艺过程分析到工步。 例题图 解：工序I 车，（见例题解答图a ），一次安装，工步为：(1)车端面； (2)粗车外圆；(3)车台阶面；(4)车退刀槽；(5)粗车孔；(6)半精车外圆； (7)精车孔； (8)外圆倒角； (9)内圆倒角。 工序Ⅱ车（见例题解答图b ），一次安装，工步为：(1)车端面；(2)车外圆；(3)车内孔；(4)倒角。 工序Ⅲ钻（见例题解答图c ），一次安装，4个工位，工步为：(1)钻4个孔；(2)攻4个螺纹孔。 工序Ⅳ磨（见例题解答图d ），一次安装，工步为：(1)粗磨外圆；(2)精磨外圆。 例题 指出例题图零件结构工艺性不合理的地方，并提出改进建议。 例题图 答：例题3. 2图a 底面较大，加工面积较大，加工量较大且不易保证加工质量，建议减少底面加工面的尺寸，如开一通槽。例题图 中孔的位置距直壁的尺寸太小，钻孔时刀具无法切入，安装也不方便，故应该增大其距离。 例 试拟定例题图所示小轴的单件小批生产和大批大量生产的机械加工工艺规程，并分析每种方案的工艺过程组成。 例题图 解：零件的机械加工工艺规程如例题表a 和b 所示。
《机械加工工艺编制》试题（A ） 专业： 机械设计与制造 年级： 2009 试题说明： 1.本试卷满分共计100分，考试时间120分钟。 2.本试卷共3页，六个大题。 一、填空题（共20个空，每空1分，共20分） 1.在制订机械加工工艺过程中， 工序 是构成工艺过程和制定生产计划的基本单元。 2.机械加工工艺过程中，在加工表面和加工刀具都不变的情况下，所连续完成的那部分工序，称为工步。 3.工艺基准是工艺过程中采用的基准，按其作用的不同可以分为工序基准、 定位基准测量基准和装配基准。 4.对于直线尺寸链，且用极值法计算时，封闭环的上偏差等于所有 増环上偏差 之和减去所有 减环下偏差 之和。 5.工件的安装可分为 夹紧 和 定位 两个过程。 6. 大批量生产箱体常采用的定位方式是一面两孔 ，它是属于基准选择原则中的 的 基准统一 原则。 7.在装配尺寸链中， 封闭环 的公差比其他组成环的公差大，为了减小其公差，应尽 量减小尺寸链的环数，这就是在设计中应遵循的 最短路线 原则。 8.加工精度的高低是以有关尺寸、形状和 位置 标准来表示的，通常在生产中，加工精 度的高低是以 加工误差 的大小来表示。 9.我们把工艺系统的误差称为原始误差，按误差性质，其主要包括工艺系统的几何误 差 、 工艺系统受力引起的误差和 工艺系统热变形引起的误差三个方面。 10. 重要的轴类零件的毛坯通常选择 锻件 。 二、单项选择题（共10小题，每小题1分，共10分） 1.当有色金属（如铜、铝等）的轴类零件外圆表面要求尺寸精度较高、表面粗糙度值较 低时，一般只能采用的加工方案为（ C ） A.粗车－精车－磨削 B.粗铣－精铣 C.粗车－精车—超精车 D.粗磨—精磨 2.基准重合原则是指使用被加工表面的（A ）基准作为精基准。 A.设计 B.工序 C.测量 D.装配 3.箱体类零件常采用（ B ）作为统一基准。 A.一面一孔 B.一面两孔 C.两面一孔 D.两面两孔 4.经济加工精度是在（ D ）条件下所能保证的加工精度和表面粗糙度。 A.最不利 B.最佳状态 C.最小成本 D.正常加工 5.为改善材料切削性能而进行的热处理工序（如退火、正火等），通常安排在（A ）进行。 A.切削加工之前 B.切削加工之后 C.磨削加工之前 D.粗加工后、精加工前 6.加工套类零件的定位基准是（ D ）。 A.端面 B.外圆 C.内孔 D.外圆或内孔 7.在车床上加工某零件，先加工其一端，再调头加工另一端，这应是（B ）。 A.两个工序 B.两个工步 C.两次装夹 D.两个工位 8.试指出下列刀具中，哪些刀具的制造误差不会直接影响加工精度（B ）。 A.齿轮滚刀 B.外圆车刀 C.成形铣刀 D.键槽铣刀 9.在大量生产零件时，为了提高机械加工效率，通常加工尺寸精度的获得方法为（ B ）。 A.试切法 B.调整法 C.成形运动法 D.划线找正安装法 10.在车床上用两顶尖装夹车削光轴，加工后检验发现鼓形误差(中间大、两头小)，其最可能的原因是（ D ）。 A ．车床主轴刚度不足 B ．两顶尖刚度不足 C ．刀架刚度不足 D ．工件刚度不足 三、判断题（共10小题，每小题1分，共10分） （错误）1.铰孔可以提高孔的尺寸精度、减小孔表面粗糙度，还可以对孔偏斜进行修正。 （错误）2.在机械加工过程中，冷作硬化与切削力和切削温度有关，与被加工材料无关。 （错误）3.加工原理误差是由于机床几何误差所引起的。 （正确）4.单件小批生产宜选用工序集中原则。 （错误）5.过定位和不完全定位的定位支承点都少于工件六个自由度数，无法保证加工 要求，在夹具设计时要力求避免。 （正确）6.在用正态分布图进行误差分析，如果σ保持不变，改变参数x ，则曲线平移而 不改变形状，这说明 x 的变化反映工艺系统调整，属于常值系统性误差。 （错误）7.表面残余应力对零件疲劳强度影响很大，很容易引起应力集中而发展成疲劳裂 纹，导致零件损坏。 （错误）8.切削过程中，积屑瘤主要发生在低速切削阶段。 密 封 线 内 不 准 答 题
1、轴类、齿轮、箱体类零件常用的材料和毛坯是什么？这三类零件加工的一般工艺路线如 何？圆棒料和锻件 2、生产类型有哪几种？不同生产类型对零件的工艺过程有哪些主要影响？ 3、常用的工艺文件有哪几种？各适用于什么场合？ 4、切削加工工序安排的原则是什么？ 5、试说明划分加工阶段的理由。 答（1）有利于保证加工质量（2）便于合理使用设备（3）便于安排热处理工序和检验工序（4）便于及时发现缺陷避免损伤已加工表面 6、确定加工余量的原则是什么？目前确定加工余量的方法有哪几种？ 7、制定工艺规程的原则是什么？应注意哪些问题？ 答：工艺规程制定的原则是优质、高产、低成本，即在保证产品质量前提下，争取最好的经济效益。（2分）在制订工艺规程时应注意以下问题：(1)技术上的先进性；(2)经济上的合理性；(3)有良好的劳动条件。（3分） 8、简述工艺规程制定的步骤 答1）分析研究产品图纸2）工艺性分析3）选择毛坯4）拟订工艺路线 5）选择设备、工装6）确定工序余量、工序尺寸7）确定切削用量、工时定额 8）技术经济分析9）填写工艺文件 9、细长轴加工的主要工艺措施有哪些。 10、套筒零件加工的技术关键是薄壁变形，简答导致变形的因素及减少套筒零件变形的工艺措施。 导致变形的因素：切削力、切削热、夹紧力、内应力 减少变形的工艺措施： （1）粗精加工分开，以减少切削力、切削热的影响。 （2）尽量采用轴向夹紧，如采用径向夹紧应使径向夹紧力均匀。 （3）热处理工序应放在粗、精加工之间。 11、简答退火、正火、时效处理、调质、淬火、渗氮处理在零件加工工序中的安排。 12、简述切削用量确定的顺序及基本方法 13、工序尺寸及公差的确定方法有哪两种，各适用于什么场合，如何计算。 14、粗基准和精基准的选择原则是什么？ 计算题 1、如图为阶梯零件图，加工表面2时应以设计基准表面3作为定位基准。若加工表面2时以表面1作为定位基准，则设计基准与定位基准不重合。为获得要求的设计尺寸，试画出以表面1为定位基准加工表面2时的工艺尺寸链图，并计算其工序尺寸及偏差。（10分）