04389ntm a2200385 i 4500952013500000952011900135999001700254003000800271005001700279006001900296008004100315020003200356040002300388090002800411100003400439245009900473264003400572264001200606300005800618336002100676336002100697337002500718337002300743338002300766338003000789347002400819500005000843502007100893504004000964520286201004610006503866650004503931650001103976942001603987  00102lcc40718REFa10000b10000d2022-02-22l0oFSTI .A45 2020 r ThesispT000001399r2022-05-19 00:00:00t1w2022-02-22yTHESIS  00102lcc40718REFa10000b10000d2022-02-22l0oCD12866pT000001400r2023-01-30 00:00:00t1w2022-02-22yTHESIS  c96757d96763MY-KuUP20251125110018.0t||||fr|||| 000 0 220222s2020    my a|||fr|||| 000 0 eng d  aTHE0009116(Local)qhardback  aUMPbengcUMPerda  aFSTI .A45 2020 r Thesis1 aAmira Alia Zulkifli,eauthor.10aIsolation of kistomin milk coagulant from calloselasma rhodostoma venom /cAmira Alia Zulkifli 1aKuantan, Pahang :bUMP,c2020 4c© 2020  axiv, 130 pages :billustrations ;c30 cm. +e1 CD-ROM  atext2rdacontent  atext2rdacontent  aunmediated2rdamedia  acomputer2rdamedia  avolume2rdacarrier  acomputer disc2rdacarrier  atext filebPDF2rda  aFaculty of Industrial Sciences and Technology  aThesis (Master of Science ) -- Universiti Malaysia Pahang – 2020  aIncludes bibliographical references3 aRennet is an enzyme used in the milk  clotting  process for cheese production. Rennet is  produced  in  the  stomach  of  ruminant  mammal  especially  calf.  However,  increasing  demand for cheese with ongoing research for innovative cheese products has encouraged  the  researchers  to  explore  the  other  new  sources  of  milk  clotting  protease.  The  new  sources of milk clotting protease include animal, plant, microbes and fungi have been  investigated. Snake venom contains  a  rich and good source of protease that needs  to be  explored  as well for its potential in milk  clotting. The objectives of this study were to  screen eleven different species of snake venoms, to isolate the protease enzyme from the  most potential venom capable to  coagulate milk  using  chromatography techniques, to  determine the enzymes milk  clotting  parameters and compare with a commercial milk  clotting protease.  The results revealed that a  crude venom from  Calloselasma rhodostoma showed the fastest  clotting  time  that is 2.83±0.17 minutes  and was chosen for further  purification  process.  The  milk  clotting  enzyme  was  purified  by  HiTrap  SP  FF  ionexchange chromatography and further separated using HiPrep 26/60 Sephacryl S 200 HR  size exclusion column. It was purified 4.41±0.06 fold and exhibited recovery activity of  54.34  %.  SDS-PAGE  analysis  indicated  a  single  band  with  a  molecular  mass  of  approximately 26 kDa.  The purified protease was completely inhibited by EDTA and  1,10 phenanthroline revealing to be a metalloprotease SVMPs P-I Kistomin confirmed  by Mass Spectrometry analysis. The specific activity of kistomin is  62.20  (U/mg). The  milk clotting activity (MCA) of kistomin under optimum conditions, was 810.44±42.45  (SU/mL)  and  the  PA  of  kistomin  was  1.39±0.01  (U/mL),  resulted  in  high  ratio  of  MCA/PA value of 585.05. The clotting  activity of kistomin on milk was the highest at  0.76  mg/mL  kistomin  concentration,  8  %  (w/v)  of  calcium  chloride  concentration,  temperature of 48 °C and stable over wide range of pH 5-7 with the peak of pH 6.5. The  addition  of  Ba2+,  Mn2+and  Ca2+ions  as  cofactor  significantly  increased  the  enzyme  activity but inhibited by Hg2+, Pb2+and Fe2+ions. The Km value of kistomin on casein is  1.153±0.08 mg/mL. The low Km value of kistomin on casein showed it has high affinity  to casein. Kistomin promoted extensive cleavage of kappa casein and low level of beta  casein hydrolysis.  From  this  preliminary  study, it can be  concluded that  kistomin has  potential  to  be  a  coagulant  in  the  dairy  industry.  However,  due  to  the  source  of  the  protease  being from a snake and always been considered lethal by people in general,  a  thorough safety investigation warranted before been utilised in any industry.20aFaculty of Industrial Sciences and TechnologyxDissertations 0aUniversities and collegesxDissertations 0aTheses  2lcccTHESIS