ISOLATION AND PURIFICATION OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE FROM THE GUT OF RHINOCEROS LARVA (Oryctes rhinoceros)

CHAPTER ONE


          1.0. INTRODUCTION AND LITERATURE REVIEW

 

1.1. INTRODUCTION


    One of the major metabolic enzymes that have gained so much interest of

scientists is 3-Mercaptopyruvate sulfurtransferase (3-MST). This enzyme occurs

widely in nature (Bordo, 2002 and Jarabak, 1981).


    It  has  been  reported  in  several  organisms  ranging  from humans  to  rats,

fishes and insects. It is a mitochondrial enzyme which has been concerned in the

detoxification of cyanide, a potent toxin of the mitochondrial respiratory chain

(Nelson et  al.,  2000).  Among  the  several  metabolic  enzymes  that  carry  out

xenobiotic  detoxification,  3-mercaptopyruvate  sulfurtransferase  is  of  utmost

importance.


        3-mercaptopyruvate  sulfurtransferase functions  in  the  detoxifications  of

cyanide; mediation of sulfur ion transfer to cyanide or to other thiol compounds.

(Vanden et al., 1967). It is also required for the biosynthesis of thiosulfate. In

combination with cysteine aminotransferase, it contributes to the catabolism of

cysteine and it is important in generating hydrogen sulphide in the brain, retina

and vascular endothelial cells (Shibuya et al., 2009). It also acquired different

functions  such  as  a  redox  regulation  (maintenance  of  cellular  redox

homeostasis)  and  defense against  oxidative  stress,  in  the  atmosphere  under

oxidizing conditions Nagahara et al (2005).


      Hydrogen sulphide  (H2S)  is  an  important  synaptic  modulator,  signalling

molecule, smooth muscle contractor and neuroprotectant (Hosoki et al., 1997).

Its  production  by  the  3-mercaptopyruvate  sulfurtransferase  and  cysteine

aminotransferase pathways is regulated by calcium ions (Hosoki et al., 1997).
      Organisms  that  are  exposed  to  cyanide  poisoning  usually  have  this

enzyme  in  them.  This  could  be  in  food as  in  the  cyanogenic glucosides being

consumed. It has been studied from variety of sources, which include bacteria,

yeasts, plants, and animals (Marcus Wischik, 1998). 


      Cyanide could be released into the bark of trees as a defence mechanism.

There are array of defensive compounds that make their parts (leaves, flowers,

stems,  roots  and  fruits)  distasteful  or  poisonous  to  predators.  In  response,

however,  the  animals  that  feed  on  them  have  evolved  over  successive

generations a range of measures to overcome these compounds and can eat the

plant  safely.  The  tree  trunk  offers  a  clear  example  of  the  variety  of  defences

available to plants (Marcus Wischik, 1998).


      Oryctes rhinoceros larva is one of the organisms that are also exposed to

cyanide toxicity because of the environment they are found.


1.2. 3-MERCAPTOPYRUVATE SULFURTRANSFERASE


        3-Mercaptopyruvate  sulfurtransferase  (EC.  2.8.1.2),  is  a  member  of  the

group,  Sulfurtransferases  (EC  2.8.1.1 – 5),  which  are  widely  distributed

enzymes of prokaryotes and eukaryotes (Bordo and Bork, 2002).


        3-Mercaptopyruvate  Sulfurtransferase  is  an  enzyme  that  is  part  of  the

cysteine  catabolic pathway.  The  enzyme  catalyzes  the  conversion  3-

mercaptopyruvate to pyruvate and H2S (Shibuya et al., 2009). The deficiency of

this enzyme will result in elevated urine concentrations of 3-mercaptopyruvate

as  well  as  of  3-mercaptolactate,  both  in  the  form  of  disulfides  with  cysteine

(Crawhall et al., 1969). It catalyzes the chemical reaction:


3-mercaptopyruvate + cyanide à  pyruvate + thiocyanate


3-mercaptopyruvate + thiol   à   pyruvate + hydrogen sulphide (Sorbo 1957).
    It  transfers  sulfur-containing  groups  and  participates  in  cysteine

metabolism (Shibuya et al., 2013). This enzyme catalyzes the transfer of sulfane

sulphur from a donor molecule, such as thiosulfate or 3- mercaptopyruvate, to a

nucleophile acceptor, such as cyanide or mercptoethanol. 3-mercaptopyruvate is

the  known  sulphur-donor  substrate  for  3-mercaptopyruvate  sulfurtransferase

(Porter & Baskin, 1995).


        3-mercaptopyruvate  sulfurtransferase  is  believed  to  function  in  the

endogenous  cyanide  (CN)  detoxification  system  because  it  is  capable  of

transferring sulphur from 3-mercaptopyruvate (3-MP) to cyanide (CN), forming

the  less  toxic  thiocyanate  (SCN) (Hylin  and  Wood,  1959). It is  an  important

enzyme  for  the  synthesis  of  hydrogen  sulphide  (H2S)  in  the  brain  (Shibuya et

al., 2009).


      The  systematic  name  of  this  enzyme  class  is 3-mercaptopyruvate:

cyanide  sulfurtransferase.  It  is  also  called beta-mercaptopyruvate

sulfurtransferase (Vachek and  Wood,  1972). It  is  one  of  three  known  H2S

producing  enzymes  in  the  body (Hylin  and  Wood,  1959).  It  is  primarily

localised in the mitochondria (Cipollone et al., 2008). 


      The expression levels of 3-MST in the brain during the fetal and postnatal

periods are higher than those in the adult brain (unpublished data) although the

promoter  region  shows  characteristics  of  a  typical  housekeeping  gene

(Nagahara et al., 2004). The observation is supported by the finding that 3-MST

expression  in  the  cerebellum  is  decreased  during  the  adult  period  (Shibuya et

al., 2013). On the other hand, its expression level in the lung decreases from the

perinatal period. These facts suggest that 3-MST could function in the fetal and

postnatal brain. It was reported that serotonin signaling via the 5-HT1A receptor

in  the  brain  during  the  early  developmental  stage  plays  a  critical  role  in  the
establishment  of  innate  anxiety  during  the  early  developmental  stage

(Richardson-Jones et al., 2011).


    In  rat,  3-MST  possesses  2  redox-sensing  molecular  switches  (Nagahara

and  Katayama,  2005).  A  catalytic-site  cysteine  and  an  intersubunit  disulfide

bond serve as a thioredoxin-specific molecular switch (Nagahara et al., 2007).

The  intermolecular  switch  is  not  observed  in  prokaryotes  and  plants,  which

emerged into the atmosphere under reducing conditions (Nagahara, 2013). As a

result, it acquired different functions such as a redox regulation (maintenance of

cellular  redox  homeostasis)  and  defense  against  oxidative  stress,  in  the

atmosphere under oxidizing conditions (Nagahara et al., 2005).


      Moreover,  3-MST  can  produce  H2S  (or  HS−)  as  a  biofactor  (Shibuya et

al.,  2009),  which  cystathionine  β-synthase  and  cystathionine  γ-lyase  also  can

generate  (Abe  and  Kimura,  1996).  Interestingly  3-MST  can  uniquely  produce

SOx in  the  redox  cycle  of  persulfide  formed  at  the  low-redox catalytic-site

cysteine (Nagahara et al., 2012). As an alternate hypothesis on the pathogenesis

of the symptoms, H2S (or HS−) and/or SOx could suppress anxiety-like behavior,

and therefore, defects in these molecules could increase anxiety-like behavior.

However,  no  microanalysis  method  has  been  established  to  quantify  H2S  (or

HS−) and SOx at the physiological level (Ampola et al., 1969).


      MCDU  was  first  recognized  and  reported  in  1968  as  an  inherited

metabolic  disorder  caused  by  congenital  3-MST  insufficiency  or  deficiency.

Most cases were associated with mental retardation (Ampola et al, 1969) while

the pathogenesis remains unknown.  


      Human  MCDU  was  reported  to  be  associated  with  behavioral

abnormalities, mental retardation (Crawhall, 1985), hypokinetic behaviour, and

grand mal seizures and anomalies (flattened nasal bridge and excessively arched
palate) (Ampola et al, 1969); however, the pathogenesis has not been clarified

since MCDU was recognized more than 40 years ago. Macroscopic anomalies

were  associated  in  1  case  (Ampola et  al,  1969);  however,  this  could  be  an

accidental  combination.  3-MST  deficiency  also  induced  higher  brain

dysfunction in mice without macroscopic and microscopic abnormalities in the

brain. 3-MST seems to play a critical role in the central nervous system, i.e., to

establish normal anxiety (Richardson et al., 2011)


1.2.1. DISTRIBUTION


      3-MST  is  widely  distributed  in  prokaryotes  and  eukaryotes  (Jarabak,

1981).  It is  localized  in  the  cytoplasm  and  mitochondria,  but  not  all  cells

contain 3-MST (Nagahara et al., 1998).


1.2.2. OCCURRENCE


      Human mercaptopyruvate sulfurtransferase (MPST; EC. 2.8.1.2) belongs

to  the  family  of  sulfurtransferases (Vanden et  al.,  1967).  These  enzymes

catalyze  the  transfer  of  sulfur  to  a  thiophilic acceptor (Sorbo  1957),  where

MPST  has  a  preference  for  3-mercapto  sulfurtransferase  as  the  sulfur-donor.

MPST  plays  a  central  role  in  both  cysteine  degradation  and  cyanide

detoxification. In addition, deficiency in MPST activity has been proposed to be

responsible  for  a  rare  inheritable  disease  known  as  mercaptolactate-cysteine

disulfiduria (MCDU) (Hannestad et al, 2006).


1.2.3. MECHANISMS OF ACTION


        3-Mercaptopyruvate  sulfurtransferase  catalyzes  the  reaction  from

mercaptopyruvate (SHCH2C (= O) COOH)) to pyruvate (CH3C (= O) COOH)

in  cysteine  catabolism (Vackek  and  Wood,  1972).  The  enzyme  is  widely

distributed in prokaryotes and eukaryotes (Jarabak, 1981).
      This disulfide bond serves as a thioredoxin-specific molecular switch. On

the other hand, a catalytic-site cysteine is easily oxidized to form a low-redox

potential  sulfenate  which  results  in  loss  of  activity (Nahagara et  al., 2005).

Then, thioredoxin can uniquely restore the activity (Nagahara, 2013).


      Thus,  a  catalytic  site  cysteine  contributes  to  redox-dependent regulation

of  3-MST  activity  serving  as  a  redox-sensing  molecular  switch (Nahagara,

2013). These findings suggest that 3-MST serves as an antioxidant protein and

partly maintain cellular redox homeostasis. Further, it was proposed that 3-MST

can  produce  hydrogen  sulphide  (H2S)  by  using  a  persulfurated  acceptor

substrate (Shibuya et al, 2009).


    As  an  alternative  functional  diversity  of  3-MST,  it  has  been  recently

demonstrated in-vitro that 3-MST can produce sulfur oxides (SOx) in the redox

cycle  of  persulfide  (S-S-)  formed  at  the  catalytic  site  of  the  reaction

intermediate (Nagahara et al, 2012).


1.2.4. MOLECULAR FORMULA AND MOLECULAR WEIGHT


      The molecular formula of 3-MST is C3H4O3S (Vachek and Wood, 1972).


3-MST  has  a  molecular  weight  of  120.127g/mol  or  23800  Daltons  (as

summarized by PubChem compound).


 


 


 


 


 


 
1.2.5. STRUCTURE OF 3-MST






















 

 

Figure 1.1: Structure of 3-mercaptopyruvate sulfurtransferase

Source: www.ebi.ac.uk/thornton-srv/databases/cgi

  bin/enzymes/GetPage.pl?ec_nnumber=2.8.1.2

 

1.2.6. AMINO  ACID  COMPOSITION  OF  3-MERCAPTOPYRUVATE

        SULFURTRANSFERASE


        3-mercaptopyruvate  sulfurtransferase  is  a  crescent-shaped  molecule

which comprises of three domains (Vachek and Wood, 1972). The N-terminal

and  central  domains  are  similar  to  the  thiosulfate  sulfurtransferase  rhodanase

and create the active site containing a persulfurated catalytic cysteine (Cys-253)

and  an  inhibitory  sulfite  coordinated  by  Arg-74  and  Arg-185 (Nahagara  and

Nishino  1996).  A  serine  protease-like  triad,  comprising  Asp-61,  His-75,  and

Ser-255, is near  Cys-253 and represents a conserved feature that distinguishes
3-mercaptopyruvate  sulfurtransferases  from  thiosulfate  sulfurtransferases

(Nahagara et al 1995).


1.2.7. CATALYTIC  ACTIVITY  OF  3-MERCAPTOPYRUVATE

        SULFURTRANSFERASE


3-mercaptopyruvate  +  cyanide  =  pyruvate  +  thiocyanate (Fiedler  and  Wood,

1956).


1.2.8. ENZYME REGULATION OF 3-MERCAPTOPYRUVATE


      Regulation is by oxidative stress and thioredoxin. Under oxidative stress

conditions, the catalytic cysteine site is converted to a sulfenate which inhibits

the  mercaptopyruvate  enzyme  activity.  Reduced  thioredoxin  cleaves  an  inter-

subunit  disulfide  bond  to  turn  on  the  redox  switch  and  reactivate  the  enzyme

(Nagahara, 2013).


1.2.9. STABILITY OF 3-MST


      3-MST  is  remarkably  stabilized  during  purification  and  storage  by  the

presence of monovalent cations. 


      Maximal stability is obtained if purification and storage are carried out at

pH 6.5-7.5 in the presence of KCN and 2-mercaptoethanol (Vachek and Wood,

1972).


      3-MST was stored at 4oC and recorded no loss of activity after 10 days

(Vachek and Wood, 1972).


1.3. PHYSICO-CHEMICAL PROPERTIES OF 3-MST

1.3.1. OPTIMAL TEMPERATURE


      Minimum temperature is at 45oC, the optimum temperature is at 45oC –

50oC, and maximum temperature is at 60oC after which there is no more activity

(Vachek and Wood, 1972).
1.3.2. OPTIMUM pH

      The  minimum  pH  is  at  9.3,  optimum  pH  is  between  9.4  and  9.5.  The

maximum pH is at 9.6 (Vachek and Wood, 1972).

1.3.3. EFFECT OF METALS/ IONS ON 3-MST

KCl: 0.02M causes 70% activation of 3-MST.

Na2SO4: 0.02M causes 70% activation.

K2SO4: 0.02M causes 70% activation.

      Furthermore,  0.5mM  arsenite  and  0.01mM  copper  acetate  has  no  effect

on 3-MST activity (Vachek and Wood, 1972).

1.3.4. SPECIFIC ACTIVITY OF 3-MST

The specific activity of 3-MST is 540mM/min/mg Vanchek and Wood, 1972).

1.3.5. INHIBITORY STUDIES OF 3-MST

The inhibitors of 3-mercaptopyruvate sulfurtransferase include:

2-mercaptoethanol: high concentration of it inhibits the activity of 3-MST.

Cyanide: it inhibits at a short-time intervals and slightly enhancement at longer

periods.

Cysteamine: it inhibits 3-MST slightly.

Mercaptosuccinamic acid: it inhibits 3-MST slightly.

Pyruvate: 17% inhibition when present in 10mM and gives 45% inihibition in

20mM.

Thioglycolic acid: it slightly inhibits 3-MST. (Vachek and Wood, 1972).

 

1.4. CYANIDE

      Cyanide  is  a  chemical  compound  that  contains  monovalent  combining

group  cyanide  (CN).  This  group,  known  as  the  cyano-group,  consists  of  a

carbon atom triple-bonded to a nitrogen atom.

      Cyanide  is  a  potent  cytotoxic  agent  that  kills  the  cell  by  inhibiting

cytochrome  oxidase  of  the  mitochondrial  electron  transport  chain.  When

ingested,  cyanide  activates  the  body  own  mechanisms  of  detoxification,
resulting  in  the  transformation  of  cyanide  into  a  less  toxic  compound  called

thiocyanate (Biller and Jose, 2007).

      The  cyanide  anion  is  an  inhibitor  of  the  enzyme  cytochrome-c  oxidase

(also known as aa3) in the fourth complex of the electron transport chain (found

in the membrane of the mitochondria of eukaryotic cells). It attaches to the iron

with this protein.  The  binding  of  cyanide  to this  enzyme  prevents  transport of

electrons from cytochrome C to oxygen. As a result, the electron transport chain

is disrupted,  meaning  that  the cell can no  longer  produce  ATP  aerobically  for

energy (Nelson et al, 2000). Tissues that depend highly on aerobic respiration,

such as the central nervous system and the heart, are particularly affected. This

is an example of histotoxic hypoxia (Biller and Jose, 2007).

      Many plants and plant products used as food in tropical countries contain

cyanogenic  glycosides (Vetter,  2000).  These  plants  include  cassava,  linseed,

beans  and  peas,  which  are  known  to  contain  linamarin  coexisting  with

lotaustralin.  Millet,  sorghum,  tropical  grass  and  maize  are  sources  of  dhurin.

Amygladin  is  found  in  plums,  cherries,  pears,  apple  and  apricots.  These

compounds  are  also  present  in  plants  such  as  rice,  unripe  sugar  cane,  several

species of nuts and certain species of yam (Osuntokun, 1981; Oke, 1979).

     In  plants,  cyanides  are  bound  to  sugar  molecules  in  the  form  of

cyanogenic  glycosides  and  defend  plants  against  herbivores. Upon  hydrolysis,

these compounds yield cyanide, a sugar and a ketone or aldehyde (Jones, 1998).

      Initial symptoms of cyanide poisoning can occur from exposure to 20 to

40 ppm of gaseous hydrogen cyanide, and  may include headache, drowsiness,

dizziness, weak and rapid impulse, deep and rapid breathing, a bright-red colour

in  the  face,  nausea  and  vomiting.  Convulsion,  dilated  pupils,  clammy  skin,

weaker and more rapid pulse and slower, shallower breathing can follow these

symptoms. Finally, the heartbeat becomes slow and irregular, body temperature

falls, the lips, face and extremities take on a blue colour, the individual falls into

a coma, and death occurs. These symptoms can occur from sub lethal exposure
to cyanide, but will diminish as the body detoxifies the poison and excretes it

primarily  as  thiocyanate  and  2-aminothiazoline-4-caarboxylic  acid,  with  other

minor metabolites.

      The  body  has  several  mechanisms  to  effectively  detoxify  cyanide.  The

majority  of  cyanide reacts  with  thiosulfate  to  produce thiocyanate in  reactions

catalyzed by sulfur transferase enzymes such as rhodanase. The thiocyanate is

then  excreted  in  the  urine  over  a  period  of  days.  Although  thiocyanate  is

approximately  seven  times  less  toxic  than  cyanide,  increased  thiocyanate

concentrations  in  the  body  resulting  from  chronic  cyanide  exposure  can

adversely affect the thyroid. 

      Cyanide  has  a  greater  affinity  for  methemoglobin  than  for  cytochrome

oxidase,  and  will  preferentially  form  cyanomethemoglobin.  If  this  and  other

detoxification  mechanisms  are  not  overwhelmed  by  the  concentration  and

duration of cyanide exposure, they can prevent acute cyanide-poisoning incident

from  being  fatal.  Other  adverse  effects  include  delayed  mortality,  pathology,

susceptibility  to  predation,  disrupted  respiration,  osmoregulatory  disturbances

and  altered  growth  patterns.  Concentrations  of  20  to  76  micrograms per  litre

free  cyanide  cause the  death of  many  species,  and  concentrations in  excess of

200 micrograms per litre are rapidly toxic to most species of fish. Invertebrates

experience  adverse  non-lethal  effects  at  18  to  43  micrograms  per  litre  free

cyanide,  and  lethal  effects  at  30  to  100  micrograms  per  litre.  (Clark,  1974; 

Azcon et al., 1987).

 

1.5. ORYCTES RHINOCEROS LARVAE


      The rhinoceros larvae  are  popular  in  oil  palm  growing  areas  of  the

rainforest and coastal areas of Nigeria. The larvae are white and soft in texture.


      The  larva,  also  called grub,  is  called osori by  the  Ijaws, tam by  the

Ogonis and utukuru by the Ibos, all of Southern Nigeria. 
 


 


 


 


 


Figure 1.2: Rhinoceros Larva


    It is either eaten raw, boiled, smoked or fried. It may be consumed as part

of a meal or as a complete meal.                    


1.5.1. TAXONOMY OF ORYCTES RHINOCEROS


Domain: Eukaryota


Kingdom: Metazoa


Phylum: Arthropoda


Subphylum: Urinamia


Class: Insecta


Order: Coleoptera


Family: Scarabaeidae


Genius: Oryctes


Species: Oryctes rhinoceros

 

1.5.2. NUTRITIONAL QUALITIES OF RHINOCEROS LARVAE

    In spite of the effects of the rhinoceros larvae on palm trunk, these insects

(Oryctes rhinoceros larvae) possess delectable and nutritional qualities that are
appealing to humans. In Nigeria, rhinoceros larvae are among the edible insect

commonly  eaten  (Banjo et  al,  2006).  They  are  well  eaten  in  the  rainforest,

riverine and coastal states where the oil palm is grown. The larvae are roasted or

fried to taste.

      The  nutritional  qualities  shows  the  percentage of  Crude  Protein    which

was  36.45%,  and  the  Lipid,  Nitrogen-free  extract  and  Crude  fibre  are  34%,

15.05% and 10.50% respectively  (Banjo et al., 2006).

It is rich in essential Amino acids which include:
 

                    Leucine Phenylalanine Methionine

                    6.30g/100g 4.65g/100g 2.085g/100g

Table 1.1: Essential amino acids present in rhinoceros larva

These rich amino acid values meet the minimum daily requirements for humans

as recommended by the WHO. It is also rich in minerals as shown in the table

below (Banjo et al., 2006).

    Iron Sodium Potassium Magnessium Zinc

    8.5mg/100g 440mg/100g 38.4mg/100g 175mg/100g 7.0mg/100g

Table 1.2: Essential Minerals in rhinoceros larva


      The  high  iron  content  of  the  larvae  of  the rhinoceros beetle  is  of

advantage  to  women  in  developing  economies  including  Nigeria  and  more  so

far  pregnant  women  who  are  reported  to  suffer  from  iron  deficiency  during

pregnancy (Banjo et al., 2006).

      Magnesium  is  useful  to  maintain  normal  muscle  and  nerve  function.  It

steadies heart rhythm, supports immune blood and regulates blood sugar levels.

Magnesium  is  needed  for  more  than  300  biochemical  reactions  in  the  human

body (Saris et al., 2000).

 

 

 
1.5.3. LIFE CYCLE OF ORYCTES RHINOCEROS LARVA

      Eggs  are  laid  and  larvae  develop  in  decaying  logs  or stumps,  piles  of

decomposing  vegetation  or  sawdust,  or  other  organic matter.  Eggs  hatch  into

larvae 8 days to 12 days, while the larvae feed and grow for another 82 days to

207 days before entering an 8 to 13 day non-feeding pre-pupa stage. 

Pupae are formed in a cell made in the wood or in the soil beneath where the

larvae feed. The pupa stage lasts 17 to 28 days.

      Adults remain in the pupa cell 17 - 22 days before emerging and flying to

palm  crowns  to  feed.  The  beetles  are  active  at  night  and hide  in  feeding  or

breeding  sites  during  the  day.  Most  mating  takes place  at  the  breeding  sites.

Adults  may  live  4-9  months  and  each female  lays  50-100  eggs  during  her

lifetime.



























 Figure 1.3: Life Cycle of Oryctes Rhinoceros Larva 


 
1.5.4. DAMAGE

      Coconut rhinoceros beetle adults damage palms by boring into the centre

of  the  crown,  where  they  injure  the  young,  growing tissues  and  feed  on  the

exuded  sap.  As  they  bore  into  the  crown, they  cut  through  the  developing

leaves. When the leaves grow out and unfold, the damage appears as V-shaped

cuts in the fronds or holes through the midrib. 


1.5.5. NATURAL ENEMIES

 Rhinoceros begtetle eggs, larvae,  pupae, and adults  may  be  attacked  by

various predators, including pigs, rats, ants, and some beetles. They may also be

killed  by  two  important diseases:  the  fungus Metarhizium  anisopliae and  the

Oryctes virus disease.
 

1.5.6. MANAGEMENT

 Rhinoceros beetles  can  be  controlled  by  eliminating the  places  where

they breed and by manually destroying adults and immature.

    In  many  countries,  the  fungus Metarhizium  anisopliae or  the

Oryctes virus  are  used  to  control  the rhinoceros beetle.  More  recently

a chemical attractant, ethyl-4-methyloctanoate, has been used in traps to attract

and  kill  the  beetles.  Both Metarhizium  anisopliae and the Oryctes virus  are

present  and  helping  to  reduce rhinoceros beetle populations  in  American

Samoa;  however,  these  pathogens  and  the attractant  have  not  yet  received

approval  from  the  United  States Environmental  Protection  Agency  for  use  as

pesticides to control the rhinoceros beetle.
 

Figure 1.4: Decaying palm trunk

 

1.5.7. ECONOMIC IMPORTANCE

    On  oil  palms,  O. rhinoceros bores  into  the  cluster  of  spears,  causing

wedge-shaped cuts in the unfolded fronds or spears. In young palms where the

spears  are  narrower  and  penetration  may  occur  lower  down,  the  effects  of

damage  can  be  much  more  severe  than  in  older  palms  (Wood,  1968a).  The

young  palms  affected  by  the  beetle  damage  are  believed  to  have  a  delayed

immaturity  period  (Liau  and  Ahmad,  1991).  Thus,  early  oil  palm  yields  could

be considerably reduced after a prolonged and serious rhinoceros beetle attack.

Although Wood et al. (1973) suggested that the damage to the immature palms

results in relatively small crop losses, field experiments conducted by Liau and

Ahmad (1991) revealed an average of 25% yield loss over the first 2 years of

production.  This  was  possibly  caused  by  the  reduction  in  the  canopy  size  of

more than 15% for moderately serious to higher damage levels (Samsudin et al.,

1993).  In  India,  the  infestation  in  oil  palm  was  more  prevalent  in  mature

plantations  (10-15  year  old)  compared  to  immature  or  younger  plantings

(Dhileepan, 1988). 


      Similarly,  on  coconut  the  reduction  in  leaf  area  of  the  palms  influences

nut production (Zelazny and Young, 1979) but the attack was more towards the

tall,  mature  trees,  from  about  5  years  of  age  onwards  (Bedford,  1976b).
Considerably serious attacks on coconut were also observed in areas adjacent to

a breeding site with a high beetle population, especially in the coastal region of

Peninsular Malaysia. Zelazny (1979) reported 5-10% damage resulting in 4-9%

yield reduction; similarly 30% damage resulted in 13% yield reduction.


1.6. THE GUT

    In zoology, the gut, also known as the alimentary canal or gastrointestinal

tract, is a tube by which bilaterian animals (including humans) transfer food to

the digestion organs (Ruppert et al., 2004). In large bilaterians, the gut generally

also has an exit, the anus by the animal disposes off solid wastes. Some small

bilaterians  have  no  anus  and  dispose  of  solid  wastes  by  other  means  (e.g.

through the mouth) (Barnes et al, 2004).


      Animals  that  have  guts  are classified  as  either  protostomes  or

deuterostomes,  as  the  gut  evolved  twice,  an  example  of  convergent  evolution.

They  are  distinguished  based  on  their  embryonic  development.  Prostotomes

develop their mouths first, while deuterostomes develop their mouths second.


Prostostomes  include  arthropods,  molluscs,  annelids,  while  deuterostomes

include  echinoderms  and  chordates. The  gut  contains  thousands  of  different

bacteria, but humans can be divided into three main groups based on those most

prominent (Zimmer, 2011).


1.7. PURIFICATION OF 3-MST

  The  purification  of  the  3-Mercarptopyruvate  Sulphur  Transferase  enzyme

from the Oryctes rhinoceros larva involves the combination of several methods

such as:

i. Ammonium Sulphate Precipitation and Dialysis.

ii. Bio-Gel P-100 and Affinity Chromatography.

iii. Protein  concentration  determination    which  is  carried  out  by  using

      Bradford Method of Protein Determination
iv. The use of Nelson and Somogyi method of assay to determine the activity

    of the enzyme in the fractions.

 

1.8. OBJECTIVES OF STUDY

      The aim and objective of this study is to:

    i. Isolate 3-MST from the gut of Oryctes rhinoceros larvae.

    ii. Purify the  3- mercaptopyruvate sulfurtransferase enzyme isolated

            from the gut of Oryctes rhinoceros larva.

 

1.9. JUSTIFICATION OF STUDY


      Rhinoceros larva feeds on woods and plants, especially the decayed palm

trees.  Plants  are  known  to  possess  defensive  but  toxic  chemical  called  cyanide

(Marcus  Wischik,  1998).  Therefore, rhinoceros larva  should  possess  a  cyanide

detoxifying enzyme of which 3-MST is one.