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


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

ABSTRACT 

      Cyanide  is  known  to  be  one  of  the  most  toxic  substances present  in  a 

wide variety of food materials that are consumed by animals. 

    One  of  the  cyanide  detoxifying  enzymes  is  3-mercaptopyruvate 

sulfurtransferase  (3-MST).  Indeed,  recent  studies  have  clearly  shown  that  3-

MST is involved in the detoxification of cyanide. 

      Rhinoceros (Oryctes rhinoceros) larva feeds on dead, decayed and living 

plants,  wood  and  palm.  Plants  are known  to  contain  cyanide  as  a  defence 

mechanism  for  intruding/pesting  organisms. Thus,  for rhinoceros larva  to  be 

able to live on plants, it must have possessed a cyanide-detoxifying enzyme. 

      3-MST,  a  cyanide-detoxifying  enzyme  was  purified  from Rhinoceros 

(Oryctes rhinoceros) larva in this work. 

      The 3-MST enzyme was isolated from the gut of Oryctes rhinoceros larvae 

and purified using Ammonium Sulphate Precipitation, Bio-Gel-P-100 Gel Filtration 

Chromatography and Reactive Blue-2-Agarose Affinity chromatography. 

      The specific activity of the enzyme was 0.22U/mg. 

      The presence of this enzyme could be exploited by including it in the diet 

of animals which  would serve  as a source  of  protein  and  3-MST. Perhaps,  these 

rhinoceros larva could be introduced on farmland with contaminated soil whereby 

they will process the dead roots and plants into soil thereby providing more space 

and manure for plants to grow healthy.  

                            TABLE OF CONTENTS 

Contents                Pages 

Title Page           i 

Certification           ii 

Dedication            iii 

Acknowledgement          iv 

Table of Content          v 

List of Figures          vi 

List of Tables          vii 

Abstract                    viii 

Chapter One 

1.0. Introduction and Literature Review      1 

1.1. Introduction         1 

1.2. 3-Mercaptopyruvate Sulfurtransferase     2 

1.2.1. Distribution of 3-MST        5 

1.2.2. Occurrence of 3-MST        5 

1.2.3. Mechanisms of Action        6 

1.2.4. Molecular Formula and Molecular Weight     7 

1.2.5. Structure of 3-MST        8 

1.2.6. Amino Acid Composition of 3-MST      8 

1.2.7. Catalytic Activity of 3-MST       9 

1.2.8. Enzyme Regulation of 3-Mercaptopyruvate sulfurtransferase  9 

1.2.9. Stability of 3-MST        9 

1.3. Physicochemical Properties of 3-MST     9 1.3.1. Optimal Temperature of 3-MST      9 

1.3.2. Optimum pH of 3-MST        10 

1.3.3. Effect of Metals/ions on 3-MST      10 

1.3.4. Specific Activity of 3-MST       10 

1.3.5. Inhibitory Studies of 3-MST       10 

1.4. Cyanide          11 

1.5. Oryctes rhinoceros Larvae       13 

1.5.1. Taxonomy of Oryctes rhinoceros 

1.5.2. Nutritional Qualities of Rhinoceros Larvae     13 

1.5.3. Life Cycle of the Rhinoceros larva      15 

1.5.4. Damage          16 

1.5.5. Natural Enemies         16 

1.5.6. Management         17 

1.6. The Gut          18 

1.7. Oryctes rhinoceros        19 

1.7.1. Description of Development Stages      19 

1.7.2. Distribution of Oryctes rhinoceros      21 

1.7.3. Hosts/Species Affected        21 

1.7.4. Economic Importance        23 

1.8. Purification of 3-MST        27 

1.9. Justification of Studies        28 

1.10. Objectives of Research        28 

Chapter Two 

2.0. Materials And Methods        29 

2.1. Materials          29 

2.1.1. Reagents          29 2.1.2. Apparatus Used         29 

2.1.3. Study Sample         30 

2.2. Method          30 

2.2.1. Preparation of Buffer and Reagents      30 

2.2.1.1. Preparation of 0.25M Potassium Cyanide    30 

2.2.1.2. Preparation of 0.5M Potassium Cyanide     30 

2.2.1.3. Preparation of 38% Formaldehyde      30 

2.2.1.4. Preparation of 0.25M Ferric Nitrates (Sorbo Reagent)   31 

2.2.1.5. Preparation of Bradford Reagent      31 

2.2.1.6. Preparation of 0.38M Tris-HCl Buffer     31 

2.2.1.7. Preparation of 0.30M Mercaptoethanol     31 

2.2.2. Preparation of Crude Extract from the rhinoceros larva gut  32 

2.2.3. Protein Concentration Determination      33 

2.2.4. Assay for 3-Mercaptopyruvate Sulfurtransferase    34 

2.2.5. Enzyme Purification        35 

2.2.6. Substrate Specificity        37 

Chapter Three 

3.0. Results 

Chapter Four 

4.0. Discussion, Conclusion and Recommendation    52 

4.1. Discussion          52 

4.2. Conclusion          52 

4.3. Recommendation         52 

References           53 

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 

Image 

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.   

ImageImage 

ImageFigure 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. 

Image 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&nbsp

.

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



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