CHARACTERISTIC OF pH AND TEMPERATURE OF CRUDE EXTRACT OF GLUKOAMILASE FROM GLIOCLADIUM KE BY USING SOLID SUBSTRATE OF SAGO HAMPAS

This research was aimed at figuring optimum pH and temperature of crude enzyme extract glukoamilase from isolate Gliocladium KE using substrate of solid sago hampas. The research was conducted by two phases, first was figuring optimum pH and second was optimum temperature. it was based on Complete Random Design. The data were statistically analyzed by using F test. If the result showed significance different, the analysis was carried out by using Duncan’s New Multiple Range Test (DNMRT) with 5% tolerance. The result showed that optimum pH of crude glukoamilase extract from sago hampas was optimum pH of glukoamilase from Gliocladium KE using solid substrate of sago hampas was pH 6,0 with activity of enzyme 76,56 U/ml with final product of solube starch 0.09%, the level of reducing sugar 62,01%, and total sugar 78,43%. Optimum temperature was 60˚C with enzyme activity 78,33 U/ml, final product of solube starch 0,13%, reducing sugar 63,04%, and total sugar 79,55%


INTRODUCTION
Waste of sago palm, especially waste of sago, was not yet optimally used by people for food. In fact, it had great potential for several regions, particularly in Papua, Sulawesi, and Sumatera. Kiat (2006) reported that waste of sago contains adventage substance such as essence and cellulose. The number of waste from sago timber was about 26% of total product while from waste of sago was about 14% of total product. The waste contains 65,7% essences and the rest were fibers, proteins, fats, and ashes.
Glucoaminase from certain isolate had to be figured in order to gain optimal activity (including temperature and pH). Different enzyme was frequently had different level of optimuml pH depends on the source of enzyme (Winarno, 2010). This microbe enzyme had wide optimum pH level (Buckle, 1997).
The fastest reaction occurs in optimal temperature (Rodwell, 1988cit Rochmawatin, 2010. Therefore, defining optimum temperature of enzyme activity was important because lower temperature will increase stability of enzyme but decrese its activity while higher temperature will increase the activity but decrese the stability (Winarno, 2010). Optimum temperature and pH of glucomilase from local isolate should be known. According to Naiola (2006), the characteristic of hard glucomilase enzyme from Saccharomycopis sp for optimum temperature activity was 40˚C-50˚C for and 6,0-7,0 for optimum pH.
Based on the research conducted by Sunaryanto (2003), glucomilase from Aspergillus niger BCS had optimum pH in 4,5 and optimum temperature in 60 ˚C.

A. Material
Microorganisme which was used in this research was isolate Gliocladium from Microbiology Laboratorium and Bioteknology Faculty of Agriculture Technology collection.

B. Method of Research
This research method uses two phases: first determine the proper pH and the second stage determine the right temperature. This study used 3 replications processes: a. Research Phase I: Determenine the optimal pH of crude glucoamylase enzyme in the substrate from 3 levels of pH.

Refreshing Gliocladium isolates (modified Sunaryanto , 2003)
Refreshing isolates was done by moving the mold culture in a the dish which contain PDA medium.
Then, it being incubated for 4 days at a temperature of 27-28 ˚ C. After the colony grew, each colony was transferred to the slant plan and each colony was coded.

Sago Hampas Drying
Sago hampas drying using solar dryers to 12 % moisture content material. Drying was done by laying out the dregs of sago in a solar dryer.

Sago Dregs Size Decreasing
Sago hampas size reduction was done using belender. After blending, sago dregs was meshed by using 80 mesh sieve. Sago dregs which do not pass 80 mesh sieve were blended again until it pass the 80 mesh sieve.

Substrate Manufacture and Production of Glucoamylase
Substrates used in this research refers to the composition used by Tani et al (1986) cit Sunaryanto, 2003, which had been modified. Its composition was as follows: 57.82 g Dregs sago, 1.67 g MgSO4.7H2O, Ca (NO3) 2 with specific treatment, and 37 mL of water. All medium of fermentation in 500 mL erlemeyer which was sterilized by autoclave at 121 ˚ C for 15 minutes. Inoculation Gliocladium KE into fermentation medium was done by using Gliocladium KE suspense in 0.1 % (v/ v) tween 80 which compose with 9 ml distilled water. After that, the fermentation was done in erlemeyer with aerobic conditions at a temperature of 27-30 ˚ C for 4 days.

Extraction and Production of Crude Enzyme Glucoamylase (modification Sunaryanto , 2003)
Extraction was done by adding acetate buffer pH wiith 4.6 that 3 times of solid substrate fermentation weight, then stirred for 30 minutes at 4˚C. Then, it was centrifuged at 11,000 × g speed for 10 minutes and filtered. The filtrate obtained was called crude enzyme. The enzyme of extracted filttrat was sendimented by adding isopropanol solvent organk cooled in a freezer at a temperature of -4 ˚ C. The filtrate was mixed with an organic solvent, solvent volume of isopropanol was added to the filtrate from the filtrate as much as 1.67 times the concentration of enzyme or 60 % (v / v). The addition of organic solvents was done bit by bit. Once mixed with an organic solvent and then stored in a cold room at 4 ˚ C for 24 hours. Once it's done 11,000 xg centrifugation for 10 min at 4 ˚ C.
Then the precipitate was separated between the filtrate. Deposition of enzyme added acetate buffer pH 4.6 to obtain a crude enzyme extract glucoamylase.

Sunaryanto 2003)
The optimum pH of glucoamylase activity was defined by conducting hydrolysis test toward soluble starch by applying method by Pandey et al., (2000) cit Sunaryanto (2003). 1.9 ml of 2 % soluble starch which had been specified with certain pH by using acetate buffer was added with 0.1 ml of the sample solution. After mixed, it was Inkubated at specified temperature for 20 minutes After that, the analysis was condeucted toward starch content, sugar content, total sugar, and glukoamise activity by measuring the amount of glucose μmol as the result of hydrolysis of soluble starch by enzyme activity per unit (min) and volume (ml).

Glucoamylase Activity Measurement
Crude enzyme extract was filtered with cotton wool and then centrifuged at 1000 rpm at a temperature of 4 ˚ C for as many as 1 ml pipetted into a test tube. After the enzyme extract was added 2 ml pipetted acetate buffer pH 4.6. To control the enzyme extract, it had to be incubated first at a temperature of 100 ˚ C for 5 minutes. Then take 0.1 ml (Vc) of the mixture and transferred it into another test tube and add 1.9 ml osoluble starch (Vsb) 2 %. Stirred well and then heated at a temperature of 60 ˚ C for 20 min (t). After heating process done, the sample will be divided into two parts; at top it will look clear and while cloudy at the bottom The clear samples were pipetted. The analysis was done by adding 0,5 ml samples with 1.5 ml DNS. After the sample mixed with DNS solution, boil it for 5 minutes. Then get the sample cool by flowing wate. The cool sample was pipetted as 0.4 ml and then added distilled water until it becomes homogenous. Then the process continued by conducting reduced sugar measurement (Cgr) at 540 nm absorbance. The research procedure was similar to sampling procdure, but in research procedure the sample was change with acetate buffer pH 4.6. Glucoamylase activity can be measured by the following formula: Glukoamilase Activity = Cgr x (Vc + Vsb) x Fp

A. Chemical composition of Solid Sago Dregs Substrate
The result of analysis toward sago dregz was reflected in the following

B. Optimum pH of Rough Glucoamylase Enzyme Extract
Based on the results of variance done, the pH of the substrate significantly affect the final starch content. Table 2 showed the higher pH used the lower starch content of soluble starch substrate.    (1985) cit Sunaryanto ( 2003) that the end product of starch hydrolysis by glucoamylase produces glucose as the main product . Glucose produced by glucoamylase was a reducing sugar. The existence of glucoamylase in starch suspensions , in particular the enzyme which can catalyze the hydrolysis of amylase, will cause the starch hydrolysed to form reducing sugar (Kusnandar, 2010).
The content of reducing sugar was in accordance with total sugar. The higher total sugar, the higher end product reducing sugar. This was because the amount of sugar consist of reducing sugars plus nonreducing sugar. The difference between total sugars and reducing sugars were shown in the Table 3 which tells that the higher pH used in the substrate, the less increment in total sugars with reducing sugars. It showed that the starch contained in soluble strach medium was dominantly hydrolyzed into reducing sugars.

Activity of Glucoamylase
The effect of pH toward glucoamylase activity had significance effect. Glucoamylase activity toward of the pH can be seen in Figure 1. The lower pH set in specified substrae, the lower glucoamylase activity was measured Figure 1. The Relationship of Glucoamylase Activity toward pH Substrate The enzyme activity at pH 4.0 was not optimal because there were many active side of active glucoamylase which not yet active in binding starch in the substrate. Glucoamylase that not yet active can be activated with the right environment. The right environment for glucoamylase activity was at pH 6.0.
The optimum activity of the crude glucoamylase extract at pH 6.0 was at 76.56 U/ml . This was in accordance with the Maggy's opinion (1989 ) which states , enzymes were proteins which composed of amino acids. Amino acids which located in the active site plays a role in binding reaction between the enzyme to the substrate with a pH neutral or slightly above or below neutral pH.
The enzyme activity was low at pH below or above the optimum pH, due to the denaturation of the enzyme protein. Denaturation of this protein led to the destruction of the molecular structure that leads to inactivation of the enzyme glucoamylase. pH can cause changes to ionized groups contained in the enzyme. This can lead to changes of structure, conformation and active side of enzyme (Creighton, 1984cit Sunaryanto, 2003. High pH can cause protein denaturation. In accordance with Winarno (2010) states that at extreme pH range, both acid and base, occurs irreversible inactivation. Denaturation of proteins occurs due to disruption of hydrogen groups and ionic groups as well as the interaction of the substrate reduced ( Campell , 2002) .
Enzymes provide plenty of proton binding. Because enzymes were proteins composed of amino acids that can ioniezed by binding and releasing protons or hydrogen ions on amine, carboxyl and other functional groups . Amino acid that plays role in binding the enzyme with substrate was amino acid with neutral pH or slightly above or below neutral pH. pH of acid or base can interfere with ionic enzyme (Maggy , 1989). The increase or decrease the pH of optimum pH can break the weak hydrogen bonds that maintain the structure of the enzyme which leads to denaturation of the enzyme and the affect the conformation of substrate and distracted to entering the active side of the enzyme (Anwar , 2008).

C. Optimal temperature Crude Glucoamylase Extract
Effect of temperature on enzyme extract of crude glucoamylase was analyzed by using parameters starch content product, reducing sugar and total soluble starch and glucoamylase activity.

Levels of Starch
Based on the results of variance substrate temperature toward significance affect the starch content product. Substrate temperature affects starch hydrolysis which done by glucoamylase. The lower starch content was in C.3 treatment (using a temperature of 60˚C) and the highest starch content was in C.1 treatment (using a temperature of 40˚C). The effect of temperature toward starch content can be seen in Table 4.  The numbers in samiliar row followed by different small letter was significant different with 5% level according to DNMRT Final product starch content was starch content that can not be hydrolyzed by glucoamylase during the enzymatic reaction. The existence of enzyme in the starch suspension, particularly the enzyme that can catalyze the hydrolysis of the amylose and amylopectin molecules, glucoamylase will cause hydrolysed the starch form saccharide with a shorter chain. The addition of glucoamylase can break the glycosidic bond α (1-4) and β (1-6) (Kusnandar, 2010).
The higher temperatures, the lower starch content , exepct at treatment that use temperature 70˚C.
The lower starch content of final product, then the glucoamylase activity. This was because the activity of glucoamylase at 70 ˚ C decreased the hydrolysis of starch which also decreases the starch content.
Sudarmadjo, (2008) states in the high heat, the enzyme which was kind of protein will denaturated.
Denaturation of this enzyme decreased the activity of the enzyme (Sukandar et al . , 2011) .

B. Reducing sugar , total sugar
Based on the results of variance effect of temperature on reducing sugar and total sugar showed significance effect. The highes level of reducing sugars and total sugars was ganied at treatment C.3 treatment ( using temperature 60˚C) they are, 79.55 % and 63.04 % . It can be seen in Table 5 . Levels of reducing sugars and total sugars had a relationship with glucoamylase activity. It can be seen in Figure 1. where the higher levels of reducing sugars and total sugars, the higher glucoamylase activity. Levels of reducing sugars formed in final hydrolisi of product of starch using glucoamylase enzyme was glucose. The final product of starch hydrolysis by glucoamylase produces glucose as the main product (Montenecourt et al , 1985cit Sunaryanto 2003.

Glucoamylase Activity
Based on the results of variance toward the effect of temperature on enzyme activity gluoamilase showed significance effect. The activity of glucoamylase product was found in treatment C.3 (using temperature 60˚C), that was 78.33 U/ml which can be seen in Figure 2.
Temperature was one of the factors that influence the development of glucoamylase. Glucoamylase activity increased from a temperature of 40˚C to 60˚C. At temperature 70˚C the result showed that the activity of glucoamylase was decreased. The highest glucoamylase activity was gained at 60˚C which was 78.33 U/ml.
Optimal temperature of glucoamylase derived from Gliocladium KE by using solid substrate of sago hampas was temperature 60˚C. Each enzyme had an optimum temperature, ie when the enzyme was working properly. The farther the optimum temperature, then enzyme will working properly. Area or range when the working temperature or rate of enzyme reaction was working properly was called optimum temperature (Sudarmadjo, 2008). According to Panesar (2010), this enzyme can withstand a range of temperatures up to 60˚C. To see the relationship between the temperature toward glucoamylase activity can be seen in Figure 2 . The increase of temperature increases the value of glucoamylase activity, but at 70˚C glucoamylase activity was decreased. It was caused at 70˚C enzymes were denaturated the protein which decrease activity of the enzyme. In general, the higher temperature the faster the rate of chemical reactions catalyzed by enzymes. However, the enzyme was a protein that will be inactivated when the ambient temperature was too high. Winarno(2010) argues that the higher the temperature of enzyme inactivation process was also increasing due to too high temperatures can accelerate the breakdown or destruction of enzymes that result in denaturation of the protein. Heating above the optimum temperature enzymes can cause damage to the conformation of the enzyme protein binding which may decrease the activity of the enzyme (Scopes , 1982cit Sunaryanto, 2003 . Suhartono (1992) cit Anwar (2008 ) states that at the begining, the reaction rate increases along with increase of temperature and the increase of reaction rate was caused by the reaction of molecules which in over high temperatures can affect the conformation of the enzyme and cause distraction in substrate to enter active side of the enzyme. At high temperatures, it will break the hydrogen bonds and hydrophobic which maintain secondary and tertiary structures, for the result restructure of threedimensional structure of the enzyme which cause denaturation of the enzyme so that the catalytic energy of enzymes was decrease (Robert et al., 2002). Sudarmadjo (2009 ) states at low temperature enzyme activity was decrease, when the temperature was increased , the speed will up to the optimum temperature. If the temperature continues to increase from the optimum temperature, the enzyme denaturated and will decrease the activities.
The destruction of enzyme bond was disrupted at 70˚C which was caused by the increasedof thermodynamic movement . As a result of the movement of thermodynamic, the enzyme wil be increased which change three-dimensional structure. Sadikin (2002) adds that three-dimensional changes in the structure of this enzyme distract the substrates to occupy the active site of the enzyme, the result the enzyme substrate was difficult to set up which decrease the number of products.
Glucoamylase activity at 40˚C was still low due to the lack of collisions between molecules of the enzyme with the substrate. If the contact between the active site of enzyme was low, then the enzyme substrate complex which formed was also low and the product was litle as well (Sadikin , 2002).

CONCLUSION
The optimal pH of glucoamylase derived from Gliocladium KE solid using sago hampas substrate was pH 6.0. The product of glucoamylase activity at pH 6.0 was 76.56 U/ml with a final concentration of soluble starch was 0.09 %, reducing sugar content was 62.01 % and total sugar content was 78.43 % .
Optimal temperature of glucoamylase derived from Gliocladium KE using solid sago hampas substrate was at 60˚C. Glucoamylase activity produced at a temperature of 60˚C was 78.33 U/ml with starch content of final product was 0.13%, reducing sugar was 63.04 % and total sugar content was 79.55 %.