Potential Marine Fungi Hypocreaceae sp . as Agarase Enzyme to Hydrolyze Macroalgae Gelidium latifolium

Agarase can degradedagarto oligosaccharide and has a lot of benefits for food, cosmetics, and others. Many species of agardegrader are marine-organism. Several agarases have been isolated from different genera of microorganisms found in seawater and marine sediments. Hypocreaceae sp. was isolated from sea water of Pari Islands, Seribu Islands, Jakarta, Indonesia. Based on the results of the 16S rDNA gene identification of 500 base pairs, A10 isolates had 99 % similarity toHypocreaceae sp. The extracellular agarase enzyme from Hypocreaceae sp. have optimum pH and temperature at 8 TrisHCl (0.148 μ.mL-1) and 50 °C (0.182 μ.mL-1), respectively. Agarase enzyme of Hypocreaceae sp. reach an optimum condition at the highest enzyme activity during incubation in 24 hours (0.323 μ.mL-1). SDS Page revealed that there are two bands of protein produced by agarase of Hypocreaceae sp. which are at molecular weight of 39 kDa and 44 kDa and hydrolisis of Gelidium latifolium obtained 0,88% ethanol.


Introduction
Agarase is one of the enzymes that is classified in two categories namely α-agarase and βagarase.So far, agarase have been isolated from several genera of microorganisms derived from sea water, sediment and marine environment (Fu and Kim, 2010).Agarase can be used in cosmetics and food industry.Another benefit of agrase is that it can hydrolyze agar into oligosaccharides.Agar is widely available in red seaweed (Rhodophyceae) including Gelidium spp.and Gracilaria spp (Kawaroe et al., 2015).Agar compositions found are around 44% for Gelidium spp.and 53% for Gracilaria spp.(Nguyen et al., 2012).In addition to the high content of agar, seaweed has a carbohydrate content ranges from 70-72% (Nahak et al., 2011).
G. latifolium has benefit as agar, raw material of paper and can also used as a raw material of bioethanol degraded by acid (Kawaroe et al., 2014).Use of G.latifolium as raw material for bioethanol has been mostly utilizing its content of starch, cellulose and hemicellulose, whereas biomass components such as agar which also have the potential to produce bioethanol has not been fully utilized.This is due to the hydrolysis process only uses cellulase enzymes that are only able to hydrolyze the cellulose fraction.Agar has potential of bioethanol raw material sincetheir components are composed by β-D-galactose and 3,6-anhydro-α-Lgalactose (Fu and Kim, 2010).The success of conversion Gelidium spp. as bioethanol raw material is determined by several different processes, hydrolysis and fermentation (Nahak et al., 2011).One of the hydrolysis processes is by using enzymes produced by micro-organisms.
Several studies have been conducted regarding agarase enzyme produced by marine microorganisms such as Bacillus megaterium (Khambhaty et al., 2008), Acinetobacter sp.(Lakshmikanth et al., 2006), Pseudomonas sp.(Gupta et al., 2013) and Alteromonas sp.(Wang et al., 2006).So far, bacteria are the only microorganisms studied and yet not researches about fungi as agarase producer.Gosh and Gosh (1992) mentioned that marine fungi has good enzyme activity in the degradation process of a compound.This study was the first research of agarase produced by a marine fungiHypocreaceae sp. to hydrolyze G. latifolium.

Material and Methods
Fungi (A10) were isolated from Pari Island, Seribu Island, Jakarta, Indonesia and obtained from microbiology laboratory of Surfactant and Bioenergy Research Center, Bogor Agricultural University, Indonesia.Isolates rejuvenation was conducted by growing isolates fungi on Potato dextrose agar (PDA) medium.Furthermore, the molds were incubated at 28 0 C for 7 days (Pervez et al., 2012).

Optimum time to produce enzyme
The optimum time to produce enzym started with determining pouring timing of inoculum.Timing of inoculum was determined by culturing 2 isolates loops in 15 mL of liquid Potato dextrose (PDL) medium and incubated in 3-5 days, then poured into 135 mL of starter medium.The cultures were incubated at 50 O C in a stirrer with agitation speed of 100 rpm.Sampling was carried out for 7 days incubation with a 24-hour time span to measure the total amount of spores.Afterwards, isolates growth curve was developed to determine the best time pouring of inoculum in media production.
In order to determine the optimum time of enzyme activity, 15 mL of potato dextrose liquid medium containing cell cultures was inoculated into 135 mL of media production.Inoculum pouring time was observed in isolates exponential growth time (logarithmic growth phase) which has been known from fungi growth curve.Sampling was done every day in 4 days during incubation, then supernatant was tested its enzyme activity by using a modified Miller method based on maximum absorbance of reagent solution (Wood and Saddler, 1988).
Agarase activity is expressed in International Units μmL -1 .One unit is the amount of enzyme needed to break down to 1 μmol of cellulose into reducing sugar per minute in test conditions.Glucose levels resulting from agar hydrolysis with agarase enzyme value based on absorbance value at λ 550 nm.

Identification of Fungi
Identification of fungi isolates (A10) based onITS1-5.8S-ITS2rDNA genes.DNA analysis was done by using primary internal transcribed spacer 1 (ITS1) 5'-CTT GGT CAT GTAA TTA GAG GAA-3 'and internal transcribed spacer 4 (ITS4) 5'-TCC TCC GCT TAT TGA TAT GC-3'.Isolation method is in accordance with the instructions of DNA Extraction Kit.The 16S rDNA gene sequence was compared with nucleotide sequences in the database by using the BLAST algorithma the NCBI site (Nursid et al., 2011).

Productionof agarase enzyme
Enzyme production was done based on highest production time on the curve that has been known previously.Media production of the enzyme was incubated in waterbath with a temperature of 50 o C and agitation speed of 100 rpm.The duration of incubation was determined based on the highest production time has been obtained previously.Production medium that contained enzyme was centrifuged at a speed of 2,500 rpm and a temperature of 4 O C for 30 minutes to separate enzyme solution or supernatant with pellets.The results of centrifugation supernatant were then stored at temperature of 10 O C as enzyme crude extract (Fu and Kim, 2010;Kawaroe et al., 2014).

Acid and temperature characteristic
Acid characteristic was known through measuring optimum pH by adding as much as 0.2 mL enzyme which was reacted with 1.8 mL of substrate.The substrate was prepared by mixing 1.8 g of seaweed powder into buffer with various levels of pH 3-9, among others, 0.05 M acetate buffer (3, 4, 5), 0.05 M citrate phosphate buffer (5, 6, 7) and 0.05 M trisHCl buffer (7,8,9).Each enzyme was incubated at 28 o C for 30 minutes.Agarase enzyme activity was measured according to previous test procedures (Fu and Kim, 2010).
Optimum temperature was measured by reacting 0.2 mL enzyme with 1.8 mL substrate in which substrate was prepared by mixing 1.8 g of macroalgae G. latifolium powder into optimum pH buffer.Enzyme that has been mixed with substrate then incubated at temperature between 28 o C to 90 o C with 10 o C range for 30 minutes of incubation time.Agarase enzyme activity was measured according to previous test procedures (Fu and Kim, 2010).

Enzyme stability test
A total of 15% (w/v) G. latifolium was dried and further added with distilled water in autoclave for 30 minutes at temperature of 121 O C and a pressure of 1 atm.Furthermore, after it was cool, enzyme in the optimum buffer was added with a concentration of 5%, and incubation conducted at optimum temperature by measuring the time span in every 24 hours along 72 hours.

Determination of molecular weight (SDS page)
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS page) of agarase was conducted in 12,5% gel as per method described by Sambrook and Russel (2001) along with standard molecular weight protein markers.

Fermentation process
Initial fermentation was done by preparing 90 mL hydrolysate and hydrolysis was performed by adding enzyme as much as 5% and then incubated at optimum temperature of this enzyme for 24 hours (taken from the enzyme stability yield).Furthermore, Saccharomyces cerevisiae inoculum was prepared in 10 mL YMGP (Yeast Malt Glucose Peptone) medium with the composition of 5 g.L -1 , 5 g.L -1 , 5 g.L - 1 and 40g.L -1 then incubated at 30 O C for 24 hours (Yanagisawa et al., 2011).Fermentation was done in anaerobic conditions at temperature of 50 O C. Hydrolysate that has been hydrolyzed then added to Saccharomyces cerevisiaeinoculum and added with 0.5% urea and 0.06% NPK from sugar as a source of nutrients (Setyaningsih et al., 2012).Fermentation process was done in 5 days.Fermentation yield was distilled, then the yield of ethanol were measured using density meter (Anton Paar).

Fungi identification
Results of ITS1-5.8S-ITS2rDNA gene identification of A10 was a species of Hypocreaceae sp. with the degree of homology of 99% (Figure 1.).
Fungi strain A10 that resulted from agarase screeningwith the highest activity was isolated from sea water of Pari Island, Seribu Islands, Jakarta.The isolates morphology is green color, massive growth on live media and has a conidia shape like a branching stem.DNA identification results showed that the isolates has similarity with the species Hypocreaceae sp. which a member of kingdom of fungi, division of Ascomycota, class of Sordariomycetes, order of Hypocreales, family of Hypocreaceae.This is a freely living species in soil environment.

Acid and temperature characteristics
Acid and temperature characteristics are parameters that can affect the activity of enzymes in hydrolysis process.Effects of pH and temperature in agarase enzyme activity can be seen in Figures 2  and 3. Figure 2 shows that agarase activity was gradually rising at pH 3-5 acetate, then decreased quite sharply at pH 5-7 phosphate and increased back sharply at pH 7-9 TrisHCl.Theoptimum agarase enzyme activity was found at pH 8.0 Tris HCl, which indicates that the agarase enzyme produced from Hypocreaceae sp.having activity at alkaline pH condition.Some studies show that optimum agarase activity of microorganisms are in the alkaline pH, 7.0 for B. cereus (Suzuki et al., 2002) and 9.0 for Pseudomonas sp.(Gupta et al., 2013).Lakshmikanth et al. (2006) stated that the average agarase will have optimal activity at pH ranging from 6.5 to 9.0.Enzymes will show the maximum catalytic activity at a certain of pH range.Changes in pH will affect the amino and carboxyl groups of the enzyme protein.Extreme pH values can cause enzyme be denatured that causes the enzyme loses its biological activity.
Effect of temperature on agarase activity can be seen in Figure 3 (Suzuki et al., 2002), B. megaterium (Khambhaty et al., 2008), and Acinetobacter sp.(Lakshmikanth et al., 2006) are all at a temperature between 30-50 O C. Temperatures can affect the enzyme activity since increased temperatures can accelerate a reaction.Decreased in enzymes activity that occurred above a temperature of 50 O C was due to interruption of the secondary bond enzyme because of the large kinetic energy of the molecules of the enzyme, resulting in the loss of the secondary and tertiary structure of the enzyme.Heat can also cause the loss of most of a less strong bond on the structure of the enzyme protein.While the decline in enzyme activity at temperatures below 50 O C was due to the low affinity between enzyme with substrate so that the hydrolysis process was not running perfectly and enzyme activity was decreased.

Enzyme stability
The enzyme stability measurement was done to see how long the enzymes can work optimally.The stability of enzymes is highest at 24 th hour and have a slow decline at 48 th and 72 nd hour.It was indicated that the enzyme will work optimally to hydrolyze a substrate at 24 th hours (Figure 4.).The best concentration of enzyme that produced highest activity was 10%.Faster stability time of agarase enzyme work has been fulfilled by the substrate at 24 th hour, so that the enzyme activity will decrease after 24 th hour.Increasing the enzyme concentration does not affect activity of enzyme produced.This is due to very high concentration of enzyme would not be meaningful because all substrate molecules have been hydrolyzed by lower concentrations.

Molecular weight (SDS page)
Determination of molecular weights was conducted based on the SDS standard curve with the equation of y= -1,5311x + 2.2827, where y= log molecular weight marker (kDa), while x= relative mobility of proteins (cm).Molecular weight was determined by taking the anti-log y value.Based on the results visualization on a gel it was known that there were two bands, 39 kDa and 44 kDa (Figure 5).Molecular weights obtained in this research are low.Several studies have shown that the agarase enzyme having such a low molecular weight of 39.5 kDa from Alteromonas sp.(Wang et al., 2006), 33 kDa from Pseudoalteromonas antarctica, 32 kDa from Pseudomonas atlantica (Morrice et al., 1983), and 20 kDa from Vibrio sp.(Aoki et al., 1990).Based on molecular weight classification agarase enzyme it was known that agarase produced by S. cucurbitacearum is included in the classification 2 (≤50 kDa) (Jonnadula and Ghadi, 2011).

Fermentation
of G.latifolium thatwas previously hydrolyzed using agarase enzyme showed that ethanol content are 0.88% compare to acid hydrolize (Figure 6.).Obtained ethanol was the result of distillation process, so it is not yet purify.The result of fermentation showed that ethanol content are 0.88% and it is higher than result of fermentation of G. latifolium by using acid (1% v/v) that produce ethanol only 0.50% (Kawaroe et al., 2015).This indicates that the use of agarase enzymes to hydrolyze G. latifolium. in the bioethanol production process is more effective than acid.In hydrolysis process, agarase role is to break down the molecules so that the β-1,4 position gives a neoagarobiose and converted into galactose.For β it istemporary, while for α will break at the α-1,3 positions resulting agarobiose and converted into galactopyranose (Khambhaty et al., 2008;Fu and Kim 2010;Chi et al., 2012).Results of the enzymatic hydrolysis then fermented by S. cerevisiae to produce bioethanol.Nguyen et al. (2012) stated Figure 6.Concentration of Bioethanol that bioethanol can be produced from the fermentation of all materials that contain sugar.The fermentation process is a biological process in which sugar molecules are converted into cellular energy and also produces ethanol as a byproduct with the help of yeast.Fermentation is basically breaking down one molecule of glucose into two molecules of pyruvate.Pyruvic acid molecules produced will be used by the yeast to produce energy.In anaerobic condition, pyruvic acid is converted to acetaldehyde and then to ethanol (Fardiaz, 1989).The fermentation process is basically influenced by medium, temperature, microorganisms, nutrients and pH substrate (Saroso, 1998).

Conclusion
Conclusion from this research is crude extract of agarase enzyme from Hypocreaceae sp.isolates has optimum activity at pH 8.0 (Tris-HCl), temperature of 50 O C, and enzyme stability at 24 th hour.Adding agarase enzyme to fermentation process is capable to produce bioethanol from G. latifolium up to 0.88% and higher than the fermentation using acid 0.50%. .

Figure 1 .
Figure 1.Phylogenic tree based on 16S rDNA sequence analysis showing the position of Hypocreaceae sp.and A10

Figure 3 .
Figure 3.Effect of temperature on agarase activity isolated from Hypocreaceae sp . The figure shows that there is a slow decrease of anzyme activity at a temperature of 20-40 O C, then increase very sharp at 50 O C and decline at 60 o C. Optimum activity of agarase enzyme isolated from Hypocreaceae sp.occurs at temperature of 50 O C. So that maximum activity of agarase enzyme was obtained at a temperature of 50 O C. Jonnadula and Ghadi (2011) stated that the average marine microorganisms has agarase activity at the optimum temperature range of 30-50 O C at intervals of 5 O C. Optimum agarase enzyme activity produced by B. cereus